Advances in Biomedical Research – from COVID to Medical Humanities

Editors: Łukasz Biały Izabela Młynarczuk-Biały

Lublin-Warszawa 2020

Wydawnictwo Naukowe TYGIEL would like to thank the Reviewers for their involvement in the reviews and substantive guidelines for the authors.

Reviewers:  prof. dr hab. Anna Jarosz-Wilkołazka  dr n. farm. Anna Biernasiuk  dr hab. Paweł Glibowski  dr Maciej Frant  dr hab. n. med. Ludmiła Grzybowska-  dr n. farm. Ewa Gibuła-Tarłowska Szatkowska  dr n. o zdr. Mariola Janiszewska  dr hab. n. med. Paweł Kalinowski  dr n. med. Łukasz Pilarz  dr hab. Roman Paduch, prof. UMCS  dr n. med. Ewa Rojczyk  dr n. o zdr. Kinga Augustowska-  dr n. med. Sylwia Smolińska Kruszyńska

All published chapters received positive reviews.

Editors:  Łukasz Biały, Department of Histology and Embryology, Medical University of Warsaw  Izabela Młynarczuk-Biały, Department of Histology and Embryology, Medical University of Warsaw

Typesetting: Monika Maciąg

Design: Marcin Szklarczyk

Publishers:

ISBN 978-83-66489-44-8 ISBN 978-83-7637-552-6

Wydawnictwo Naukowe TYGIEL sp. z o.o. Warszawski Uniwersytet Medyczny, ul. Głowackiego 35/341, ul. Żwirki i Wigury 61, 02-091 20-060 Lublin Warszawa www.wydawnictwo-tygiel.pl

Table of Contents

The experience on coronavirus disease 2019 and lung cancer from Regional Center of Pulmonology in Bydgoszcz ...... 7

MALDI-TOF Serotyping of Bacterial Bfo1 Variants...... 16

Modern technologies in forensic medicine ...... 35

Neuromyelitis optica (NMO) – a challenging demyelinative disorder ...... 43

Gluten intolerance – the complexity of the problem, the heterogeneity of clinical picture – diagnosis and treatment ...... 52

Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview ... 60

Redox homeostasis in melanoma cells treated with caffeic acid ...... 88

Applications of alginates in the biomedical field ...... 97

The effect of periodontal disease on acute cardiovascular events: a preliminary report ...... 113

Implementation of Geneticin in the in vitro cell culture and in vivo studies ...... 122

Cardiac fibrosis, its causes and consequences ...... 134

Review: Role of pregnancy in eye functioning...... 142

Angiogenesis and pulmonary vascularisation disorders in lung hypoplasia in the course of Congenital Diaphragmatic Hernia ...... 149

The role of melatonin and noradrenaline interaction in REM sleep phase modulation ...... 161

Annexin 5 as a multifunctional marker in biomedical research ...... 173

Body donation as the basis for advance in medicine in past and today – meaning, principles, controversies ...... 187

An image of a first-year student at the Faculty of Medicine. Qualitative research based on written work...... 199

Index of authors ...... 212

The experience on coronavirus disease 2019 and lung cancer from Regional Center of Pulmonology in Bydgoszcz

Grzegorz Przybylski1*, Marta Pilaczyńska-Cemel1, Anna Kozanecka1 1 Department of Respiratory Medicine and Tuberculosis, Nicolaus Copernicus University in Torun, Collegium Medicum in Bydgoszcz *Corresponding author: Dr hab. Grzegorz Przybylski, Prof. UMK, Departament of Respiratory Medicine and Tuberculosis, Nicolaus Copernicus University in Torun, Collegium Medicum in Bydgoszcz, Seminaryjna Street 1, 85-326 Bydgoszcz, Phone number: 052-3256781, fax: 052-3256606, Mobile phone: 602-573638, e-mail: [email protected] ABSTRACT In December 2019, an outbreak of 2019 novel coronavirus disease (COVID-19) occurred in Wuhan, Hubei, which has been linked to the severe adult respiratory syndrome coronavirus 2 (SARS-CoV-2). The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related disease (coronavirus disease 2019 [COVID-19]) has spread rapidly to a pandemic proportion. It is characterized by rapid human-to-human transmission from droplet contamination. As of May 21, 2020, 20143 COVID-19 cases and 972 deaths have been reported in Poland, representing a global share of 0.41% and 0.29% for incidence and mortality, respectively. We extracted data from the World Health Organization's progress reports. In addition, we retrieved clinical data on patients with cancer and with confirmed COVID-19 in Regional Center of Pulmonology in Bydgoszcz. Here, we report the incidence and outcomes of SARS- CoV-2 infection in cancer patients who were treated at our institution.We reviewed the medical records, including demographic, clinical, and treatment data of 23 patients who were admitted to the Regional Center of Pulmonology, from March 25, 2020, to May 21, 2020 (data cutoff date). COVID-19 pneumonia was diagnosed based on the updated COVID-19 Diagnostic Criteria. Outcomes of COVID-19 among patients with lung cancer were reported and was confirmed in 6 cases (6/23). The median age of infected patients was 69 years (range, 51 to 92 years); 4 of 6 patients (66.7%) were older than 60 years. Cancer has been reported as a major risk factor for adverse outcomes of and death from COVID-19. Keywords: Cancer, COVID-19, SARS-CoV-2, Coronavirus INTRODUCTION Coronavirus disease 2019 (COVID-19), caused by the newly identified strain of the coronavirus family severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly evolved into a worldwide pandemic and caused a public health emergency of major international concern (Shi, Han et al. 2020, Chen, Zhou et al. 2020). In December 2019, an outbreak of respiratory disease caused by a novel coronavirus was first detected in China and has now spread to more than 150 countries (Woldometer, 2020). The virus was named severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and has a phylogenetic similarity to SARS‐CoV‐1 that caused the SARS pandemic in 2002 (Guan, Ni, 2020). This new type of respiratory illness is characterized by rapid human‐to‐human transmission, having achieved pandemic spread (Yu, Ouyang et al. 2020). There are currently no therapeutics or vaccines available and, presumably, no pre‐existing immunity in the population. With the World Health Organization (WHO) declaring the novel coronavirus outbreak a pandemic, focus is needed on the impact of this rapidly spreading viral infection on

Grzegorz Przybylski, Marta Pilaczyńska-Cemel, Anna Kozanecka cancer patients (Anderson, Heesterbeek et al. 2020, WHO Director, 2020, WHO, Coronavirus, 2020). Patients with cancer are more susceptible to infection than individuals without cancer, because malignancy and anticancer therapy result in an immunosuppressive state (Liang, Guan 2020). In a retrospective study during the 2009 influenza A (H1N1) virus pandemic, the cancer patient population was at higher incidence of pneumonia (66%) and 30‐day mortality (18.5%) compared with the general population (Dignani, Costantini et al. 2014) 10. A recent small case series study that evaluated SARS‐CoV‐2 in cancer patients found that patients with cancer had worse outcomes from SARS‐CoV‐2 than other individuals without cancer (Liang, Guan et al. 2020). This comprehensive reallocation of health resources is of particular concern in patients such as those with underlying chronic diseases, including cancer. In this context, the threat of COVID-19 infection might also factor into decision making – a role which could possibly be lessened by knowledge of the COVID-19 status of patients suitable for anticancer therapy (Ueda, Martins et al.2020). This already dismal scenario seems to be even more severe for patients with lung cancer because of the high risk of interference of COVID-19 with their effective diagnostic and therapeutic management by treating physicians. Limited studies and research regarding preparedness plans for the patients with during an infectious pandemic exist (Battershill, 2006, ASCO, 2020). In this review, we aim specifically to address challenges associated patients with COVID-19 and concomitant lung cancer during the COVID‐19 pandemic. MATERIALS AND METHODS PATIENTS We reviewed the medical records, including demographic, clinical, and treatment data of 23 patients who were admitted to the Regional Center of Pulmonology, from March 25, 2020, to May 21, 2020 (data cutoff date). COVID-19 pneumonia was diagnosed based on the updated COVID-19 Diagnostic Criteria. We had patients with pneumonia of unknown cause, which was identified as SARS- CoV-2 soon after. In this program, all consecutive patients with confirmed COVID-19 admitted to the Regional Center of Pulmonology in Bydgoszcz from March 25, 2020, to May 21, 2020 were enrolled. All patients with COVID-19 enrolled in this study were diagnosed and admitted in accordance with the guideline of the polish recommendations (Flisiak, Horban et al. 2020, Zalecenia, 2020). The final date of follow-up was May 21 2020. Of these 23 patients with Covid-19, 6 confirmed lung cancer, which was diagnosed in four patients during current hospitalization, in two with this diagnosis established earlier.

8 The experience on coronavirus disease 2019 and lung cancer from Regional Center of Pulmonology in Bydgoszcz

DATA COLLECTION We reviewed clinical charts, nursing records, laboratory results for all patients. Epidemiological, clinical, imaging, and serological records and treatment and outcomes data were collected from the electronic medical network of hospital. REAL-TIME REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION (RT-PCR) TESTS The confirmation of COVID-19 is achieved by RT-PCR detection of throat swab samples of suspected patients. by the Polish recommendation (Zalecenia, 2020). STATISTICAL ANALYSIS Categorical variables were reported as number and percentages, and comparisons between groups were made using the Chi-squared test. Nominal two-tailed statistical significance was set at 0.05. All analyses were performed using programmes Statistica and Excel. RESULTS The enrolled 23 patients were all confirmed infected with SARS-CoV-2 with PCR tests of throat swabs. The median age of the patients was 66,6 years (35.0-92.0) (tab. 1). Median age were similiar in both group. The median age of lung cancer group was 64 years, whereas the median age without lung cancer group was 67 years. 9 patients (39%) were men, and 14 patients (61%) were women. In group patients with lung cancer domniated women (83%). Of the 23 patients, almost all had comorbidities (21 [91%]), including malignancy (6 [26%]), hypertension (9 [39%], cardiovascular disease (8 [35%]), and diabetes (7 [30%]), and so forth. Patients of malignancy group showed more underlying comorbidities when compared non malignancy group, such as hypertension (4 [67%] vs 5 [29%], P < 0,001). The incidence of other comorbidities was comparable in both groups.

Table 1. Characteristics and Symptoms of Patients Infected With SARS-CoV-2 All Patients (N With lung cancer Without Lung cancer P = 23) (n = 6) (n = 17) Value Age (y) 66,6 (35-92) 64,0 (51- 92) 67 (35 - 90) NS

Sex

Male 9 (39%) 1 (17%) 8 (47%) <.001

Female 14 (61%) 5 (83%) 9 (53%)

Comorbidity 21 (91%) 6 (100%) 15 (88%) NS

Malignancy 6 (26%) 6 (100%) 0 (0%) <.001

Hypertension 9 (39%) 4 (67%) 5 (29%) <.001

Grzegorz Przybylski, Marta Pilaczyńska-Cemel, Anna Kozanecka

All Patients (N With lung cancer Without Lung cancer P = 23) (n = 6) (n = 17) Value Cardiovascular disease 8 (35%) 3 (50%) 9 (53%) NS

Diabetes 7 (30%) 2 (33%) 6 (35%) NS

Chronic obstructive Pulmonary 4 (17%) 2 (33%) 4 (24%) NS disease hepatopathy 1 (4%) 0 (0%) 1 (6%) <.001

Fever at onset of illness 17 (74%) 4(67%) 13 (76%) NS

Symptoms at onset of illness

Cough 20 (87%) 5 (83%) 15 (88%) NS

Dyspnea 20 (87%) 5 (83%) 15 (88%) NS

Fatigue 18 (78%) 3 (50%) 15(88%) .499

Sputum production 11 (48%) 3 (50%) 8 (47%) NS

Chest pain 6 (26%) 3 (50%) 3 (18%) <.001

Oppression in chest 6 (26%) 4 (67%) 2 (12%) <.001

Anorexia 4 (17%) 3 (50%) 1 (6%) <.001

Myalgia 4 (17%) 1 (17%) 3 (18%) NS

Headache 3 (13%) 1(17%) 2 (12%) NS

Palpitation 3 (13%) 1 (17%) 2 (12%) NS

Diarrhea 2 (9%) 1(17%) 1 (6%) NS

Vomiting 2 (9%) 1 (17%) 1 (6%) NS

The most common clinical feature at the onset of illness was fever (17 [76%]). Other common clinical manifestations included cough (20 [87%]), dyspnea (20 [87%]), and fatigue (18 [78%]). Less common symptoms were sputum production, oppression in chest, dyspnea, diarrhea, headache, and so forth (tab. 1). Compared with the non lung cancer group, patients of the lung cancer group tend to show more frequency of chest pain, oppresion in chest and anorexia. The frequency of these symptoms among patients with lung cancer can also be explained by the advancement of cancer. The blood counts of patients with lung cancer on admission showed decrease in white blood cells, neutrophils and lymphocytes concurrently to patients of non lung cancer group. The patients with lung cancer showed increase in Lactate dehydrogenase and decrease of aspartate aminotransferase, C-reactive protein compare to patient without lung cancer (tab. 2).

10 The experience on coronavirus disease 2019 and lung cancer from Regional Center of Pulmonology in Bydgoszcz

Table 2. Laboratory Records of Patients Infected With SARS-CoV-2 on Admission All Patients With lung cancer Without Lung cancer P (N = 23) (n = 6) (n = 17) Value White blood cell count, 3.82 (2.98-3.57) 3.57(2.96-4.93) 6.52 (4.30-7.73) .006 ×109/L

Neutrophil count, ×109/L 2.35(1.62-367) 2.16(1.60-2.70) 5.24 (2.90-6.44) <.001

Lymphocyte count, ×109/L 1.15 (0.82-1.46) 0.61 (0.37-1.00) 1.19 (0.95-1.46) .002

Monocyte count, ×109/L 0.31 (0.23-0.44) 0.31 (0.24-0.46) 0.27 (0.14-0.41) NS

Eosinophil count, ×109/L 0.01 (0.00-0.02) 0.01 (0.00-0.02) 0.00 (0.00-0.01) NS

130.00 (118.00- 131.00 (121.00- Hemoglobin, g/L 128.00 (117.00-136.00) NS 140.00) 141.00)

171.00 (142.00- 172.00 (138.00- Platelet count, ×109/L 167.00 (144.00-215.00) NS 211.00) 206.00)

Alanine aminotransferase, 25.00 (17.00-40.00) 24.00 (16.00-40.00) 31.50 (23.00-52.00) NS U/L

Aspartate aminotransferase, 28.00 (22.00-42.00) 26.00 (21.00-39.00) 40.50 (24.00-62.00) .03 U/L

224.00 (183.00- 517.50 (267.00- Lactate dehydrogenase, U/L 207.00 (181.00-274.00) .001 291.00) 549.00)

C-reactive protein, mg/L 13.20 (6.78-49.00) 11.30 (6.53-26.30) 81.55 (48.85-105.90) <.001

Procalcitonin, μg/L 0.13 (0.13-0.15) 0.13 (0.13-0.15) 0.13 (0.13-0.15) NS

So they were not enrolled in the analysis of treatment and prognosis in table 3. The median time from onset of symptoms to admission was 6.0 days. The all of patients needed oxygen suport. All patients received in accordance with Polish recommendations. Death occurred significantly more frequently in the group of patients with lung cancer.

Table 3. Treatments and Outcomes of Patients Infected With SARS-CoV-2 All Patients With lung cancer Without Lung cancer P Value (N = 23) (n = 6) (n = 17) Onset of symptom to 6.0 (4.0-9.0) 6.0 (4.0-9.0) 7.0 (4.0-9.0) NS admission

Oxygen support 23 (100%) 6 (100%) 17 (100.0%) NS

Death 7 (30%) 3 (50%) 4 (24%) <.001

Grzegorz Przybylski, Marta Pilaczyńska-Cemel, Anna Kozanecka

DISCUSSION Our experience with these 23 patients confirms that COVID-19 is a kind of epidemic pneumonia with fever, dry cough, and fatigue as the most common onset symptoms. Most patients have mild manifestations and excellent prognosis. However, in our group of patients with lung cancer and COVID, the prognosis was significantly worse. Cancer patients with SARS-CoV-2 infection may have increased morbidity and mortality from COVID-19 than noncancer patients with SARS-CoV-2 infection. Accumulating evidence suggests that cancer patients are at higher risk of COVID‐19 infection and more likely to have higher morbidity and mortality than the general population. In a study with a total of 1,524 patients with cancer, cancer patients had a twofold increased risk of COVID‐19 infection when compared with the general population (Yu, Ouyang et al. 2020). In another series from a single institution in the Wuhan region, the infection rate of SARS‐CoV‐2 in patients with cancer was 0.79% (95% CI = 0.3-1.2), which was higher than the cumulative incidence of all diagnosed COVID‐19 cases that was reported over the same time period (0.37%, 41,152/ 11,081,000 cases, data cutoff on February 17, 2020) (Yu, Ouyang et al. 2020). The Chinese Center for Disease Control and Prevention described the epidemiological characteristics of 72,314 COVID‐19 cases in mainland China as of February 11, 2020. They reported that 107 patients (0.5%) had cancer, and 6 of them died. The case fatality was 5.6%, which is higher than the overall reported case fatality (2.3%) from COVID‐19 (Novel, 2020). Similarly, the WHO-China Joint Mission on COVID‐19 identified significantly higher case fatality amongst patients with pre‐existing malignancy (7.6%) compared with patients without comorbid conditions (1.4%) (Report,2020). In the series by Liang et al., cancer was associated with higher risk of severe events (i.e., admission to the intensive care unit, invasive ventilation, or death seen in 7 of 18 patients [39%] with cancer vs. 124 of 1,572 patients [8%] without cancer; p = .0003) (Liang, Guan et al. 2020). These findings have been corroborated internationally, as an Italian study assessing the case fatality of COVID‐19 found that amongst 355 patients who died and underwent detailed chart review, 72 (20.3%) had active cancer (Onder, Rezza et al. 2020). While these analyses are preliminary and require validation from larger international cohorts, several factors could account for an elevated risk for acquiring COVID‐19 and consequent complications amongst cancer patients, including frequent hospital visits and admissions, immunocom- promised state, advanced age, and poor functional status (Yu, Ouyang et al. 2020). In our study the small study sample size, patients in cancer group characterized similiar results a higher death rate and more severe course. While having cancer and receiving certain cancer therapies remain plausible risk factors for both contracting SARS-CoV-2 infections and having more severe COVID-19 outcomes, existing data do not yet answer these questions. Notably, the early publications in this area include data from a very small number of patients, but have nonetheless had substantial effects. The cited study by Liang et al. is often used as justification for ‘a possible increased risk’ associated with chemotherapy, despite only two patients in this analysis receiving systemic chemotherapy within the month before COVID-19 diagnosis. Data from Yu et al. have been used to support universal

12 The experience on coronavirus disease 2019 and lung cancer from Regional Center of Pulmonology in Bydgoszcz screening of patients with lung cancer for SARS-CoV-2 infections, even though this cohort contained seven patients with lung cancer, six of whom did not have laboratory- confirmed COVID-19 (Yu, Ouyang, 2020). It is difficult to imagine any other context in which data from such small, highly selected, and often flawed case series would be published in major journals and have such a substantial influence on clinical practice and policy. Patients with lung cancer usually have compromised lung function with associated dyspnea, cough, and polypnea. They might be at higher risk of severe forms of COVID‐19 infection due to decreased pulmonary function. In the aforementioned studies that evaluated COVID‐19 in a small cohort of patients with cancer, the investigators found that lung cancer was the most frequent type of malignancy in this cohort of COVID‐19 – infected patients (five [28%] of 18 patients). Whether this reflects a true increase in the susceptibly of the lung cancer population to SARS‐CoV‐2 infection or is simply due to the fact that lung cancer is the most cancer in China is yet to be determined (Feng, Zong, 2019). Lastly, other potential causes for the respiratory deterioration of NSCLC patients, which could mimic COVID‐19 symptoms, must be considered; these may include obstructive pneumonia, pleural or pericardial effusion, pulmonary embolism, and heart failure; therefore, rapid access to SARS‐CoV‐2 assays is of utmost importance. Postponing anticancer treatment should be considered according to the patient's risk of infection, performance, and clinical status. Currently, there are no clinical trials to examine the safety and efficacy of antiviral prophylaxis for SARS‐CoV‐2 in cancer patients. COVID-19 and the cancer patient is based on the latest information and knowledge available to the medical community at this time. As the COVID-19 pandemic continues to evolve and unfold, it is likely that the health care community will be faced with additional, yet unknown challenges. It is imperative that we stay abreast of all deve- lopments with COVID-19 to provide our most vulnerable COVID-19 and the cancer patient with the care needed for their best chance of to optimal health care. As the size of this cohort is limited, the statistical analysis results should be interpreted with caution, and the P value without statistical significance does not necessarily reflect the exact situation of the whole population. Larger sample size of clinical studies is needed to elucidate the epidemiology, clinical characteristics and prognostic factors of COVID-19. Moreover, due to the outcomes data of patients have shown that, the prognosis comparation between the lung cancer group and the nonlung cancer group is much worse. CONCLUSIONS The study groups infected with SARS-CoV-2 (with and without lung cancer) differed significantly in several aspects. In group of patients with lung cancer dominated women. Patients of malignancy group showed more underlying comorbidities, such as hypertension and hepatopathy. Patients of the lung cancer group tend to show more frequency of chest pain, oppression in chest and anorexia. Another difference was blood laboratory analysis. The lung cancer group showed decrease in white blood cells, neutrophils, lymphocytes, aspartate aminotransferase and C-reactive protein compared

Grzegorz Przybylski, Marta Pilaczyńska-Cemel, Anna Kozanecka to the non-lung cancer group. The most important difference is the mortality rate in the studied groups. Death occurred significantly more frequently in the group of malignancy patients. Considering the above aspects, it is safe to say that the coexistence of COVID-19 and lung cancer may change the diagnosis, treatment and prognosis of both diseases. SUMMARY Our review on COVID-19 and the cancer patient is based on the latest information and knowledge available to the medical community at this time. As the COVID-19 pandemic continues to evolve and unfold, it is likely that the health care community will be faced with additional, yet unknown challenges. Lung cancer associated with COVID-19 is a disease that significantly worsens prognosis. Therefore, patients should be isolated and protected against potentially possible coronavirus disease infection (COVID-19). Early diagnosis, timely isolation, and appropriate treatment are the keys in fighting this infection. REFERENCES Anderson R.M., Heesterbeek H., Don Klinkenberg, Déirdre Hollingsworth T., How will country‐based mitigation measures influence the course of the COVID‐19 epidemic?, Lancet, 2020; 395: 931-934. ASCO COVID‐19 Clinical Oncology Frequently Asked Questions (FAQs), Last updated – March 12, 2020. Available at https://www.asco.org/sites/new‐www.asco.org/files/content‐files/ blog‐release/pdf/COVID‐19‐Clinical%20Oncology‐FAQs‐3‐12‐2020.pdf. Accessed March 14, 2020. Battershill P.M.. Influenza pandemic planning for cancer patients, Curr Oncol, 2006; 13: 119-120. Chen N., Zhou M., Dong X., Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study, Lancet, 2020;395:507-513. Dignani M.C., Costantini P., Salgueira C., Jordán R., Guerrini G., Valledor A. et al., Pandemic 2009 influenza A (H1N1) virus infection in cancer and hematopoietic stem cell transplant recipients; a multicenter observational study, F1000Res, 2014; 3: 221. Feng R.M., Zong Y.N., Cao S.M., Xu R.H., Current cancer situation in china: Good or bad news from the 2018 global cancer statistics?, Cancer Commun (Lond), 2019; 39: 22. Flisiak R., Horban A., Jaroszewicz J., Kozielewicz D., Pawłowska M., Parczewski M. et al., Management of SARS-CoV-2 infection: recommendations of the Polish Association of Epidemiologists and Infectiologists as of March 31, 2020, Pol Arch Intern Med., 2020; 130: 352-357. doi:10.20452/pamw.15270. Guan W.J., Ni Z.Y., Hu Y., Liang W.H., Ou Ch., He J. et al., Clinical characteristics of coronavirus disease 2019 in China, N Engl J Med., 2020 Feb 28 : NEJMoa2002032. Published online 2020 Feb 28. doi: 10.1056/NEJMoa2002032. Liang W., Guan W., Chen R., Cancer patients in SARS‐CoV‐2 infection: A nationwide analysis in China, Lancet Oncol, 2020; 21: 335-337. Novel Coronavirus Pneumonia Emergency Response Epidemiology Team. The epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (COVID‐19) in China [in Chinese], Zhonghua Liu Xing Bing Xue Za Zhi 2020;41:145-151. Onder G, Rezza G, Brusaferro S., Case‐fatality rate and characteristics of patients dying in relation to COVID‐19 in Italy, JAMA, 2020 [Epub ahead of print].

14 The experience on coronavirus disease 2019 and lung cancer from Regional Center of Pulmonology in Bydgoszcz

Report of the WHO–China Joint Mission on Coronavirus Disease 2019 (COVID‐19), Available at https://www.Who.Int/docs/default‐source/coronaviruse/who‐china‐joint‐mission‐on‐covid‐19‐final‐report.p df. Accessed March 12, 2020. Shi H., Han X., Jiang N., Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study, Lancet Infect Dis, 2020; 20:425-434. Ueda M., Martins R., Hendrie P.C., Managing cancer care during the COVID-19 pandemic: agility and collaboration toward a common goal, J Natl Compr Canc Netw. 2020 doi: 10.6004/jnccn.2020.7560. published online March 20. World Health Organization. WHO Director‐General's opening remarks at the media briefing on COVID‐19, March 11, 2020. Available at https://www.who.int/dg/speeches/detail/who‐director‐general‐s‐opening‐remarks‐at‐the‐media‐briefing‐on‐ covid‐19—11‐march‐2020. Accessed March 11, 2020. World Health Organization. Coronavirus disease (COVID‐19) Pandemic, Available at https://www.who.int/emergencies/diseases/novel‐coronavirus‐2019, Accessed March 11, 2020. Worldometer. COVID‐19 Coronovirus Pandemic, Available at https://www.worldometers.info/coronavirus/#countries. Accessed March 16, 2020. Yu J., Ouyang W., Chua M.L.K., SARS‐CoV‐2 transmission in cancer patients of a tertiary hospital in Wuhan, JAMA Oncol, 2020 [Epub ahead of print]. Zalecenia w COVID-19 Polskie zalecenia diagnostyczno-terapeutyczne oraz organizacyjne w zakresie opieki nad osobami zakażonymi lub narażonymi na zakażenie SARS-CoV-2, Agencja Oceny Technologii Medycznych i Taryfikacji z 25 kwietnia 2020 r.

MALDI-TOF Serotyping of Bacterial Bfo1 Variants

Piotr Franciszek Burzynski1* 1 Medical Biochemistry BSc (Hons), Medical School, Swansea University, Singleton Park, Sketty, Swansea SA2 8PP, United Kingdom *Corresponding author: Piotr Franciszek Burzynski, Medical School, Swansea University, [email protected] ABSTRACT The diversity of bacteria is very poorly known and it modulates rapidly, forming new species and new serotypes. Correct identification of bacterial strains and serotypes comes with several benefits, such as the ability to choose a specified healthcare in case of specific bacterial infection. Serotyping of bacteria can be time-consuming and expensive, and that is why this research aimed to find a new method, with Matrix- assisted laser desorption/ionization – Time of flight mass spectrometry (MALDI-TOF MS) as the main analytical apparatus. The same equipment was used before to address this purpose but with different methods used, which have produced inaccurate results. Research described here also faced several limitations as the experiments were completed within the limits of the undergraduate research-project module. This approach was based on the analysis of bacterial ribosomal proteins by the whole cell analysis. Two serotypes, "FoA" and "FoWT2", of bacterial symbiont BFo1 were used as the experimental organisms, and relative peak intensities were chosen as potential differing factors. Unexpectedly, this experiment produced significant results, which led to the conclusion that those two serotypes can be distinguished by comparing obtained mass spectra. However, other experiments proved that MALDI-TOF cannot be always used for serotyping, and even some of the BFo1 serotypes produce similar mass spectra. That is why I propose three novel potential experimental approaches, for sample preparation prior to the MALDI-TOF analysis. Analysed BFo1 serotypes proved that MALDI-TOF mass spectrometry can be considered as a potential analytical method for serotyping of bacteria. INTRODUCTION The diversity of bacteria which are present on planet earth is very poorly known (Dykhuizen, 1998). Different types of bacteria can be found in different environments, and within various organisms. Daniel E. Dykhuizen have estimated that thirty grams of forest soil may contain more than half a million species of bacteria (Dykhuizen, 1998). Some of those microorganisms have a pathogenic effect on humans, thus the identification of those types of bacteria should be cheap and quick, so the correct therapy can be applied. Different strains present different antibiotic resistance abilities. Nevertheless, ability of serotyping those microorganisms can be important. Here I present how mass spectrometry can be potentially used to serotype different variants of the same bacteria, BFo1. Bacterial symbionts BFo1 were analysed in this experiment, and those microorganisms derive from Frankliniella occidentalis, the western flower thrips (Facey et al., 2015). Bacterial symbionts can manipulate host’s reproduction, and can affect interactions with other species (Ferrari & Vavre, 2011). There are many variants of this bacteria, and the two analysed here are FoA, and FoWT2. Matrix-assisted laser desorption/ionization – Time of flight mass spectrometry (MALDI-TOF) was used before for the analysis and comparison of bacterial strains (Holland et al., 1999), but also for bacterial serotyping (Nakano et al., 2015). In this experiment bacterial proteins were observed from the whole cell analysis. Two analysed bacteria types were Shigella flexneri and Escherichia coli, and those two

MALDI-TOF Serotyping of Bacterial Bfo1 Variants types can be distinguished by comparing relative intensities of signals on MALDI-TOF mass spectra (Holland et al., 1999). Experiment described above was carried out over twenty years ago and today there are many other methods which can be used, to determine which species are present in the given sample. However, it is much harder to determine which serotype of the same bacteria is in the given sample. This experiment aimed to estimate whether it is, or it is not possible to distinguish different bacterial strains with the use of MALDI-TOF, by whole cell analysis, and by observing ribosomal proteins. It is important to note that this paper was originally written as a Dissertation describing a final year scientific project at the university, which was accomplished under the supervision of the academic staff at Swansea University. This report has several faults as the research was completed within the limits of the undergraduate research-project module (module code: PM-304, module name: Biomolecular Research Project (2020)). Nonetheless, described experiments proved that MALD-TOF can be potentially used for the serotyping of certain bacteria, but most importantly, here I propose certain experimental approaches for sample preparation prior to the MALDI-TOF analysis which are hypothesised to increase the accuracy and efficiency of the MALDI-TOF- based bacterial serotyping. Further and more comprehensive experiments are required to evaluate this method. MATERIALS AND METHODS Two analysed variants, FoA and FoWT2, were bacteria symbionts BFo1 of Frankliniella occidentalis. All samples were obtained as bacterial cultures, from microbiological lab, on petri dishes with unknown media. Those samples were stored in the fridge at temperature of four degrees Celsius, in the mass spectrometry laboratory. Matrix solution was prepared in the lab, in volume of 150 μl. 15 μg of 99% pure α-Cyano-4-hydroxycinnamic acid [α-CHCA] was added to the plastic vial. α-CHCA crystals were partially dissolved by adding 15 μl of Trifluoroacetic acid [TFA] to the vial, and by mixing. Then, 60 μl of distilled water, and 75 μl of 99.8% Acetonitrile [ACN] were added to the vial. The solution was mixed again, and then left on the stand undisturbed, to let the remaining α-CHCA crystals fall down to the bottom of the vial. The bacterial cells were carefully transferred from different spots of bacterial cultures in petri dishes, onto the four no-etched wells, of the target plate (stainless steel with 10 X 10 numbered sample positions), for each variant. Plastic tips for automatic pipette were used to transfer those cells. Bacterial spots on MALDI plate were covered with matrix solution droplets of 1 μl volume. Solution was taken from the top of the mixture without disturbing it, so undissolved crystals remained on the bottom of the vial, and so the transferred solution was pure and did not contain any solid fragments. One extra droplet was placed onto the ninth well to form a control sample. MALDI plate was left for the samples to dry out. Applied Biosystems Voyager DE-STR MALDI-TOF MS and the laser attached had a specified instrument settings. Default calibration, was completed with Sinapinic acid as calibration matrix. Number of laser shots, with laser intensity of 2104, and with

Piotr Franciszek Burzynski

Laser Rep Rate of 3.0 Hz, was set to 25 per spectrum. General Acquisition mass range was set from 1000 to 20000 Da. Low mass gate was 900 Da. Those settings were stored for each mass spectra in the saved results file, and were the same for each sample. Applied Biosystems Voyager DE-STR MALDI-TOF mass spectrometer was prepared for analysis, by setting the linear mode of operation, with delayed extraction mode. MALDI target plate with prepared samples was fixed in the apparatus. Samples were analysed by accumulation of the mass spectrums of three different spots with 25 shots each. That gave 75 shots per each final mass spectrum. Accumulated results were saved. Control sample was analysed in order to ensure that matrix solution was not contaminated, but the results were not saved. Saved results were analysed with "Applied Biosystems Sciex Data Explorer" software. X axis display range was changed for each result. New range is from 5000 to 15000 Daltons. Noise Filter was applied with Correlation Factor of "1" to all of the samples. Peak properties were saved and copied into the "Excel – Microsoft" file, and then statistical analysis was performed with the use of "SPSS – IBM" software. RESULTS Sample analysis with the MALDI-TOF provided sufficient data for the comparison of two serotypes. Blank spot on MALDI plate proved that there was no contamination in the prepared matrix. Accumulated data from other eight samples provided eight final results, which gives four for each serotype (fig. 1, fig. 2). Just by comparing visual properties of the mass spectrum, of each serotype, clear difference in relative peak intensities between FoA and FoWT2 can be noticed. Based on visual analysis, three protein signals were chosen for further analysis. Those signals are labelled as "A", "B", and "C" with corresponding average ion masses of 8296.64 Da, 8803.44 Da, and 9523.08 Da, respectively. BFo1 proteins which are represented by those peaks were not identified, and are not described in detail, in any database. With the use of Applied Biosystems Sciex Data Explorer software, values for the peaks were collected and reorganised in the table (tab. 1). Centroid Masses were formatted to whole numbers, as linear data in this mass range must be reported as whole mass unit. Two factors were chosen for further analysis, area under the peak, and relative intensity for each peak. For the two samples (FoA_3, and FoWT2_1) relative intensities were recalculated in relation to protein "A", as originally relative intensity of protein A in those two samples was not equal to 100, possibly due to the poor sample preparation at the transfer stage. Average values were calculated, using values of FoA analysis, for each protein for each factor, which are: area under the peak, relative intensity, and centroid mass. Same calculation process was applied for FoWT2 (tab. 1). Average centroid mass was calculated in order the prove that compared proteins are indeed the same in FoA, and in FoWT2. It was done by calculations in table 2 (tab. 2). Centroid mass was subtracted across the two serotypes. It was also done for other factors to determine the difference between FoA, and FoWT2 (tab. 2). It is clearly visible that relative intensities of protein "B", and "C" in relation to protein "A", are different for analysed BFo1 variants (tab. 2)

MALDI-TOF Serotyping of Bacterial Bfo1 Variants

Figure 1. Four Mass Spectrum results of BFo1 variant FoA, produced with "Applied Biosystems Voyager DE-STR MALDI-TOF" mass spectrometer

Piotr Franciszek Burzynski

Mass range is 5000 to 15000 Da. Noise filter was applied with Correlation Factor of 1, to all samples. Three proteins were chosen for analysis and are labelled as "A", "B", and "C".

Figure 2. Four Mass Spectrum results of BFo1 variant FoWT2, produced with "Applied Biosystems Voyager DE-STR MALDI-TOF" mass spectrometer

MALDI-TOF Serotyping of Bacterial Bfo1 Variants

Mass range is 5000 to 15000 Da. Noise filter was applied with Correlation Factor of 1, to all samples. Three proteins were chosen for analysis and are labelled as "A", "B", and "C".

Table 1. MS derived chosen parameters of FoA and FoWT2

Three chosen factors, regarding mass spectrums of the samples, were collected. Average values were calculated for each protein for each factor which are, area under the peak, relative intensity, and centroid mass, using values of FoA analysis. Same calculation process was applied for FoWT2. *For the two samples (FoA_3, and FoWT2_1) relative intensities were recalculated in relation to protein "A", as originally relative intensity of protein A in those two samples was not equal to 100.

Table 2. Mean values of MS derived chosen parameters of FoA and FoWT2 and the difference between serotypes

Centroid mass was subtracted across the two serotypes. It was also done for other factors to determine the difference between FoA, and FoWT2. Similar Centroid mass proved that analysed proteins are the same. First statistical analysis was performed for area under the peak, for each sample, comparing two BFo1 variants. By performing the T-test, with T.TEST function in Microsoft Excel, p-values were generated (tab. 3). The p-values for those three proteins, labelled as before, proved that area under the peak is not a significant factor for bacterial serotyping as values were higher than threshold of significance (< 0.05). That is the reason why area under the peak was not included or manipulated in following steps (tab. 3). Isomeric effect, and excessive "signal noise" which could be avoided by purification, are possible explanations, why the area under the peak was not significant as a factor.

Piotr Franciszek Burzynski

Table 3. Analysis results of the significance for difference in the area under the beak between FoA, and FoWT2

Statistical analysis of results for area under the peak, for each sample, comparing two BFo1 variants. p-values for three proteins labelled as "A", "B", and "C", proved that area under the peak is not a significant factor for bacterial serotyping, as values were greater than threshold of significance (< 0.05). Second step of statistical analysis compared "Relative Intensity" between FoA, and FoWT2. By performing the T-test, with T.TEST function in Microsoft Excel, p-values were generated (tab. 4). The p-values for this factor for proteins "B", and "C", proved that relative intensity is a significant differing factor for bacterial serotyping of FoA, and FoWT2 variants. Calculated p-values are lower than threshold of significance (< 0.05). Those results are visualised by box plots, generated by the use of "descriptive statistics" in SPSS software (fig 3). The p-value for protein "A" was not calculated at this stage of analysis as intensity of this protein is equal to 100 for each sample. This approach evolved to create new term "Calibration Protein" which will be used in the next sections of this paper. "Calibration Protein" is a protein with clear, and intensive signal on mass spectrum, to which the intensities of other protein signals are related to. Recalculating relative intensities, in relation to different Calibration Proteins are artificial ratios, and can exceed the value of 100. Use of Calibration Proteins may provide information about which protein is over- or under- expressed in which serotype, or sample.

Table 4. MS peak intensities for protein "B" and "C" in relation to protein "A"

MALDI-TOF Serotyping of Bacterial Bfo1 Variants

Statistical analysis of results for relative intensity, for each sample, comparing two BFo1 variants. p-values for three proteins labelled as "B", and "C", in relation to protein labelled as "A", proved that relative peak is significant as factor for bacterial serotyping. p-values of relative intensities for two proteins are smaller than threshold of significance (< 0.05).

Figure 3. Box plots of descriptive statistics in SPSS software as visualisation of the results in table 4. Box Plot A – Relative peak intensity (range) of protein labelled as "B" in relation to protein labelled as A. Box Plot B – Relative peak intensity (range) of protein labelled as "C" in relation to protein labelled as "A"

Relative intensities were recalculated by changing the Calibration Protein from protein "A", to "B". Recalculated relative intensity of protein "C" in relation to protein "B" provided useful information, for future conclusions, and data manipulations. By performing the T-test, with T.TEST function in Microsoft Excel, p-value was generated for new relative intensities of protein "C" (tab. 5). The p-value was much higher than threshold of significance (< 0.05), thus it was concluded that those two proteins (labelled "B" and "C") are expressed with similar relative ratios, for two analysed serotypes. Results were visualised with use of box plot (fig. 4).

Table 5. Recalculated MS peak intensities for protein "C" in relation to protein "B"

Recalculated relative intensity of protein labelled as "C" in relation to protein labelled as "B". p-value was generated for new relative intensities of protein "C". Obtained p-value was much greater than the threshold of significance (< 0.05).

Piotr Franciszek Burzynski

Figure 4. Box plot for relative intensity (range) of protein labelled as "C" in relation to protein labelled as "B" for both BFo1 variants

Taking the fact into consideration, that possibly two proteins labelled as "B" and "C", are expressed with similar relative ratios, for two analysed serotypes. It was concluded that it is protein "A" that is overexpressed in FoA serotype, or is under expressed in FoWT2 serotype. New relative intensities were calculated. Calibration Protein is the protein labelled as "B", thus the new ratios of protein "A" were recalculated in relation to protein "B" (tab. 6). The results were visualised with a bar graph (fig. 5).

Table 6. Recalculated MS peak intensities for protein "A" in relation to protein "B"

New relative intensities of protein labelled as "A" in relation to protein labelled as "B". Protein labelled as "B" is considered to be the Calibration Protein in this case. Those are four accumulated measurements for both FoWT2 and FoA.

MALDI-TOF Serotyping of Bacterial Bfo1 Variants

Figure 5. Visualisation of results in table 6. Protein labelled as "B" is a Calibration protein in this case. Graph represents relative intensities of protein peaks for both serotypes of BFo1

DISCUSSION ANALYSED RESULTS In the world, there are many bacterial species, and each contains various serotypes. In some way those serotypes are different, but the difference is usually not specified. Genetic analysis of bacteria may provide accurate results, when the serotype is being analysed, but it does not always provide detailed information about the structure, or contained compounds. MALDI-TOF mass spectrometry, on the other hand, may provide the data with partially detailed information about the difference between serotypes. Some of the chosen proteins, those labelled as "A", "B", and "C", are one of the most abundant proteins on the obtained mass spectrum (fig. 6). Thus it was concluded that analysed proteins are ribosomal proteins, as usually those are the most abundant in bacterial cells [Milo & Phillips, 2015]. It was estimated that in Escherichia coli (E. coli) one ribosomal protein (RplL) can exceed the number of 100,000 copies per cell. What is more, there are different, other proteins which are forming the structure of bacterial ribosomal subunits [Milo & Phillips, 2015]. This fact provided an approach for further analysis of the given serotypes. As seen in the "Results" section of this article, there are a few ways to analyse, and compare serotypes with the given data. Protein "A" had the highest initial relative intensity, meaning that it was the most abundant protein in the analysed mass range. Initially other relative intensities were calculated by Applied Biosystems Sciex Data Explorer software, automatically, in relation to this protein labelled with letter "A". Those results already proved that peak relative intensities of BFo1 variants are significant serotyping factors. However, comparing relative intensities between only two proteins does not provide information about which protein is over- or under- expressed. That is the reason why the third protein was investigated. Comparing the ratios between three or more different proteins may provide information about which protein is most likely to be over- or under- expressed. When comparing the relative

Piotr Franciszek Burzynski intensities between even more proteins, this structural relation can be estimated even more accurately. The chances are much higher that it is one protein that is under- or overexpressed, rather than other proteins are under- or overexpressed in the same ratio, when comparing them to this one protein. That is the reason why the term "Calibration Protein" was formed. Changing the protein to which other protein peaks have calculated relative intensity may provide more detailed results. In case of this experiment the ratio between protein "B", and "C" was calculated for each variant and it seems that those two proteins are most likely equally expressed in case of those two serotypes. Concluding this, third artificial ratio was calculated. Calibration Protein was protein labelled as "B", thus the new ratios of protein "A" were recalculated in relation to protein "B" (tab. 6). It provided results which are most likely to be true, but there was not enough number of proteins in each mass spectrum to compare, in order to determine that. Possible conclusion points out that it is the protein labelled as "A" that represents abnormal expression in one of the two serotypes.

Figure 6. Visual representation of the original peak relative intensities, of chosen proteins labelled as "A", "B", and "C". Bar Graph A – relative intensities for FoA. Bar Graph B – relative intensities for FoWT2. Peak intensity for each serotype was calculated in relation to protein labelled as "A" automatically, with exception of FoA_3 and FoWT2_1. For samples (FoA_3 and FoWT2_1) relative intensities were recalculated in relation to protein labelled as "A", as originally relative intensity of protein labelled as "A", in those two samples, was not equal to 100

Perhaps if the analysed samples could be purified prior to the experiment, for the protein analysis, area under the peak could be used to determine which protein, is under or overexpressed. As this experiment was based on the whole cell analysis, comparison of the area under the peak has not provided significant results. Peak areas did not follow the same trends as the relative intensities, possibly due to the isomeric shift of mass spectrum. It is worth mentioning that the experiment should be quick, cheap, and easy to carry out. That is why pre-analytical purification with affinity chromatography is not a preferable option. This research is focused on the whole cell analysis alone, thus purification would only be included if the planned steps failed to produce significant results. Perhaps, after purification, the experiment could provide significant results for area under the peak, when serotyping bacterial variants. If other variants would have been analysed, relative intensities could be compared further to determine whether protein is under- or overexpressed by chance. If five serotypes would have been analysed, and four of them would produce same relative intensities of peaks for protein "B" and "C" in relation to protein "A", and all five would produce

MALDI-TOF Serotyping of Bacterial Bfo1 Variants same relative intensity of peak for protein "C" in relation to protein "B", it would mean that one serotype is characterised by naturally overexpressed protein "A". In summary, analysing and comparing relative intensities of more protein peaks in one spectrum may provide information about which protein is under- or overexpressed in this one serotype, while, again, level of expression remains unknown. Then, by comparing more serotypes, it could be determined whether the known protein in question is under- or overexpressed in particular serotype. It can all be done just by comparing relative intensities for one mass spectrum, and by comparing protein ratios for different serotypes. For example, if a third serotype produced same ratios between proteins "A", "B" and "C" as serotype FoWT2, it would mean that, most likely, it is serotype FoA that represents abnormal, overexpression of protein labelled as "A". This potential result could be noted as differing characteristic factor for distinguishing FoA serotype from other BFo1 serotypes. If the similar factors could be found for other serotypes on the same basis, and then included in a table, a chart of differences between BFo1 serotypes could be formed. However, as other serotypes seemed to be the same when comparing the relative intensities between protein peaks, it would be necessary to apply additional steps for sample preparation. Planned experiment includes three stages for further experiments, of MALDI-TOF serotyping. Analysis of the mass spectra of this experiment suggests that FoA and FoWT2 serotypes can be distinguished. It was proved by statistical analysis. Those two BFo1 variants provide different results, and the abundance of ribosomal proteins in those variants is characterised by different ratios. It is a positive result, but it was not expected to be the case. Different variants of BFo1, which were analysed (results not shown), provided opposite results. Serotyping other variants of BFo1 may not be accurate or significant when using described simple method. This experiment had a second stage which was not completed due to the coronavirus outbreak (COVID-19), thus other variants of BFo1 were not properly analysed. Despite that, the research plan was completed, and this plan is focused on the manipulation of the ribosomal protein abundance in bacteria. Used equipment provided limited accuracy. What is more, there is a minor difference in the centroid mass for each analysed sample, for each protein. It is possibly the reason why the identification of those proteins, by comparing centroid mass with the masses from databases for BFo1 proteins, is impossible. Identification could provide a specified approach of targeting those proteins, in order to determine how different serotypes respond to the targeting compound. Otherwise, the plan includes a wider range of compounds which possibly can be used, and if one provides expected result, by binding target protein, it is no longer important what kind of protein it is. The most important part of this experiment is relative intensity between free target protein and bound target protein. The result of this kind would provide a significant approach of serotyping bacteria. Full understanding and analysis of observed proteins is not essential, but can provide ideas for the treatment or further action, if protein is investigated further. Results for FoA/FoWT2 comparison are still very useful, because various and known data manipulation methods could be applied in the following experimental stages.

Piotr Franciszek Burzynski

(FUTURE OUTLOOK) EXPERIMENTAL APPROACH 1 It is the simplest method for further analysis. It is also the method which, most likely, will not produce significant results. This experimental approach is a Heat Shock Response Test, and includes heating of the samples as a preparation method. This method requires preparation of the bacterial cell solutions in glass tubes. The same amount of bacterial cells would be added to the liquid solution and mixed, and the remaining media would be dissolved at the same time. Each sample would contain the same amount of compounds in the mixture, providing solutions with equal volume and density. After this preparation the "Specific Heat Capacity" of the control solution could be measured, by determining how much heat energy is required to rise one gram of solution one degree Celsius. As each tube would contain the solution of the same volume and density, the "Specific Heat Capacity" would be the same for any sample with different serotypes. It could be then calculated how much heat energy each portion of the cells has received, and thus it could be calculated how much heat energy was applied, per gram of analysed protein in MALDI-TOF. Having this noted, and conducted for all serotypes, but with different temperatures, it could be determined how different serotypes respond to certain amount of energy. In theory, one serotype could show different mass spectrum shift of proteins in relation to its control group, than different serotype in relation to its own control group, after the same amount of heat energy per gram of protein was applied to both of the analysed samples. Being more specific, it is possible that after certain amount of heat energy would be applied, one serotype could provide the results, presenting aggregated protein with centroid mass, twice as high as the centroid mass of unaggregated protein. It is possible that ratio of aggregation, or degradation in one serotype would be higher, than in other serotype, due to the different structure (different heat resistance). If specific heat resistance of the proteins could be determined, and would be different for each serotype. This would be a significant serotyping method with use of the MALDI-TOF mass spectrometry. (FUTURE OUTLOOK) EXPERIMENTAL APPROACH 2 The ribosome is the most complex enzyme. There are more than fifty different proteins and RNA components, which are forming the final structure of the ribosome [Douthwaite & Poehlsgaard, 2005]. Complexity and high abundance of those key- important structures provides multiple sites where the action or assembly of the ribosome can be disturbed. Correct protein synthesis is essential for all living organisms, thus breaking this process may easily cause lethal effects. That is why the ribosome, made of independent components with required particular arrangement to function, is considered to be the target for many antibiotic-based treatments [Douthwaite & Poehlsgaard, 2005]. Ribosome-targeting antibiotics lodge between crucial components, disrupting the mechanisms of operation. That is how those compounds interfere the synthesis pathways of new proteins, including the ribosomal proteins, and structural proteins for cell division [Douthwaite & Poehlsgaard, 2005]. Different antibiotics have different binding sites, but the mechanism of inhibition is often similar. Those binding sites are located within functionally important structures, in the ribosomal proteins or rRNA.

MALDI-TOF Serotyping of Bacterial Bfo1 Variants

Action of the antibiotics on bacteria, in general, is often efficient. Different bacterial species or even different bacterial serotypes, are presenting different antibiotic resistance. Among those abilities is the resistance to some of the widely used ribosome-binding antibiotics. It does not mean that, despite resistance, there is no efficiency of antibiotic-ribosome binding reaction. It may be entirely eliminated, but it may be also partially decreased. Luckily there is a wide range of ribosome-binding antibiotics available, and thus, the efficiencies can always be compared. This experimental approach is based on targeting ribosomal proteins directly, with the use of ribosome-binding antibiotics, and the hypothetical method is called "Trial with antibiotic-ribosome binding efficiency". In theory, different serotypes may present different resistance to some of the widely used ribosome-binding antibiotics. Most importantly, those antibiotics are targeting specified sites of specified structures. Antibiotic resistance can be correlated to the mutations of ribosomal proteins, regardless whether those were caused by previous ribosome-binding antibiotic presence, or not. When the binding site of antibiotic in ribosome is modified, the efficiency of reaction may not be as high as in more casual cases. Antibiotic resistance can also be correlated to base substitution, or methylation at the binding sites in tRNA [Douthwaite & Poehlsgaard, 2005]. Wobble hypothesis explained why modified nucleosides are paying crucial role in the protein assembly [Yarian et al., 2002]. Different organisms, are characterised by different amounts of certain modified nucleosides. Even for one organism, there may be a shift in amount or type of contained modified nucleosides, in certain specified conditions, such as cancer [Omran et al., 2019]. It is possible that different serotypes contain ribosomes which are made of partially modified proteins, and different rRNAs with different modified nucleosides. If this theory is true, each and different serotype may present different ribosome-antibiotic resistance, because, as mentioned before, action sites of those antibiotics are specified. This novel approach is based on the sample preparation by antibiotic application and incubation. After preparations, samples should be analysed with the use of MALDI- TOF. It is also possible that some of the chosen antibiotics are not going to penetrate the bacterial membrane and cell wall. This possibility is also included as a potential differing factor. The designated differing factor is a ratio between free analysed ribosomal protein and bound analysed ribosomal protein. As the molecular mass of all antibiotics used is known (tab. 7), the peak of bound ribosomal protein could be easily identified. If certain antibiotic would bind example protein "F", with centroid mass of 7500.00, and antibiotic would be added with corresponding mass of 300 per one molecule, then the peak for bound protein (ribosomal protein – antibiotic) would be equal to 7800.00. New peak would form, and thus, using relative intensities, the ratio of bound to free protein could be calculated. Calculated ratio would be related to ribosomal protein binding efficiency of the antibiotic in question. This ratio would be calculated for each antibiotic sample. If ratios were different, constant, and specific for each serotype, for all tested antibiotics, it would mean that analysed serotypes can be distinguished by "Trial with antibiotic-ribosome binding efficiency".

Piotr Franciszek Burzynski

Ribosome-binding antibiotics were chosen, regarding their different binding sites, and different mechanisms of action (tab. 7). Chosen antibiotics should be prepared in equal concentrations, as solutions. Bacterial cells should be separated from the given medium, and equal mass of those whole bacterial cells should be added to the plastic vial. This process should be performed in four repeat samples, for each ribosome- binding antibiotic, and for each serotype of BFo1 (see the table of groups for approach number two) (tab. 8). Also control groups should be prepared by composing the solutions with no antibiotics. Antibiotic solutions, with equals concentrations, should be added to the plastic vials with bacterial cells, in volumes of 1ml. Closed vials should be left for incubation in the room temperature for three hours. This incubation time, should be long enough for the antibiotics to penetrate cell walls, and bind ribosomal proteins, but short enough to prevent bacteria degradation. After incubation bacterial cells should be transferred from the plastic vial onto the wells, on the MALDI plate. Prepared matrix solution of α-Cyano-4-hydroxycinnamic acid, which was prepared for the completed stages of analysis, should be added onto the cells on wells. Analysis of mass spectrum with the use of MALDI-TOF should be performed, with the same method as the one described in the "Materials and Methods" section of completed experiment. Data manipulation methods and statistical analysis, which were used in the completed experiment, apply in this hypothetical stage. Linezolid is a synthetic antibacterial reagent of the oxazolidinone class of antibiotics, distributed as a solution with 200 mg/100 ml initial concentration. It selectively inhibits bacterial protein synthesis by binding ribosomal proteins. This action prevents a functional 70S-initiation complex formation, by specific binding to the bacterial 23S ribosomal RNA of the 50S subunit (DrugBank, 2020b). Neomycin inhibits bacterial ribosomes by 30S ribosomal subunit inhibition, disrupting the translational mechanism of bacterial protein synthesis. This antibiotic prevents mRNA binding to 30S ribosomal subunit (DrugBank, 2020c). Chloramphenicol, which is lipid-soluble, diffuses through bacterial cell membrane. This feature should be included when the antibiotic solutions are prepared. While some other antibiotics can be dissolved in water, this antibiotic should be dissolved in a lipid solution. Final concentrations of the antibiotics remain the same. This antibiotic reversibly binds to the L16 protein of the 50S subunit of bacterial ribosomes, thus transfer of amino acids for protein synthesis is prevented. Peptide bond formation is inhibited possibly by suppression of peptidyl transferase activity (DrugBank, 2020a). Puromycin is toxic to both eukaryotic and prokaryotic cells at appropriate doses. This antibiotic prevents bacterial protein translation by binding to multiple ribosomal proteins in humans, but also in bacteria, which makes this reagent promising for comparison of a wide range bacterial proteins. In bacterial cells it also binds to the Leucyl/phenylalanyl-tRNA--protein transferase (DrugBank, 2020d).

MALDI-TOF Serotyping of Bacterial Bfo1 Variants

Piotr Franciszek Burzynski

Retapamulin inhibits bacterial protein synthesis by binding to the specific site on the 50S subunit of bacterial ribosome (domain V of 23S rRNA). This mechanism involves ribosomal protein L3, and the binding site is in the ribosomal P site, and peptidyl transferase centre. By blocking P-site interactions, normal formation of active 50S ribosomal subunit is prevented (DrugBank, 2020e). This approach can provide a functional MALDI-TOF MS serotyping method. If the difference in the ribosome binding efficiency for each serotype could be proved, the table for "Trial with antibiotic-ribosome binding efficiency" would be created, with constant ratio values. Then for an actual serotyping method, this ratio could be calculated and compared with the prepared chart. Same characteristic ratio would prove that certain serotype is in the analysed sample. (FUTURE OUTLOOK) EXPERIMENTAL APPROACH 3 Final approach is associated with controlled growth of bacterial cultures, and with several ribosomal protein-regulation factors applied. It is possible that this method would be more time-consuming, and would require both greater funding and access to microbiological laboratory with biosafety level three. This makes this method optional, and could theoretically be used for serotyping of bacterial variants when other methods failed, or are not available. On the positive side, this approach may provide more detailed information regarding regulation of bacterial ribosomal proteins, and may provide results which are not expected to be observed. It was proved before that several ribosomal proteins, show extra-ribosomal functions (Aseev & Boni, 2011). Those proteins modulate various cell processes, such as the regulation of mRNA stability, DNA repair, transcription, translation. Ribosomal proteins are also associated with the regulation of phage-specific processes. Bacterial ribosomal protein genes are located in operons which also include genes coding for other proteins, including translation factors (Aseev & Boni, 2011). Among those functions, when constant growth is considered, ribosomal proteins are also regulating the process that leads to the synthesis of the new ribosomal proteins. It is very important for each cell to perform natural processes of ribosomal proteins undisturbed. Knowing this, artificial disturbance of the ribosomal protein expression can be applied by disturbing and targeting the existing ribosomal proteins. In theory, each bacterial serotype may respond differently to those ribosome targeting factors, providing differentiated mass spectra, and a significant serotyping method. Ribosomal proteins in growing cultures, which are proposed, include: targeting the ribosome regulation protein Gemin5 (Francisco-Velilla et al., 2016), and action of several viruses which are interacting with several ribosomal proteins (Warner & McIntosh, 2009), (Aseev & Boni, 2011). RNA-binding protein Gemin5 plays crucial role in all organisms (Francisco-Velilla et al., 2016), and yet the exact structure of this protein remains unknown (Moreno- Morcillo et al., 2019). Gemin5 regulates small nuclear ribonucleoproteins (snRNPs) assembly, translation control through distinct domains, and ribosome binding. The interactions between Gemin5-cleavage product p85, a viral-induced Gemin5 cleavage fragment, and whole Gemin5 protein were observed, with the use of mass spectrometry

MALDI-TOF Serotyping of Bacterial Bfo1 Variants

(Moreno-Morcillo et al., 2019). The possibility of using mass spectrometry and Gemin5-viral interactions are the factors that led to this idea. Several viruses also regulate the ribosomal proteins (Warner & McIntosh, 2009), but also Gemin5 as described above. Bacteriophages are interacting with ribosomal subunits such as S1 (Aseev & Boni, 2011), and are modulating several ribosomal processes. This approach is based on the Gemin5 targeting with Gemin5-specific antibodies and viruses. It is also based on the several bacteriophage applications which could have been applied on different bacterial growth stages. Finally, MALDI-TOF MS would be used to determine how the expression of certain ribosomal proteins was modulated by those factors. If different serotypes would provide different mass spectra, with the same factor applied, efficient serotyping method would be found. This approach is optional, and is still at the early stage of development. CONCLUSIONS The experiment which was carried out proved that two BFo1 variants FoA, and FoWT2 can be distinguished with the use of Matrix-assisted laser desorption/ionization – Time of Flight mass spectrometry (MALDI-TOF MS). This analytical method was proved to be significant and accurate for serotyping of certain variants, by protein analysis from the whole cells. Purification steps are not essential. Downside is that not every serotype can be distinguished. Some of those serotypes proved to produce similar mass spectra, when proteins were analysed. Nevertheless, FoA and FoWT2 experiment proved that some changes can be visualised. That is why, the next step should be focused on specific sample preparation, to cause the mass spectrum changes of the analysed samples. Those changes can be compared and are expected to be different for each serotype. In theory, the analysis of more serotypes in one experiment, with some of the proposed methods applied, can provide a significant serotyping method. It could be very important in the world of science, and public health, as the blood samples from infected patients could be analysed quickly and cheaply, to determine which serotype of bacteria infected its host. With this information provided, correct care, or antibiotic treatment could be applied without the risk that serotype will gain new antibiotic resistance due to the poor choice of treatment. ACKNOWLEDGMENTS This paper was originally written as Dissertation, that only resembles a scientific paper, which describes a final year scientific project, and which was accomplished by me (an undergraduate student of Medical Biochemistry (Hons) Swansea University, (module code: PM-304, module name: Biomolecular Research Project)). That is why this research project faced several financial limitations, and that is why other BFo1 variants were not compared. More advanced experimental approaches were not applied due to the project complexity restrictions. Despite the fact that described methods are not approved, this work may guide and lead to significant conclusions for future work. I am very grateful for significant support, I have received from my personal project supervisor, Senior Research Officer, Dr Mark Wyatt. MALDI-TOF mass spectrometry

Piotr Franciszek Burzynski training and equipment availability was provided by my supervisor. I am very grateful for that as well. Many thanks to Dr Ricardo Del Sol Abascal for providing BFo1 samples. REFERENCES Aseev L.V., Extraribosomal Functions of Bacterial Ribosomal Proteins. In: Boni I.V. (ed.). 2011. Douthwaite J.P.S., The bacterial ribosome as a target for antibiotics. Nature, 2005. DrugBank, Chloramphenicol, DrugBank, 2020a, Available at: https://go.drugbank.com/drugs/DB00446. DrugBank, Linezolid, DrugBank, 2020b, Available at: https://www.drugbank.ca/drugs/DB00601. DrugBank, Neomycin, DrugBank, 2020c, Available at: https://www.drugbank.ca/drugs/DB00994. DrugBank, Puromycin, DrugBank, 2020d, Available at: https://www.drugbank.ca/drugs/DB08437. DrugBank, Retapamulin, DrugBank, 2020e, Available at: https://go.drugbank.com/drugs/DB01256. Dykhuizen D.E., Santa Rosalia revisited: Why are there so many species of bacteria?, Springer, 1998. Facey P.D., Méric G., Hitchings M.D., Pachebat J.A., Hegarty M.J., Chen X., Morgan L.V.A., Hoeppner J.E., Whitten M.M.A., Kirk W.D.J., Dyson P.J., Sheppard S.K. and Sol R.D., Draft Genomes, Phylogenetic Reconstruction, and Comparative Genomics of Two Novel Cohabiting Bacterial Symbionts Isolated fromFrankliniella occidentalis, Genome Biology and Evolution, 7(8), pp. 2188- 2202. Ferrari J. and Vavre F., Bacterial symbionts in insects or the story of communities affecting communities, Philosophical Transactions of the Royal Society B: Biological Sciences, 366(1569), 2011, pp. 1389-1400. Francisco-Velilla R., Fernandez-Chamorro J., Ramajo J. and Martinez-Salas E., The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation, Nucleic Acids Research, 2016, 44(17), pp. 8335-8351. Holland R.D., Duffy C.R., Rafii F., Sutherland J.B., Heinze T.M., Holder C.L., Voorhees K.J., Lay Jr J.O., Identification of Bacterial Proteins Observed in MALDI TOF Mass Spectra from Whole Cells, Analytical Chemistry, 1999, 71(15), pp. 3226-3230. Moreno-Morcillo M., Francisco-Velilla R., Embarc-Buh A., Fernández-Chamorro J., Ramón-Maiques S., Martinez-Salas E., Structural basis for the dimerization of Gemin5 and its role in protein recruitment and translation control, Nucleic Acids Research, 2019. Nakano S., Matsumura Y., Ito Y., Fujisawa T., Chang B., Suga S., Kato K., Yunoki T., Hotta G., Noguchi T., Yamamoto M., Nagao M., Takakura S., Ohnishi M., Ihara T., Ichiyama S., Development and evaluation of MALDI-TOF MS-based serotyping for Streptococcus pneumoniae, 2015, 34(11), pp. 2191-2198. Omran M.M., Rashed R.E., Darwish H., Belal A.A., Mohamed F.Z., Development of a gas chromatography–mass spectrometry method for breast cancer diagnosis based on nucleoside metabolomes 1‐methyl adenosine, 1‐methylguanosine and 8‐hydroxy‐2′‐deoxyguanosine, Biomedical Chromatography, 2019. Phillips R.M.R., Cell biology by the numbers, 2015. Warner J.R., McIntosh K.B., How Common Are Extraribosomal Functions of Ribosomal Proteins?, Molecular Cell, 2009, 34(1), pp. 3-11. Yarian C., Townsend H., Czestkowski W., Sochacka E., Malkiewicz A. J., Guenther R., Miskiewicz A., Agris P.F., Accurate Translation of the Genetic Code Depends on tRNA Modified Nucleosides, 2002, 277(19), pp. 16391-16395.

Modern technologies in forensic medicine

Jędrzej Siuta*, Wojciech Golema Department of Forensic Medicine, Wroclaw Medical University, ul. J. Mikulicza- Radeckiego 4, 50-345 Wrocław Poland. *Corresponding author: Jędrzej Siuta, Department of Forensic Medicine, Wroclaw Medical University, e-mail: [email protected] ABSTRACT Forensic medicine is a specific field of medical sciences, which is situated between medicine and law. It cooperates with many medical sciences such as biochemistry, genetics, microbiology and anatomopathology. Advances in all of that sciences are strongly associated with progress in forensic medicine research and practical methods. Also so called classical forensic medicine – which consist of forensic thanatology and traumatology – uses modern technology for its purposes, such as determining the cause of death, detection of traumatic lesions in crime victims, determining of the time of death, reconstruction of the crime scene or better visualisation of the medical evidence to the justice system. Some methods of modern technology – like computed tomography for the post mortem examination – are quite widely used in forensic diagnostics. Other methods – such as ultrasound examination to determine subcutaneous bruises or use of the bioimpedance parameters measurement to determine the time of death – are pioneer methods. They are tested at the Department of Forensic Medicine of Wroclaw Medical University. The result of other research conducted in this unit, containing innovative connection of medicine and architecture was creation of a spatial model of crime reconstruction. Results were very promising so the 3D Analysis Laboratory was formed in order to conduct further research. In this review we will present modern research methods used in forensic medicine with particular emphasis on the methods being developed at the Department of Forensic Medicine of Wroclaw Medical University. INTRODUCTION Forensic medicine is a science situated between medicine and law, which cooperates with many medical sciences such as biochemistry, genetics, microbiology and anatomopathology. Advances in all of these sciences are strongly associated with progress in forensic medicine research and practical methods. Also so called classical forensic medicine – which consist of forensic thanatology and traumatology – uses modern technology for its purposes. This work presents the introduction of modern technological solutions within the most important issues of forensic medicine, with a short presentation of their development from historical times to the present, as well as their possible development in the future. SEARCH STRATEGY AND SELECTION CRITERIA The history and development directions of the most important forensic medicine issues were presented based on the authors' own professional experience and a query of Polish and international professional literature. Both historical and contemporary literature was used – from the beginning of the 20th century to the present. REWIEV One of the basic and also the oldest branches of forensic medicine is forensic thanatology, generally called the science of death. Its purpose is not only to learn about the process of dying itself and the biochemical and physiological reactions that accompany it, but above all putting this knowledge into practice for law enforcement and justice purposes. Besides the process of dying, thanatology also analyses among

Jędrzej Siuta, Wojciech Golema others possible causes of death and posthumous transformation of corpses. In terms of describing the injuries present on corpses, thanatology is closely related to another basic field of forensic medicine – forensic traumatology, called the study of injury. Determining the cause of death is done by means of forensic medical examination of the corpse and its autopsy, so the activities most often associated with forensic medicine. Medical examination consists in the description of externally visible injuries, disease lesions and special signs of the examined person. The autopsy involves opening the corpse, i.e. incision of the skin, muscles and some bones and opening of the pleural cavity, abdominal cavity and head (sometimes also limbs) well as incision and examination of internal structures (not only organs, but also e.g. veins, arteries, nerves, ligaments, etc.). The described activities are accompanied by the collection of materials (swabs, body fluids, tissue sections) for further laboratory tests (Knight, 2016; Wyatt, 2015; Chróścielewski, 1990). One of the biggest problems when performing a classic autopsy is their "devastation", which means that the corpse is significantly damaged as a result of the cuts made. This problem is particularly important among others in Poland, where it is customary during the funeral to keep the casket with the corpse open, so that the family of the deceased and other persons participating in the funeral say goodbye not only by sight, but also often by a direct "last touch" of a dead person, which – although often not in accordance with applicable sanitary regulations – results from strongly rooted regional traditions. Corpses after the section are "repaired", which includes not only stitching of the skin, but also various treatments in the field of thanathopraxia, as well as corpse clothing and posthumous make-up (so-called thanatocosmetics). The effectiveness of these treatments, and above all the amount of time-consuming work needed to perform them, depends on the degree of injury, so it is advisable to keep it as low as possible. This can be achieved by carrying out, among others the so-called. targeted cuts in specific, previously designated places, e.g. to confirm the suspected occurrence of internal injury or to locate a bullet in the body that is not always stuck within the gunshot wound channel, undergoing displacement e.g. with a current of blood within the blood vessel. The development of a sectional technique focused on this aspect particularly requires the use of modern technologies (Okas, 2000; Knight, 2016; Wyatt, 2015; Chróścielewski, 1990). The classical sectional technique itself, although having its roots in ancient times, has undergone gradual development since the time of Andreas Vesalius in the sixteenth century. The first documented "forensic autopsy” in Poland was coducted in 1586 by Mikołaj Bucella and Jakub Gosławski autopsy of King Stefan Batory (Szumowski, 1961; Bilikiewicz, 1978). Modern sectional technique is based primarily on the work of nineteenth-century researchers such as prof. Rudolf Weigl, Karl von Rokitansky, Friedrich Albert von Zenker and Maurice Letulle (Skowronek, 2010). The first Polish manuals of sectional technique were: "Anatomo-Pathological Diagnostics" by dr Zdzisław Dmochowski (Dmochowski, 1903), "Corpse Section Technique" by prof. Leon Wachholz and prof. Stefan Ciechanowski (Wachholz, 1919) and "The autopsy technique" of prof. Ludwik Paszkiewicz (Paszkiewicz, 1923). Since then, the sectional technique has undergone only slight modifications, including mainly additional manual procedures (consisting mainly of specific incision of appropriate anatomical structures) in specific cases, such as, for example, the death of a newborn

Modern technologies in forensic medicine baby or a person being hit by a car (Chróścielewski, 1990; Teresiński 2019). The use of more modern manual tools during classical autopsy, e.g. rigid bronchoscopes, for the diagnosis of channels of wounds penetrating deep into the body (Śliwka, 1994) remains a marginal phenomenon. Imaging diagnostics is an area of modern technologies used during autopsy, which has been developing intensively in the last 20 years. Although in its most classic form – X-ray images – it was used in forensic medicine as early as the 1930s (Skowronek, 2010), it was the development of computed tomography and magnetic resonance imaging that created the possibility of a revolution in sectional technique. It gives not only the possibility of fairly non-invasive accurate localization of traumatic and disease changes occurring on the corpse and foreign objects stuck in it, but also, in combination with other methods, such as spectroscopy, laser surface scanning or 3D photogrammetry – on the possibility of conducting a virtual autopsy, called Virtopsy (Bolliger 2008; Thali, 2007; Skoworonek, 2010). This method – including on the creation of a spatial computer model of the examined corpse – it is minimally invasive, and thus not damaging the body. Additional advantages of such a method are: the possibility of using it in situations where (e.g. for moral or religious reasons) it is difficult to carry out a classical corpse opening, or the possibility of proceeding several autopsies of the same body by various independent specialists (Skowronek, 2010). The latter advantage is especially important because during the classic so-called "section after section", especially after a long time after the death of the deceased and with the necessity of exhumation, the body is heavily damaged both as a result of previously performed cuts and post-mortem processes (e.g. decomposition), and thus the diagnostic possibilities during such a section are extremely limited in relation to section carried out originally. The disadvantages of this method, besides its very high technical requirements and significant costs, is still its lower evidential credibility compared to the classical method. It results from the fact that, although the first textbook for Virtopsa was published in 2009 (Thali, 2009), and courses of this method are organized in Switzerland (Skowronek, 2010), it still remains largely an experimental method. However, it sets a certain direction for the development of "forensic medicine of the future". Another method of diagnostic imaging used in forensic medicine is medical ultrasound – ultrasonography. Although this method in terms of sectional diagnostics cannot be compared to the methods previously described, such as computed tomography (Farina, 2002), it has significant advantages: it is cheap, easily available and can be used without any problem in the medical examination of living persons (Susło, 2014). The latter issue is particularly important, because it is often forgotten that forensic medicine is not only autopsies or assessment of medical proceedings in terms of making medical errors, but also forensic examination of live victims of crimes against life and health (e.g. beatings, sexual crimes). A commonly known problem is the occurrence of bruises in the subcutaneous tissue or muscles, that are not visible on the outer layer of the skin. During autopsy, this problem can be solved by layer – cut of the corpse and exposing the inner layers of the tissues gradually, but for obvious reasons this solution cannot be used when examining a living person (Knight, 2016; Wyatt, 2015; Chróścielewski, 1990). In this situation, ultrasonography examination – which may show blood located in the subcutaneous or deeper layers of the tissue at the site of injury – may be really helpful.

Jędrzej Siuta, Wojciech Golema

(Susło, 2014). Currently, the Department of Forensic Medicine of the Medical University in Wrocław (with the authors of this work) is working on developing this method in the described context. The scientific work which deserves a separate mention is the use of the Ultra-high field magnetic resonance 7.0 Tesla to examine developmental defects of the 19th century, preserved in preservative fluids human fetuses, which are being exhibited at the Museum of Forensic Medicine in Wroclaw. It shows that modern technology can also be used for non-standard scientific research in forensic medicine. The results of the study are currently being developed and will be published soon. Another field in which forensic medicine together with other forensic sciences uses modern technology is crime scene imaging. Crime scene investigation comprises not only of sample collection and (if present) corpse description but also includes documentation of the scene (including the layout, sample location, mutual arrangement of the evidence). It is sometimes a key piece of evidence in the investigation, allowing validation of the perpetrator’s testimony (Millet, 2014). Blood trace pattern examination is a known and recognised branch of criminology. This evidence can also be lost in time so preserving in detail the image of the crime scene for a longer period of time is very useful. In the beginning of forensic sciences investigators had only their eyes and memory. In time they were able to draw the sketch of the crime scene. First revolution was the invention of photography which allowed taking pictures of the place. It allowed not graphically talented investigators to also participate in the process. First cameras were of course not sophisticated and required time and good light to record the picture. Further development of photography introduced better optics, films, video cameras and then digital photos. In modern times taking pictures of the crime scene is considered to be the golden standard of investigation. Photography and video recording had also one big disadvantage – the captured image was 2-dimentional. Sometimes it was hard to visualise the crime scene and it’s arrangement to the jury and the court. This was especially when the scene was complicated with many small details. Video recording allowed to show many aspects of the scene layout but it was time taking – films sometimes lasted for few hours to show all aspects of the place. Modern technologies allowed the 3D reconstruction of the crime scene with the use of simple digital pictures but it always included some sort of graphic interference with the source material which could be then used by the defence to file a motion to reject the evidence as compromised (Maksymowicz, 2014). 3D scanning nowadays use the technique of contact and non-contact scanners. The first ones are used mostly in specified branches of industry because they require contact between scanned object and the probe. This requirement made them useless for crime scene investigators. Non- contact scanner however works similar to photo cameras. They can be either passive (only register the light of the crime scene) or active (emit some kind of radiation and then acquire the reflected ray). Placing the scanner in different points of the scene (allowing it to "see" the whole spot) enables the technician to obtain enormous amount of information for further processing. This is the opportunity to save the exact image of the crime scene and then display it for the use of legal persons and other authorities. Development of modern 3D non-contact scanners using laser or visual light opened new possibilities for crime scene image preservation (Ebert, 2014). The common thing about 3D scanners and "normal" photography is the possibility of acquiring data that

Modern technologies in forensic medicine are visible to the camera – this means that they are not covered from the lens. Laser light is emitted by the scanner and the ray is controlled by a set of mirrors and lenses. The laser ray than reflects from the scanned object and returns to the scanner like normal visible light in photo cameras. It is recorded on CCD. In addition to information collected by the photo camera, 3D scanners collect much more data. Apart from colour, light and 2D position they gather the information about the distance between the scanner and the object. This is possible by measuring the most lighted points of the object with connection to mirror position. Recorded data contains information about location of the measured points in 3 dimensional matrix (X, Y, Z axis). This allows to reconstruct the 3D image of the scene, object using computer software but it does not interfere with the image itself (Direct Dimensions, 2016). Information about the depth of the registered point is measured by the device which cannot be the ground for evidence denial due to illegal manipulation. 3D reconstruction of initially flat object – like digital photo – implied the "making" of depth information which is not allowed by the criminal trial standards. Any alteration done to raw source material can be the ground for evidence not to be allowed by the court. Potential use of data acquired by the 3D scanner is almost limitless. Not only it preserves the complete schematic of the scene but also can be transformed and enhanced without destruction of raw data. Scans of skulls can be used for facial image reconstruction – layers of muscles skin and subcutaneous tissue can be added on the initial data. Objects registered by 3D scanner could be set in motion or given specific physical properties to simulate mutual interactions. Such models can be used to analyse arrangement of the crime scene than in further steps be used to form opinion about possible participant’s behaviour their interactions and line of sight. This is used to establish whether situation description given by the perpetrator and the victim are even possible. This reconstruction does not destroy the acquired data and all changes can be reversed so in most cases such opinion will be allowed by the court (Maksymowicz, 2014). Modern scanners allow also high definition imaging thanks to sophisticated lens systems and better software of the scanner itself. This is very useful in terms of scene reconstruction and detail preservation. It also decreases time needed for complete scan – it can be performed from longer distance and covers wider field. 3D reconstruction is very often applied in computer tomography (CT) or magnetic resonance imaging (MRI). These machines are also considered scanners (active and non-contact) and are used to perform described in this article virtual autopsies. Introduction of all modern technologies to forensic medicine has to be very responsible due to possible errors that could destroy life of innocent people found guilty of the crime they did not commit (Direct Dimensions, 2016; Milliet, 2014). 3D laser scanning also requires interdisciplinary team of specialists which can be challenging. The possible use of the data gathered by this technique more than enough compensates the labor needed for applying this tool to common forensic medicine practice (Ebert, 2014). One of the modern 3D Labs is a part of Forensic Medicine Department of Wroclaw Medical University (Maksymowicz, 2014). Attempts to use modern technologies also concern another significant issue of forensic medicine – determining the time of death. How important it is to determine the most accurate date and time of death for an investigation, especially in the event of a murder, needs no explanation. Often, only this information allows, for example, to

Jędrzej Siuta, Wojciech Golema verify the alibi of a person suspected of committing a crime or selecting a suspect on the basis of secured recordings from monitoring (Marcinkowski, 2010; Pounder 2005; Kaliszan 2019). Meanwhile, determining the time of death is still based primarily on methods developed in the nineteenth and early twentieth centuries based on the observation of the properties of the so-called signs of death. These include, first of all, the assessment of the appearance, movement and pallor after squeezing of lividity (livor mortis), the place of occurrence and the degree of severity of rigor mortis together with the ability to return after breaking, as well as the assessment of the degree of post mortem fall of the body temperature. Interlethal reactions (e.g. posthumous muscle responses to physical and pharmacological factors) and the stage of food digestion in the stomach are also assessed. The disadvantage of this type of methods is primarily their not very high accuracy, significantly decreasing with the increase of post mortem interval. Even within the first 6 hours of death, it is difficult to determine the time of death with accuracy greater than about 1.5-2 hours, and in the case of decomposed corpses, this accuracy usually does not exceed several months, with the progression of posthumous changes reaching even several years (Pounder, 2005; Kaliszan, 2019). For this reason, attempts are constantly being made to develop more accurate methods for determining the time of death, both based on the development of natural sciences (forensic entomology and microbiology) as well as modern technology. The methods using the latter include, among others the so-called biochemical methods using laboratory tests of body fluid and tissue samples taken from corpses. With biochemical methods among others changes in enzyme activity or changes in the biochemical composition (e.g. potassium, calcium, creatinine or lipids level) of the internal fluids (blood, cerebral liquid, vitreous humor of the eye) in post mortem period can be checked. Some of these studies give quite good results up to 35 days after death, and some of them, such as determining the level of potassium in the vitreous humor of the eye, are very well developed (Pounder, 2005; Knight, 2016; Kaliszan, 2019). Other methods include the development of genetic testing technology which, in addition to the use of, for example, physical evidence and identification of people, is used – by assessing posthumous DNA breakdown – to determine the time of death. Magnetic resonance spectroscopy (H-MRS) was also used to determine the time of death by assessing brain tissue breakdown products (Pounder, 2005; Knight, 2016; Kaliszan, 2019). A modern method, the use of which is development in the Department of Forensic Medicine of the Medical University in Wrocław, is the use of post-mortem measurements of changes in the parameters of bioimpedance of the human body to determine the time of death (Siuta, 2012). Tests of post-mortem tissue resistance to electric current were already carried out in the 1930s (Freiberger, 1934), however bioimpedance is a modern method that allows, apart from resistance, to determine the so-called reactance (capacitive resistance) of the body and calculate the phase angle between them. The method is commonly used for so-called body composition analysis, it allows, among others for determining the water and fat content in the body, however, it is used in various fields of science, including medicine to assess, for example, the state of the circulatory system, water management in kidney diseases, wound healing assessment, the degree of malnutrition, and even to detect cancer (Siuta, 2012; Mao, 2011). The potentially large usefulness of this method in forensic medicine is indicated

Modern technologies in forensic medicine by studies carried out about 20 years ago on body fragments (Querido, 1999). The research conducted by the authors of this work, aimed at developing the official method for determining the time of death by means of posthumous measurements of bioimpedance parameters, also give very promising results. SHORT CONCLUSION The development of modern technologies enables progress in all scientific fields. This also applies to forensic medicine. Creating research methods using modern technologies allows for the refinement of previously existing methods as well as for supplementing (and sometimes replacing) them with new ideas. Some methods, such as virtual autopsy or crime scene imaging, set completely new directions in the development of forensic medicine. These methods, due to the importance of the cases in which they are used, still require detailed elaboration, but their future wide application, until recently treated almost as Science Fiction, now seems more and more likely. The rapid increase in demand for telemedicine services and the use of non- invasive research methods in connection with the current pandemic by virus Sars-CoV- 2 have a large impact on this. Since this type of epidemic, with a potential for a much more serious course than the current pandemic, is likely to occur in the future, perhaps it is virtual treatments – such as virtual autopsy – that will become the gold standard of medicine wherever possible. REFERENCES Knight B., Saukko P., The forensic autopsy, In: Knight B., Saukko P., Knights forensic pathology 4th edition, CRC Press (Boca Raton, USA), 2016, ISBN: 978-1-4441-6508-1. Wyatt J., Squires T., Norfolk G., Payne-James J., Forensic autopsy In: Wyatt J., Squires T., Norfolk G., Payne-James J., Oxford handbook of forensic medicine, Oxford University Press (Oxford, UK), 2015, ISBN: 978-0-19-922994-9. Chróścielewski E., Raszeja S., Sekcja zwłok, PZWL (Warszawa), 1990. Okas W., Obrzędy i zwyczaje pogrzebowe towarzyszące ostatnim chwilom człowieka, 2000; Niedziela Łódzka, 46/2000. Available online: https://www.niedziela.pl/artykul/898/nd/Obrzedy-i-zwyczaje- pogrzebowe [accessed on 07.07.2020]. Szumowski W., Historia medycyny, PZWL (Warszawa), 1961. Bilikiewicz T., Rys historii anatomii, In: Bochenek A., Reicher M., Anatomia człowieka T.1., PZWL (Warszawa), 1978. Skowronek R., Chowaniec Cz., Ewolucja techniki sekcyjnej – od Virchova do Virtopsy, Arch. Med. Sad. Krym., 2010l LX: 48-54. Dmochowski Z., Dyagnostyka anatomo-patologiczna, Wydawnictwo Gazety Lekarskiej (Warszawa), 1903. Wachholz L., Ciechanowski S., Technika sekcyi zwłok, Gebethner i Wolff, 1919. Paszkiewicz L., Technika sekcji zwłok, Wyd. II. PZWL (Warszawa), 1953. Śliwka K., Berent J., Miścicka-Śliwka D., Bloch-Bogusławska E., Tyloch F., Application of rigid endoscopes for forensic examination of the stab wounds, Acta Medicinae Legalis, 1994; XLIV: 312- 315. Teresiński G., Mechanizmy obrażeń stawów kończyn dolnych, In. Teresiński G., (Editor) Medycyna sądowa T.1., PZWL (Warszawa), 2019, ISBN: 978-83-200-5856-7.

Jędrzej Siuta, Wojciech Golema

Freiberger H., Der elektrische Widerstand des menschlichen Körpers gegen technischen Gleich- und Wechselstrom, Verlag von Julius Springer (Berlin), 1934. Bolliger S. A.,. Thali M. J., Ross S., Buck U., Naether S., Vock P., Virtual autopsy using imaging: bridging radiologic and forensic sciences. A review of the Virtopsy and similar projects, Eur Radiol., 2008; 18: 273-282. Thali M.J., Jackowski C., Oesterhelweg L., Ross S.G., Dirnhofer R., VIRTOPSY – The Swiss virtual autopsy approach, Leg. Med., 2007; 9: 100-104. Thali M.J., Ross S., Oesterhelweg L., Grabherr S., Buck U., Naether S. et al., Virtopsy Working on the future of forensic medicine, Rechtsmedizin, 2007; 17: 7-12. Thali M.J., Dirnhofer R., Vock P., The virtopsy approach: 3D optical and radiological scanning and reconstruction in forensic medicine, CRC Press (Boca Raton, USA), 2009, ISBN: 9780849381782. Farina J., Millana C., Jesus Fdez-Acenero M., Furio V., Aragoncillo P., Martin V. G. et al., Ultrasonographic autopsy (echopsy): a new autopsy technique, Virchows Arch., 2002; 440: 635-639. Susło R., Trnka J., Drobnik J. Kawecki J., Siuta J. et al., Zastosowanie badania ultrasonograficznego w wykrywaniu, weryfikowaniu i dokumentowaniu niewidocznych zewnętrznie zmian urazowych, Fam. Med. & Pirm. Care Rev., 2014, 16, 4:376-377. Maksymowicz K., Tunikowski W., Kościuk J., Crime Event 3D Reconstruction Based on Incomplete or Fragmentary Evidence Material: A Case Report, Forensic Science International., 2014, 242: e6-e11. Direct Dimensions, Almost Everything You Always Wanted to Know About 3D Scanning (but Were Afraid to Ask), 2016, http://www.dirdim.com/lm_everything.htm (accessed 13th June 2020). Milliet Q., Delémont O., Margot P.A., Forensic Science Perspective on the Role of Images in Crime Investigation and Reconstruction, Science and Justice, 2014, 54: 470-480. Ebert L.C., Nguyen T.T., Breitbeck R., Braun M., Thali M.J., Ross S., The Forensic Holodeck. An Immersive Display for Forensic Crime Scene Reconstructions, Forensic Science Medicine Pathology, 2014, 10: 623-626. Marcinkowski T., Medycyna sądowa dla prawników, WSPOL (Szczytno, Poland), 2010, ISBN 978-83- 7462-250-9. Pounder D.J., Postmortem interval, In: Payne-James J., (editor) Encyclopedia of Forensic and Legal Medicine., Elsevier (Oxford, UK), 2005, ISBN 9780128000342. Kaliszan M., Teresiński G., Metody ustalania czasu zgonu, In: Teresiński G., (editor) Medycyna sądowa T.1., PZWL (Warszawa), 2019, ISBN: 978-83-200-5856-7. Knight B., Saukko P., The pathophysiology of Death, In: Knight B., Saukko P., Knights forensic pathology 4th edition, CRC Press (Boca Raton, USA), 2016, ISBN: 978-1-4441-6508-1. Siuta J., Susło R., Trnka J., Gęsicki M., Komputerowo wspomagane pomiary zmian impedancji narządów ciała ludzkiego jako wykładnik stanów fizjologicznych i patologicznych organizmu. In: Zarzycki J (editor) Komputerowe wspomaganie badań naukowych. T.19., Wrocławskie Towarzystwo Naukowe (Wrocław), 2012, ISBN 978-83-7374-081-5. Mao S., Xiuzhen D., Fu F., Seese R., Zhenyuan W., Estimation of postmortem interval using an electric impedance spectroscopy technique: A preliminary study, Science and Justice, 2011, 51: 135-138. Querido D., A preliminary study of changes in scalp impedance during the early post-mortem period in rats, Forensic Science International, 1999, 101: 123-130.

Neuromyelitis optica (NMO) – a challenging demyelinative disorder

Justyna Chojdak-Łukasiewicz Department and Clinic of Neurology, Wrocław Medical University, ul. Borowska 213, 50-556 Wrocław, tel. +48 71 734 31 00, fax. +48 71 734 31 09, Poland Corresponding author: Justyna Chojdak-Łukasiewicz, Department and Clinic of Neurology, Wrocław Medical University, email: [email protected] ABSTRACT Neuromyelitis optica (NMO, Devic’s disease) spectrum comprises a range of rare autoimmune inflammatory diseases of the central nervous system (CNS), mainly affecting optic nerves and the spinal cord. For many years, NMO was considered a variant of multiple sclerosis (MS), until antibodies against aquaporin 4 (AQP4) were discovered in 2004 and recognised as the specific background for this disease. AQP4 antibodies are detected in 60-90% of patients with NMO. Recent studies have revealed a range of clinical presentations of NMO spectrum and a possibility of their relapsing course. Current diagnostic criteria include the typical clinical manifestation, lesions in brain and spinal cord shown in magnetic resonance imaging (MRI) and positive test for antibodies against AQP4. Recommended therapeutic options for acute attacks of NMO include high doses of corticosteroids and/or plasma exchange. However, differentiation between NMO and MS is crucial because of substantial differences in the long-term treatment. The prognosis in NMO used to be unfavourable, but there is evidence from recent clinical trials that monoclonal antibodies (rituximab, eculizumab, inebilizumab, satralizumab) may prevent relapses and allow a better outcome. INTRODUCTION NMO and neuromyelitis optica spectrum disorders (NMOSD) are rare autoimmune inflammatory diseases of the CNS. NMO is characterised by a monophasic course of bilateral optic neuritis (ON) and transverse myelitis (TM) occurring concomitantly or within a short interval (Drori, 2014, Wingerchuk, 2007). The first case of NMO was reported by Albutt in 1870, who described optic nerve disease followed by myelitis in approximately 3 months (Albutt 1870). In 1894, a French neurologist Devic and his student Gault used the term "neuro-myelite- optique" to denote a novel syndrome characterised by acute myelitis and ON. They documented 17 patients with a monophasic course of bilateral (or rapidly sequential) ON and myelitis. In 1907, a Turkish physician Acchioté suggested the name "Devic’s syndrome" for the disorder characterised by monophasic and relapsing NMO (Jarius, 2013). For many years, it was originally considered a form of opticospinal MS. (Wingerchuk, 1999). In 2004, the discovery of specific IgG antibodies against aquaporin 4 (Anti-AQP4-Ab) (Lennon, 2004) helped to classify NMO as a separate disease and not a variant of MS. AQP4 is expressed strongly in astrocytes in the brain, spinal cord and optic nerve, but also in epithelial cells outside the CNS, such as stomach and kidney (Papadopoulos, 2012). AQP4 is the dominant water channel in the CNS and its disturbed function due to immune-mediated attack results in severe

Justyna Chojdak-Łukasiewicz neurological deficits. Anti-AQP4-Ab are characterised by 73% sensitivity and 91% specificity for clinically defined NMO (Wingerchuk, 2007). The term NMOSD is a newly disease spectrum with or without anti-AQP4-Ab and includes symptoms of NMO. At first, the term was used in 2015 by an international group of experts (IPND, International Panel for NMO Diagnosis) (Trebst, 2014). A better insight into neuroimmunological issues improved the perspective of a specific therapeutic approach. AIMS OF THE STUDY The goal of this review is to collect and analyse the available material on the aetiology, pathogenesis, clinical symptoms, diagnosis, treatment and prognosis of the course of NMO and NMOSD. MATERIALS AND METHODS To carry out the study, the following databases were used: PubMed (NCBI), Google Scholar, Web of Science and Scopus, using the following key words: Devic’s disease, neuromyelitis optica, neuromyelitis optica spectrum, optic neuritis, transverse myelitis, epidemiology, pathogenesis, treatment, prognosis. The articles were selected through original study, case studies, and reviews published between 1980 and 2020. The results of the study include alternative keywords submitted to put together a complete picture of this disease. EPIDEMIOLOGY The epidemiology of NMO remains poorly characterised. In the past, many patients (> 20%) with NMO were misdiagnosed with MS (Jarius, 2014). The incidence of NMO per 100,000 population ranged from 0.053 to 0.40, while the prevalence per 100,000 population ranged from 0.52 to 4.4 in Europe and North America (Bizzoco, 2009). The NMO has been reported all over the world, but clinic-based series have suggested that the risk of NMO is higher among non-whites than whites (Cree, 2002, Mealy, 2012). NMO is more prevalent in areas with Black, Asian and Indian population, where MS prevalence is usually low (Kira, 1996). All of the evaluated material confirmed that women were affected more often than men (Wingerchuk, 2009). The prevalence among women is about 8 times higher than among men (Bizzoco, 2004, Ghezzi, 2009, Wingerchuk, 2007). The median age of onset is 39.7 years, which differs from most patients with MS (Wingerchuk, 2007, Jarius, 2014). Comparatively, multiple sclerosis has a median age of onset of 24 years and an estimated female to male incidence of 2.3:1 (Kingwell, 2013, Harbo, 2013). However, NMO occurs in < 20% in children and the elderly (Jarius, 2014, Lotze, 2008, Barbieri, 1989). Several studies have shown that women with NMO have an increased risk of relapse postpartum; the relapse risk during pregnancy is unknown (Mao- Draayer, 2020, Fragoso, 2013) . Most cases of NMO are sporadic (Matiello, 2010), although a few familial cases have been described in literature (Pittock, 2008, Braley, 2007, Cabrera-Gomez, 2009).

Neuromyelitis optica (NMO) – a challenging demyelinative disorder

Despite these clues, the role of genetic factors in NMO is not known. Several HLA (human leukocyte antigens) variants and other polymorphisms have been reported in association with NMO (Zéphir, 2009; Wang et al., 2011; Asgari et al., 2012, Deschamps, 2011, Brum, 2010), and about 3% of patients have affected relatives (Matiello, 2010). NMO has been associated in approximately 10-40% with other autoimmune disorders, including myasthenia gravis, systemic lupus erythematous, Sjögren’s syndrome, and celiac disease (Pittock, 2008; Bergamaschi, 2004; 2009, Jarius, 2011; Leite, 2012). PATHOGENESIS The immunopathology of NMO is different from that of multiple sclerosis (Wingerchuk, 2010). The aquaporin-4 (AQP4) is a water channel is expressed by astrocytes and plays the main role in the pathogenesis. Production of the antibody against AQP4 is triggered by an unknown process in a genetically susceptible person. Anti-AQP4-Ab enters the CNS when the blood brain barrier is made permeable and has high affinity for orthogonal array particles of AQP4. Anti-AQP4-Ab causes extensive astrocytic loss in specific regions of the CNS through complement mediated cytotoxicity (Jasiak-Zatonska, 2016). A wide range of inflammatory cytokines have been associated with the pathogenesis of NMO. Especially Interleukin-6 (IL-6) contributes to the production of antibodies against aquaporin 4 by the plasmoblasts and play the important role and in the enhancement of inflammation in the central nervous system. CLINICAL CHARACTERISTICS The classic clinical manifestations of NMO are recurrent acute attacks of TM and/or uni- or bilateral ON. The course of the disease could be monophasic (no relapses in the future), recurrent (attacks of TM,ON), or both at the same time (Nandhagopal, 2010, Wingerchuk, 1999). In 80-90% of patients, NMO follows the relapsing course. This form of the disease is associated with predisposing factors such as female sex, older age of onset, less pronounced motor symptoms after the first myelitis attack, longer periods of remission and coexistence of systemic autoimmune diseases (Weinshenker, 2006). A number of studies have shown that monophasic NMO with simultaneous bilateral ON and TM occurs only in a minority of cases, approximately 10%. In these cases, men and women are equally affected (Wingerchuk, 2007, O’riordan, 1996). A secondary progressive course of the disease is seen in 2% (Wingerchuk, 2007). Typical symptoms of ON include ocular pain exacerbated by eye movement, loss of central vision and positive visual phenomena called photopsias (spontaneous flashing black squares, flashes of light or showers of sparks, sometimes precipitated by eye movement). Visual field defects manifest usually as central scotoma, bitemporal hemianopsia or paracentral scotoma. Patients with ON related to NMO have a more severe course than in MS. They had recurrent ON more often and tended to have simultaneous bilateral ON involvement at their first ON attack. Most MS patients

Justyna Chojdak-Łukasiewicz recover significant visual acuity following ON, NMO patients often manifest severe visual loss (Trebst, 2014). Spinal cord involvement in the course of the disease occurs usually in the form of TM with symmetric paraparesis, sensory loss below the level of the spinal cord lesion and sphincter dysfunction. Typically, the changes affect the cervical and upper cord segments. Radicular pain, paroxysmal tonic spasms, persisting itching and Lhermitte’s symptom occur in about 30% of the recurrent form of myelitis in NMO. In 10% of cases, NMO demyelinating and inflammatory changes affect the hypothalamus and brainstem region (area postrema). It is related to a high expression of AQP4 in these regions (Shosha, 2018, Guo, 2017). Brainstem syndromes are manifested by nystagmus, dizziness, persistent hiccups, vomiting, trigeminal neuralgia, facial paralysis, hearing loss and even breathing problems (area postrema syndrome). Respiratory failure due to cervical myelitis of brainstem is the most common cause of death related to NMO.The typical course of NMO is associated with gradual deterioration of function as a result of acute attacks. Generally, the 5-year survival rate is approximately 90% for patients with monophasic disease and < 80% for patients with relapsing disease (Wingerchuk, 2006, Pittock, 2006). Clinical consequences of relapsing type of NMO in more than 50% include visual deficits or paraplegia. DIAGNOSIS OF NMO The criteria defined by Devic’s and Galut included bilateral NMO and acute myelitis, which appear at the same time or within a few weeks apart. The diagnosing of NMO presented by Wingerchuk et al. in 1999 was based on laboratory tests, clinical signs and imaging results. In 2006, the diagnostic criteria for NMO were revised after anti- AQP4-Ab were detected. Simultaneous ON and TM are considered as the absolute criterion for diagnosing NMO (Sellner, 2010). The diagnosis of NMO is now based on the criteria from 2015. The diagnosis requires that two of the three following supportive criteria be fulfilled: • contiguous spinal cord MRI lesion extending over three or more vertebral segments; • brain MRI not meeting diagnostic criteria for MS according to Paty; • anti-AQP4-Ab seropositive status. Anti-AQP4-Ab allows also the identification of condition called NMOSD. The diagnostic criteria include NMO-IgG positive and one of the conditions listed below: 1. Limited forms of NMO: • idiopathic single or recurrent events of LETM (longitudinally extensive transverse myelitis); • isolated, recurrent or simultaneous bilateral ON; 2. ON or LETM associated with systemic autoimmune disease. 3. ON or myelitis associated with brain lesions typical of NMO.

Neuromyelitis optica (NMO) – a challenging demyelinative disorder

These diagnostic criteria may be modified or replaced in the nearer future. NMO can be either a fulminating monophasic disease (20%) or more typically, one that is polyphasic (80%), with long-term survival being related to the number of attacks in the early period after the initial event. Diverse neurological diseases including inflammatory, infectious, malignant, vascular, and hereditary aetiologies can mimic the NMO and NMOSD. In differential diagnosis the following should be taken into account: acute disseminated encephalomyelitis (ADEM), idiopathic acute transverse myelitis (ATM), inflammatory diseases associated with antibody to myelin oligodendrocyte glycoprotein (anty-MOG), sarcoidosis, systemic autoimmune disease (Sjogren’s syndrome, systemic lupus erythematous, Neuro-Behçet’s disease), infections neurosyphilis. Also, rare infections such as Herpes Virus, human T-lymphotrophic virus 1 (HTLV-1), dengue virus, Borrelia burgdorferi (Lyme), tuberculosis, Mycoplasma pneumoniae and Streptococcus pneumonia can manifest as LETM and/or ON (Kim, 2017). Despite some similarity in their course, they are distinct from each other in their pathogenesis, prognosis, and most importantly treatment. TREATMENT Although NMO cannot be definitely cured, there are some therapeutic options (Collongues, 2011, Patterson, 2017, Akaishi, 2017, 25. Kimbrough, 2012, Palace, 2012). Treatment for NMO can be divided into three categories: treatment of acute attacks as they occur, treatment to reduce the activity of the disease and symptomatic therapy. Recommended therapeutic options for acute attacks of NMO include high doses of intravenous corticosteroids (methylprednisolone 1 g daily for three or five days) as the initial treatment. If the patient’s condition does not improve, therapeutic plasma exchange should be done (five to seven cycles every other day). Plasma exchange is therapy for patients suffering from an acute, severe loss of vision which is resistant to corticosteroid therapy (Wingerchuk, 2007). In most cases, NMO takes a relapsing course with stepwise disability deterioration, so long-term prophylactic treatment should be started after NMO or anti-AQP4-Ab positive NMOSD has been confirmed. The most commonly used first-line therapies in NMO are azathioprine and rituximab (Jacob, 2008). If the treatment has no effect or in case of side effects, the treatment can be switched to mycophenolate mofetil, methotrexate or mitoxantrone (Weinstock-Guttman, 2006). In patients with NMOSD anti-AQP4-Ab negative, therapy initiation depends on the severity and remission of the first attack and the clinical course. Based on a better knowledge of NMO immunology, there are novel drugs being studied, targeting complement activation, interfering with Interleukin-6 receptor (IL6R) activation and depleting antibody-producing plasma cells. Also tocilizumab (IL-6 receptor-blocking antibody) may be a therapeutic option. Treatment with tocilizumab resulted in decrease of the relapse rate, reduced EDSS (Expanded Disability Status Scale) score and relieved neuropatic pain (Araki, 2014). Satralizumab, a humanised monoclonal antibody that binds IL-6R receptors, thereby suppressing inflammation mediated by IL-6 signalling pathways is effective in preventing relapses of NMO.

Justyna Chojdak-Łukasiewicz

Several new potential therapeutic options have resulted, including complement (eculizumab-humanised antibody that binds to the complement component C5 and inhibits the formation of C5b-induced membrane attack complex) and neutrophil elastase inhibition (sivelestat) or blocking of anti-AQP4-Ab (aquaporumab) (Tradtrantip, 2012, Saadoun, 2012). Aquaporumab eliminates complement- and cell-mediated cytotoxicity. Another antibody used in NMO therapy is inebilizumab, a humanised mAb of IgG1 subtype directed against the extracellular B cell marker CD19 leading to depletion of a broad range of B cells, including autoantibody-secreting plasmablasts and CD19- expressing plasma cells (Cree, 2019). The novel therapies are very effective. Eculizumab reduced the risk of NMOSD relapse by 94.2 per cent compared to placebo (p < 0.0001) (Pittock, 2013). Satralizumab together with immunosuppressive therapy significantly reduced the risk of a relapse by 62%. Immunomodulatory therapy used for prophylactic treatment of relapsing-remitting MS appears ineffective for the treatment of NMO and may even increase the course of the disease and relapse rate. Interferon β may aggravate the course of NMO (Shimuzu 2010, Palace 2010). The treatment with natalizumab and fingolimod should be avoided. A retrospective study reported clinical exacerbation after treatment with their use (Barnett 2012, Jacob 2012, Min 2012). Also, alemtuzumab worsens the course of NMO (Gelfand 2014). CONCLUSION NMOSD are rare inflammatory diseases of the CNS. Hallmark features of NMOSD include acute attacks characterised by ON (leading to visual loss), acute TM and the area postrema syndrome. anti-AQP4-Ab are pathogenic and are specific for the disease. The prognosis in NMO is poor, but there are a lot of new potential therapeutic approaches, which may prevent relapses and improve the course of the disease. Understanding the detailed clinical, serological, immunological, and prognostic factors of NMO will improve the proper treatment as well as diagnosis of patients. REFERENCES Akaishi T., Nakashima I., Sato D.K., Takahashi T., Fujihara K., Neuromyelitis Optica Spectrum Disorders, In: Neuroimaging Clin N Am. 2017;27(2):251-265. Allbutt T.C., On the ophthalmoscopic signs of spinal disease, In: Lancet 1870;1:76-78. Araki M., Matsuoka T., Miyamoto K., Kusunoki S., Okamoto T., Murata M. et al., Efficacy of the anti- IL-6 receptor antibody tocilizumab in neuromyelitis optica A pilot study, In: Neurology, 2014;82(15):1302-1306. Asgari N., Owens T., Frøkiaer J., Stenager E., Lillevang S.T., Kyvik K.O., Neuromyelitis optica (NMO)- an autoimmune disease of the central nervous system (CNS), In: Acta Neurol Scand. 2011;123(6):369-384. Barnett M.H., Prineas J.W., Buckland M.E., Parratt J.D., Pollard J.D., Massive astrocyte destruction in neuromyelitis optica despite natalizumab therapy, In: Mult Scler. 2012;18(1):108-112. Barbieri F., Buscaino G.A., Neuromyelitis optica in the elderly, In: Acta Neurol. (Napoli).1989;11:247-251. Bergamaschi R., Ghezzi A., Devic's neuromyelitis optica: clinical features and prognostic factors, In: Neurol Sci. 2004;25 Suppl 4:S364-S367.

Neuromyelitis optica (NMO) – a challenging demyelinative disorder

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Justyna Chojdak-Łukasiewicz

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Neuromyelitis optica (NMO) – a challenging demyelinative disorder

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Gluten intolerance – the complexity of the problem, the heterogeneity of clinical picture – diagnosis and treatment

Emilia Królewicz, Irena Kustrzeba-Wójcicka Department of Medical Biochemistry, Wroclaw Medical University, Chałubinskiego 10, 50-368 Wrocław, Poland *Corresponding author: Emilia Królewicz, PhD, Department of Medical Biochemistry, Wroclaw Medical University, e-mail: [email protected] ABSTRACT Autoimmune diseases and allergies are considered one of the most important factors affecting the quality of human life in industrialized communities. Gluten intolerance attracts the increasing interest of both the researches and physicians because of heterogeneity of its pathomechanisms and a great variety of clinical symptoms. This paper summarizes current knowledge about this phenomenon. Three main forms of irregular reactions to gluten are discussed in detail: autoimmune reaction (coeliac disease, Dühring disease, and gluten ataxia), allergic reaction to gluten – hypersensitivity to protein fraction present in wheat and non-specific gluten intolerance – gluten sensitivity. The pathogenesis of these diseases and clinical differences between them are shown. A diagnostic algorithm for gluten intolerance based on genetic, serological and histopathological tests is proposed. It was emphasized, that the diagnosis of autoimmune and allergic gluten intolerance is easier compared to the diagnosis of non-specific gluten sensitivity. Apart from the lack of knowledge about biomarkers enabling the diagnosis of non-specific hypersensitivity to gluten, the reasons for this phenomenon and the possible level of gluten tolerance in people with gluten sensitivity are also unknown. The diagnosis must be based on clinical criteria, supported by observing the patient during a challenge with gluten-rich foods and during a gluten-free diet. In order to gain a new knowledge in the area of gluten intolerance diagnosis, further detailed research and analyzes are necessary. INTRODUCTION World Health Organization (WHO) and Food Agriculture Organization (FAO) define gluten as a fraction of storage proteins contained in grasses and cereals, called prolamines, soluble in ethanol and insoluble in water and 0.5% NaCl (Arendt et al., 2011). Prolamines cause enteropathy in patients with genetic predisposition: atrophy of the intestinal villi, enterocolitis, and digestive and absorption disorders. Gluten is defined as a family of proteins found in some grains (wheat, rye, barley, oats). Guten includes two main proteins: gliadin and glutenin. Also, similar proteins such as secalin in rye, harden in barley and avenues in oats contribute to the definition of "gluten" (Biesiekierski, 2017). Gluten proteins are characterized by high proline and glutamine content, which are resistant to proteolytic enzymes in the gastrointestinal tract (Ortiz et al., 2017). Excessive response of the immune system to these protein fractions in predisposed individuals leads to damage and even complete disappearance of intestinal villi, whose physiological role is the absorption of small-molecule digestive products. This phenomenon is also accompanied by a significant decrease in the number of cells producing intestinal hormones, which results in disorders in the process of digestion and absorption of food. As a result of the described irregularities, there is an impairment of the absorption of nutrients, manifested in chronic diarrhea and deficiencies of micro- and macroelements, necessary for the proper functioning of the body (Sapone et al., 2012).

52 Gluten intolerance – the complexity of the problem, the heterogeneity of clinical picture – diagnosis and treatment

SEARCH STRATEGY AND SELECTION CRITERIA The aim of this work was to present a literature review about three known types of gluten intolerance. We described gluten intolerance due to genetic causes: coeliac disease (CD), wheat allergy (WA) and non-coeliac gluten sensitivity (NCGS). Particular attention has been paid to presenting the complexity of the problem, the heterogeneity of clinical picture, diagnostics and treatment of each disease. To prepare a paper, the following data bases were used: PubMed (NCBI), Google Scholar, Web of Science, Scopus, HUGO Gene Nomenclature Committee database, UniProt Know- ledgebase. We searched through primary original articles and reviews published from 2014 until 2019. COELIAC DISEASE Nowadays there are three known types of gluten intolerance. The first of them is coeliac disease (CD), known as a genetic disease. This disease is permanent gluten intolerance, conditioned by the occurrence of HLA class II alleles (human leukocyte antigens) encoding HLA DQ2 and/or HLA DQ8 antigens (Al-Toma et al., 2019). The HLADQ gene is part of the MHC class II major histocompatibility complex, consisting of a set of proteins responsible for presenting antigen to T lymphocytes. Due to the presence of HLADQ, the human immune system can distinguish between the body’s own cells and foreign cells (Glissen Brown, Singh, 2019). Some literature data indicate that coeliac disease is now recognised as a global disease affecting about 0.7-1% of the world's population (Glissen Brown, Singh, 2019; Lebwohl et al., 2018). According to several authors, CD prevalence based on the serological results, is estimated to be 1.1% to 1.7% worldwide (Tanveer, Ahmed, 2019; Singh et al., 2018). Symptoms are often unusual or resemble symptoms characteristic of other diseases (Mavrinac et al., 2014). In the case of overt and full-blown coeliac disease, patients experience constant fatigue as a result of chronic gastric disorders such as diarrhea, flatulence or constipation (Levy et al., 2014). Deficiencies of iron, folic acid and vitamins are the cause of weight loss, and often also the appearance of apathy or depression in patients suffering from coeliac disease (Sams, Hawks, 2014). Unlike the explicit form, scanty symptomatic and latent form of coeliac disease is much more difficult to diagnose. This is due to the occurrence of non-specific parenteral symptoms, which include, but are not limited to, fertility disorders, bone changes, neurological disorders, and decreased immunity (Tonutti, Bizzaro, 2014). The disease can manifest itself at any age. Epidemiological data indicate that the geographical distribution of coeliac disease is more related to genetic background and high consumption of cereal products than to race, or age (Schuppan et al., 2009). An important aspect in the pathogenesis of coeliac disease is the presence of specific markers in the blood serum, such as: tissue transglutaminase antibodies class A (tTG), endomysial antibodies (EmA) and antibodies against deaminated gliadin peptides (DGP) (Gianfrani et al., 2007). Endomysial antibodies are a type of immunoglobulin against tissue transglutaminase (tTG), which is an enzyme involved in many biochemical processes. The substrate for this enzyme is, inter alia gliadin (one of the gluten fractions), which is modified by tTG as a result of deamidation or transamidation

Emilia Królewicz, Irena Kustrzeba-Wójcicka reactions (Gianfrani et al., 2007). It has been proved that new forms of antigens resulting from these reactions are characterized by high affinity for HLA DQ2 and HLA DQ8 heterodimers, and thus are presented to CD4+ helper lymphocytes, which induces a strong inflammatory response of the immune system (Hujoel et al., 2019). About 10% of people with coeliac disease have neurological disorders called cerebellar ataxia, in which there is usually no histological and serological changes typical of coeliac disease. However, there are other determinants of coeliac disease, such as the presence of IgA anti-transglutaminase deposits and anti-type 6 transglutaminase antibodies (so-called neuronal tissue transglutaminase) in the patient's blood serum (Nikpour, 2012). In the case of ataxia in the patients, states of imbalance, uncoordi- nated gait and rapid movements are characteristic as a consequence of the body's intolerance to gluten (Nikpour, 2012). In 1884, Louis Dühring described the cutaneous form of coeliac disease, also referred to as the entero-cutaneous syndrome (dermatitis herpetiformis), as chronic skin lesions occurring mainly around the knees, elbows, buttocks, shoulder blades and on the face and head (Dühring, 1884). However, the relationship between the symptoms of Dühring's disease and the consumption of gluten and the treatment of this disease with a gluten-free diet was discovered only in 1969 (Dühring, 1983). The cutaneous form of coeliac disease most often manifests later than coeliac disease, but it affects both adults and children. It is estimated that about 10% of patients, in addition to multiform skin lesions (vesicles, papules, erythema) also experience gastrointestinal complaints caused by partial or total atrophy of the small intestine villi (Abdelmaksoud, 2017). Studies confirming the diagnosis of Dühring's disease are based on a biopsy of healthy skin from the buttock to detect granular deposits of immunoglobulins class A. These antibodies, moving in the papillary layer of the skin cause the occurrence of microtuns. Similarly to celiac disease diagnosis, IgAEmA level determination and genetic testing for the presence of specific haplotypes of the HLA DQ2 and DQ8 system are ordered. The basic treatment for entero-cutaneous syndrome is strict adherence to a gluten-free diet. The first effects of therapy noticeable as the disappearance of skin lesions are often felt after six months. In order to accelerate the results of the diet, additional pharmacological treatment is also included. An important aspect is also the reduction of iodine intake (iodized salt, fish, seafood, drugs containing this element) as a factor exacerbating symptoms in Dühring's disease (Abdelmaksoud, 2017). WHEAT ALLERGY Wheat protein allergy (WA) is another type of gluten intolerance. Depending on the time of onset of symptoms, an allergic reaction to gluten can be immediate (occurs within a few minutes or up to an hour after eating food) or delayed (manifests itself within a few hours or after 1-2 days) (Ortiz et al., 2017). In the case of an immediate reaction, we are dealing with an IgE-dependent allergy, involving antibodies associated with the mast cell membrane. With delayed-type hypersensitivity, which also occurs with gluten allergies, cell response mechanisms are of primary importance. T-cells, which secrete cytokines and growth factors, play a key role in this type of reaction, thus activating macrophages to participate in the inflammatory process (Cianferoni, 2016).

54 Gluten intolerance – the complexity of the problem, the heterogeneity of clinical picture – diagnosis and treatment

The clinical manifestation of wheat allergy differs depending on age. Children usually have atopic dermatitis after ingesting even milligram doses of gluten (Mäkelä et al., 2014). Urticaria, angioedema, gastrointestinal symptoms, and in some cases even anaphylactic shock are observed in adults (Inomata, 2009). The incidence of wheat protein allergies ranges from 0.4% to 1% among societies of different countries and depends not only on the age but also on the geographical region (Zuidmeer et al., 2008; Inomata, 2009). Wheat allergy among children occurs with a frequency of 0.4-9% (Ortiz et al., 2017; Elli et al., 2015). It is known from clinical experience that most children grow out of this type of allergy, while in adults, gluten allergy lasts for years, so observing a gluten-free diet is an important aspect in its treatment (Mäkelä et al., 2014). COELIAC DISEASE DIAGNOSTICS Coeliac disease defined as an inflammatory enteropathy of the small intestine with an autoimmune basis is a completely different disease compared to allergic gluten intolerance. Detailed and properly conducted diagnostics makes it possible to accurately diagnose these diseases and introduction of proper treatment in people with an incorrect reaction to gluten products. The algorithm for diagnosing gluten intolerance begins with the exclusion or confirmation of coeliac disease. For this purpose, several types of clinical criteria are considered, starting from genetic testing. The presence of HLA DQ2 and/or DQ8 haplotypes is observed in almost all coeliac patients (Megiorni et al., 2009). Using the PCR (EUROArray) method, the HLA DQ2 and/or DQ8 genes may be detected, but this is not a clear indication that the patient is suffering from coeliac disease. It is estimated that about 30% of the healthy population also has this genotype, which only indicates a certain genetic predisposition to this disease (Gallegos, Merkel, 2019). Nonetheless, negative HLA DQ2 and/or DQ8 haplotype testing results can rule out celiac disease. Serological tests performed using immunoenzymatic (ELISA) or immunofluorescent methods are also an important element in the diagnosis of coeliac disease. Within them, the concentration of antibodies against smooth muscle endomysium (EmA), against tissue transglutaminase (tTG) and deamidated gliadin peptide (DGP) is measured. Increased levels of these markers several times inclines towards expanding diagnostics with histopathological tests in order to explicitly confirm coeliac disease. In biopsy specimens of the small intestine, the degree of atrophy of intestinal villi is assessed according to the Marsh scale. Microscopic evaluation of material collected during the biopsy at the level of 2-3 on the Marsh scale and positive results of genetic and serological tests clearly indicate coeliac disease (Tonutti, Bizzaro, 2014). WHEAT ALLERGY DIAGNOSTICS Diagnosis of gluten allergy is based on the implementation of the skin prick test and determining the specific IgE antibodies circulating in the blood serum of the patient (Cianferoni, 2016). It is worth noting that the predictive value of skin prick tests for protein wheat is lower than 75%, mainly due to the occurrence of a cross-allergy to grass pollen. Furthermore, allergen extracts available on the pharmaceutical market are characterized by low sensitivity, because they are mostly water-soluble mixtures

Emilia Królewicz, Irena Kustrzeba-Wójcicka of salts of proteins present in wheat grains and do not contain the insoluble fraction of gliadin (Sapone et al., 2012). Due to the questionable reliability of skin tests in the diagnosis of food allergy, immunoenzymatic methods that allow the determination of allergen-specific gluten IgE should be used in further diagnostics. In laboratories, the Phadia ImmunoCAP system is used for this purpose. NON-COELIAC GLUTEN SENSITIVITY In recent years, scientists have been particularly interested in the third type of abnormal reaction to wheat protein, known as non-coeliac gluten sensitivity (NCGS). In this disease, despite the negative results of genetic tests for the HLA DQ2 and/or DQ8 genotype and normal results of tTG, EmA levels, as well as negative skin tests for gluten and the lack of an elevated level of specific IgE antibodies, the patient has adverse reactions on the part of the digestive system. There is virtually no atrophy of intestinal villi (Marsh histopathological examination at 0 or 1). In the clinical picture of people suffering from hypersensitivity to gluten, in addition to typical gastric abnormalities, rash, constant tiredness, numbness and pain in the limbs, headache and anxiety, and often also a burning sensation in the esophagus or glossitis are observed (Cabrera-Chávez et al., 2017; Mansueto et al., 2014). In 2011, the first international conference on gluten intolerance took place in London, during which world-renowned scientists and doctors recognized gluten hypersensitivity as a new disease entity (Catassi et al., 2013). Due to an absence of diagnostic markers and population studies, the prevalence of NCGS is not well established (Ortiz et al., 2017; Barbaro et al., 2018). Although studies have been conducted by several authors, this problem is still insufficiently explored. Literature data indicate that this disease mainly affects adults on a scale of 0.6% up to 13% of the general population (Carroccio et al., 2017; Cabrera-Chávez et al., 2017; DiGiacomo et al., 2013; Van Gils et al., 2016; Aziz et al., 2014). Non-coeliac gluten sensitivity was reported more often among women than in men (Cabrera-Chávez et al., 2017; DiGiacomo et al., 2013; Van Gils et al., 2016), adults in the fourth decade of life (Aziz et al., 2014; Volta et al., 2014) and individuals coming from urban areas (Van Gils et al., 2016). Currently, due to the lack of specific markers, the diagnostics of NCGS must be based on clinical criteria, supported by the observation of the patient during a challenge with gluten-rich foods and during a gluten-free diet. Based on scientific research, it is known that the presence of the HLA DQ2 and/or DQ8 genotype may be observed only in about 50% of people hypersensitive to gluten, therefore this disease, unlike coeliac disease, has no genetic background (Sapone et al., 2012; Ashat, Kochhar, 2014). When determining the gluten-free diet, should take into account that natural gluten-free products are: corn, rice, potatoes, soy, millet, buckwheat, tapioca, amaranth, cassava, lentils, beans, sago, sorghum, nuts, as well as meat, fruit and vegetables. Processed products are also considered gluten-free, in which, according to the World Health Organization (WHO), the gluten content does not exceed 20 ppm (20 mg per kg of dry matter) and they are marked with the international mark of the crossed-out ear of wheat. There are reports in the literature of an impressive increase in the use of a gluten-free diet. People who decide to take this step do so by trial and error to see if gluten

56 Gluten intolerance – the complexity of the problem, the heterogeneity of clinical picture – diagnosis and treatment products are harmful to them. Others, however, follow a fashionable trend that has spread in recent years, especially among Americans, and is based on the supposition that switching to a gluten-free diet guarantees effective weight loss (Lundin, 2014). CONCLUSIONS The treatment of all three forms of gluten intolerance presented in the work is based on the exclusion of this protein from the patient's diet. The differences, however, are that in coeliac disease, the diet must be used throughout life, and in the case of allergies and hypersensitivity to gluten, the diet may be used temporarily. However, it is not possible to determine how long patients should exclude gluten from their diet. Each case of hypersensitivity is individual and requires a separate therapeutic procedure. NCGS is a much more common phenomenon than coeliac disease and wheat protein allergy. Apart from the lack of knowledge about specific biomarkers enabling diagnostics of non-coeliac gluten sensitivity, the reasons for this phenomenon and the possible level of gluten tolerance in individuals with gluten sensitivity are also unknown. In order to gain new knowledge in the above-mentioned areas of diagnosis of gluten hypersensitivity, further detailed research and analyzes are necessary. CONFLICT OF INTEREST The contribution of the authors in the order from affiliation. Authors Emilia Królewicz and Irena Kustrzeba-Wójcicka declare that they have no conflict of interest. ABBREVIATIONS CD – Coeliac Disease DGP – Deaminated Gliadin Peptides EmA – Endomysial Antibodies HLA – Human Leukocyte Antigens IgA – Immunoglobulin class A IgE – Immunoglobulin class E NCGS – Non-Coeliac Gluten Sensitivity tTG – tissue TransGlutaminase WA – Wheat Allergy REFERENCES Al-Toma A., Volta U., Auricchio R., Castillejo G., Sanders D.S., Cellier C. et al., European Society for the Study of Coeliac Disease (ESsCD) guideline for coeliac disease and other gluten-related disorders, In: United European Gastroenterol J. 2019; 7(5): 583-613, https://doi.org/10.1177/2050640619844125. Abdelmaksoud A., Dermatitis herpetiformis (Duhring’s disease): a therapeutic challenge, In: Int J Dermatol. 2017; 56(10): e192-3, https://doi.org/10.1111/ijd.13641.

Emilia Królewicz, Irena Kustrzeba-Wójcicka

Arendt E., Kelly A., Moore M., New product development: the case of gluten-free food product, In: Arendt E., Dal Bello F. (Editors), Gluten-free cereal products and beverages, 2011; Elsevier; ISBN: 978-0-12-373739-7. Ashat M., Kochhar R., Non-celiac gluten hypersensitivity, In: Trop Gastroenterol.2014; 35(2): 71-8. https://pubmed.ncbi.nlm.nih.gov/25470868. Aziz I., Lewis N. R., Hadjivassiliou M., Winfield S. N., Rugg N., Kelsall A. et al., A UK study assessing the population prevalence of self-reported gluten sensitivity and referral characteristics to secondary care, In: Eur J Gastroenterol Hepatol. 2014; 26(1): 33-9, https://doi.org/10.1097/01.meg.0000435546.87251.f7. Barbaro M. R., Cremon C., Stanghellini V., Barbara G., Recent advances in understanding non-celiac gluten sensitivity, In: F1000Res. 2018; 7: F1000 Faculty Rev- 1631, https://doi.org/10.12688/f1000research.15849.1. Biesiekierski J.R., What is gluten?, In: J Gastroenterol Hepatol. 2017; 32 (Suppl 1): 78-81. https://doi.org/10.1111/jgh.13703. Cabrera-Chávez F., Dezar G.V.A., Islas-Zamorano A.P., Espinoza-Alderete J.G., Vergara-Jiménez M.J., Magaña-Ordorica D., Prevalence of Self-Reported Gluten Sensitivity and Adherence to a Gluten-Free Diet in Argentinian Adult Population, In: Nutrients. 2017; 9(1): 81, https://doi.org/10.3390/nu9010081. Carroccio A., Giambalvo O., La Blasca F., Iacobucci R., D’Alcamo A., Mansueto P., Self-reported non- celiac wheat sensitivity in high school students: Demographic and clinical characteristics, In: Nutrients. 2017; 9(7): 771. https://doi: 10.3390/nu9070771. Catassi C., Bai J.C., Bonaz B., Bouma G., Calabrò A., Carroccio A., Non-Celiac Gluten sensitivity: the new frontier of gluten related disorders, In: Nutrients. 2013; 5(10): 3839-53, https://doi.org/10.3390/nu5103839. Cianferoni A., Wheat allergy: diagnosis and management, In: J Asthma Allergy. 2016; 9: 13-25. https://doi.org/10.2147/JAA.S81550. DiGiacomo D.V., Tennyson C.A., Green P.H., Demmer R.T., Prevalence of gluten-free diet adherence among individuals without celiac disease in the USA: results from the Continuous National Health and Nutrition Examination Survey 2009-2010, In: Scand J Gastroenterol. 2013; 48(8): 921-925. https://doi.org/10.3109/00365521.2013.809598. Duhring L.A., Landmark article, Aug 30, 1983: Dermatitis herpetiformis. By Louis A. Duhring, In: JAMA. 1983; 250(2): 212-6, https://doi.org/10.1001/jama.250.2.212. Duhring L.A., Dermatitis herpetiformis, In: JAMA. 1884; 3(9): 225-9. Elli L., Branchi F., Tomba C., Villalta D., Norsa L., Ferretti F. et al., Diagnosis of gluten related disorders: Celiac disease, wheat allergy and non-celiac gluten sensitivity, In: World J Gastroenterol. 2015; 21(23): 7110-9. https://doi.org/10.3748/wjg.v21.i23.7110. Gallegos C., Merkel R., Current Evidence in the Diagnosis and Treatment of Children With Celiac Disease, In: Gastroenterol Nurs. 2019; 42(1): 41-8, https://doi.org/10.1097/SGA.0000000000000365. Gianfrani C., Siciliano R.A., Facchiano A.M., Camarca A., Mazzeo M.F., Costantini S. et al., Transamidation of wheat flour inhibits the response to gliadin of intestinal T cells in celiac disease, In: Gastroenterology, 2007; 133(3): 780-9, https://doi.org/10.1053/j.gastro.2007.06.023. Glissen Brown J.R., Singh P., Coeliac disease, In: Paediatr Int Child Health, 2019; 39(1): 23-31. https://doi.org/10.1080/20469047.2018.1504431. Hujoel I.A., Reilly N.R., Rubio-Tapia A., Celiac Disease: Clinical Features and Diagnosis, In: Gastroenterol Clin North Am. 2019; 48(1): 19-37, https://doi.org/10.1016/j.gtc.2018.09.001. Inomata N., Wheat allergy, In: Curr Opin Allergy Clin Immunol. 2009; 9(3): 238-43. https://doi.org/10.1097/ACI.0b013e32832aa5bc.

58 Gluten intolerance – the complexity of the problem, the heterogeneity of clinical picture – diagnosis and treatment

Lebwohl B., Sanders D.S., Green P.H.R., Coeliac disease, In: Lancet. 2018; 391 (10115): 70-81, https://doi.org/10.1016/S0140-6736(17)31796-8. Levy J., Bernstein L., Silber N., Celiac disease: an immune dysregulation syndrome, In: Curr Probl Pediatr Adolesc Health Care. 2014; 44(11): 324-7, https://doi.org/10.1016/j.cppeds.2014.10.002. Lundin K.E.A., Non-celiac gluten sensitivity – why worry?, In: BMC Med. 2014; 12: 86, https://doi.org/10.1186/1741-7015-12-86. Mäkelä M.J., Eriksson C., Kotaniemi-Syrjänen A., Palosuo K., Marsh J., Borres M., Wheat allergy in children - new tools for diagnostics, In: Clin Exp Allergy. 2014; 44(11): 1420-30. https://doi.org/10.1111/cea.12393. Mansueto P., Seidita A., D’Alcamo A., Carroccio A., Non-celiac gluten sensitivity: literature review, In: J Am Coll Nutr. 2014; 33(1): 39-54, https://doi.org/10.1080/07315724.2014.869996. Mavrinac M.A., Ohannessian A., Dowling E.P., Dowling P.T., Why celiac disease is so easy to miss, In: J Fam Pract. 2014; 63(9): 508-13, https://pubmed.ncbi.nlm.nih.gov/25353024. Megiorni F., Mora B., Bonamico M., Barbato M., Nenna R., Maiella G. et al., HLA-DQ and risk gradient for celiac disease, Hum Immunol. 2009; 70(1): 55-9, https://doi.org/10.1016/j.humimm.2008.10.018. Nikpour S., Neurological manifestations, diagnosis, and treatment of celiac disease: A comprehensive review, In: Iran J Neurol. 2012; 11(2), 59-64, https://pubmed.ncbi.nlm.nih.gov/24250863. Ortiz C., Valenzuela R., Lucero A.Y., Celiac disease, non celiac gluten sensitivity and wheat allergy: comparison of 3 different diseases triggered by the same food, In: [Article in Spanish] Rev Chil Pediatr. 2017; 88(3): 417-423, https://doi.org/10.4067/S0370-41062017000300017. Sams A., Hawks J., Celiac disease as a model for the evolution of multifactorial disease in humans, Hum Biol. 2014; 86(1): 19-36. https://doi.org/10.3378/027.086.0102. Sapone A., Bai J.C., Ciacci C., Dolinsek J., Green P.H.R., Hadjivassiliou M. et al., Spectrum of gluten- related disorders: consensus on new nomenclature and classification, In: BMC Med. 2012; 10: 13, https://doi.org/10.1186/1741-7015-10-13. Schuppan D., Junker Y., Barisani D., Celiac disease: from pathogenesis to novel therapies, In: Gastroenterology. 2009; 137(6): 1912-33, https://doi.org/10.1053/j.gastro.2009.09.008. Singh P., Arora A., Strand T.A., Leffler D.A., Catassi C., Green P.H. et al., Global Prevalence of Celiac Disease: Systematic Review and Meta-analysis, In: Clin Gastroenterol Hepatol. 2018; 16(6): 823-836.e2, https://doi.org/10.1016/j.cgh.2017.06.037. Tanveer M., Ahmed A., Non-Celiac Gluten Sensitivity: A Systematic Review, In: J Coll Physicians Surg Pak. 2019; 29(1): 51-57, https://doi.org/10.29271/jcpsp.2019.01.51. Tonutti E., Bizzaro N., Diagnosis and classification of celiac disease and gluten sensitivity, In: Autoimmun Rev. 2014; 13(4-5): 472-6, https://doi.org/10.1016/j.autrev.2014.01.043. Van Gils T., Nijeboer P., IJssennagger C.E., Sanders D.S., Mulder C.J.J., Bouma G., Prevalence and Characterization of Self-Reported Gluten Sensitivity in The Netherlands, In: Nutrients. 2016; 8(11):714, https://doi.org/10.3390/nu8110714. Volta U., Bardella M.T., Calabrò A., Troncone R., Corazza G.R., An Italian prospective multicenter survey on patients suspected of having non-celiac gluten sensitivity, In: BMC Med. 2014; 12: 85, https://doi.org/10.1186/1741-7015-12-85. Zuidmeer L., Goldhahn K., Rona R.J., Gislason D., Madsen C., Summers C. et al., The prevalence of plant food allergies: a systematic review, In: J Allergy Clin Immunol. 2008; 121(5): 1210-1218.e4, https://doi.org/10.1016/j.jaci.2008.02.019.

Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview

Aleksandra Zarek*, Aleksandra Kroll Aleksandra Zarek: Department of Medical Humanities and Social Science, Wroclaw Medical University, ul. J. Mikulicza-Radeckiego 7, 50-368 Wrocław, Poland Aleksandra Kroll: Department and Clinic of Psychiatry, Pomeranian Medical University, ul. Broniewskiego 26, 71-460 Szczecin, Szczecin, Poland *Corresponding author: Aleksandra Zarek, Department of Medical Humanities and Social Science, Wroclaw Medical University, ul. J. Mikulicza-Radeckiego 7, 50-368 Wrocław Poland, e-mail: [email protected] ABSTRACT Introduction: In this theoretical article the process of interviewing the patient will be presented in the medical and psychotherapeutic contexts. Aim: The presented study aims to find the main similarities and differences between the medical and psychotherapeutic approach to the first interview with the patient. Materials and methods: Comparative analysis of this process is performed with the use of three models of communication: the First Interview in Positive Psychotherapy created by Nossrat Peseschkian; Three Functional model of the medical interview described by S. Cole and J. Bird and Calgary-Cambridge Guide model of the authorship of S. Kurtz, J. Silverman and J. Draper. The analysis is conducted using seven categories: building the relationship, content, structure together with questioning style, communication skills applied and their function, and time. Results: Presented models of medical and therapeutical interviews share many similarities in the areas of building relationship, providing structure and questioning style. They differ mostly in content and duration of the visit, but not approach to time quality. Both the therapist and the physician are advocated to use the same 10 groups of skills to: create the safe personal environment; show their understanding and acceptance for the patient as a person; reveal interest in the patient’s perspective; create emotional bond between them and the patient; get the broad picture of the patient’s situation and adequately direct the interview; enhance precision of obtained information; enhance active attitude and engagement of the patient in the healing process; prevent overburdening the patient and facilitate their learning; broaden patient’s perception. Conclusions and recommendations: While conducting an interview the therapist and the physician apply similar communication skills especially concerning building relationship, providing structure and questioning style and those skills serve simultaneously diverse instrumental and relational purposes. According to the presented models building a safe relationship is considered as essential to the efficiency of the specialists’ assistance. A physician, like a therapist, is expected to properly respond to patients' emotions, but may be not enough equipped for this task. It is then recommended to include interpersonal training in the curriculum of medical education. Keywords: communication skills, psychotherapy, Positive Psychotherapy, doctor-patient communication, Calgary-Cambridge Guide, Three Functional model of medical interview INTRODUCTION Interpersonal communication is one of the most important factors in every medical profession, which lacks results in e.g. an increase in health care costs and negative patients outcomes (Ratna, 2019). Communication skills can be trained and therefore

60 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview are part of the medical professions curriculum in many countries to ensure these skills are adequately developed in students (Yedidia et al., 2003). One of the key communication skills in all medical occupations including general medical practice, as well as psychotherapy, is interviewing the patient (Hatem et al., 2007). In a variety of its forms, psychotherapy is a method of treatment applied in people with mental health disturbances that helps them to recognize and change (reorganize) their intrapsychic qualities influencing inadequate, and in turn, insufficient ways of realization of their own needs. In all psychotherapeutic approaches, those difficulties are explained as resulting from negative life experiences and corrected in psychotherapeutic contact (Czabała, 2016). Polish therapists declare working with patients being diagnosed with: neurotic disorders (87%), personality disorders (74%), psycho- somatic disorders (69%), affective disorders (68%), victims (51%), and perpetrators (16%) of violence, eating disorders (46%), somatic illnesses (31%), sexual dysfunctions (22%), schizophrenia (22%) and psychotic disorders other than schizophrenia (21%), organic disorders (12%) and mental retardation (7%). The main psychotherapeutic approach that they apply in their work is psychodynamic (53%), then systemic (21%), existential (20%), and cognitive-behavioral (17%) approach (Suszek, 2017; Szymańska, 2017a). Independently of the approach-oriented techniques, the results of psychotherapy are influenced by the quality of a therapeutic interaction – a therapeutic alliance established between the patient and their therapist, which includes therapeutic goals and tasks, and leads to the development of the professional bond (positive affective attachment) (Cooper, 2010; Szymańska, 2017). Positive Psychotherapy is an integrative method of therapy, rooted in psychodynamic and humanistic approaches to psychotherapy, but encompassing also elements of cognitive-behavioral and systemic approaches, and at the same time, it is the school of therapy established in 1968 in Germany by Nossrat Peseschkian (Remmers, 2020). It’s founder, born in Iran in 1933, finished medical school in Germany, where he lived and worked till the end of his life in 2010. He was a neurologist, psychiatrist, and psychotherapist – and (possibly due to his multicultural personal and professional experiences) also especially sensitive to the cultural context of psychological difficulties experienced by his patients (Ciesielski, 2015; Dobiała, 2016). Peseschkian defined Positive Psychotherapy as a psychotherapeutic method resting upon differentiation analysis and also a metatheory of a universal character aiming at thoroughly understanding the patient – as a unique individual having the capacity for both illness and health. He believed that the concepts of Positive Psychotherapy – especially the actual capabilities understood as descriptive categories of human behavior and experience – were neither class nor culture-specific, and therefore presented an effective basis for communication (Peseschkian, 1987). Three Functional Model of the medical interview was developed by Steven Cole and Julian Bird. According to the authors providing the medical interview efficiently and effectively requires from the physician incorporating three components into every meeting with the patient: 1) building the relationship; 2) assessing and understanding and 3) collaboration for management. Each of them has its specific function and requires using certain communication skills and techniques, but they are comple-

Aleksandra Zarek, Aleksandra Kroll mentary altogether and serve the purpose of recognizing and improving the patient’s medical situation (Bird & Cole, 2014). The authors of Calgary-Cambridge Guide – Suzanne Kurtz, Jonathan Silverman, and Juliet Draper – intended to make a conceptualization that would correspond with the organization of a real medical situation and aimed at integrating three aspects of medical interview understood as clinical competences: the content of the medical interview, the process of gathering information and the patient’s perspective. They put special attention to the structure of the medical interview, as the overall comprehension of the structure enables the students, facilitators, and practicing physicians to apply the various communication skills intentionally and deliberately at all stages of medical consultation (Kurtz, 2009; Silverman, 2018). All three models have been developed as tools for conducting the first interview with patients for the purpose of engaging proper healing procedures. We investigate what they have in common and how they differ? Although these models have been elaborated for work with persons suffering from various health disturbances (psychic vs somatic), their authors were both practitioners and scientists, educated in the field of medicine and their recommended methods of communication with patients are based on long-term personal experience. AIM The presented study aims to find the main similarities and differences between the medical and psychotherapeutic approach to the first interview with the patient by comparison and analysis of the three different models of the interview with the patient dedicated to the psychotherapists and general practitioners. MATERIALS AND METHODS In this theoretical article the process of interviewing the patient in two contexts – psychotherapeutic and medical – is presented and analyzed. Comparative analysis of these two forms of contact with a patient using three models of communication is conducted. Three models of interviewing the patient are presented, in the sequence corresponding with the time of their first publication: The first Interview in Positive Psychotherapy created by Nossrat Peseschkian in 1987, Three Functional models of the medical interview described by Steven Cole and Julian Bird in 1991, and the Calgary-Cambridge Guide model of medical communication of the authorship of Suzanne Kurtz, Jonathan Silverman and Juliet Draper first published in 1998. These models are analyzed according to seven categories recognized as vital in the organization of a real encounter with a patient: building the relationship, content, structure together with questioning style, communication skills applied and their function, and time. RESULTS The results of comparative analysis are shown below in three separate sections divided into seven above mentioned categories.

62 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview

THE FIRST INTERVIEW IN POSITIVE PSYCHOTHERAPY (PPT) As Nossrat Peseschkian described it, the first interview is usually the first encounter between a psychotherapist and a patient and it serves various purposes. Firstly, it enables data gathering as the basis for a professional opinion or scientific work, subsequent counseling, decision making whether to start psychotherapy, and in what form. Secondly, it already contains therapeutic elements that are to be pursued in future contact with the patient. As it has a predominantly diagnostic function, it aims at the comprehensive operationalization of the patient’s conflict areas. Like any other interaction, it can be described in correspondence to three phases: fusion, differentiation, and breakaway (Peseschkian, 1987). BUILDING THE RELATIONSHIP In the First Interview, the phase of fusion aims at the establishment of a relationship of trust with the patient, which is necessary to enhance their honesty and openness in the further steps of the encounter. In this phase, the main task of the therapist is to connect emotionally to their patient and create conditions for establishing a therapeutic alliance. The therapist takes the role of the observer, putting the patient’s perspective and their view of the situation (their explanatory model) in the center of attention. To encourage the patient’s disclosure, the therapist presents a non-judgmental and nondirective attitude, as well as appropriate listening behaviors, adjusting to the patient’s tempo. When asking questions, the therapist should pay attention to the flow of the interview and give the patient enough time to answer them at length, justifying, and explaining any interruption (Peseschkian, 1987). By using these skills the therapist ensures their patient conditions to speak freely in an emotionally safe atmosphere and at the same time presents active interest by reflecting the situation and feelings of the clients and noting down their expressions (Remmers, 2020). While listening to their patient’s narration, the therapist gets informed about the patient’s reasons for the consultation but also makes a hypothesis about their unconscious motives and expectations (Ciesielski, 2016). CONTENT The aspect of the content pertains to the question of the kind and range of exchanged information. In the first part of the interview (fusion phase), the therapist’s goal is to attune to the patient’s emotional needs of being understood and accepted and establish the ground for future contact. In the first visit to a therapist the patient might feel abashed, anxious, unsure or ashamed, and at the same time still have hope to receive help. Others may feel doubtful and for some patients, it would be very difficult to trust anyone. The therapist’s task is to recognize the patient’s hope and motivation for change, but also their need and ability to trust. Another task is to gather information about the patient, which includes a portrayal of the patient’s complaints, together with the main complaint, symptom profile (a catalytic situation, severity) and its genesis, social anamnesis (age, marital status, occupation, partner’s occupation, parents, siblings, religion), somatic anamnesis (physical development and prior illness), previous psychiatric and psychotherapeutic treatment. The therapist puts special attention to the context in which the symptoms have developed and the patient’s view of their situation.

Aleksandra Zarek, Aleksandra Kroll

The phase of differentiation centers around the psychological background of the conflict. It has two goals: 1) the determination and isolation of the conflicts and the developmental possibilities and 2) formulating a content-related diagnosis of the conflicts and symptoms presented (actual capabilities involved, actual conflict, conflict situations, conflict partners, basic conflict). That is realized with the use of the Differentiation-Analytic Inventory (DAI) and the modes of the basic capacities as Model Dimensions. The therapist also aims at understanding the background of the preferences given to some values and behaviors over the others and recognizing the patient’s cognitive style, as well as the style of coping with conflict. In the phase of breakaway/integration, the main tasks of the therapist are oriented at integrating gathered information and formulating diagnoses for the patient together with proper recommendations. From the therapist’s standpoint, this includes forming a diagnosis for himself, responding to the patient’s cognitive style and preferred form of communication ("therapeutic entreѐ") to show empathy, building a well-fitting treatment plan, and explaining the diagnosis in the form adjusted to the patient (Peseschkian, 1987, Remmers 2020). STRUCTURE AND THE QUESTIONING STYLE The categories of structure and questioning style correspond with the way the therapist’s questions and comments are formulated and in what sequence they are presented. At the beginning of the interview, the therapist asks open-ended questions to recognize the patient’s motivation and get the portrayal of the patient’s complaints (Who has referred you to me? What brings you to see me?/What complaints do you have?). They might be followed by a series of closed questions for clarification, in case the patient would not include the necessary details in his narration. Then, the anamnesis is undertaken (usually in the form of closed questions) to gather data about the patient’s social and somatic situation. It interrupts the continuum of the interview, but also neutralizes fixations in the patient’s perspective and helps to associate relevant events. Observation of the manner the patient responds to questions of the anamnesis, as well as the overall context of their response serves the therapist as situational hints and enhances the interpretation of the patient’s situation. And finally, the therapist asks about the context in which the symptoms have developed (e.g.: "You said your ailments have been increasing for about two and a half years. Can you tell me what happened to you at about that time?"). It is also possible to gather socio-demographic data at the very beginning of the interview, which some patients find unburdening. As Remmers and Peseschkian suggest, detailed information of this kind is needed for expertly crafted case, but cannot be gathered within the time of the first session (Remmers, 2020). In the next stage of the interview (in the procedure of DAI application) the therapist takes a more directive role and asks questions concerning actual capabilities and at the same time is taking notes of the patient’s spontaneous responses (both wording and behaviors), as well as their interpretations. This process should not interrupt the flow of communication, either be experienced as invasive by the patient, so it requires flexibility on the part of the therapist (giving priority to following patient’s verbali-

64 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview zations instead their of own theoretical expectations). When the data from DAI is gathered, the conflict potentials are presented to the patient and then their task is to decide which are considered to be most important. To avoid overloading the patient the therapist usually selects three focal conflict potentials to be discussed and presented as goals for subsequent treatment. Understanding the background of the preferences given to some values and behaviors over the others requires from the therapist asking questions about the initial social relationships of the patient, which is connected to the patient’s past life (Model Dimensions). That includes the attitudes of the reference persons (parents) and the siblings (also the similarly aged playmates) towards the patient when he/she was a child; the parents’ attitudes toward one another, their environment and religion, or philosophy. To recognize the patient’s cognitive style and coping with conflict, as well as their disturbances, the therapist can either directly ask questions about instrumenta- lities of senses, reason, tradition, and intuition, or derive this information from the content of the conversation. In the phase of breakaway/integration, the therapist synthesizes for himself the findings of the interview to form the diagnosis. This is done not to provide labels, but rather offer initial information concerning optimal procedures (starting psychotherapy, counseling or some form of self-help, formulating the aim of psychotherapeutic work and prognosis considering its achievement, suggesting cooperation with other specialists, writing a referral to the hospital). The therapist’s tasks at this point are: 1) integrating the obtained information in the form of a preliminary report; 2) recognizing the patient’s dominating cognitive style and preferred phase of interaction (to show empathy); 3) building a treatment plan (establishing a recommended form of treatment and expectations regarding prognosis), and finally 4) translating the diagnosis for the patient, which requires eliciting patient's perspective and explanation of the process (Peseschkian, 1987). SKILLS AND THEIR FUNCTION As many of the skills have been discussed in detail in the previous sections, the summary of the therapist’s tasks together with required communication skills in three phases of the interaction of the First Interview is presented in table 1.

Table 1. Therapist’s tasks and required communication skills in stages of fusion, differentiation, and breakaway/integration Therapist’s tasks Therapist's behaviors (verbal and non-verbal) FUSION (20 min) Establishment of a trusting relationship, which provokes a patient’s honesty Presenting appropriate non-verbal behaviors and listening behaviors, asking pointed questions Responding to patient’s needs of being understood (from open to close), giving the patient time to and accepted answer the therapist’s questions, justifying and explaining interruptions, observing patient's Recognizing the patient’s perspective and view of reactions their situation (their explanatory model)

Aleksandra Zarek, Aleksandra Kroll

Establishing a first contact and patient’s motivation, justifying their presence, and recognizing their trust Who has referred you to me? (both as a need and an ability) What brings you to see me?/What complaints do Establishing a portrayal of the patient’s complaints you have? If necessary, closed questions for clarification: Getting the main complaint, symptom profile (a where? when? to what degree? since when? first catalytic situation, severity), and its genesis time/previously appeared? Getting social anamnesis (age, marital status, occupation, partner’s occupation, parents, siblings, pointed (closed) questions religion) Gettig somatic anamnesis (physical development and prior illness), previous psychiatric and pointed (closed) questions psychotherapeutic treatment Establishing the context in which the symptoms have What happened to you at about that time? developed DIFFERENTIATION Asking questions concerning actual capabilities (DAI) and at the same time taking notes of patient’s spontaneous responses (both wording Determination and isolation of the conflicts and the and behaviors) and their interpretations developmental possibilities (data gathering) Giving priority to following patient’s verbalizations instead of their theoretical expectations Formulating content-related diagnosis of the Presenting conflict potentials (usually not more conflicts and symptoms presented (actual capabilities than 3) to the patient and establishing which are involved, actual conflict, conflict situations, conflict the most important partners, basic conflict) Understanding the background of the preferences Asking pointed questions concerning modes given to some values and behaviors over the others of the capacity to love (Model Dimensions) Asking pointed questions concerning modes Recognizing the patient’s cognitive style and coping of the capacity to know (or deriving it from the with conflict style content of the conversation) BREAKAWAY/INTEGRATION Forming a diagnosis for himself Preparing a summarizing report Recognizing (and at first adjusting to) the Recognizing "therapeutic entreѐ" to show empathy patient's dominating modes of the capacity to know and their type of interaction pattern Establishing a recommended form of treatment (medication/consultation/therapy) and Building a treatment plan expectations regarding prognosis (achievable results and time required) Translating the diagnosis for the patient

66 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview

Eliciting patient's perspective (clarifying if Has anything become clear to you? necessary) Using concise, easily understood language Relating explanation to patient's perspective (previously elicited ideas, concerns and expectations) Explanation of the process Informing about the further steps concerning the form of therapy, its timeframe, patient's tasks, the involvement of other parties, major issues/goals of therapy Closing a session Asking for corrections or questions source: own elaboration based on Peseschkian, 1987

TIME Nossrat Peseschkian pointed out that the First Interview may be realized in more than one session in order not to overburden the patient and other authors state it is used either in the first encounter or during the early sessions (Peseschkian, 1987; Remmers, 2020). According to Peseschkian, the first part of the interview (fusion phase) should last on average about 20 minutes. The time range of the differentiation and integration phase would depend on the nature of the patient’s problem and the quality of the therapeutic alliance (influenced both by the psychological qualities of the patient and the therapist, and the therapist’s professional skills), as well as the aim of the consultation. The therapist should respect patient’s tempo of disclosure and find balance between the diagnostic purposes (gathering information) and patient’s emotional comfort. He/she needs to be flexible in directing this process and aware of the reasons of giving time priority to certain diagnostic aspects and omit others (Peseschkian, 1987). THREE FUNCTIONAL MODEL (3F) In the Three Functional model of the medical interview, Cole and Bird present interviewing strategies efficient in assessing patient’s physical condition in the purpose of indicating adequate actions enhancing their health, but also suggest that it is the doctor’s role to create conditions for the patient to disclose all medically relevant information, as well as to motivate them to accept and follow treatment recommendations. BUILDING THE RELATIONSHIP Building the relationship with the patient is the first function of the medical interview and the foundation of effective cooperation. It is based upon a set of emotional response skills considered as uniquely important for practitioners of medicine. These are: 1) nonverbal skills indicating the doctor’s interest/engagement in the conversation with the patient and an attitude of concern and warmth (appropriate eye contact and body posture, body movements, facial expression, tone and rate of speech, touch, the space between the physician and the patient, attentive silence); 2) empathy expressed nonverbally, but also through reflection and legitimation of patient’s feelings; 3) support expressed openly as sincere readiness and willingness to be helpful; 4) partnership shown by the acknowledgment of patient’s importance in the process

Aleksandra Zarek, Aleksandra Kroll of treatment and finally 5) respect presented in a form of open appreciation of patient’s actions or personal qualities directed at improving their health. By using those skills properly a physician can address patients’ expectations and needs to relay on someone who is not only a knowledgeable and technically competent specialist, but also a person that can listen to them, is supportive and emotionally available. It is important for the clinician to strive for consistency between their verbal and nonverbal behaviors, as verbal assurance of being attentive, empathetic and respectful can only be con- vincing and by that helpful for the patient, if it is honest. CONTENT When realizing the first function of the medical interview, described in the previous section, the physician aims at creating an atmosphere of safety and trust. This is done by presenting adequate nonverbal skills and empathy, as well as support and respect for a patient, together with readiness to treat their patient as a partner in their encounter. Building the relationship is the prerequisite of a successful realization of two other functions. Assessing and understanding the patient’s problems is the second function of the medical interview and the basis for constructing a valid diagnosis. It aims at getting a broad view of the patient’s medical problem. The physician’s role is to present behaviors encouraging the patient to reveal comprehensive and reliable information, which requires him/her using abilities like empathetic listening, facilitation, clarification and paraphrasing, direction and summarizing and to adjusting proper questioning style. The third function of the medical interview – Collaboration for management – aims at strengthening the patient's activity and self-reliance and involves: 1) educating the patient about their condition, 2) motivating them to follow a treatment plan, and 3) offering self-management support. Cole and Bird advocate adjusting the spirit of motivational interviewing to influence the patient’s motivation in incorporating necessary health behaviors’ changes in their lives. That means presenting the attitude towards behavioral change that encompasses four elements: 1) evocation – ideas for behavioral change comes from the patient, the physician can only facilitate it; 2) partnership – the clinician and the patient are equals in the change process; 3) acceptance/autonomy support – the role of the medical team is to provide the necessary information, but it is the patient’s decision whether to change; 4) compassion – in the process of change patient’s needs have to be taken into consideration. STRUCTURE AND THE QUESTIONING STYLE The key role in the process of building the relationship with the patient plays the way the clinician responses to the emotional needs and difficulties of the patient. The physician is encouraged to be attentive and present an attitude of concern and warmth from the very beginning of the encounter. To adequately assess and understand the patient’s problem the physician is recommended to 1) start the interview with open questions (opening statement) and then move to closed ones; 2) present proper listening behaviors as indicators of doctor’s attention; 3) avoid leading questions (which means formulating questions in the way

68 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview suggesting what answer is expected, e.g. "How helpful was the medication I gave you?"); 4) facilitate – that it to use expressions and non-verbal signals inducing the patient to continue with talking (nodding, saying "go on", repeating patient’s words); 5) use clarification and direction; 6) ask "what else?" to help the patient in complementing their list of complaints and 7) use checking/summarizing. When informing and educating the patient about their condition – the doctor should take into account the patient’s emotional state, as well as the difference between the patient’s and their medical knowledge. For successful realization of this task it is recommended to follow six stages: 1) elicit patient's baseline understanding of the problem (explanatory model and expectations); 2) tell the patient the core message (diagnosis); 3) ask the patient for their understanding of the condition and their reaction; 4) respond to the emotional impact; 5) counsel the patient about the details of the educational message; 6) check patient’s understanding (ask the patient to paraphrase the core details of the message). Especially in the situation of breaking bad news or passing information that is possibly unexpected by the patient, the authors underline the importance of responding to patient’s emotional reaction before educating them about their condition. To support a patient’s self-management in the process of health behaviors change it is recommended to use Brief Action Planning. That should be realized successively in 5 steps: 1) elicit patient’s ideas for change; 2) use SMART Behavioral Planning (ask questions like What? When? Where? How often? to check if the patient’s plan of health behaviors is Specific, Measurable, Achievable, Relevant, and Time-specific); 3) elicit the commitment statement (what the patient is planning to do); 4) estimate patient’s confidence to perform their plan, and 5) arrange accountability (inform that you will ask about patient’s plan performance). SKILLS AND THEIR FUNCTION The summary of the skills of the three functions of the medical interview together with examples of their use is presented in table 2.

Table 2. Skills Examples Function One: BUILDING THE RELATIONSHIP Attentive listening (eye-contact, forward lean, appropriate pause and silence, Non-verbal behavior avoid interruptions). Reflection "I can see this is very difficult for you." or "You seem very sad." "I can understand why you feel this way." or, "I think most people would feel Legitimation the same way." Support "I want to do whatever I can to help." or "I care about what happens to you." Partnership "We are in this together." or "Let's work on this together." I'm impressed with how well you're coping under the circumstances." or Respect (Affirmation) "I think you're doing a great job managing your illness and keeping up with everything else at the same time."

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Function Two: ASSESSING AND UNDERSTANDING Questioning style (open-to-closed cone) "How can I help you today?" or "What brought you to the hospital?" (open- Opening statement ended question)

Direction/ "Can you tell me more about the back pain?" (directed question) Clarification "Does the pain go down your leg?" (closed question) "Tell me more about…" or "Okay" (attentive silence) or "Uh-huh" (pause, head Facilitation nod) Checking/ Summarizing "Let me review what you've been saying to make sure I've gotten it right..." "What else concerns you?" or "Are there any other ways I can be of help?" or SurveyProblems "Is there anything else on your mind?" "There are several issues you have brought up. Which one would you like to Negotiate agenda talk about first?" Develop a narrative of "Okay, you said the back pain started about three months ago. Why don't we the problem start from that point…what happened next?" Explore patient perspective Ideas: "What ideas gave you had about what might be causing your problems?" Concerns: "What concerns you most about these problems?" Expectations: "What are your expectations for this visit?" "I'd like to know how these problems have affected your day-to-day – your Impact of illness home life, your work, how you feel in general…" Function Three: COLLABORATIVE MANAGEMENT elicit the patient's baseline understanding of the problem Tell the patient the first chunk of information (just for culture and literacy)

Educate (use Ask about understanding and concerns regarding the first chunk eTACCT) Care by using relationship skills as emotions appear Counsel patient regarding the information needed Tell back - ask the patient to review his or her understanding of information 1. "Is there anything you'd like to do about your health in the next week or two?" a. SMART Behavioral Planning - specific, measurable, relevant, time-specific Support Self- (what? where? when? how often?) Management (by using b. elicit the commitment statement e.g. "Would you mind repeating back what Brief Action Planning you just decided to do?" – 3 core questions and 5 associated skills) c. For patients needing or wanting or ideas, offer a behavioral menu 2. "About how confident do you feel that you can carry out your plan, on a 0 to 10 scale, where 0 means you are not at all confident and 10 means you are very confident?"

70 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview

d. If confidence is less than 7, problem-solve ways to overcome barriers 3. When would you like to come back to review how you've been doing with your plan?" e. Follow-up, e.g. "So, how did you go with your plan?"

Motivational 1. Elicit and resolveambivalence Interviewing – advanced skills 2. Develop the discrepancy source: Cole, 2014, pp. 294-295 (with some modifications)

TIME In the Three Functional model, the length of a whole visit is not much discussed, however, the authors advocate to pay special attention to the issue of time – and offer the patient enough time – particularly in two types of situations. First, when the patient is answering the opening statement, the clinician should wait at least 90 seconds without interruption, because usually in that time the patient mentions the most important complaints. Secondly, the physician should respond with patience when breaking bad news and discussing other issues that could be potentially emotionally sensitive (Cole, 2014). CALGARY-CAMBRIDGE GUIDE (CCG) Suzanne Kurtz, Jonathan Silverman, and Juliet Draper intended to create a model of doctor-patient communication that on one hand adequately represents reality of such encounter, but on the other hand is suitable for educational purposes. The model is therefore very structured, precise and concrete. BUILDING THE RELATIONSHIP According to the Calgary-Cambridge Guide model, building a relationship with a patient is a special kind of task, in that it does not appear as a next part of the interview, but instead is incorporated in the whole process of interviewing. Building a relationship with a patient requires presenting adequate non-verbal behaviors, building rapport, and involving the patient. Kurtz, Silverman, and Draper mention non- verbal behaviors like appearance and posture, body movements, gestures, and facial expression, eye contact and vocal signals, proxemics, and touch. They also consider environmental signals (location of the office, an arrangement of space and furniture, lighting and temperature) and operating with time (being early, on time or late, exceeding time, being in a hurry or being slow) as elements of non-verbal communication. To successfully build rapport with their patient, a physician should be able to respond with acceptance, empathy, support, and sensitivity. On the other hand, the idea of involving the patient corresponds with the authors’ intention to build a relationship based on partnership and can be reached with sharing thinking (e.g. "Sometimes it is difficult to decide if the stomachache is a symptom of a disease or rather a result of stress…") and offering explanation of procedures or questions for transparency.

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CONTENT According to the Calgary-Cambridge Guide model, the medical doctor has 7 basic tasks to fulfill (also described as the stages of the interview). Five from them should be realized in sequence – in chronological order, those are: initiating the session, gathering information, physical examination, explanation, and planning and closing the session. The other two tasks are realized continuously throughout the meeting with a patient; and these are: building the relationship and providing structure. For each of the 6 communication tasks (apart from physical examination) several goals have been identified, which realization requires the use of specific process skills, described in the model in detail (Silverman, 2018). Calgary-Cambridge Guide includes 56 basic skills, two additional skills (serving to understand a patient’s perspective), and another 15 optional skills used at the stage of explanation and planning. Altogether the specialist has 73 skills at their disposal, which should be adjusted to the patient’s situation and the actual stage of the interview (Silverman, 2018, Kurtz, 2009). STRUCTURE AND QUESTIONING STYLE As it was mentioned in the introduction, the authors of Calgary–Cambridge Guide put special attention to the structure of the medical interview. Providing structure is a separate task, incorporated in the whole process of interviewing. It consists of two elements: making the organization overt and attending to flow. The first aspect pertains to openly and directly informing the patient about the process of directing the interview (e.g. introducing certain topics, procedures, changes of the subjects of inquiry), which creates comfortable conditions for the patient to follow. The second element – attending to the flow of the interview – includes the ability of the physician to adjust the structure of the visit to the individual patient and also be able to realize their tasks and simultaneously attend to timing (Silverman, 2018; Kurtz, 2009). Physician’s tasks differs depending on the stage of the interview. Before starting the next consultation, the doctor prepares himself/herself for it, which means eliminating those aspects of the situation that could distract the specialist from being focused on the patient – including one’s comfort. Though it seems to be an obvious element, it should be considered very important, as it’s practical realization requires a special work attitude, which could potentially prevent future job burnout (balancing work engagement and self-care). It can be noticed that at the stages of initiating the session, gathering information, and explanation and planning a similar scheme is used. The physician starts with recognizing the patient’s perspective using an open question, then obtains or gives more precise and specific information (using convergent techniques) and finally responds to the patient’s reaction (checks their understanding and personal impact of the information on the patient). Closing the session the physician makes sure that the patient accepts the plan of care and is well informed of what to do next. This includes explaining possible unexpected outcomes and giving suggestions when and how to seek help. Kurtz, Silverman, and Draper argue that the way the interviewer formulates their questions and statements has a vital influence on both the quality and quantity of the information obtained from the patient. Close (convergent) techniques limit the patient’s

72 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview response to the area strictly defined by the interviewer. They are constructed in the way that requires from a patient giving a certain answer, sometimes limited only to one word (yes/no) (e.g. "Do you have a headache every morning?"). On the other hand, open (divergent) techniques are formulated in such a way to introduce the subject of inquiry, but without directly shaping the answer or focusing on one specific aspect of the subject. They indicate that additional information is both adequate and valued (e.g. "Please, tell me more about your headaches"). Some statements or questions are built in the "in-between style" – they are directed, but still give the patient a lot of possibilities of shaping their answer (e.g. "What makes the headaches increase?"). It is crucial to mention that the authors of the Calgary-Cambridge model give no priority to either open or closed questioning techniques, but rather suggest the interviewer get acknowledged with various forms of them, so they can be later used deliberately following patient’s medical situation (Silverman, 2018). SKILLS AND THEIR FUNCTION Kurtz, Silverman, and Draper assume that in the effective communication between doctors (or other healthcare professionals) and their patients crucial are three types of skills, interrelated but having different functions in the process of the diagnosis and treatment. These are 1) content skills (what they communicate – the substance of their questions and responses, the information that they gather and give, the treatments they discuss); 2) process skills (how it is done – in what way they formulate their questions or provide information, what verbal and non-verbal skills they use, how they develop the relationship with the patient, the way they organize and structure communication) and finally 3) perceptual skills (what healthcare professionals are thinking and feeling – their internal decision-making, clinical reasoning, and problem-solving skills; their attitudes and intentions, values and beliefs; their awareness of feelings and thoughts about the patient, about the illness and about other issues that may be concerning them; their awareness of their self-concept and confidence, of their own biases and distractions). The authors of Calgary-Cambridge Guide argue that content skills are acknowledged by doctors in professional training, and so presenting their model of communication, they pay special attention to process skills, and to a lesser extend perceptual skills, but still emphasize mutual influence between content, process, and perception. An example of that would be a situation when a doctor is irritated with patients’ personality features recognized as unacceptable (perceptual skills), which makes it difficult for her to attentively listen to the patient’s complaints (process skills) and in consequence, she misses medically important information (content skills). In another example, a doctor finds their patient sexually attractive, which makes him feel uncomfortable (perceptual skills) and because of that, he is inclined to omit questions about sexual issues in the interview (content skills) or avoids performing a physical examination (Kurtz, 2009). The interviewer’s communication tasks together with their goals and corresponding 58 process skills are presented in table 3.

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Table 3. Interviewer's tasks, their goals, and corresponding skills INITIATING THE SESSION 1. Finish the last task and pays attention to their comfort Preparation 2. Focus attention and prepare for the consultation 3. Greet the patient and obtain the patient’s name Introduce yourself, role and nature of interview; obtain consent 4. Establishing initial rapport if necessary Demonstrate respect and interest; attend to patient’s physical 5. comfort Identify the patient’s problems or the issues the patient wishes 6. to address with an appropriate opening question Listen attentively to the patient’s opening statement, without 7. Identifying reason(s) for the interrupting or directing the patient’s response consultation 8. Confirm list and screen for further problems Negotiate agenda taking both patient’s and your needs into 9. account GATHERING INFORMATION Encourage patient to tell the story of the problem(s) from when 10. first started to the present, in own words Use open and closed questioning techniques, appropriately 11. moving from open to closed Listen attentively, allowing the patient to complete statements 12. without interruption 13. Facilitate patient’s responses verbally and non-verbally Exploration of patient’sproblems 14. Pick up verbal and non-verbal cues Clarify patient’s statements that are unclear or need 15. amplification 16. Periodically summarise Use concise, easily understood questions and comments; avoid 17. or adequately explain the jargon 18. Establish dates and sequence of events Actively determine and appropriately explore the patient's 19. Understanding the ideas, concerns, expectations, symptoms' effects patient'sperspective 20. Encourage patient to express feelings

PROVIDING STRUCTURE 21. Summarise at the end of a specific line of inquiry Makingorganizationovert Progress from one section to another using signposting, 22. traditional statements

74 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview

23. Structure interview in a logical sequence Attending to flow 24. Attend to timing and keeping interview on task BUILDING THE RELATIONSHIP 25. Demonstrate appropriate non-verbal behavior Using appropriate non-verbal If you read or write, do it so it doesn't interfere with 26. behavior dialogue/rapport 27. Demonstrate appropriate confidence Accept legitimacy of patient’s views and feelings; be not 28. judgemental Use empathy to communicate understanding and appreciation 29. of the patient’s feelings or predicament Developing rapport Provide support: express concern, understanding, willingness 30. to help; acknowledge coping efforts; offer partnership Deal sensitively with embarrassing and disturbing topics and 31. physical pain Share thinking with the patient to encourage patient’s 32. involvement Explain the rationale for questions or parts of a physical Involving the patient 33. examination that could appear to be non-sequitur During physical examination – explain the process, ask 34. permission EXPLANATION & PLANNING Chunk and check: give information in assimilable chunks; 35. check for understanding Assess the patient’s starting point: ask for the patient’s prior 36. Providing the correct amount and knowledge early on when giving information type of information Ask the patient what other information would be helpful e.g. 37. etiology, prognosis 38. Explain at appropriate times Organize explanation: divide into discrete sections; develop 39. a logical sequence 40. Use explicit categorization or signposting 41. Use repetition and summarising to reinforce information Aiding accurate recall and Use concise, easily understood language; avoid or explain the understanding 42. jargon 43. Use visual methods of conveying information Check patient’s understanding of the information given or 44. plans made by e.g. asking to restate in own words Relate explanation to the patient’s perspective: to previously Achieving a shared 45. understanding: incorporating the elicited ideas, concerns, and expectations

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patient’s perspective Provide opportunities and encourage a patient to contribute: to 46. ask questions, seek clarification, or express doubts Pick up and respond to verbal and non-verbal cues (patient’s 47. need to ask questions, information overload, distress) Elicit patient’s beliefs, reactions, and feelings regarding 48. information given, terms used Share own thinking as appropriate: ideas, thoughts process, and 49. dilemmas Involve patient: offer suggestions and choices rather than 50. directives, encourage the patient to contribute their ideas 51. Explore management options Planning: shareddecisionmaking Ascertain the level of involvement patient wishes in deciding 52. on hand Negotiate a mutually acceptable plan (determine patient's 53. preferences and options, inform about yours) Check with the patient if he/she accepts the plan and if their 54. concerns have been addressed CLOSING THE SESSION Contract with the patient regarding the next steps for patient 55. and physician Planning Create safety nets explaining possible unexpected outcomes, 56. what to do if the plan is not working, when and how to seek help 57. Summarisethe session briefly and clarify the plan of care Ensuring appropriate point of Finally check that the patient agrees and is comfortable with closure 58. the plan and ask if there are any corrections, questions

Source: Kurtz, 2009; Silverman, 2018

TIME In Calgary-Cambridge model the time management is discussed in the context of providing structure in the interview (skill no 24: Attend to timing and keeping interview on task), which suggest that the clinician needs to be flexible when deciding how much time to offer to a particular patient. The authors of the model indicate that efficiency in time management is influenced by skilfulness in three areas: building relationship, negotiating agenda at the beginning of the interview and responding to patient’s emotional signals. They also underline the issue of time perception from the perspective of the patient, as some research results suggest that when the doctor is "present" for their patient, both verbally and nonverbally, then even in case of relatively short visits the patient is left with an impression that the doctor offered them enough time.

76 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview

RESULTS The results of the analysis of the three presented models – First interview in Positive Psychotherapy (PPT), Three Functional mode (3F) and Calgary-Cambridge Guide (CCG) – in seven categories are summarized below as a list of similarities and differences. 1. Building the relationship Similarities – in all three models the quality of relationship: • is recognized as influencing patent's honesty and by that influential for data gathering; • is related to the character of nonverbal signals influencing patient's feeling of safety; • is based on partnership; • is based on open and friendly (emotionally warm) attitude (revealed by interest, empathy, respect, concern and sensitivity); • is based on proper level of transparency (understood both as making the process of interview explicit and understandable and revealing one's emotions and thoughts); • is based on the interviewer’s ability to respond to patient's emotions. Differences: • for the general practitioner the touch is a way of gathering clinical information (physical examination), but the therapist is usually very careful with this form of contact. 2. Content Similarities – in all three models the following type of information is collected and given: • demographic data; • portrayal of complaints/symptoms; • patient’s motivation; • social situation; • general health; • diagnosis (the type of health disturbance) and treatment/recommendations. Differences: • in PPT in the therapists is additionally interested in the patient’s trust and hope as an ability; • in PPT the observation of the encounter as a "scene" helps the therapist to acknowledge the patient’s interaction patterns;

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• in PPT, apart from social situation, the therapist aims at recognizing patient’s sociogenesis (the genesis of relational patterns); • in PPT the therapists analyses their countertransference – which in CCG is recognized as an aspect of perceptual skills; • in PPT the diagnosis includes the content of main psychological conflict and the function of the symptoms; • in PPT the therapist aims at recognizing the patient’s cognitive style and coping with conflict style. 3. Structure Similarities: • PPT and 3F models of interview are structured according to three consecutive parts of different objectives (first – connecting to the patient, second – gathering information from the patient, third – giving the patient proper information: summarizing and offering recommendations); • in all three models at the stage of informing the patient about their condition (formulating diagnosis), the interviewer asks the patient about their perspective. Differences: • in CCG the interview is realized in 4 consecutive parts and 2 additional tasks. 4. Questioning style Similarities: • in all three models the interviewer starts the process of gathering information with opening question (formulated as an open-ended question) and moves to closed ones depending on the patient’s narration; • in all three models the questioning style corresponds with the role taken up by the interviewer in the process of communication – from nondirective to directive; • in all three models the interviewer is recommended to use concise, easily understood language. Differences: • specific for PPT is taking notes of spontaneous patient’s responses and in 3F model the interviewer is advice to avoid leading questions. 5. Communication skills Similarities – in all three models the interviewer is encouraged to use the following groups of skills: • (1) self-awareness, observation of the patient’s, adequate nonverbal signals indicating attention, engagement and an attitude of concern and warmth, sensitivity, congruency;

78 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview

• (2) showing empathy (acceptance), reflection, legitimation, respect (appreciation), partnership-like attitude, support; • (3) encouraging patient to present their perspective (ideas, doubts, questions); • (4) encouraging patient to express their feelings and responding to them; • (5) (formulating diverse forms of questions from open (divergent) to closed (convergent) ones, speaking concise, understandable language (avoiding jargon); • (6) adequate verbal and nonverbal listening behaviors, facilitation (in various forms); • (7) clarification, paraphrasing, checking, summarizing, asking for feedback; • (8) negotiation (of agenda, issues included, undertaken actions, treatment plan); • (9) explaining or educating (procedures, forms of treatment, mechanism of disturbances) if necessary and according to patient’s needs; • (10) using visual methods of conveying information (in PPT and CCG models). Differences: • specifically in 3F the interviewer is encouraged to avoid leading questions; • specifically in CCG the interviewer is encouraged to take care of one’s own comfort, show proper level of confidence and use explicit categorization or signposting; • in PPT observation of the patient includes patient’s interaction patterns (ability to view an encounter as a "scene"). 6. Function Similarities: • in all three models the functional aspect is revealed by relating particular groups of skills to more general task or/and part of the interview or – (in CCG) presenting diverse forms of realization of continues tasks as dependable on the stage of the interview; • using the first group of skills allows both the therapist and the physician to create the safe personal environment for the patient; with the second group of skills the interviewer may show their understanding and acceptance for the patient as a person; using the third group of skills the specialist reveals interest in the patient’s perspective; with the fourth groups of skills the therapist and the physician show that they care and enable to create emotional bond between them and the patient; the fifth and sixth groups of skills serve to get the broad picture of the patient’s situation and adequately direct the interview; using the seventh groups of skills the interviewer enhances precision of obtained information; with negotiation the specialist enhances active attitude and engagement of the patient in the healing process; adjusting explanation and education to patient’s needs prevents overburdening them and facilitates patient’s learning; and finally using visual methods

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of conveying information, broadening patient’s perception, facilitates acquisition of new concepts; • one skill can serve simultaneously diverse purposes (e.g. asking for feedback decreases the chance of misunderstanding and is a way to show interviewer’s interest in the patient’s perspective). 7. Time Similarities: • in all three models it is recommended to start an interview with an open question and allow the patient to answer in their own way, without interruption. Differences: • actual duration of the medical and psychotherapeutic encounter; • in 3F the interviewer is encouraged to use emotional response skills to influence patient’s perception of the quality of time offered; • in CCG the interviewer is encouraged to enhance their skillfulness in building relationship, negotiating agenda at the beginning of the interview and responding to patient’s emotional signals, as well use their verbal and nonverbal signals to show that they are "present"; • specifically in PPT, apart from being attentive, the interviewer is encouraged to adjust to the patient’s tempo. DISCUSSION BUILDING A SAFE RELATIONSHIP AS A CRUCIAL FACTOR In all three presented models of interview building a relationship in which the patient feels safe enough to reveal all the relevant information is considered as essential to the efficiency of the specialists’ assistance. It is assumed, that the patient chooses to visit the specialist primarily for instrumental reasons – to receive professional help in a form of advice, explanation, recommendation, procedure. However, apart from that they simply need to feel safe. To enable this the interviewer is advised to present adequate nonverbal behaviors and open, friendly attitude. Neglecting this basic need would make fulfilling all the other needs impossible, therefore concerning it is in line with the main assumptions of the holistic approach (Royen et al., 2010). According to Stephen W. Porges, the author of the polyvagal theory, what induces the feeling of being safe with someone, is how they act nonverbally in our presence. If a therapist or a physician keeps eye contact, smiles gently, moves in moderate tempo, speaks with emotional prosody and in a relaxed, calm tone of voice – that signals activation of their social engagement system, that in turn diminishes sympathetic activation and keeps the activity of the parasympathetic nervous system and abdominal system in homeostasis. Being in such a neurophysiological state, it is unlikely to present aggressive behavior, so when the patient detects them through neuroception, they can feel relaxed and be more open to the exchange of information. Interestingly,

80 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview to present such signals, the interviewer needs to have a sense of safety himself/herself. Porges points out that safety cannot be defined as a lack of danger, but rather is an active state of feeling safe. To have a sense of safety in the presence of others, humans require specific signals from the social environment (Porges, 2020). It is the interviewer’s role to build a trusting relationship with their patient, which is also connected with the specialist’s ability to respond to the patient’s emotional needs. In PPT the therapist is expected to reflect the situation and feelings of the client to connect emotionally to them. According to CGG the physician should "communicate understanding and appreciation of the patient’s feelings or predicament". Similarly, in model expressing empathy using reflection of a patient’s feelings is needed as a part of building a doctor-patient relationship that is often found challenging (Hardavella et al. 2017). According to the theory of constructed emotion of the authorship of Lisa Feldman Barrett, emotions are not reactions to stimuli coming from the world, but active constructs of the human brain. From actual sensory input coming from outside and inside of the body, with the use of concepts acquired from past experiences (similar enough to the actual situation and the bodily state), the brain constructs the meaning and prescribes action. This meaning is an instant of emotion. "Sadness", "sorrow", "happiness" or "guilt" are examples of emotion categories that can be experienced very differently from person to person and also differ by the same person, depending on the situation and the state of their body. Emotion concepts stem from individual experiences that are grounded in the contexts of language and culture. Emotions cannot be detected or recognized in someone, only their expressions can be perceived and categorized by the observer. The ability to distinguish between very many concepts of emotions in oneself and other people (and label them with different words) is called emotional granularity. People exhibiting high emotional granularity use hundreds or even thousands of various emotional concepts – words like "disgruntlement", "irritation", "frustration", "hostility", would be experienced by them as different states. Individuals with a moderate level of this quality might have dozens of emotion concepts, and those with low emotional granularity – will have only a few (Barrett, 2017; Barrett, 2017a). The theory of constructed emotion implicates, that discussing and soothing other persons’ emotional states, requires not only emotional granularity but also attentiveness, flexibility, as well as personal and cultural sensitivity. Any form of labeling emotions should be done with great caution and gentleness, not to mention linguistic proficiency. Psychotherapists are expected to present high competence in responding to the emotional needs of their clients and patients. Their emotional abilities are being enhanced due to professional education and constant training – e.g. emotional self-awareness resulting from obligatory attendance in self-experience groups and supervisions (Kodeks Etyczny PTP Sekcji Psychoterapii, 2007). Whereas among medical professionals the high interpersonal competencies are not always considered as necessary (Johnson, 2017).

Aleksandra Zarek, Aleksandra Kroll

Medical professionals are not always obliged to take part in structured training concerning psychological and interpersonal abilities. They may voluntarily participate in Balint groups training (originally developed for doctors) or other workshops, and benefit from them, but in Poland, it is not a part of their obligatory education (Wasi- lewski, 2011). For that reason, some of them may have difficulties with effectively dealing with patients’ emotions, not to mention their emotionality – which has been revealed in a recently published qualitative study aiming at identifying family physicians' work-difficulties and work-related emotions (Bankiewicz-Nakielska, 2020). THE DIFFERENCES IN THE AREA OF CONTENT Helping to solve a patient’s health problems is a role shared among both the therapist and the medical doctor (Kodeks Etyki Lekarskiej, 2003; Template for a National Psychotherapy, 2009). Successful fulfillment of this task requires from the helper professional knowledge and expertise, as well as communication skills (Cooper, 2010a; Doroszewska, 2017; Knapp, 2009; Witkowska-Łuć, 2016; Wróblewska, 2013). The instrumental task of the interview – an exchange of information – pertains to the content and in that area are found the most differences between the medical and therapeutical interview. This is possibly related to the particular goals of psychotherapeutic and medical contact. For example, both specialists need to get the main complaint, symptom profile, somatic anamnesis, and to some extent also social anamnesis, but apart from that a positive therapist aims at recognizing the genesis of relational patterns of their patient. They would also ask their patient about the functions of the symptom. The physician is usually not interested in that aspect. Additionally, patient’s behaviors presented during the visit and corresponding interviewers reactions are considered by the therapist not only as an interpersonal situation, but as a "scene" which is infor- mative for understanding patient’s interaction patterns. The therapist is more interested in the context of the symptom’s development than the medical doctor and additionally aims at recognizing the patient’s cognitive style and coping with conflict style. Generally, the positive therapist asks more questions that are not directed at the concrete problem or the symptom itself, but rather help to describe the patient as the person and their life situation. IMPORTANCE OF THE PERCEPTUAL AND PROCESS SKILLS In the profession of a physician, especially the general practitioner, interviewing the patient is recognized as the main tool to gather necessary information and that realize their diagnostic tasks (Doroszewska, 2017; Jankowska, 2013). In Positive Psycho- therapy the initial interview is crucial for building a setting for the whole psychotherapeutic process (Peseschkian, 1987; Remmers, 2020). Both the therapist and the physician have to use specific communication skills to realize their main instrumental task, that is to be able to operationalize the patient’s problem and recognize the problem solution, and then offer professional advice that is applicable for the patient. One part of this process is explicit for both the patient and the specialist and another part is internalized by the interviewer (e.g. thinking about alternative explanations, the therapist’s/doctor’s uncertainty, their feelings concerning the patient), who decides whether and how to share it with the patient. In the CCG,

82 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview these two aspects of communication are named respectively process skills and perceptual skills. In a conversation between a therapist and the patient, already in the first interview, diagnostic and therapeutic aspects are combined (Cooper, 2010; Cooper, 2010a; Knapp, 2009). In the preliminary interview, a therapist aims at realizing and balancing three objectives: 1) obtaining information about the actual problem, as well as comprehensive knowledge about the client’s health and their social situation; 2) building cooperation based on mutual trust, and 3) meeting the client’s needs. At this point, the crucial skill of the therapist is to create an atmosphere of safety (to encourage the client’s honesty) and communicate understanding (express ability to take the client’s perspective of the problem), which is a prerequisite of any therapeutical cooperation. Communication skills useful in that process are: informing the client about the therapist role and ethics of confidentiality, adjusting to the client’s communication style (wording, vocal qualities, body language), avoiding intimate questions, showing respect to the clients own boundaries, dealing with silence, starting therapeutic work from the stage indicated by the client, using non-judgmental comments (Knapp, 2009). Basic communication and psychological competencies considered crucial in the medical doctor’s work include: listening, leading, adjusting (synchronizing nonverbal behaviors and wording), reflecting, creating adequate personal distance, presenting neutral (non-judgmental attitude), confronting, informing and educating, supporting, intervening in crisis, solving problems and decision making, negotiating, motivating, modeling behaviors, empathy, assertiveness, reflectiveness and self-awareness, congruency and self-presentation (Bankiewicz-Nakielska, 2017). The way the doctor conducts an interview matters for their patients and is potentially influential – the majority of Polish patients favorably evaluate diagnostic (69%) and therapeutic competence (64%) of medical doctors, but to a lesser extent are satisfied with physicians’ work involvement (43%), their ability to listen to patient’s problems (46%) or inform the patient about their medical condition (51%) (Biostat, 2018). Apart from the Internet (91,2%), the patients indicate the general practitioner (72,8%) and the medical specialist (62,9%) as the main source of medical information (ibidem). For the practitioner of medicine, continual self-reflection over the quality of the provided interview is helpful and necessary to improve one own’s communication skills (Perkowska-Klejman, 2013). This, in turn, not only leads to satisfying cooperation for both the patient and the interviewer but may enhance patient’s health literacy, influence their health behaviors, and that has an impact on their medical outcomes (TavakolySany, 2020). IMPORTANCE OF THE APPLIED SKILLS’ FUNCTION Whenever the therapist or the physician formulates their question or comment in a certain way (concerning the content, wording, and the tone of voice), introduces the moments of silence or prevents himself/herself from sharing certain information, they should always be aware of their reasons. It is important to underline that the interviewer needs to comprehend the function of the communication skill applied. There may be several functions involved. For example, in all three described models of communication, the interviewer starts the questioning process with an open

Aleksandra Zarek, Aleksandra Kroll question. It serves at least two purposes: enables to understand the patient’s problem and their perspective in a relatively short time and simultaneously – if proper listening behaviors are presented – it satisfies the patient’s needs to express himself/herself and to be accepted. Formulating closed-ended questions helps the interviewer to gather specific information necessary in the operationalization of the problem, but also shows that they were listening to the patient’s narration. Asking questions about other issues than the symptom itself enables broadening the interviewer’s knowledge about the context of the problem/symptom and potentially about their cause, but also broadens the patient’s perspective. Summarizing the gathered information helps to check on their accuracy, but also proves that the interviewer was attentive. Simultaneously summarizing helps to educate the patient about their condition and broaden their perspective. Asking patients for feedback serves in two ways: firstly it decreases the chance of misunderstanding, and secondly is a way to show respect and the interviewer’s interest in the patient’s perspective. It is noticeable, that the above mentioned communication skills used in medical and therapeutical interviews, simultaneously serve instrumental and relational purposes, which reveals the two functions of the interview encounter. STRUCTURE AND QUESTIONING STYLE The way of structuring the visit of the patient in a therapeutic and medical interview, as well as the questioning styles used by the interviewer, shares many similarities. The session starts with establishing initial contact, then the interviewer should recognize the patient’s problem and gradually get informed about the details on that subject (formulating questions from open to close-ended ones, asking for clarification, checking, and paraphrasing, facilitating patient narration if necessary). The patient’s perspective (their explanatory model) must be taken into consideration to clarify their needs and expectations. The interviewer should summarize the gathered information before they operationalize the case for themselves and formulate a diagnosis. The next step is to inform or educate the patient about their situation in a form suited to the patient’s perspective, then negotiate a treatment plan (or make other recommendations), ask for feedback, and finally close the visit. Looking at a broader perspective, the structure of both types of interviews reveals the natural dynamics of communication in every encounter that holds the potential for cognitive or behavioral change. Firstly you build interpersonal connections, than exchange relevant information, and finally end the conversation, adapting acquired knowledge in a new way. This process is especially noticeable in the first interview in PPT and 3F. TIME QUALITY AND DURATION OF THE INTERVIEW As it was mentioned previously, in PPT the interview can be conducted in one session, but also take several visits and it is the task of the therapist to find balance between the acquisition of information and patient’s emotional comfort, and to adjust to the patient’s tempo (Peseschkian, 1987; Remmers, 2020). The authors of both models of medical interview also underline the necessity to show patience when listening to

84 Communication skills applied in the first interview in Transcultural Positive Psychotherapy compared to Calgary-Cambridge Guide and Three Functional model of the medical interview the patient’s response to the opening statement and pay special attention to those moment of the interview that appear emotionally difficult for the patient, be "present" (Cole, 2014; Silverman, 2018). It suggest that patient’s perception of the quality of time offered is connected with the interviewer’s ability of being attentive. The biggest difference between the therapeutical and medical interview is in the duration of the visit. A therapeutic session usually lasts about 50 minutes (Will, 2015). During that time a general practitioner in Poland would realize on average 5 visits with different patients (an average consultation length differs across the world, ranging from 48 s in Bangladesh to 22.5 min in Sweden) (Irving, 2017). For that reason, the physician needs to be more skillful in attending to timing and keeping the interview on tasks than the therapist. CONCLUSIONS AND RECOMMENDATIONS In the First Interview in Positive Psychotherapy, Three Functional model of the medical interview and Calgary-Cambridge Guide model, while conducting an interview with a patient the therapist and the physician apply very similar communication skills especially concerning building safe relationship, providing structure and questioning style. Those skills serve simultaneously diverse instrumental and relational purposes. Therapeutical and medical interview differ mostly in the area of content and duration of the visit, but not advocated time quality. According to the presented models of interview building a safe relationship is considered as essential to the efficiency of the specialists’ assistance. A physician, like a therapist, is expected to properly respond to patients' emotions, but may be not enough equipped for this task. It is then recommended to include group interpersonal training in the curriculum of medical education, to enable the students to enhance their emotional competence. ACKNOWLEDGE Presented research results have been realized within the subject about the number SUB.A150.20.001 according to the records of Simple system. REFERENCES Badanie zrealizowane przez firmę BioStat za pomocą Panelu Badania Opinii na reprezentatywnej grupie 1000 Polaków, w dwóch falach: od 30 maja do 7 czerwca, od 20 do 28 grudnia 2018 roku (pobrano 24.06.2020: https://www.badanie-opinii.pl/raporty/zestawienie-badan-polak-u-lekarza). Bankiewicz-Nakielska J., Tyszkiewicz-Bandur M., Walkiewicz M., Karakiewicz B., Kompetencje psychologiczne w pracy lekarza, Med Dypl 2017; 26 (1): 108-114. Bankiewicz-Nakielska J., Walkiewicz M., Tyszkiewicz-Bandur M., Family physicians’ problems with patients and own limitations – a qualitative study, Family Medicine & Primary Care Review. 2020; 22(1): 18-21. Barrett L.F., How emotions are made: the secret life of the brain, 2017; Macmillan (UK); ISBN: 978-1- 5098-3750-2. Barrett L.F., The theory of constructed emotion: An active inference account of interoception and categorization, SocCognAffectNeurosci. 2017a; 12 (1): 1-23.

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Ciesielski R., Model Równowagi i Trzy Etapy Interakcji w ujęciu Psychoterapii Pozytywnej, 2016; Wydawnictwo Continuo (Poland); ISBN: 978-83-62182-55-8. Ciesielski R., Transkulturowa Psychoterapia Pozytywna jako zintegrowany system terapeutyczny, 2015; Wydawnictwo Continuo (Poland); ISBN: 978-83-62182-54-1. Cole S., Bird J., The medical interview: three-function approach, (3rd ed). 2014; Elsevier Saunders (ISBN:978-0-323-05221-4). Cooper M., Czynniki związane z relacją: to relacja uzdrawia… a może nie?, In: Cooper M. (Editor). Efektywność psychoterapii i poradnictwa psychologicznego, 2010; Instytut Psychologii Zdrowia PTP (Poland); ISBN: 978-83-60747-59-9, (pp.139-173). Cooper M., Czynniki związane z terapeutą: kto pracuje na co? In: Cooper M. (Editor) Efektywność psychoterapii i poradnictwa psychologicznego, 2010a; Instytut Psychologii Zdrowia PTP (Poland); ISBN: 978-83-60747-59-9, (pp. 117-138). Czabała J., Poradnictwo psychologiczne a psychoterapia, Annals of Psychology, 2016; 19 (3): 519-533. Dobiała E, Winkler P., ‘Positive Psychotherapy’ according to Seligman and ‘Positive Psychotherapy’ according to Peseschkian: A Comparison, Int J Psychotherapy. 2016; 20(3): 5-17. Doroszewska A., Ogólne zasady komunikowania się lekarza i pacjenta. Etyka komunikacji, In: Ostrowska A. (Editor) Jak rozmawiać z pacjentem? Anatomia komunikacji w praktyce lekarskiej, 2017; Fundacja Polska Liga Walki z Rakiem (Poland); ISBN: 978-83-7683-140-4, (pp. 15-39). Hardavella G., Aamli-Gaagnat A., Frille A., Saad N., Niculescu A., Powell P., Top tips to deal with challenging situations: doctor–patient interactions, Breathe, 13(2), 2017, 129-135, doi:10.1183/20734735.006616. Hatem D.S., Barrett S.V., Hewson M. et al., Teaching the Medical Interview: Methods and Key Learning Issues in a Faculty Development Course, J GEN INTERN MED 22, 1718-1724, 2007, https://doi.org/10.1007/s11606-007-0408-9. Irving G., Neves A.L., Dambha-Miller H., Oishi A., Tagashira H., Verho A. et al., International variations in primary care physician consultation time: a systematic review of 67 countries, BMJ Open 2017; 7:e017902, (doi:10.1136/ bmjopen-2017-017902). Jankowska A., Jak poprawnie prowadzić wywiad?, In: Steciwko A., Barański J. (Editors) Relacja lekarz-pacjent. Zrozumienie i współpraca, 2013; Elsevier Urban & Partner (Poland); ISBN: 978-83- 7609-866-1, (pp. 51-56). Johnson M., Do Physicians Have an Ethical Duty to Repair Relationships with So-Called "Difficult" Patients?, AMA J Ethics, 2017;19(4):323-331. doi: 10.1001/journalofethics.2017.19.4.ecas1-1704. Knapp H., Komunikacja w terapii, 2009; Wydawnictwo Naukowe PWN (Poland); ISBN: 978-83-01- 15771-5. Kodeks Etyczny Sekcji Psychoterapii Polskiego Towarzystwa Psychologicznego z roku 2007 (pobrano 24.06.2020 http://www.sekcjapsychoterapii.pl/kodeks-etyczny). Kodeks Etyki Lekarskiej znowelizowany w roku 2003 (pobrano 26.06.20220: https://nil.org.pl/uploaded_images/1574857770_kodeks-etyki-lekarskiej.pdf). Kurtz S., Silverman J., Draper J., Teaching and learning communications skills in medicine, 2nd ed. Oxford, San Francisco: Radcliffe Publishing; 2009. Perkowska-Klejman A., Modele refleksyjnego uczenia się, Teraźniejszość – Człowiek – Edukacja 2013; 1(61): 75-90. Peseschkian N., Positive Psychotherapy. Theory and Practice of a New Method, 1987; Springer-Verlag (Germany, USA); ISBN: 3-540-15794-8, 0-387-15794-8.

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Porges S.W., Teoria poliwagalna, 2020; Wydawnictwo Uniwersytetu Jagiellońskiego (Poland); ISBN: 978-83-233-4676-0. Ratna H., The importance of effective communication in healthcare practice, Harvard Public Health Review. 2019;23. Remmers A., Theoretical foundations and roots of Positive Psychotherapy, In: Messias E., Peseschkian H., Cagande C. (Editors) Positive Psychiatry, Psychotherapy and Psychology, 2020; Springer (Switzerland); ISBN: 978-3-030-33263-1, (pp. 297-307). Remmers A., Peseschkian H., The First Interview in Positive Psychotherapy, In: Messias E., Peseschkian H., Cagande C. (Editors) Positive Psychiatry, Psychotherapy and Psychology, 2020; Springer (Switzerland); ISBN: 978-3-030-33263-1, (pp. 309-329). Royen P., Beyer M., Chevallier P., Eilat-Tsanani S., Lionis Ch., Peremans L., Petek D., Rurik I., Soler J. K., Stoffers H. E., Topsever P., Ungan M. & Hummers-Pradier E., The research agenda for general practice/family medicine and primary health care in Europe. Part 3. Results: Person centred care, comprehensive and holistic approach, European Journal of General Practice, 2010; 16(2): 113-119, DOI: 10.3109/13814788.2010.481018 Silverman J., Kurtz S., Draper J., Umiejętności komunikowania się z pacjentami, 2018; Medycyna Praktyczna (Poland); ISBN: 978-83-7430-558-7. Suszek H., Grzesiuk L., Styła R., Krawczyk K., Kto i w jaki sposób prowadzi psychoterapię w Polsce?, Część II, Wyniki ogólnopolskiego badania, Psychiatria, 2017; 14(2), 90-100. Szymańska A., Dobrenko K., Grzesiuk L., Cechy i doświadczenia pacjenta z przebiegu psychoterapii oraz skuteczność psychoterapii. Podejście strukturalne, Psychiatr Pol. 2017; 51(4): 619-631. Szymańska A., Gresiuk L., Suszek H., Doberenko K., Rutkowska M., Krawczyk K., Badania polskich psychoterapeutów – z jakimi pacjentami pracują i jakie stosują metody psychoterapii?, Psychiatr Pol. 2017a; 76, 1-21. TavakolySany S.B, Behzhad F, Ferns G, Peyman N., Communication skills training for physicians improves health literacy and medical outcomes among patients with hypertension: a randomized controlled trial, BMC Health Serv Res. 2020; 23; 20 (1): 60, (doi: 10.1186/s12913-020-4901-8). Template for a National Psychotherapy Law formulated by the European Association of Psychotherapy in 2009 (pobrano 24.06.2020:https://www.europsyche.org/app/uploads/2019/05/National_PTLaw_modified- version-21Feb2009_voted-by-Board.pdf). Wasilewski B., Engel L., Grupowy trening Balintowski: teoria i zastosowanie, 2011; ENETEIA Wydawnictwo Psychologii i Kultury (Poland); ISBN: 978-83-61538-05-9. Will H., Das Konzept der 50-Minuten-Stunde, Forum Psychoanal 2015; 31(3): 267-281 (doi.org/10.1007/s00451-015-0208-5). Witkowska-Łuć B., Znaczenie komunikacji niewerbalnej w komunikacji terapeutycznej, Psychiatria i Psychoterapia. 2016; 12 (4): 16-28. Wróblewska I., Steciwko A., Zaufanie jako jeden z najistotniejszych czynników umożliwiających prawidłowe porozumiewanie się pomiędzy lekarzem a pacjentem. In: Steciwko A., Barański J. (Editors) Relacja lekarz-pacjent. Zrozumienie i współpraca, 2013; Elsevier Urban & Partner (Poland); ISBN: 978-83-7609-866-1, (pp. 13-24). Yedidia M.J., Gillespie C.C., Kachur E. et al., Effect of communications training on medical student performance, JAMA. 2003; 290(9):1157-1165, doi:10.1001/jama.290.9.1157.

Redox homeostasis in melanoma cells treated with caffeic acid

Magdalena Kimsa-Dudek*, Agata Krawczyk, Agnieszka Synowiec-Wojtarowicz Department of Nutrigenomics and Bromatology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia, Katowice, Jednosci 8, 41-200 Sosnowiec, Poland *Corresponding author: Magdalena Kimsa-Dudek, Department of Nutrigenomics and Bromatology, Medical University of Silesia, Jednosci 8, 41-200 Sosnowiec, Poland Tel.: +48 32 364 11 72, e-mail address: [email protected] ORCID: 0000-0002-6113-7006 ABSTRACT Melanoma is a type of skin cancer that prognosis depends on the stage of disease. Its characteristic feature is the difficulty in treating it in the advanced stages of the disease, which translates into a high mortality rate. Hence, new strategies for supporting treatment are continuously being sought. Natural plant compounds such as phenolic acids have many biological and pharmacological properties. However, the mechanism of their action is not yet fully understood. One of the promising compounds is caffeic acid (CA). However, there are inconclusive research results regarding the protective properties of CA on cells. Therefore, the aim of this study was to assess the effect of caffeic acid on the culture growth and antioxidant status of melanoma cells. The cytotoxicity of CA (at concentrations between 1 and 1,500 µmol/l) was tested against two human cell lines: human melanoma cells and normal human dermal fibroblasts using an MTT assay. In the next step, the gene expression of SOD1 (superoxide dismutase 1), SOD2 (superoxide dismutase 2), GPx1 (glutathione peroxidase 1), GSR (glutathione reductase) and CAT (catalase) were determined using real-time RT-qPCR. The SOD, GPx and CAT activity was also analyzed in the cell lysates. The concentration of 1,000 µmol/l, which had only reduced the viability of the melanoma cells, was selected for the further studies. The expression of SOD1, GPx1 and CAT was significantly lower in the CA-treated cells compared to the control. Furthermore, CA also reduced the antioxidant enzyme activity of GPX and CAT. The redox imbalance that is caused by caffeic acid and its cytotoxicity effect at a concentration of 1,000 µmol/l on melanoma cells (but not on normal cells) implies the usefulness of this compound as a supporting treatment for cancer therapy. INTRODUCTION Malignant melanoma is a type of skin cancer that prognosis depends on the stage of disease. Its characteristic feature is the difficulty in treating it in the advanced stages of the disease, which translates into a high mortality rate. The main treatment for melanoma, but only in its early stages, is surgery. In more advanced stages of the disease, systemic therapies are used such as: immunotherapy (e.g. treatment with PD-1 and CTLA4 inhibitors), targeted therapy (e.g. treatment with BRAF and MEK inhibitors) and chemotherapy (Garbe, 2016; Domingues, 2018). Research is also being conducted on the effectiveness of specific vaccines that might improve the immune

Redox homeostasis in melanoma cells treated with caffeic acid response to melanoma (Ozao-Choy, 2014). However, the above-mentioned therapies are often associated with numerous side effects, and above all, do not result in satisfactory health results. Hence, new strategies for supporting treatment are continuously being sought. Natural plant compounds such as phenolic acids have many biological and pharmacological properties (Kumar, 2019). They also act as bioactive molecules that have the ability to regulate gene expression including the genes that are responsible for cell proliferation (Nicholson, 2008; Anantharaju, 2016). However, the mechanism of their action is not yet fully understood. In their reports, Wu (2016) and Zhou (2014) proposed that dietary antioxidants may regulate the gene expression through epigenetic modifications. There are also reports of the effects of polyphenols on cancer stem cells (Mileo, 2016). One of the promising compounds is caffeic acid (CA) because of its properties. In the human diet, it is found in coffee and also in vegetables, fruits and herbs (Meinhart, 2019). Caffeic acid and its natural derivatives have strong antioxidant properties and protect cells against oxidative stress even at low concentrations. These compounds have been reported to have the ability to efficiently remove reactive oxygen species (ROS), to chelate metal ions and to inhibit lipid peroxidation. The number of its hydroxyl groups and their locations in the structure are responsible for the antioxidant properties of phenolic acids (Kołodziejczyk-Czepas, 2015; Cizmarova, 2020). Because of the activity of caffeic acid, it is considered to be an effective component of cosmetics or as a support for pharmacological therapy in the treatment of skin diseases (Magnani, 2014). However, there are inconclusive research results regarding the protective properties of CA. Therefore, the aim of this study was to assess the effect of caffeic acid on the culture growth and antioxidant status of melanoma cells. MATERIALS AND METHODS Human melanoma cells (the amelanotic C32 cell line) were obtained from ATCC (CRL-1585; Manassas, VA, USA) and were routinely maintained in a DMEM medium (Dulbecco's Modified Eagle Medium, Lonza, Basel, Switzerland), which was supplemented with 10% fetal bovine serum, penicillin (10,000 U/mL) and amphotericin B (0.25 mg/mL) at 37˚C in a 5% CO2 incubator (Heraeus, Hanau, Germany). Normal human dermal fibroblasts (the NHDF cell line) were obtained from Clonetics (CC-2511; San Diego, CA, USA) and cultured as was previously described (Kimsa- Dudek, 2018). Both the number of cells and their viability were monitored by cell counting in a Countess TM Automated Cell Counter (Invitrogen, Carlsbad, CA, USA) after staining with 0.4% trypan blue. The experiment was performed on cells that were in the logarithmic phase of growth under conditions of ≥ 98% viability, which was assessed by trypan blue exclusion.

Magdalena Kimsa-Dudek, Agata Krawczyk, Agnieszka Synowiec-Wojtarowicz

CYTOTOXICITY The cytotoxicity of caffeic acid (at concentrations between 1 and 1,500 µmol/l) was tested against two human cell lines: human melanoma cells (the C32 cell line) and normal human dermal fibroblasts (the NHDF cell line) using an MTT assay. The caffeic acid (Sigma-Aldrich, St. Louis, MO, USA) was prepared as a stock solution in phosphate buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) and then diluted in the culture medium. The viability of the cells was evaluated after 24 h and 48 h of exposure to the CA in two independent assays as was previously described (Pawłowska-Góral, 2016). TREATMENT OF MELANOMA CELLS WITH CAFFEIC ACID In the next step, the C32 cells were seeded into 25 cm2 cell culture flasks (Sarstedt, Nümbrecht, Germany) at a density of 3 x 106 cells. The cells were then subjected to CA at a concentration of 1,000 µmol/l because at a higher concentration, this compound also induces a strong cytotoxic effect on normal cells. The cultures were maintained under these conditions for 24 hours at 37°C in a 5% CO2 incubator (Heraeus). Next, the cells were washed with PBS and the cell numbers were monitored by cell counting in a Countess TM Automated Cell Counter (Invitrogen, Carlsbad, CA, USA) after staining with 0.4% trypan blue. The biochemical analyses were performed in the cell lysates and the cells were pelleted and frozen at -70˚C for 24 hours until RNA extraction for the molecular analysis. RNA EXTRACTION Total RNA was extracted using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The quality of the extracts and the RNA concentration were determined as was previously reported (Kimsa-Dudek, 2018). REAL-TIME RT-QPCR ASSAY The mRNA level of SOD1 (superoxide dismutase 1), SOD2 (superoxide dismutase 2), GPX1 (glutathione peroxidase 1), GSR (glutathione reductase), CAT (catalase) and β-actin were evaluated using real-time RT-qPCR and SYBR Green I chemistry (SYBR Green QuantiTect RT-PCR Kit; QIAGEN, Valencia, CA, USA) as was previously described (Pawłowska-Góral, 2016). All of the samples were tested in triplicate. β-actin was also included as an endogenous positive control of the amplification and the integrity of the extracts in order to monitor the RT-qPCR efficiency. Wells that did not contain a template were run as the negative controls. The thermal profile for the one-step RT-PCR was as follows: reverse transcription at 50°C for 30 min, denaturation at 95°C for 15 min and 40 cycles consisting of the following temperatures and time intervals: 94°C for 15 s, 60°C for 30 s and 72°C for 30 s.

Redox homeostasis in melanoma cells treated with caffeic acid

ASSAY OF THE ACTIVITY OF SUPEROXIDE DISMUTASE (SOD), GLUTATHIONE PEROXIDASE (GPX) AND CATALASE (CAT) SOD activity was measured using a Superoxide Dismutase Assay Kit (Cayman Chemical, Ann Arbor, MI, USA), the GPx activity was measured using a Glutathione Peroxidase Assay Kit (Cayman Chemical) and the CAT activity was measured using a Catalase Assay Kit (Cayman Chemical), according to the manufacturer’s instructions. Absorbance at wavelengths of 450 nm, 340 nm and 540 nm, respectively, was read on a Wallac 1420 VICTOR microplate reader (PerkinElmer, Waltham, MA, USA). STATISTICAL ANALYSES The obtained results of the RT-qPCR assay were recalculated per 1 µg of total RNA and all of the biochemical parameters that were studied were recalculated for 106 cells. Statistical analyses were performed using Statistica 10.0 software (StatSoft, Tulsa, OK, USA). The values are expressed as the means and standard deviation (SD) of three independent experiments. Comparative analyses between the control group and the CA-treated cells (1,000 µmol/l) were performed using the Student’s t-test, while the one-way ANOVA test and Dunnett’s test were used to evaluate any significant differences among the groups in the cytotoxicity assays. The results were considered statistically significant when the significance level was p < 0.05. RESULTS EFFECT OF CAFFEIC ACID ON CELL VIABILITY Melanoma cell viability, which is expressed as the percentage in relation to the control group, was significantly reduced by 25±11% for the cells that had been treated with 500 µmol/l CA (Dunnett’s test, p = 0.005); by 28±8% for the cells that had been treated with 1,000 µmol/l CA (Dunnett’s test, p = 0.003) and by 36±8% for the cells that had been treated with 1,500 µmol/l CA (Dunnett’s test, p < 0.001) after a 24 h incubation. It was also shown that caffeic acid significantly inhibited the melanoma cell proliferation at concentration of 1,000 µmol/l by 24±14% (Dunnett’s test, p = 0.027) and 1,500 µmol/l by 55±10% (Dunnett’s test, p < 0.001) after 48 h (fig. 1). Compared to the melanoma cell proliferation, only the concentration of 1,500 µmol/l CA was significantly cytotoxic to the NHDF cells (normal human dermal fibroblasts) after 24 and 48 h (Dunnett’s test, p < 0.001) (fig. 2). Therefore, a concentration of 1,000 µmol/l, which had only reduced the viability of the melanoma cells, was selected for the further studies.

Magdalena Kimsa-Dudek, Agata Krawczyk, Agnieszka Synowiec-Wojtarowicz

Figure 1. Cell viability in the melanoma cell cultures that had been exposed to caffeic acid for 24 and 48 h. The results are presented as the mean ± SD; *p < 0.05 vs. the control (C)

Figure 2. Cell viability in the normal human dermal fibroblast cultures that had been exposed to caffeic acid. The results are presented as the mean ± SD; *p < 0.05 vs. the control (C)

Redox homeostasis in melanoma cells treated with caffeic acid

EFFECT OF CAFFEIC ACID ON THE ANTIOXIDATIVE DEFENSE PARAMETERS There was a statistically significant decrease in the SOD1, GPx1 and CAT gene expression levels between the CA-treated and control cells (Student’s t-test, p = 0.033; p = 0.007 and p = 0.038, respectively). However, the mRNA levels of SOD2 and GSR were not statistically significant. Furthermore, caffeic acid reduced the antioxidant enzyme activity of GPX and CAT (Student’s t-test, p = 0.029 and p < 0.001, respectively) (tab. 1).

Table 1. Effect of caffeic acid on the antioxidative defense parameters mRNA copy number/µg RNA SOD1 SOD2 GPx1 GSR CAT C 222.4±27.67 2802.3±5416.94 261568.8±86481.01 23.4±17.79 5239.4±2580.98 CA 156.22±39.19* 117.09±16,70 87781.25±8926.30* 14.73±9.38 1565.50±1003.17* antioxidant enzyme activity CAT (nmol/min/106 SOD (U/106 cells) GPX (nmol/min/106 cells) cells) C 1.948±0.41 20.635±2.91 3864.685±536.50 CA 1.704±0.17 7.278±1.48* 496.448±203.654*

The results are presented as the mean ± SD; *p < 0.05 vs. the control; C – untreated control cells; CA – the melanoma cells that had been treated with caffeic acid (1,000 µmol/l)

DISCUSSION A redox imbalance in cells leads to oxidative stress (Trachootham, 2008). Cancer cells are characterized by an altered antioxidant enzyme activity compared to normal cells. Although there was a reduction in the SOD and CAT activity, in most cases, there was an increase in the GSH (glutathione)-dependent enzyme activity, which enables cells to adapt to oxidative stress conditions (Ścibior-Bentkowska, 2009). However, maintaining conditions of oxidative stress in cancer cells can also be helpful during treatment. It has been indicated that natural compounds can support traditional therapy via their pro- oxidative activity (Abotaleb, 2020). This is probably related with the inhibition of the NRF2 (nuclear factor, erythroid 2 like 2) signal path, which is activated in response to oxidative stress and plays a role in generating the antioxidant enzymes (DeNicola, 2011; Sznarkowska, 2017). Because melanoma is difficult to treat, especially in the advanced stages, in this study, we used the C32 cells that were derived from a human male with skin melanoma. Moreover, melanoma cells are characterized by an altered oxidative status (Obrador, 2019). Our research showed that treating melanoma cells with caffeic acid at a concentration of 1,000 µmol/l inhibited the activity of the antioxidative enzymes, which suggests the induction of oxidative stress. We used this concentration because it did not have a cytotoxic effect on the normal cells. However, the results of the research on the effect of caffeic acid on cancer cells are still not clear. There are reports that point to an enhanced carcinogenesis by CA. Hirose (1998) observed that low doses of phenolic compounds including caffeic acid may affect the carcinogenesis process. In

Magdalena Kimsa-Dudek, Agata Krawczyk, Agnieszka Synowiec-Wojtarowicz turn, Khan (2016) showed that a low dose of CA had cytostatic properties and that a high dose could be cytotoxic for cells. Additionally, the International Agency for Research on Cancer (IARC) has classified caffeic acid in group 2B – possibly carcinogenic to humans (Magnani, 2014). However, many current studies have shown its potential use in the therapy for different types of cancer, which is similar to our research concerning melanoma. This may suggest that its action depends on the type of cells. It has been demonstrated that a derivative of caffeic acid (caffeic acid phenethyl ester, CAPE) can be a potential therapeutic agent in cancer treatment for prostate cancer (Tolba, 2013; Lin, 2015), oral cancer (Kuo, 2015), lung cancer and melanomas (Ozturk, 2012) by affecting many of the cellular signaling pathways that are associated with cell proliferation or apoptosis. Additionally, the anticancer effect of caffeic acid has also been observed. Rajendra Prasad (2011) suggested that caffeic acid disturbs the redox balance in HT-1080 cells (human fibrosarcoma cell line), inhibits their proliferation and thus has a potential anticancer effect. It has also been shown that CA can increase the effectiveness of anticancer drugs such as cisplatin, which is used in the treatment of epithelial malignancies (Koraneekit, 2018). Moreover, CA together with CAPE can reduce the toxicity of the drugs that are used in cancer therapy such as irinotecan in the treatment of colorectal cancer (Kalthoff, 2020). Espíndola (2019) emphasized that caffeic acid may be also used in the therapy for hepatocarcinoma. They indicated that the molecular mechanism of its anticancer effect involved, among others, the inhibition of angiogenesis and the down-regulation of metalloproteinases such as MMP2 (Matrix Metallopeptidase 2) and MMP9 (Matrix Metallopeptidase 9), which are responsible for metastasis. In addition, it seems to be that the phenolic compounds in food may be important as chemopreventive agents as the properties of caffeic acid (including the regulation of the cholesterol metabolism and neoplastic development) have been recognized as chemopreventive factors for colon cancer (Rezaei-Tavirani, 2019). To summarize, the results of our research showed the potential anticancer effects of caffeic acid. However, further research into the mechanisms of such action is needed, which will contribute to the development of experimental oncology. SHORT CONCLUSION The redox imbalance that is caused by caffeic acid and its cytotoxicity effect at a concentration of 1,000 µmol/l on melanoma cells (but not on normal cells) implies the usefulness of this compound as a supporting treatment for melanoma therapy. CONFLICT OF INTEREST The authors declare that they have no conflict of interest. ACKNOWLEDGMENTS This study was supported by grant No KNW-2-I13/N/8/K from the Medical University of Silesia, Katowice, Poland.

Redox homeostasis in melanoma cells treated with caffeic acid

REFERENCES Abotaleb M., Liskova A., Kubatka P., Büsselberg D., Therapeutic Potential of Plant Phenolic Acids in the Treatment of Cancer, Biomolecules. 2020; 10(2):221. Anantharaju P.G., Gowda P.C., Vimalambike M. G., Madhunapantula S.V., An overview on the role of dietary phenolics for the treatment of cancers, Nutr J. 2016; 15(1):99. Cizmarova B., Hubkova B., Bolerazska B., Marekova M., Birkova A., Caffeic acid: a brief overview of its presence, metabolism, and bioactivity, Bioactive Compounds in Health and Disease. 2020; 3(4):74-81. DeNicola G.M., Karreth F.A., Humpton T.J., Gopinathan A., Wei C., Frese K. et al., Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis, Nature. 2011; 475(7354):106-109. Domingues B., Lopes J.M., Soares P., Pópulo H., Melanoma treatment in review, Immunotargets Ther. 2018; 7(7):35-49. Espíndola K., Ferreira R.G., Narvaez L., Silva Rosario A., da Silva A., Silva A. et al., Chemical and Pharmacological Aspects of Caffeic Acid and Its Activity in Hepatocarcinoma, Frontiers in oncology. 2019; 9: 541. Garbe C., Peris K., Hauschild A., Saiag P., Middleton M., Bastholt L. et al., Diagnosis and treatment of melanoma. European consensus-based interdisciplinary guideline – Update 2016, Eur J Cancer. 2016; 63:201-217. Hirose M., Takesada Y., Tanaka H., Tamano S., Kato T., Shirai T., Carcinogenicity of antioxidants BHA, caffeic acid, sesamol, 4-methoxyphenol and catechol at low doses, either alone or in combination, and modulation of their effects in a rat medium-term multi-organ carcinogenesis model, Carcinogenesis, 1998; 19(1):207-212. Kalthoff S., Paulusch S., Rupp A., Holdenrieder S., Hartmann G., Strassburg C.P., The coffee ingredients caffeic acid (CA) and caffeic acid phenylethyl ester (CAPE) protect against irinotecan induced leukopenia and oxidative stress response, Br J Pharmacol. 2020; 10.1111/bph.15162. Khan M.S.S., Iqbal M.A., Majid A.M.S.A., Effect of crystallization of caffeic acid enhanced stability and dual biological efficacy, Cogent Biology. 2016; 2(1):1243460. Kimsa-Dudek M., Synowiec-Wojtarowicz A., Derewniuk M., Gawron S., Paul-Samojedny M., Kruszniewska-Rajs C. et al., Impact of fluoride and a static magnetic field on the gene expression that is associated with the antioxidant defense system of human fibroblasts, Chem Biol Interact. 2018; 287:13-19. Kołodziejczyk-Czepas J., Szejk M., Pawlak A., Żbikowska H.M., Właściwości przeciwutleniające kwasu kawowego i jego pochodnych, ŻYWNOŚĆ. Nauka. Technologia. Jakość. 2015; 3(100):5-17. Koraneekit A., Limpaiboon T., Sangka A., Boonsiri P., Daduang S., Daduang J., Synergistic effects of cisplatin-caffeic acid induces apoptosis in human cervical cancer cells via the mitochondrial pathways, Oncol Lett. 2018; 15(5):7397-7402. Kumar N., Goel N., Phenolic Acids: Natural Versatile Molecules with Promising Therapeutic Applications, Biotechnol Rep (Amst). 2019; 24:e00370. Kuo Y.Y., Jim W.T., Su L.C., Chung C.J., Lin C.Y., Huo C. et al., Caffeic Acid phenethyl ester is a potential therapeutic agent for oral cancer, Int J Mol Sci. 2015; 16(5):10748-10766. Lin H.P., Lin C.Y., Huo C., Hsiao P.H., Su L.C., Jiang S.S. et al., Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1, Oncotarget. 2015; 6(9):6684-6707. Magnani C., Isaac V.L.B., Correa M.A., Salgado H.R.N., Caffeic acid: a review of its potential use in medications and cosmetics, Analytical Methods. 2014; 6(10):3203-3210.

Magdalena Kimsa-Dudek, Agata Krawczyk, Agnieszka Synowiec-Wojtarowicz

Meinhart A.D., Damin F.M., Caldeirão L., de Jesus Filho M., da Silva L.C., da Silva Constant L. et al., Study of new sources of six chlorogenic acids and caffeic acid, Journal of Food Composition and Analysis, 2019; 82:103244. Mileo AM, Miccadei S., Polyphenols as Modulator of Oxidative Stress in Cancer Disease: New Therapeutic Strategies, Oxid Med Cell Longev. 2016; 2016:6475624. Nicholson S.K., Tucker G.A., Brameld J.M., Effects of dietary polyphenols on gene expression in human vascular endothelial cells, Proc Nutr Soc. 2008; 67(1):42-47. Obrador E., Liu-Smith F., Dellinger R.W., Salvador R., Meyskens F.L., Estrela J.M., Oxidative stress and antioxidants in the pathophysiology of malignant melanoma, Biol Chem. 2019; 400(5):589-612. Ozao-Choy J., Lee D.J., Faries M.B. Melanoma vaccines: mixed past, promising future. Surg Clin North Am. 2014; 94(5):1017-1030. Ozturk G., Ginis Z., Akyol S., Erden G., Gurel A., Akyol O., The anticancer mechanism of caffeic acid phenethyl ester (CAPE): review of melanomas, lung and prostate cancers, Eur Rev Med Pharmacol Sci. 2012; 16(15):2064-2068. Pawłowska-Góral K., Kimsa-Dudek M., Synowiec-Wojtarowicz A., Orchel J., Glinka M., Gawron S., Effect of static magnetic fields and phloretin on antioxidant defense system of human fibroblasts, Environ Sci Pollut Res. 2016; 23(15):14989-14996. Rajendra Prasad N., Karthikeyan A., Karthikeyan S., Reddy B.V., Inhibitory effect of caffeic acid on cancer cell proliferation by oxidative mechanism in human HT-1080 fibrosarcoma cell line, Mol Cell Biochem. 2011; 349(1-2):11-19. Rezaei-Tavirani M., Rezaei Tavirani M., Zamanian Azodi M., The bioinformatics aspects of gene screening of HT-29, human colon cell line treated with caffeic acid, Gastroenterol Hepatol Bed Bench. 2019; 12(3):246-253. Ścibior-Bentkowska D., Czeczot H., Komórki nowotworowe a stres oksydacyjny, Postepy Hig Med Dosw. (online), 2009; 63:58-72. Sznarkowska A., Kostecka A., Meller K., Bielawski K.P., Inhibition of cancer antioxidant defense by natural compounds, Oncotarget. 2017; 8(9):15996-16016. Tolba M.F., Esmat A., Al-Abd A.M., Azab S.S., Khalifa A.E., Mosli H.A. et al., Caffeic acid phenethyl ester synergistically enhances docetaxel and paclitaxel cytotoxicity in prostate cancer cells, IUBMB life. 2013; 65(8):716-729. Trachootham D., Lu W., Ogasawara M.A., Nilsa R.D., Huang P., Redox regulation of cell survival, Antioxid Redox Signal. 2008; 10(8):1343-1374. Wu J.C., Lai C.S., Lee P.S., Ho C.T., Liou W.S., Wang Y.J. et al., Anti-cancer efficacy of dietary polyphenols is mediated through epigenetic modifications, Current Opinion in Food Science, 2016; 8: 1-7. Zhou H., Chen J.X., Yang C.S., Yang M.Q., Deng Y., Wang H., Gene regulation mediated by microRNAs in response to green tea polyphenol EGCG in mouse lung cancer, BMC Genomics. 2014; 15:S3.

Applications of alginates in the biomedical field

Magdalena B. Łabowska1, Agnieszka M. Jankowska1*, Izabela Michalak2, Jerzy Detyna1, Julita Kulbacka3 1 Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering, Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-370 Wroclaw, Poland; 2 Department of Advanced Material Technologies, Faculty of Chemistry, Wroclaw University of Science and Technology, Smoluchowskiego 25, 50-372 Wroclaw, Poland 3 Department of Molecular and Cellular Biology, Faculty of Pharmacy, Wroclaw Medical University, 50-367 Wroclaw, Poland *Corresponding author: Agnieszka M. Jankowska, Department of Mechanics, Materials and Biomedical Engineering, Faculty of Mechanical Engineering, Wroclaw University of Science and Technology, e-mail: [email protected] ABSTRACT Alginate is a polysaccharide naturally occurring in brown seaweeds cell walls. Unique properties such as ease of gelation, biodegradability, biocompatibility, and non-toxicity of alginates make them widely employed in a variety of industries. Mentioned here properties and versatility of alginate resulted in approval by U.S. FDA (Food and Drug Administration), thereby initiating its application in the biomedical sector on an industrial scale. The chemical modifications of this biopolymer allow to obtain different forms (e.g. hydrogels, films, fibers), as well as to receive the desired characteristic. The challenge is to match the physical properties to the specific application requirements. Alginates are often utilized in the pharmaceutical industry in the controlled drug delivery and as protein carriers. Moreover, alginates can be a component of drugs for obesity and reflux. They are also used in wound healing due to water retention capability, anticancer and antimicrobial properties. On account of the similarity of alginate hydrogels to living tissues, elasticity and soft structure, their successful application in vitro, in tissue engineering or regenerative medicine is possible. Alginate hydrogel can be widely used as a bioink for 3-D bioprinting. The manufactured alginate scaffolds may be a matrix that provides a suitable substrate for cell growth and cell protection against possible external damages by customization of mechanical properties. Although it is feasible to control the mechanical properties at the alginate extraction stage or during hydrogel cross- linking process, alginate hydrogels have very limited overall physical properties, mainly mechanical stiffness. The possibility to obtain accurately regulated chemical and physical properties of alginate based material may lead to a revolution in the biomedical sciences. Keywords: alginate, hydrogels, biomaterial, drug delivery, cell culture, wound healing, tissue engineering INTRODUCTION Marine brown seaweeds are commonly known to be a rich source of bioactive compounds used in many industries, especially in the medical field. One of them is alginate, anionic polysaccharide available in the cell wall of algae, present as a mixed salt of alginic acid (sodium and/or calcium, magnesium, potassium). This natural polymer is linked by 1-4 glycosidic bonds, including three structure block types: G block (α-L-guluronic acid), M block (β-D-mannuronic acid) and MG block (composed of both polyuronic acids), as shown in Figure 1 (Venkatesan, 2015; Theagarajan, 2019). Block quantity may vary depending on the origin and the seaweed species, their maturity level and the algal harvesting period. The ratio of M and G blocks, their location and molecular weight affect the alginate physical properties. Alginates are able to bind large amount of water and gelling in the presence of divalent cations (e.g., Ca2+, Ba2+, Mg2+) (Skjåk-Bræk, 2012; Şenel, 2015).

Magdalena B. Łabowska, Agnieszka M. Jankowska, Izabela Michalak, Jerzy Detyna, Julita Kulbacka

Figure 1. Structure of alginates a) fragment of the polysaccharide chain with different MG sequences, b) monomer units: β-D-mannuronic acid and α-l guluronic acid, c) M and G blocks distribution in the alginate chain (based on Davis 2003; Fraczyk 2020)

Alginate has unique biological and pharmaceutical properties, as well as it is non-toxic and not immunogenic and therefore, has been approved by the U.S. FDA (Food and Drug Administration). Consequently, it makes alginate suitable for a wide range of industrial applications. Due to its rheological properties, it can be often applied as a stabilizer and thickener in food (e.g. jellies, ice creams, sauce thickener, animal food) and beverages (e.g., in the instant milk desserts, as a stabilizer beer foam), but also in the food packaging (Cosenza, 2017; Theagarajan, 2019). Other industrial applications of alginates include textile printing and ceramics production, fertilizers in agriculture, as well as the manufacture of welding rods. Their ability to bind metal ions, make them appropriate for wastewater treatment (Abhilash, 2017). Properties such as biodegradability and biocompatibility enable its use in the medical sector, including pharmacy – drug delivery systems, biomedical engineering, tissue engineering – cell carriers (such as liver, nerve, heart, skin, cartilage and bone) (Lee, 2001; Catoira, 2019) and cell cultures (Ravi, 2015), orthopedics, medical textiles – wound dressing, but also dentistry (Venkatesan, 2015; Afewerki, 2019). The biomaterial structure and its properties (e.g. biodegradation, mechanical strength, gelling properties and cellular affinity) can be achieved by physical or chemical cross- linking, the incorporation of ligands such as peptides and carbohydrates, and the connection with other biomaterials (Sun, 2013). Mechanical properties of alginate hydrogel are determined by cross-linking density, molecular weight of the monomer and the number and sequence of its units (M and G) as well as chain stiffness (Kong, 2002). The mechanical stiffness of ion-crosslinked alginate hydrogels is also dependent on the molecular weight of the alginate and the concentration of binding cations (Mancini, 1999). Also appropriately selected rheological properties of pre-gel solution, as well as concentration and molecular weight distribution in the polymer affect the mechanical stiffness of post-hydrogel (Kong, 2002). It is possible to obtain different structures and properties of the material, as alginate can be easily modified by chemical and physical reactions (Lee, 2001).

Applications of alginates in the biomedical field

Molecular weight of alginate affects the rate of degradation and mechanical properties of biomaterials – the higher the molecular weight, the smaller number of reactive sites available for hydrolysis (slower degradation) (Mancini, 1999). Biomaterial degradation affects mechanical properties due to structural changes at both molecular and macro- scopic level (Sun, 2013). Alginate plays an important role in the long-term stability and performance of in vitro culture (Hospodiuk, 2017). Alginate applied in a biomedicine is manufactured as hydrogels, microspheres, microcapsules, sponges, foams, fibers, porous scaffolds and increasingly used bioinks (Sun, 2013). Hydrogels consist of hydrophilic polymers with high water content forming three-dimensionally cross-linked networks (Lee, 2001; Tan, 2010). Alginate hydrogels can be classified according to their gelling mechanisms into physically cross-linked and covalently cross-linked alginate gels. Alginates can easily form gel and solid- microspheres using appropriate methods. Alginate gel spheres are produced by ionic cross-linking under aqueous conditions (Chang, 1998), while alginate solid-spheres are produced by emulsion solvent evaporation technique. Both gel and solid alginate-based microspheres show good biocompatibility and can be successfully used in regenerative medicine (Sun, 2013). Gel spheres are suitable for cell encapsulation, while solid alginate-based microspheres are mainly used for drug administration (Kong, 2003; Chen, 2006). Microspherical delivery systems can be used to supply cells, growth factors, proteins, genes and drugs in tissue engineering (Basmanav, 2008). Alginate can be manufactured as a porous scaffold in various forms (spheres, sponges, foams, fibers and rods) for cell cultures (Yang, 2001). These structures can be used as carriers of bioactive molecules and as a three-dimensional skeleton, organizing cells to accommodate and stimulate new tissue growth (Kuo, 2012). Many of the mentioned structures are currently used in tissue engineering and regenerative medicine (Tan, 2012). The different types of alginate have been gathered in table 1, including a description of their structure and examples of potential applications in the biomedical field.

Table 1. General description of used alginate forms Form of Structure Manufacture Application in the References alginate method biomedical field Hydrogel highly swollen soft cross-linked via drug delivery, tissue Sun 2013; material, capable chemical (e.g. engineering, of retain a significant ionic bonding) or regenerative Ahmed 2015; amount of water, but physical reaction medicine, wound will not dissolve in (e.g. photo cross- healing water linking) Fiber rough longitudinal electrospinning controlled drug Zhu 2009; surface with grooves, (nanofibers), wet- release, tissue and irregular serrated- spun technology engineering, Bonino 2011; shaped cross section regenerative Sun 2013; with a dense and medicine, wound homogeneous structure healing Szparaga 2020;

Magdalena B. Łabowska, Agnieszka M. Jankowska, Izabela Michalak, Jerzy Detyna, Julita Kulbacka

Foam highly porous, flexible freeze-dried cross- surgical products, Aderibigbe and pliable solid linked the alginate tissue engineering 2018; structure with pore size microbubbles (scaffolds), wound Karimpoor 100 μm to 250 μm healing 2018; Micro- micrometric size internal or external delivery systems Chan 2002; sphere spherical particles often gelation using (bioactive proteins, filled with various emulsification cells, genes, growth Sun 2013; substances methods factors) Micro- small solid particles or coaxial air or cell therapy, drug Mørch 2008; capsule liquid droplets coated by liquid flow, delivery systems, Al-Rammah thin alginate based emulsification, treatment of Type 1 coating forming extrusion through diabetes 2004; spherical structure a needle, rotating Hamizah preserved by freeze jet breakage, 2016; drying; homogenous vibrating jet surface breakage Holkem 2016 Sponge elastic and flexible most often occurs drug delivery Han, 2006; porous structure with in combination systems, wound pore size between 100 with chitosan, healing, tissue Sun 2013; μm and 500 μm gelatine, graphene; engineering (e.g., Jiang 2019 scaffolds) chemical foaming, freeze-drying step

Moreover, universal properties of this natural biopolymer enable it to be utilized as a bioink in Additive Manufacturing (AM) technology. Alginate can be used as bioink in bioprinting effectively at a range of temperatures in order to achieve the appropriate mechanical properties and desired structure (Hospodiuk, 2017; Zhu, 2018) 3D cell cultures are applied in differentiation studies, pharmacological applications (drug discovery, cancer research), gene and protein expression studies, they also allow a better understanding of complex physiological cellular mechanisms (Ravi, 2015). The unique properties of discussed polysaccharide and the possibility of achieving different forms by physico-chemical modifications of alginates result in a wide range of their applications. The main purpose of this chapter is to present alginates properties and their current use in the biomedicine sector. EXTRACTION METHODS Brown seaweeds mainly from the genus Ascophyllum, Alaria, Cystoseira, Ecklonia, Eisenia, Fucus, Laminaria, Lessonia, Macrocystis, Nereocystis, Sargassum are the natural resources of alginates. The main representatives, used in the industry are Ascophyllum nodosum and Macrocystis pyrifera (Pereira, 2020). Production of sodium alginate from brown seaweeds was described in many publications (e.g. McHugh, 1987; Sanaa, 2013; Łabowska, 2019), and in particular in a series of publications of Hernandez-Carmona (1998, 1999, 2002) and McHugh (2001) who described all production steps such as pre-extraction treatment of seaweed biomass (Macrocystis pyrifera); selection of extraction conditions and methods of separation of the alkaline- insoluble residue after extraction (filtration, centrifugation, flocculation and their

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Applications of alginates in the biomedical field combinations); precipitation and conversion of calcium alginate into alginic acid and finally its conversion into sodium alginate, drying and milling. General scheme of sodium alginate production is presented in figure 2.

Figure 2. General scheme of sodium alginate production (based on McHugh 1987; Sanaa, 2013)

Considering the use of alginates in biomedical field, it is important to prepare the algal biomass before extraction in order to prevent the contamination of the isolated polysaccharide. This preparation includes seaweeds washing with distilled water to remove salt and impurities (Dobrinčić, 2020). Other pre-treatment involves removal of lipids, pigments and compounds with low molecular weight from brown seaweeds using solvents and their mixtures, that do not cause any structural changes of extracted polysaccharides. For this reason usually acetone, methanol, petroleum ether, methanol- dichloromethane are used (Łabowska, 2019; Dobrinčić, 2020). Brown seaweeds contain alginic acid mainly as the calcium salt of alginic acid, although magnesium, potassium and sodium salts may also be present. In the first step, these insoluble salts are converted into soluble sodium alginate using alkaline

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Magdalena B. Łabowska, Agnieszka M. Jankowska, Izabela Michalak, Jerzy Detyna, Julita Kulbacka extraction. But this extraction may be more efficient if first seaweed are treated with dilute mineral acid (marked with (*) in figure 2)). There are two routes to obtain sodium alginate – calcification and acidification process. The obtained sodium alginate forms white or pale yellowish-brown powder, which is slowly soluble in water forming a viscous, colloidal solution. The quantity and quality of the sodium alginate depend on many factors such as brown seaweed species, the used extraction method and chemical modifications. The extraction parameters determine physico-chemical and mechanical properties of sodium alginate and its potential applications (Łabowska, 2019). This biomaterial has numerous applications in biomedicine mainly as hydrogel due to its favorable properties, such as ease of gelation (e.g. ionic cross-linking, covalent cross- linking, thermal gelation, cell cross-linking) and biocompatibility (Lee, 2012). The type of cross-linking, as well as the molecular weight and chemical composition of the polymer influence the physico-chemical properties of hydrogels (Varghese, 2006). The physical properties of the obtained hydrogels can be improved by the increase in the alginate molecular weight. But, on the other hand the alginate solution received from polymers with a high molecular weight can be greatly viscous, which is often undesirable (LeRoux, 1999). Before application of alginate in biomedical field, several requirements must be met. According to the European Pharmacopeia, sodium alginate should contain: chlorides – max. 1.0%, calcium – max. 1.5%, sulfated ash from 30.0 to 36.0%, heavy metals – max. 20 mg/kg, loss on drying – max. 15.0%, microbial contamination: total aerobic microbial count – acceptance criterion 103 CFU (a colony-forming unit)/g, total yeast/moulds count – acceptance criterion 102 CFU/g, absence of Escherichia coli and Salmonella (European Pharmacopeia 2011). Sodium alginate extracted from natural brown seaweeds can contain various impurities such as polyphenolic compounds, heavy metals, endotoxins and proteins (Lee, 2012; Fertah, 2017). Therefore, purification is crucial for the application of alginate in the biomedical field. For this purpose, sodium alginate can be precipitated with ethanol and finally purified twice with ethanol, then with methanol and acetone before drying (Fertah, 2017). BIOMEDICAL APPLICATIONS OF ALGINATES Biomaterials from natural origin have the potential to be widely used in the biomedical field, due to their abundance in the environment, gelling capacity and bioactivity. Algi- nates are a one of these materials, which exhibit unique biological and pharmacological properties. Physico-chemical modification of alginate gives the possibility of obtaining mechanical characteristic suited to its utilisation. The role of this polysaccharide as a scaffold for tissue engineering, wound healing systems, drug delivery systems and implantable medical devices, as well as the possibility of bioprinting with alginates are widely explored by many researchers. Certain applications of alginate will be described below. DRUG CARRIER Oral medications are one of the simplest forms of drugs administration. The requirements for oral form of drugs include their high quality, effectiveness in treating diseases and safety in use. Alginate fulfils all these criteria and is consequently readily

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Applications of alginates in the biomedical field used as a binding agent in oral products, tablet disintegrant, thickening agent and stabilizer (Sachan, 2009). In addition, its application also include the use as a dietary supplement or pills in obesity treatment – gelling properties of alginate cause that it swell in the stomach, providing a sense of satiety. Another application includes prevent gastroesophageal reflux and even the possibility of being a promising candidate for type II diabetes (Szekalska, 2015; Joshi, 2018). Moreover, studies conducted by Idota et al. (2016) showed hypocholesterolemic activity of calcium alginate, which makes it suitable for the use in reducing plasma cholesterol level (Venkatesan, 2015). Compared to traditional tablets, the drug carriers are focused on delivery a specific dose through better biodistribution. Oral therapeutic products are mainly designed for the immediate-release of active pharmaceutical ingredient (API) for rapid absorption. Their coating may affect delays in the release of the medical substance. Control of API release from the carrier should be reproducible and kinetically predictable (Nahar, 2017). The application of alginate hydrogel allows to control the release of the drug from the carrier, due to its biodegradable properties (it disintegrates into non-toxic products). The ease of gelation enables the use of alginate in the pharmaceutical industry to produce drug carriers in different forms such as tablets, micro spheres, beads and films, but recent research has focused mainly on microparticles and nanoparticles. They are a promising research direction, due to their fast release rate, compared to traditional delivery systems (Sosnik, 2014). Additionally, control of drug release from an alginate-based carrier can give the possibility to incorporate into alginate antibiotics, antigens, enzymes, polysaccharides (e.g. heparin) and peptide hormones (e.g. growth hormone, insulin) (Sachan, 2009; Venkatesan, 2015). The hydrophilic nature of alginate enables different release methods of active pharmaceutical ingredients (API) from carrier. Weakly water-soluble drugs are released by matrix erosion, while water-soluble drugs are released by diffusion (Sosnik, 2014). Furthermore, its possibility of degradation or hydrolysis in alkali or strong acids in increased temperatures gives a chance to determine the specific location of the drug release (Tønnesen, 2002). WOUND HEALING The moisturizing, antibacterial, antiviral and antifungal properties of alginate makes it suitable for contact with skin, hereby alginate is widely used in cosmetics (e.g. as a face and body treatment, cream ingredient, mask additive). Besides its moisturizing properties, alginate is also applied as an anti-aging and anti-wrinkle agent (Joshi, 2018). Moreover, its haemostatic efficacy, soothing and anaesthetic effects ensure its application in wound management materials (Szekalska, 2015). Alginate occurs in the dressing products most often in the form of calcium alginate. The merger of alginate with the wound exudate causes ion exchange between calcium in the alginate and sodium in the wound exudate, which leads to the formation of a gel on the wound surface. The created gel absorbs excess exudate and maintains a moist micro- environment that promotes faster wound healing (Abhilash, 2017). Alginate wound dressing showed a potential to activate macrophages and possesses the ability to initiate a proinflammatory signal, which contributes to the granular tissue formation (Thomas, 2000).

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Alginate-based dressings are recommended for the treatment of moderate to highly exuding wounds, as well as post-operative and traumatic wounds, partial thickness burns, donor sites, venous leg ulcers, pressure ulcers and diabetic foot ulcers. The dressing can be removed easily without causing pain or trauma due to its gel-like consistency. Compared with normal gauze, the use of alginate-based bandages showed a considerably reduced chronic wound area in a shorter period after use (Chaby 2007; Clark, 2012; Paul, 2015; Cherng 2018). However, there are some restrictions on the use of alginate-based dressings. They cannot be applied to dry wounds or minimal exudate wounds, third degree burns, surgical implantations or in case of allergy to any of the dressing ingredients. The duration of the dressing is 5-7 days, but it should be changed when the secretion from the dressing edges leaks. The majority of alginate- based dressings need additional dressing as well (Clark 2012). Depending on the type of wound, alginate is used in various forms such as hydrogel, fiber, sponge, foam and film (Clark 2012, Paul, 2015). These wound dressings are often supported by additives that possess antimicrobial properties. Among such additives are: silver (Opasanon, 2010), zinc oxide (Monandas, 2015), gold (Faoucher, 2009), iodine (Thorn, 2009) or antibiotics, such as ciprofloxacin, gentamicin, octenidine, tetracycline, vancomycin (Yang, 2018). Commercially available hydrogels used in wound healing applications are for example: AQUACEL Hydrofibre Wound Dressing, AlgiDERM®, CarraSorb H®, CarraSorb H® SILVERCEL™ (Gray, 2009; Leveriza-Oh, 2012; Paul, 2015). CELL CULTURES IN 3-D TECHNOLOGY The recent study revealed an increased interest in the application of 3D culture models. 3D culture involves physiological processes as cell adhesion, migration, immune signal activation, antioxidative response etc. (Smidsrød, 1990; Andersen, 2015). Thus, the transposition from 2D to 3D culture enables development of more realistic models as organoids or spheroids that can imitate real tissues or organs. 3D cultures can be created independently using "hanging drop models" or special culture dishes (Langhans, 2018), but there are several biomaterials that are suitable for more effective 3D cells cultivation. There are available hydrogels (e.g. protein hydrogels, extracellular matrix (ECM) based, peptide hydrogels or polymer hydrogels i.e. synthetic materials) or mesoporous structures. Three dimensional cell cultivation utilizes scaffold or matrix substructure. The initiation of 3D culture might be performed by (1) the injection of cells suspension to the previously selected scaffold material, (2) cultivation on the material, or (3) layer by layer to put cells within the matrix as a stratum. It is important to deliver all nutrients and supplements to cells during the development of the unified 3D structure. Here we focus on the application of alginate hydrogels in 3D cultures. This material is stable for a long time and widely used in this application and "ready to use systems" (e.g. NovaMatrix®-3D, AlgiMatrix®) are commercially available (Szekalska, 2016; Hunt, 2017). It was noted that cells do not have receptors recognizing alginate, thus cellular processes do not require signalling molecules and matrix interaction (Cavo, 2018). There are several types of cell lines verified to grow in alginate hydrogels. We can find normal (muscle, kidney, fibroblast) and cancer cell models e.g. cervix, ovarian, colon, lung prostate, pancreas, leukemia or breast cancer cells. The type of the cells/tissue determines what level of elasticity of alginate hydrogel must be used. An interesting approach of the cell immobilization was implemented in pancreatic islet

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Applications of alginates in the biomedical field cells. Authors applied calcium alginate matrices to encapsulate pancreatic cells. Then, alginate gel beads were coated by poly-L-lysine, poly-L-ornithine, or chitosan. The aim of these polycations was to strengthen the coating surface and to control the porosity (Lim, 1980; Smidsrød, 1990). This method of cell encapsulation by alginate hydrogels was widely developed and applied to implantation in e.g. diabetic patients for insulin production (Tuch, 2009). The other method is "delayed gelation system" where alginate gels are formed inside the body and enable its implantation to avoid invasive surgery (Andersen, 2015). For larger constructions, macroporous scaffolds are used. These materials become gels after rehydration and cell seeding. There are numerous methods for 3D culture, and future application determines this type. TISSUE ENGINEERING/REGENERATIVE MEDICINE Cell constructs based on alginate hydrogels are good candidates for tissue engineering and regenerative medicine applications. The existing studies demonstrate stem cells blended with biomaterial scaffold for in vitro and in vivo research. There are different biomaterial formulations for tissue engineering e.g. microspheres, hydrogel or electrospun nanofibers. Alginate-based material was recommended for hydrogels and electrospun method (Willerth, 2019). It was indicated that this material was efficiently used with adipose-derived adult stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) to generate replacement cartilage (Awad, 2004) or with neural tissue (Razavi, 2015). In another study, calcium alginate gels were mixed with gelation. The gels were injectable and could be applied as immobilization matrices for human MSCs (Schmitt et al. 2015). Other application involved the transplantation of cells into injured tissues. The authors used human Wharton's jelly-derived mesenchymal stem cells (HWJ-MSCs) to load them into alginate hydrogel and support cell proliferation (Azandeh, 2019). Alginate-based materials have also found application in the regenerative medicine. The main utilization involves implants based on hydrogel scaffolds, which contain an appropriate type of cells to stimulate tissue regeneration processes. The available studies indicate an effective usage of alginate-based material on cartilage repair, bone regeneration, wound healing, as well as drug delivery and also cell encapsulation (Ghidoni, 2008; Sun, 2013; Kaczmarek-Pawelska, 2019). Pandolfi et al. verified that alginate-based microencapsulation can be efficiently applied for transplantation in patients that exclude immunosuppressive therapies (Pandolfi, 2017). Commercially available hydrogels for tissue engineering and regenerative medicine applications are for example AlloDerm, DermaMatrix (Becker, 2009). ALGINATE AS A BIOINK FOR BIOPRINTING Bioprinting is a rapidly growing field of biomedicine that combines tissue engineering, material science and additive technologies to obtain biological structures with high cell viability (Hospodiuk, 2017). Although synthetic materials have properties suitable for supporting cell and tissue growth (ease of modification, printability, viscosity and mechanical strength), they have limited bioactivity (limited cell adhesion and limited ability to support the biological components of cell growth) and low biodegradation (O'Brien, 2011). Due to the role of natural biomaterials – cell growth promoters, their

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Magdalena B. Łabowska, Agnieszka M. Jankowska, Izabela Michalak, Jerzy Detyna, Julita Kulbacka use in biomedicine is promising. Furthermore, they are characterised by a good bioactivity, biocompatibility, biodegradability and mechanical stability (Gershlak, 2017). The material used to produce bioink must be liquid enough to allow extrusion and, at the same time, should maintain a stable structure after printing to permit the overgrowth and viability of cells and tissues on the printed scaffold. The matching of these two conditions often poses problems and obtaining the right material is a time consuming process (Jovic, 2019). 3D bioprinters (Figure 3), with precisely printing biomaterials without putting cells at risk, have an advantage over other scaffolding production technologies (i.e. melt molding, gas foaming, electrospinning, solvent casting, particular leaching, phase separation and freeze drying) (Hölzl, 2016; Mohseni, 2019).

Figure 3. Graphic representation of the bioprinting procedure (Li, 2018)

Alginate hydrogels are commonly utilized due to their low price and their mechanical and rheological properties suitable for 3D bioprinting (Hölzl, 2016), which should be considered each time depending on the specific application (Hospodiuk, 2017). The viscoelastic properties should also be taken into consideration in 3D bioprinting technology (Jovic, 2019). Alginates are used in the extrusion based bioprinting process (requires a fast gelation), they can be also enriched with additives to improve cell adhesion to alginate scaffolds (Axpe, 2016). The bioprinting technology may be used in tissue engineering, implant manufacturing, development of cell-based sensors, drug screening models and cancer models (Jia, 2014; Markstedt, 2015). Commercially available bioink is CELLINK, which combines alginate and nanocellulose. It has the ability to shearing and fast cross- linking, which makes it valuable in soft tissue engineering applications (Hölzl, 2016). DENTISTRY In dentistry, alginates can be applied in prosthetics for the fabrication of primary casts, testing models in orthodontics and reproducing impression castings. Alginate imprez- sions can be also used to make indirect restorations with a chamfer finish lines (Datla, 2015). The benefits of using discussed biomaterial include low cost, very simple Manu- facturing technique, short time to manufacture and ease of manipulation (Cervino,

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2019). Dental impressions based on alginates are easy to manage for both, the dentist and the patient because they are cost-effective and quickly set at room temperature (Abhilash, 2017). Although alginate is the most commonly used impression material in dentistry, it does not mean that it is ideal because the alginate impression has poor dimensional stability, low tear strength and no possibility of long storage or repeated use (Onwubu, 2019). For better customized impressions, alginate should be integrated with additives. Alginates available on the market are of two types: fast hardening (time 1-2 min) or normal (time between 2-5 min) (Cervino, 2019). Alginate impression material is contraindicated in some patients because cases allergic reactions to alginate material (containing crystalline silica, calcium sulphate or potassium titanium fluoride) and in such cases, non-alginate impression material should be used (Gangemi, 2009). Alginates utilized in dentistry are in the form of powder and should be mixed with water in appropriate doses (in order to turn into a soft paste) and then placed on a tray and finally introduced into the oral cavity for an impression. The setting time depends on the composition and temperature at which mixing takes place, where increasing the amount of powder accelerates the hardening reaction and increasing the temperature, reduces the setting time (Cervino, 2019). The restoration of accurate and correct impressions of the dental arch from alginate impression material depends on strict adherence to the art of taking impressions (Onwubu, 2019). CONCLUSION To successfully use natural materials in medicine, many requirements must be taken into account. In particular, this material must be biocompatible and non-toxic, to avoid damage in contact with the tissue or during oral intake. Depending on the application, the biodegradation properties are also desired. Alginate, as a natural biomaterial, fulfils a number of medical criteria, therefore, has been approved by the FDA and is widely used in medicine. The flexibility of its physico-chemical modification enables obtaining alginate in various forms, but most often it occurs in the form of hydrogels. Alginate-based biomaterials are promising substrates for drug delivering, wound healing, regenerative medicine and tissue engineering, cell culture and also dentistry. Its ability to gel forming in aqueous systems, cross-linking with various factors and its ability to mix with other polymers, both natural and synthetic, makes it the most widely used polymer. Possibility of adapting the different structure depending on the application, achieving various mechanical properties by physico-chemical modification and printability give the possibility to applied alginate as a bioink in bioprinting, which enables obtain the accurate and complex shapes not achievable by other technologies.

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The effect of periodontal disease on acute cardiovascular events: a preliminary report

Katarzyna Dębska-Łasut1, Małgorzata Kobusiak-Prokopowicz2, Marek Ziętek3, Andrzej Mysiak2 1 Katedra i Zakład Periodontologii UM we Wrocławiu Division and Department of Periodontology, Wroclaw Medical University, Wrocław, Poland 2 Katedra i Klinika Kardiologii UM we Wrocławiu Division and Department of Cardiology, Wroclaw Medical University, Wrocław, Poland 3 Katedra Stomatologii Doświadczalnej UM we Wrocławiu Department of Experimental Dentistry, Wroclaw Medical University, Wrocław, Poland ABSTRACT Objectives: The aim of the study was to assess the effect of periodontal disease on systemic diseases, and particularly on acute cardiovascular events, in patients aged 25 to 50 years. We also evaluated the association of periodontal disease as a chronic inflammatory state and poor oral hygiene with the risk of cardiovascular disease (CVD). Material and methods: We enrolled patients aged 25 to 50 years with acute cardiovascular events and periodontal disease and without any other known comorbidities. The group was compared with healthy individuals matched for age. The analysis of oral hygiene parameters, periodontal status, the community periodontal index of treatment needs (CPITN), as well as the bacterial profile of the periodontal pockets and peripheral blood was performed to develop a set of simple diagnostic tests for assessing the risk of acute cardiovascular events in patients with periodontal disease. Results: The preliminary findings show that the level of oral hygiene in both groups (with cardiovascular and periodontal disease) is not satisfactory in the context of World Health Organization goals. Both the depth of the periodontal pockets and the parameters of oral hygiene and bleeding on probing largely exceeded the reference values. Most patients in both groups had high CPI scores, with CVD patients showing lower scores than patients with periodontal disease. Conclusions: Patients with periodontal disease and acute cardiovascular events show poorer oral hygiene and worse oral health; thus, they have higher oral health and periodontal treatment needs. Moreover, cardiovascular patients showed lack of education regarding the risk of systemic diseases due to poor or no oral hygiene. They also lacked motivation for the treatment of periodontal disease. Keywords: periodontal disease, oral hygiene, STEMI, NSTEMI, acute cardiovascular events, atherosclerosis INTRODUCTION Contemporary medicine is facing a number of challenges, including civilization diseases. The link between periodontal and cardiovascular disease (CVD) is usually discussed in the context of patients aged over 50 years. We decided to investigate this association in a younger population, namely, individuals between 25 and 50 years of age, who were not burdened with any comorbidities apart from acute cardiovascular events. Due to the high incidence of CVD, there is a wide range of diagnostic tools that are used to identify at-risk populations and develop preventive measures. However, there is still no cost-effective screening method for a quick and reliable identification of individuals with cardiovascular risk factors. As triggers of acute cardiovascular events are well known, including also the impact of comorbidities, preventive measures based on a more active lifestyle, healthy diet,

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Katarzyna Dębska-Łasut, Małgorzata Kobusiak-Prokopowicz, Marek Ziętek, Andrzej Mysiak quitting smoking, and stress reduction are widely discussed. However, there are no easy-to-obtain markers of increased cardiovascular risk in young patients with periodontal disease without other comorbidities. THE PATHOMECHANISM OF ATHEROSCLEROSIS As periodontal disease has been widely studied, its molecular pathomechanism is generally well known. Similarly, research into CVD has provided sufficient evidence to elucidate the causes of acute conditions, understand emergencies and risk factors, as well as develop treatments and recommendations on postdischarge management. Studies have demonstrated a link between periodontal disease and acute cardiovascular conditions. The underlying mechanisms include chronic inflammation in the oral cavity that causes changes to the immune system as well as direct and indirect effects of oral bacteria that are responsible for the periodontal disease. In addition, the factors responsible for chronic inflammation in periodontal tissue are also involved in the development of vascular endothelial inflammation by way of a similar pathomechanism (Smith, 2018). Atherosclerosis is a chronic disease involving the formation of atherosclerotic plaques due to the disturbance of laminar flow at branching sites (Smith, 2018). It is also defined as a chronic progressive unresolved inflammation associated with local deposits of cholesterol inside the subendothelial layer of the vascular wall (Hanson, 2005, Hafiane 2019). Although cholesterol accumulation has potential beneficial effects by promoting inflammation in response to infection, it worsens the course of diseases that are associated with chronic metabolic inflammation, including atherosclerosis (Toll, 2015). There are six stages of atherosclerotic plaque formation. The first three stages are asymptomatic and involve the intra- and extracellular accumulation of lipids and formation of the so-called lipid core. Clinical symptoms occur at a later stage when the lipid core becomes surrounded by the fibrotic layer, followed by further plaque thickening, formation of thrombotic lesions, and plaque rupture. Generally, stage 1 involves low-density lipoprotein (LDL) binding to the subendothelial arterial matrix. Stage 2 is characterized by the chemical modification of LDL (LDL oxidation), damage to endothelial cells and macrophages, and continuous leukocyte recruitment. Monocyte–macrophage recruitment to the arterial wall and adhesion of inflammatory cells to the endothelial surface occurs in stage 3 (Hafiane, 2019; Legler, 2017). Stage 4 involves the formation of the lipid core. Macrophages undergo phagocytosis and build up in the atherosclerotic plaque (consume LDL and form foam cells). Intensive cholesterol accumulation in macrophages leads to formation of foam cells and subsequent fatty streaks (Wollard, 2010). In stage 5, fibrous cap is formed, which promotes progressive narrowing and hardening of the arteries (Moreno, 2010). Microcal- cifications and spotty calcifications represent an active stage of vascular calcification correlated with inflammation (Xuan, 2020). The final stage 6 is advanced plaque with the possibility of plaque-associated thrombosis and erosion or rupture (Hafiane, 2019). Heart disease remains the leading cause of death, and acute cardiovascular events such as acute myocardial infarction and sudden cardiac death account for the majority of mortality cases. The gradual growth of plaque results in the formation of collateral

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The effect of periodontal disease on acute cardiovascular events: a preliminary report circulation, but when the plaque tears or ruptures, this may lead to sudden total occlusion and, consequently, to acute coronary syndrome (ACS). Acute coronary syndrome results from an imbalance between myocardial oxygen supply and demand. Based on electrocardiographic findings patients are classified as having one of the following: 1) persistent ST-segment elevation (ie, ST-segment elevation ACS). Most patients in this group develop ST-segment elevation myocardial infarction (STEMI). 2) acute chest pain without persistent ST-segment elevation. Patients may develop non–ST-segment elevation myocardial infarction (NSTEMI) or unstable angina (UA). In UA, myocardial ischemia without cell loss and negative cardiac biomarkers are observed (Task, 2016). On the basis of clinical symptoms and test results (electrocardiographic and cardiac biomarkers), five conditions are distinguished: myocardial infarction, STEMI, NSTEMI, UA, and sudden cardiac death (Thygesen, 2012). PERIODONTAL DISEASE Periodontal disease is associated with chronic inflammation of the periodontal tissues in response to the presence of bacteria. In some individuals, the immune response leads to tissue damage as well as alveolar bone and connective tissue loss, which in turn results in dental loosening and loss of teeth (Larsson, 2017). According to the latest epidemiological data, periodontitis affects almost 50% of individuals aged above 30 years and 60% of those between 35 and 44 years (Thornton-Evans, 2013; Konopka, 2015; Górska, 2012). Frencken et al. reported that severe periodontitis was the sixth most prevalent condition worldwide in 2010 and was observed in 10.8% of people (743 million) aged 15 to 99 years, with a similar prevalence and incidence among men and women. The prevalence of severe periodontitis increased with age, reaching peak values at the age of 40 years (Frencken, 2017). Other studies also consistently show that the incidence and severity of periodontal disease increases with age (Thornton- Evans, 2013; Konopka, 2015; Górska, 2012; Górska, 2019). In 2015, the American Academy of Periodontology started an update of the classification of periodontitis, which was finalized in 2017. Initially, periodontal disease was classified as mild, moderate, and severe. The diagnostic criteria for the severity of periodontitis included the pocket depth during probing, presence or absence of bleeding during probing, signs of bone loss on X-ray, and clinical attachment loss (Górska, 2015). The current classification includes the degree, extent, and rate of progression of the disease. There are four stages of periodontitis based on disease severity and complexity of management: stage I, initial periodontitis; stage II, moderate periodontitis; stage III, severe periodontitis with the risk of additional tooth loss; and stage IV, severe periodontitis with the risk of loss of dentition. The extent and distribution of the disease are classified as localized, generalized, and molar-incisor. Finally, there are three grades for evaluating the risk of disease progression and response to treatment: grade A, slow rate of progression; grade B, moderate rate of progression; and grade C, rapid rate of progression (Caton, 2018).

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Periodontal disease is a chronic condition. It may develop slowly over the years proportionately to the amount of plaque, but its progression may also be very quick and aggressive, leading to defects in the vertical alveolar bone, which means that there is no connection with the plaque. For periodontal disease to develop, the simultaneous presence of several risk factors is required, which are classified as follows: 1) environmental; 2) local; 3) dependent on host immune responses; and 4) genetic (Toczewska, 2019; Donos, 2018; Cafferata, 2020). The presence of plaque along with the full spectrum of bacteria is not sufficient to lead to full-blown periodontitis, but still it may account for as much as 20% of cases (Toczewska, 2019; Esteves-Lima, 2016). However, the presence of plaque is essential for the interaction between periodontal pathogens and the immune response of the host and the susceptibility gene. A chronic pathogen causes local inflammation, initially gingivitis and then periodontitis, which provides the gateway for bacterial virulence factors and their metabolites to enter the bloodstream of the host. Hence, chronic periodontal inflammation caused by inflammatory mediators secreted by red periopathogen complexes, such as tumor necrosis factor α (TNF-α) and interleukins IL-6 and IL-1β, is considered a risk factor for coronary artery disease (Davĕ, 2008; Zaremba, 2009; Cafferata, 2020; Esteves- Lima, 2016; Wang, 2016). These factors may exacerbate or induce atherosclerosis through their activities in the subgingival aera or by penetrating into the blood (Davĕ, 2008; Zaremba, 2008; Zaremba, 2009). The inflammatory response, on the other hand, is dependent on the monocytic phenotype of the host, and patients with severe inflammatory phenotypes of monocytes activated by periodontal pathogens or lipopolysaccharides may develop excessive inflammatory reactions. Numerous studies have shown that these patients are more predisposed to periodontal disease and are more likely to develop atherosclerosis (Zaremba, 2009; Naiff, 2020; Smith, 2018; Khumaedi, 2019). Both periodontal disease and atherosclerosis, which are directly associated with the risk of ACS, share common risk factors. These include non-modifiable factors (e.g. age, sex, family history, and genetic and environmental factors) as well as modifiable factors (such as diet, stress, psychosocial status, tobacco use, obesity, increased levels of proinflammatory cytokines, and the presence of chronic inflammation) (Moutsopoulos, 2006; Aguilera, 2020). In addition, cross-serological reactions (molecular mimicry) between heat shock proteins (HSPs) are observed in the human body (HSP60) and in certain types of bacteria responsible for periodontitis (GroEL – HSP60 equivalent in Actinobacillus action- mycetemcomitans and Porphyromonas gingialis) and bone loss (Affek, 2007; Davĕ, 2008; Tabeta, 2001). A report from 2016 found that P. gingivalis HSP60 accelerates the development of experimental atherosclerosis by cross-reactivity of the immune response to bacterial HSPs. Moreover, it described a strong correlation between high HSP60 levels and the risk of CVD (Wu, 2016). Smoking is a risk factor with significant impact on the human body. It increases blood density, which strongly affects arterial endothelium by indirectly disrupting the laminar flow and exerting a negative effect on plasma, which, in turn, enhances the severity of periodontitis. At the same time, this process masks symptoms by reducing blood flow in the gums and leads to bleeding during probing (Moutsopoulos, 2006; Tabeta,

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2001; Aguilera, 2020). Reduced blood supply leads to hypoxia and malnutrition of the gums, which hinders soft tissue repair and exacerbates periodontal inflammatory conditions, thus facilitating the penetration of bacteria and inflammatory mediators into the blood. PRELIMINARY RESULTS OF THE STUDY So far, 90 participants aged 25 to 50 years have been recruited to the study, including 30 patients with acute cardiovascular events without other comorbidities; 30 patients with periodontal disease without other comorbidities, and 30 aged-matched healthy individuals without periodontal disease and CVD (control group). The exclusion criterion was the presence of any comorbidities apart from periodontitis in the periodontal group, any comorbidities apart from CVD in the cardiovascular group, and any comorbidities in the control group. It should be noted that it is nowadays difficult and time consuming to identify individuals that would meet all these criteria to ensure a sample size with sufficient statistical power. All participants gave their written informed consent to take part in the study. The study protocol was in line with the ethical standards of the 1975 Declaration of Helsinki, as revised in 2000, and was approved by the local Ethics Committee (KB-582/2012). On physical examination, cardiovascular patients showed no knowledge on the link between oral health and CVD, while their oral hygiene needs did not meet the minimum recommendations. Patients with periodontal disease showed better oral hygiene and treatment needs, but their daily oral hygiene was insufficient to achieve a satisfactory outcome. In both groups, the education level was secondary or voca- tional. In the control group, oral hygiene was correct, which may be associated with the fact that they had higher education level and, in consequence, higher awareness of oral health. Our preliminary analysis shows that the level of oral hygiene in all groups is unsatisfactory in terms of the objectives set by the World Health Organization. The mean Aproximal Plaque Index (API) was 67% in patients with periodontal disease, 50% in healthy individuals, and 66% in patients after an acute cardiovascular event. Of note, in some patients with periodontal disease and cardiovascular event, the API was 100%. The mean depth of the periodontal pockets on first examination was 5 mm in the periodontal group, 4 mm in the cardiovascular group, and 2 mm in the control group, with almost full dentition in all groups. The critical probing depth was 14 mm in the periodontal group, 10 mm in the cardiovascular group, and 4 mm in the control group. Almost one-third of the teeth in the periodontal group showed a varying degree of mobility on the 3-point Hall scale, while the other groups showed no abnormal tooth mobility. The community periodontal index (CPI) of 4 was noted in 60% of the sextants in the periodontal group, as compared with 10% in the cardiovascular group, and in 1 sextant in the control group. The CPI of 3 was noted in 21% of the sextants in the periodontal group and 56% of those in the cardiovascular group.

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The assessment of periodontal treatment needs in all groups showed that both patients with periodontal disease and those with a history of a cardiovascular event required specialized preventive periodontal treatment as well as nonsurgical and surgical interventions. The control group required only nonsurgical periodontal treatment based on oral hygiene interventions. This shows that all patients should be informed about their treatment needs and should receive comprehensive instructions on oral hygiene. DISCUSSION A similar study to that performed by our group was published by Górska et al. in 2019, although the age of patients ranged from 25 to 70 years and less stringent exclusion criteria were applied (Górska, 2019). Both studies showed that periodontal treatment needs are lower in patients with CVD than in those with periodontal disease, which may be related to the age of these patients. In contrast, patients with periodontal disease required comprehensive periodontal treatment irrespective of age. Numerous meta-analyses showed an increased incidence of acute cardiovascular events in patients with periodontal disease, and the association was confirmed by randomized controlled trials (Bui, 2019). Moreover, Groves et al reported that periodontal disease duration and periodontal pocket depth, but also concomitant type 1 diabetes, were directly linked to a higher risk of CVD (Groves, 2015). Angeli et al also reported that the severity of periodontitis, evaluated on the basis of the critical periodontal pocket depth, was correlated with left ventricular mass and stiffness, which, in turn, was associated with acute cardiovascular events. Cardiovascular patients were classified using the CPITN according to the World Health Organization (Angeli, 2018). Hamilton et al. assessed the effect of periodontitis on atherosclerotic complications, acceleration of atherosclerotic plaque formation, and the risk of unstable atherosclerotic plaque (Hamilton, 2017). In cardiovascular patients, smoking contributes to worse periodontal status as well as to masking of disease symptoms. Patients with periodontal disease, despite determination and willingness to undergo treatment, were moderately motivated to perform daily hygiene activities, while smokers were unwilling to quit smoking despite receiving regular instructions to do so. This status may involve dental anxiety, which is firmly ingrained since childhood and early adolescence and is observed even among medical staff and students (Górska, 2012; Kaczmarek, 2010; Kaczmarek, 2011; Wang, 2019). The findings from the above studies are in line with our own hypotheses and results. CONCLUSIONS In conclusion, regardless of overall health, the results of the oral hygiene survey are highly unsatisfactory. In comparison with healthy individuals, patients with periodontal disease and those who have suffered cardiovascular events show poorer oral hygiene and health, and thus have greater medical treatment needs. Moreover, cardiovascular patients demonstrated an alarming lack of awareness about the systemic risks related to poor or no oral hygiene, along with lack of motivation in terms of their oral treatment needs. It seems to be important to raise the awareness about the risks of smoking both among healthy individuals and those burdened with disease, particularly with perio-

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The effect of periodontal disease on acute cardiovascular events: a preliminary report dontal and cardiovascular conditions. Smoking obviously has detrimental effects, and lack of cooperation in terms of reducing smoking negatively affects treatment outcomes in patients with CVD. It also worsens periodontal tissue healing after nonsurgical and surgical procedures, and the effects of treatment are maintained for a shorter time in smokers. Considering the above findings, it seems absolutely vital to educate all patients about treatment needs, the necessity of daily oral hygiene, as well as regular dental visits and interventions. Patients should be informed about the negative effect of smoking on general and oral health. The available evidence points also to the negative effect of periodontitis on CVD, including the increased risk of ACS. Further studies are needed to fully elucidate this relationship and develop simple tests for early identification of at-risk individuals. With proper prevention and early treatment of periodontal disease, the risk of CVD may be substantially reduced. REFERENCES: Affek K., Jagusztyn-Krynicka E.K., Molekularna charakterystyka czynników wirulencji Actinobacillus actinomycetemcomitans, In: Post. Mikrobiol. 2007: 46 (2): 113-123. Aguilera E.M., Suvan J., Buti J., Czesnikiewicz-Guzik M., Ribeiro A.B., Orlandi M., Guzik T.J., Hingorani A.D., Nart J., D’Aiuto F., Periodontitis is associated with hypertension: a systematic review and meta-analysis, In: Cardiovascular Research 2020 116 (1): 28-39, https://doi.org/10.1093/cvr/cvz201. Angeli F., Verdecchia P., Pellegrino C., Pellegrino R.G., Pellegrino G., Prosciutti L. et al., Association between periodontal disease and left ventricle mass in essential hypertension, In: Hypertension 2003; 41: 488-492. doi: 10.1016/j.hlc.2018.05.102. Bui F.Q., Almeida-da-Silva C.L.C., Huynh B., Trinh A., Liu J., Woodward J., Ojcius D.M., Association between periodontal pathogens and systemic disease, In: Biomedical Journal 2019; 42; 27-35, doi:10.1016/j.bj.2018.12.001. Cafferata E.A., Terraza-Aguirre C, Barrera R et al., Interleukin-35 inhibits alveolar bone resorption by modulating the Th17/Treg imbalance during periodontitis, In: J Clin Periodontol. 2020 Mar 11, doi: 10.1111/jcpe.13282. Caton G.J., Armitage G., Berglundh T., Chapple I.L.C., Jepsen S., Kornman K.S., Mealey B.L., Papapanou P.N., Sanz M., Tonetti M.S., A new classification scheme for periodontal and peri‐implant diseases and conditions – Introduction and key changes from the 1999 classification, In: J Clin Periodontol. 2018; 45: 45 (Suppl 20): S1-S8. doi: 10.1111/jcpe.12935. Davĕ S., Van Dyke T.E., The link between periodontal disease and cardiovascular disease is probably inflammation, In: Oral Dis. 2008, 14: 95-101. Donos N., The periodontal poczet, In: Periodontol 2000, 2018 ; 76 (1): 7-15, doi: 10.1111/prd.12203. Esteves Lima R., Cota L., Silva T., Cortelli S., Cortelli J., Costa F., Periodontitis and type 2 diabetes among woman with previous gestational diabetes: epidemiological and immunological aspects In a follow up three years, In: J App Oral Sci. 2017; 25 (2): 130-139, doi: 10.1590/1678-77572016-0367. Esteves-Lima R.P., Costa F.O., Cota L.O.M., Cyrino R.M., Impact of different settings in periodontitis prevalence, In: Arq Odontol, Belo Horizonte 2016,52(4):215-220, Frencken Jo. E., Sharma P., Stenhouse L., Green D., Dietrich T., Global epidemiology of dental caries and severe periodontitis – a comprehensive review, In: J Clin Periodontol 2017; 44 (Suppl. 18): S94- S105, doi: 10.1111/jcpe.12677

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Tabeta K., Yoshie H., Yamazaki K., Characterization of serum antibody to Actinobacillus actinomycetemcomitans GroEL-like protein in periodontitis patients and healthy subjects, In: Oral Microbiol. Immunol. 2001; 16: 290-295. Tall A.R., Yvan-Charvet L., Cholesterol, inflammation and innate immunity, In: Nat. Rev. Immunol. 2015; 15: 104-116. doi: 10.1038/nri3793. Task Force for the Management of Acute Coronary Syndromes in Patients Presenting without Persistent ST-Segment Elevation of the European Society of Cardiology (ESC). 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. European Heart Journal 2016; 37: 267-315. Thornton-Evans G., Eke P., Wei L., Palmer A., Moeti R., Hutchins R., Borrell L.N., Periodontitis among adults aged ≥30 years – United States, 2009-2010, In: MMWR Suppl., 2013; 62: 129-135. Thygesen K., Joseph S.A., Allan S.J., Maarten L.S., Bernard R.C., Harvey D.W., The Writing Group on behalf of the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction. In: Third universal definition of myocardial infarction, European Heart Journal 2012; 33: 2551–2567. Toczewska J., Konopka T., Activity of enzymatic antioxidants in periodontitis: A systematic overview of the literature, In: Dent Med Probl. 2019; 56 (4): 419-426, doi: 10.17219/dmp/112151. Wang J., Geng X., Sun J., Zhang S., Yu W., Zhang X., Liu H., The risk of periodontitis for peripheral vascular disease: a systematic review, In: Rev Cardiovasc Med. 2019; 20 (2): 81-89, doi: 10.31083/j.rcm.2019.02.52. Woollard K.J., Geissmann F., Monocytes in atherosclerosis: subsets and functions, In: Nat. Rev. Cardiol. 2010; 7: 77-86. Wu C., Guo S., Niu Y., Yang L., Liu B., Jiang N., Su M., Wang L., Heat-shock protein 60 of Porphyromonas gingivalis may induce dysfunction of human umbilical endothelial cells via regulation of endothelial-nitric oxide synthase and vascular endothelial Catherine, In: Biomedical Reposts 2016 5(2)243-247, doi: 10.3892/br.2016.693. Xuan S., Jie G., Qiushi L., Haodi C., Fang W., Ruidong Y., Xinfeng L., Calcification in Atherosclerotic Plaque Vulnerability: Friend or Foe?, In: Front Physiol. 2020; 11: 56, doi: 10.3389/fphys.2020.00056. Zaremba M.L., Choroby przyzębia a wzrost odpowiedzi zapalnej, In: Czas. Stomatol., 2009; 62 (7): 531-548. Zaremba M., Górska R., Choroba przyzębia jako potencjalny czynnik ryzyka chorób sercowo- naczyniowych, In: Kardiol. Pol. 2008: 66: 1102-1106.

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Anna Sobiepanek1,*, Patrycja D. Kowalska1,2, Marta Soszyńska3, Aneta Ścieżyńska3,4* *corresponding authors: [email protected] and [email protected] 1 Laboratory of Biomolecular Interactions Studies, Chair of Drug and Cosmetics Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Warsaw, Poland 2 Polish Stem Cells Bank, Warsaw, Poland 3 Department of Histology and Embryology, Medical University of Warsaw, Warsaw, Poland 4 Department of Regenerative Medicine, Military Institute of Hygiene and Epidemiology, Warsaw, Poland ABSTRACT Geneticin, widely known as G418, is an aminoglycoside antibiotic produced by a bacteria Micro- monospora rhodorangea. Geneticin blocks the polypeptide synthesis in both prokaryotic and eukaryotic cells by inhibiting the elongation step. In consequence, rapidly dividing cells are affected more than those with a small proliferation rate. Therefore, geneticin is frequently used in molecular biology as a selective agent for mammalian cells with a different time of population doublings and during cell transfection. The dose of the G418 that is used for the selection of the transfected eukaryotic cells depends on many factors like: the type of the cells and organism from which they were isolated, as well as the growth conditions and the selected culture media. Which is why, the concentration of this antibiotic should be selected for each cell line individually. Here, we present an overview regarding the spectrum of applications, in which geneticin can be implemented as a general selective agent in normal and cancerous cell culture studies. Keywords: Geneticin G418, cells’ selection, cell culture, cancer INTRODUCTION In 1974 a new aminoglycoside produced as the major component by a new species of a gram-positive and spore-forming bacteria Micromonospora rhodorangea was introduced to the public and named Geneticin. The production of geneticin was carried out by the submerged fermentation in a soybean-dextrin medium, and next subsequent adsorption from the fermentation broth by an ion-exchange procedure (Wagman et al., 1974). Geneticin is composed of three rings functionalized with ammonium, hydroxyl and methyl groups, thus its structure is similar to gentamicin (especially gentamicin B1 and gentamicin A – other aminoglycoside antibiotics known since 1944; Aubrecht et al., 2011), therefore it may be called as gentamicin G or by a well-known abbreviation G418 (Vicens and Westhof, 2003). The lyophilized base is a white powder soluble in water or methanol, whereas sulfate is soluble only in water. A typical stock solution is of a 50 mg/ml concentration (Wagman et al., 1974). Although with some limitations geneticin retains a high biological and pharmaceutical compound of interest. Geneticin blocks the polypeptide synthesis due to the irreversible binding to the 70S and 80S cell ribosomes and inhibition of the protein elongation. In consequence, geneticin presents broad toxicity against bacteria, yeast, protozoa, helminths and mammalian cells. Resistance to G418 is conferred by the bacterial gene for aminoglycoside-3’-phosphotransferase (APH(3’)) that can be expressed in eukaryotic cells (Vicens and Westhof, 2003; Prokhorova et al., 2017).

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SEARCH STRATEGY AND SELECTION CRITERIA For the literature analysis performed with the use of the PubMed and Google Scholar databases, the combination of the search terms "geneticin – G418 – cell culture – transfection – cell selection" has been implemented. As the selection criteria we have chosen articles in which geneticin was used as a means for the cell selection or treatment purposes. Moreover, during hand-search some additional articles were also investigated for a better comprehension of the subject. STATE OF ART Geneticin presents a strong influence on all types of cells from prokaryotic to the eukaryotic, especially for those with a high proliferation rate. G418 mode of action mainly results in the inhibition of protein synthesis, the activation of phosphati- dylinositol phospholipase C (which leads to the release of GPI-anchored proteins), as well as the increase of dihydroxyacetone phosphate acyltransferase and the peroxisomal β-oxidation activity (Jin et al., 2004). Some of these functions, however, may be reduced. For example, the irreversible binding of G418 to ribosomes may be compromised by the bacterial aminoglycoside phosphotransferases APH(3’)II and APH(3’)I encoded by the genes on transposons Tn5 and Tn601 (903), respectively. As a consequence, cells transfected with genes of neomycin resistance (neo) from transposon Tn5 or Tn601 gain resistance to G418 and grow in media supplemented with this antibiotic. The selection strategy should be applied individually for various types of cells, as geneticin’s effective concentration differs according to the growth medium, culture conditions as well as metabolic rate of the cells (Davies and Jimenez, 1980; Scholar, 2007). Response to G418 depends on cell metabolism, i.e. some cell lines or clones are able to better tolerate a metabolic load than others. Moreover, geneticin may differently influence cell growth and their proliferation rate depending on the composition of the culture media (serum, glutamine and insulin concentration). As an example, the addition of G418 may cause the change of the flux of glucose from the lactate production towards either the TCA cycle to provide energy or other biosynthetic pathways (Yallop and Svendsen, 2001). Further action of geneticin on cells can include caspase-3-dependent apoptosis (initiated by at least two pathways: cytochrome c release and endoplasmic reticulum (ER) stress) with cell shrinkage and nuclear fragmentation (Jin et al., 2004). REVIEW AND DISCUSSION In general, the use of geneticin may be implemented in case of one of these three applications: (i) for selecting stable clones after transfection, (ii) to eliminate the quickly dividing cells from the mixed cell culture and (iii) as a potential treatment for several diseases. Each application requires an individual analysis regarding the optimal concentration of G418, time of treatment, as well as the application of the culture medium and other conditions.

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G418 FOR THE SELECTION OF STABLE TRANSFECTANTS Transfection is a process of introducing exogenous genetic material in the form of DNA, RNA, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) and short hairpin RNA (shRNA) into mammalian cells, that is widely used in the study of genes and protein expression. Two types of transfection can be obtained depending on the applied methodology: transient (chemical or physical) and stable (biological and physical). In principle the physical method is based on the direct injection of the genetic material, biolistic particle delivery, electroporation or laser- fection; whereas the biological method is connected with the virus-mediated gene transfer (the so-called transduction). On the other hand, the chemical method requires the usage of cationic polymers, cationic lipids or calcium phosphate. The selected approach depends on the cell type and the object (gene or protein) of the experiment (Sheikh et al., 2017). The usability of geneticin as a selective agent after transfection has been known since 1980, because only positively transfected cells and resistant to geneticin are able to grow in culture media supplemented with this antibiotic (Davies and Jimenez 1980). Typically, 24 or 48 hours after cell transfection the old medium is replaced by the full growth medium with the appropriate G418 concentration and the incubation is carried out though 1-4 weeks with the G418-medium exchange every 3-4 days. Articles describing various protocols of the cell selection methods after transfection were gathered in Table 1, e.g. the physical method – electroporation (Fountain et al. 1988), biological – with virus (Belsham et al. 2004) and chemical – with Lipofectamine 2000 reagent (Yang et al., 2019). Due to the fact, that the optimal dose of G418 for the selection of transfected eukaryotic cells depends on numerous factors (e.g. cell type, culture medium, growth conditions and the cells’ metabolic rate), the most suitable approach is to self-determine the sensitivity of each cell line to geneticin before performing the transfection (tab. 2). Several concentrations of G418 ranging from 100 μg/ml to up to even 2 mg/ml should be tested. The treatment should be carried out for at least 48 hours. Next, the cytotoxic effect of G418 on cells can be checked with the MTT test (the conversion of tetrazolium salt into formazan by living cells and calculation of the half maximal inhibitory concentration – IC50) (Sehati et al., 2019, Sadeghiyeh et al., 2019). If the antibiotic concentration is too low or the cell seeding density is too high, the cells can escape the selection. Thus, the selected drug concentration should be approximately 25-50% higher than the minimal concentration required to kill 100% of the cells within 7-14 days (Mally et al., 1992). Another matter to discuss is the growth medium used for the cell culture. Some medium components (e.g. serum, glutamine, insulin) may significantly influence the effect of geneticin (tab. 3), which is why the experiment aimed at the selection of a proper G418 concentration should be performed in the medium appropriate for the study (Yallop and Svendsen, 2001).

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Table 1. Sample conditions for cell transfection and next clone selection with geneticin (***shortcuts: Biol.TM – biological transfection method, Chem.TM – chemical transfection method, Phys.TM – physical transfection method; CF – calf serum, HS – horse serum) Clone Cell type used for selection with Transfection method transfection Growth medium G418 Reference (TM) (cell line) (concentration and time) Human skin Phys.TM: 400 µg/ml Fountain et EMEM+15% FBS fibroblasts (HSF) electroporation for 4 weeks al., 1988 Spontaneously EMEM+10% FBS, immortalized Chem.TM: 100 U/ml penicillin Boukamp human skin the Ca2+-phosphate 800 µg/ml and 50 µg/ml et al., 1990 keratinocytes precipitation method streptomycin (HaCaT) 1000 µg/ml Mouse murine Phys.TM: Armstrong DMEM+5% CS melanoma (HFH18) electroporation for 10-14 et al., 1994 days Human primary 800 µg/ml Chem.TM: Tomizawa lung cancer cell RPMI+10% FBS for 12-21 et al., 2001 lines DMRIE-C reagent days

Human cervical 50 µg/ml cancer (HeLa); Phys.TM: (WM9), Leaman et (not mentioned) Human melanoma electroporation 500 µg/ml al., 2002 (A375, WM9) (HeLa, A375) DMEM+10% FCS, 2 mM glutamine, Human melanoma Chem.TM: Sounni et 10 mM HEPES, 400 µg/ml (A2058) Lipofectamine al., 2002 penicillin-streptomycin (100 IU/ml, 100 µg/ml) DMEM+10% FBS, 400-600 Primary fetal mouse 10% HS, 1% Belsham et Biol.TM: virus µg/ml hypothalamus cells penicillin-streptomycin al., 2004 for 2-3 weeks and 20 mM D-glucose RPMI+10% FBS, Human colon 100 µg/ml Chem.TM: 1000 µg/ml Wang et al., cancer (Lovo) streptomycin and LipofectAMINE2000 for 4 weeks 2007 100 µg/ml penicillin

Human melanoma RPMI+10% FCS and Chem.TM: 700 µg/ml Alexaki et (SK28, 1205Lu) antibiotics FuGENE reagent for 3 weeks al., 2010 Coon’s modified Rat epithelial cells F-12 medium + 5% CS Chem.TM: Di Palma et (Fischer rat thyroid for 2 weeks and 6- hormone al., 2013 line 5; FRTL-5) FuGENE reagent mixture

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DMEM+10% FCS, Chem.TM: 15 mM HEPES, 500 mg/ml Wang et al., Human melanoma (A375) 2 mM glutamine, Lipofectamine for 2 weeks 2014 100 U/ml penicillin 2000 and streptomycin DMEM+10% FCS, 100 μg/ml Human neuroblastoma Chem.TM: 500 µg/ml Pirou et al., penicillin- (SH-SY5Y) for 4 weeks 2017 streptomycin and Lipofectin reagent 1% Glutamax (A431) RPMI+10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin; Human skin cancer (A431), (SK-MEL-28) Chem.TM: 600 µg/ml Lee et al., MEM+10%FBS, Lipofectamine® human melanoma for 2 weeks 2018 1 mM sodium 3000 reagent (SK-MEL-28) pyruvate, 1× non- essential amino acid, 100 U/ml penicillin and 100 μg/ml streptomycin DMEM-F12+10% FBS and 1% Chem.TM: Mouse inner medullary 800 ng/μl Mirvis et penicillin- Lipofectamine® collecting duct 3 (IMCD3) for 5-6 days al., 2019 streptomycin- 3000 reagent kanamycin cocktail Chem.TM: 50 mg/ml Sadeghiyeh Human lung cancer (A549) RPMI+10% FBS jetPEI® solution for 2 weeks et al., 2019

Chem.TM: 460 µg/ml Sehati et al., Human melanoma (A375) RPMI+10% FBS jetPEI® solution for 72 h 2019 Chem.TM: Human melanoma (A375, 500 µg/ml Yang et al., DMEM+10% FBS Lipofectamine SK-MEL-28) for 4 weeks 2019 2000 reagent

Table 2. Sample of the self-determined IC50 concentration of geneticin for the used cell lines Cell type (cell Growth Selection of G418 IC Reference line) medium concentration 50 MTT assay (100, 200, 400, RPMI+10% 460 melanoma (A375) 600, 800, 1000, 1200 µg/ml) Sehati et al., 2019 FBS µg/ml for 72 h lung cancer RPMI+10% MTT assay (100, 200, 400, 494.1 Sadeghiyeh et al., (A549) FBS 600, 800, 1000, 1200 µg/ml) µg/ml 2019

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Table 3. The IC50 concentrations of geneticin used after cell transfection in different growth media (TOKU-e Company, information materials)

Cell type (cell line) Growth medium IC50 DMEM 500 µg/ml colon cancer (Caco-2) MEM 1000 µg/ml DMEM 600 μg/ml

hamster, Chinese ovary DMEM + F12 medium 150 μg/ml (CHO-K1) EMEM 500 μg/ml Ham’s F-12 nutrient mixture 1000 μg/ml DMEM 1000 μg/ml cervical cancer (HeLa) DMEM + F12 medium 600 μg/ml EMEM 400 μg/ml

G418 FOR ELIMINATING CELLS FROM MIXED CULTURE Except for cells transfected with the neomycin resistance gene, other type of cells naturally resistant to geneticin are melanocytes. These slowly dividing cells (origi- nating from the neural crest) are present in the epidermis and are responsible for the production of melanin – the UV photon-absorbing pigment (Sobiepanek and Kobiela, 2020). Their mitosis in vivo has been rarely observed, but during the melanocytes culture in vitro the doubling time varies from 48 hours to up to even 10 days depending on the growth conditions (De Luca et al., 1988; Hoerter et al., 2012). These cells are important for the research connected with their maturation, migration, melanin production as well as cell transformation to melanoma, the deadliest type of skin cancer (Satyamoorthy and Herlyn, 2002; Sobiepanek et al., 2017). During melanocyte isolation from skin mainly two types of contaminant cells may occur: keratinocytes (other components of the epidermis) and fibroblasts (components of the dermis) (Halaban and Alfano, 1984; Sobiepanek et al., 2020). Keratinocytes can be easily separated either from melanocytes as well as from fibroblasts based on differential trypsin digestion (Ścieżyńska et al., 2019), however, separation of melanocytes and fibroblasts causes a lot of difficulties. The first attempts were made with the addition of cholera toxin (CT) or 12-O-Tetradecanoylphorbol-13-acetate (TPA) to the culture medium, but fibroblasts remained insensitive to these components (Halaban, 2005). In 1984 Halaban and Alfano showed that the antibiotic G418 at the concentration of 100 µg/ml applied for 2 days can help in receiving a pure culture of normal melanocytes. From that time, geneticin has been used frequently for these purposes; especially due to the fact that this method requires minimal efforts to obtain a good success rate (Tomonobu et al., 2019). The elimination of fibroblasts, however, may not be immediate and may require some time to observe the expected effect. Moreover, the exposure of melanocyte-fibroblast coculture to G418 has to be repeated in some cases in order to entirely eliminate fibroblasts (Halaban, 2005). A few examples of the implementation of geneticin prior to fibroblast eradication from melanocyte culture are gathered in Table 4. Similar approach can be included for the elimination of fibroblasts from melanoma cell culture (Chapman et al., 2009).

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Table 4. Examples of the G418 usage on cell coculture to eliminate fibroblasts and receive pure melanocytes culture (***shortcuts: IBMX – isobutylmethyl xanthine, EGF – epidermal growth factor, BPE – bovine pituitary extract) G418 dose and Origin of cells Growth medium incubation Research focus Reference time Ham F-10 medium + 10% Nu- to establish Halaban Human neonatal serum, 2% FCS, penicillin- 100 µg/ml a pure human and foreskin streptomycin, 48 nM TPA, for 48 h melanocyte Alfano, 2.5 nM CT and 0.1 mM IBMX culture in vitro 1984 Dulbecco-Vogt Eagle's and Ham's F12 media (3:1) + 5% to gain FCS, 5 μg/ml insulin, 5 μg/ml knowledge of transferrin, 0.18 mM adenine, the dependencies De Luca Human skin from 0.4 mg/ml hydrocortisone, 100 µg/ml between et al., biopsy 1 nM CT, 20 pM for 2-4 days melanocytes and 1988 triiodothyronine, 10 ng/ml keratinocytes EGF, 10 ng/ml PMA, cultured in vitro 50 μg/ml BBE and 50 IU/ml penicillin/streptomycin to establish isolation and Ham's F12 medium + 10% 100 µg/ml cultivation Hu et al., Human eyes FBS, 100 nM TPA, 0.1 mM for 3-7 days methods 1993 IBMX and 10 ng/ml CT of human uveal melano-cytes what is the role EMEM + 10% FBS, 0.2 µg/ml of amelanotic CT, 50 nM PMA, 0.05 mg/ml (details not hair-follicle Tobin et Human scalp gentamicin, 2.5 μg/ml mentioned) melanocytes in al., 1995 fungizone and keratinocyte hair growth and serum-free medium diseases DMEM + 2.5% FBS, 2 mM investiga-ting glutamine, 1 mM sodium melanin pyruvate, 1 % non-essential Amna Hanwoo cattle 100 µg/ml production and amino acids, 2.5 ng/ml EGF, et al., skin from muzzle for 3-4 days gene expression 25 µg/ml BPE, 10 ng/ml TPA, 2012 (MC1R, Tyr, 100 U/ml penicillin, 100 µg/ml Tyrp-1, Tyrp-2) streptomycin investiga-ting Clonetics MGM-4 Melanocyte melanin Gledhill Human neonatal 150 µg/ml Growth Media-4 with 100 nM production, et al., foreskin for 48 h endothelin-3 (EDN3) transfer and 2015 uptake

G418 FOR POTENTIAL TREATMENT OF VARIOUS DISEASES While being an antibiotic, geneticin may be used in medicine as an antiparasitic agent, but with some limitations due to its toxicity to human ear and kidney (Vincens and Westhof, 2003). The in vivo research of Aubrecht et al. performed on mice (with the lethal dose of 153 mg/kg) confirmed that the primary target organs of G418 toxicity are kidney and liver. This was shown by the serum biochemistry analysis (a significant increase in the parameters: blood urea nitrogen (BUN) and creatinine (CRE) – markers of the kidney functionality) and histopathological data (present signs of mild

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Implementation of Geneticin in the in vitro cell culture and in vivo studies nephrotoxicity) (Aubrecht et al., 2011). In case of the in vitro study of normal rate kidney cells treated with 400 µg/ml of G418 for 3 days, the cells presented obvious apoptotic features: cell rounding and shrinkage as well as nuclear condensation and fragmentation. Apoptosis was activated in those cells by the caspase-3 pathway (Jin et al., 2004). In general, G418 acts through different mechanisms depending on the number of positive charges on the antibiotic, its concentration and the organ where the toxicity develops. Known mechanisms also include: the production of hydroxyl radicals by geneticin/metal ion complexes and the disturbance of membrane functionalities by the interaction with phospholipids and the inhibition of the phospholipases activity (Vincens and Westhof, 2003). Furthermore, in case of patients with cancer, the treatment of the occurring infection may cause a risk to the patient. An example was shown during the in vitro study of the human breast cancer cell (MCF-7) treatment with geneticin (10 µg/ml), which allowed the cancer cell survival (by suppressing apoptosis) despite all glucose consumption from the medium under hypoxia (1% O2). At the same time the untreated cells after glucose consumption died. As it is well-known, in solid tumors there are regions where oxygen supply is very low (hypoxic conditions), thus the addition of G418 to treat the infection may cause a side effect of the cancer cells’ resistance as well as the limitation of the action of the drugs dedicated to cancer treatment (Lee et al., 2002). On the other hand, the anti-apoptotic activity of geneticin may be used against perinatal hypoxic-ischemic (H-I) brain injury to cause a neuroprotective effect. The in vivo study was performed on an animal model of Sprague-Dawley rat pups and an intraperitoneal injection of 0.1 µg/kg G418 was made before and after the rats were placed in the hypoxic chamber for a 2.5-hour period (92% N2 and 8% O2). In case of mice with H-I brain injury and treated with G418, a decreased number of apoptotic cells was observed as well as a decrease of the Bax/Bcl-2 expression ratio and a decrease of caspase-3 activity (Ju et al., 2008). Administration of geneticin has proven helpful also in the treatment of patients suffering from several genetic disorders (Vincens and Westhof, 2003). A genetic abnormality may be a discrete mutation not influencing our organism or of a major significance (e.g. insertion or deletion), which in consequence leads to genetic diseases. Some disorders may be inherited, while others occur randomly or because of some environmental influence. Genetic disorders are caused by the nonsense or frameshift mutation, which induces the premature termination codons (PTCs) (Miller and Pearce, 2014). The most common human genetic disease is cancer, where the appearance of a PTC in a tumor suppressor gene results in the loss of the protein or the synthesis of a truncated protein unable to inhibit cell proliferation or to promote apoptosis. The known examples are mutations in p53 and APC suppressor genes present in 50% of human cancers. The first application of G418 in disease-causing nonsense mutation was in 1985 (Nudelmann et al., 2010). Aminoglycosides (such as geneticin and gentamicin) induce the readthrough of PTCs by binding to ribosomes, which restores the synthesis of a full-length functional protein in the cultured in vitro mammalian cells

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Anna Sobiepanek, Patrycja D. Kowalska, Marta Soszyńska, Aneta Ścieżyńska and in vivo animal models (Floquet et al., 2011; Bidou et al., 2017). An example is the human cancer cell line containing a PTC (the non-small-cell lung cancer cell line, H1299; p53-null), for which high levels of the readthrough were obtained in the presence of G418 during the in vitro study. Also, the viability of cancer cells with the nonsense-mutated p53 gene was significantly decreased after the aminoglycoside treatment (Floquet et al. 2011). About 12% of human genetic disorders involve the PTCs (Kuschal et al., 2013), where various aminoglycosides have presented a therapeutic potential for the treatment of cystic fibrosis, Duchenne muscular dystrophy, dystrophic epidermolysis bullosa, the Werner syndrome as well as the Hurler syndrome (Heier and DiDonato, 2009; Prokhorova et al., 2017). Recent development also emphasizes the use of translational read-through inducing drugs as a strategy for treating nonsense mutation-based on retinal disorders (Nagel-Wolfrum et al., 2014; Samanta et al., 2019). Another therapeutical focus of G418 is on the spinal muscular atrophy (SMA) disease, where it demonstrated an ability to induce the readthrough of the SMN target and to increase the SMN protein level. Moreover, geneticin in the dose of 14 mg/kg administered by the intraperitoneal injection into a SMA mouse increased its motor functions during the in vivo experiment (Heier and DiDonato, 2009). In general, this antibiotic and the derivatives of aminoglycoside antibiotics offer a rational basis for developing new personalized strategies of treatment for various diseases.

SHORT CONCLUSION Undeniably, geneticin is a very useful antibiotic with a wide range of application possibilities. For cell biology it allows to eliminate the slowly dividing cells from the culture. In melanocyte-fibroblast coculture the concentration of 100 µg/ml of G418 applied for 2 days can help in receiving a pure culture of normal melanocytes. In case of molecular biology, it helps in selecting only those cells which underwent transfection. The applied concentration of geneticin has to be determined experimentally by using the cells of interest exposed to the treatment of broad G418 concentrations in an appropriate medium and via the MTT assay. The selected drug concentration should be approximately 25-50% higher than the minimal concentration required to kill all the cells within 7-14 days after transection. Concerning the medical field geneticin can be used against various diseases but when taking proper safety precautions. So far, it has been utilized as an antiparasitic agent, neuroprotective compound in the (H-I) brain injury or it has been used to treat patients with genetic disorders caused by nonsense or frameshift mutations.

ACKNOWLEDGEMENT This work was supported by the Warsaw University of Technology, the Medical University of Warsaw and the National Science Centre (Poland) – grants no. 2016/23/N/NZ5/02588 and 2017/27/N/ST4/01389.

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ORCID Anna Sobiepanek https://orcid.org/0000-0003-3186-7582 Patrycja D. Kowalska https://orcid.org/0000-0003-0635-7214 Marta Soszyńska Aneta Ścieżyńska https://orcid.org/0000-0003-0438-2678 REFERENCES Alexaki V.-I., Javelaud D., Van Kempen L.C.L., Mohammad K.S., Dennler S., Luciani F., Hoek K.S., Juàrez P., Goydos J.S., Fournier P.J., Sibon C., Bertolotto C., Verrecchia F., Saule S., Delmas V., Ballotti R., Larue L., Saiag P., Guise T.A., Mauviel A., GLI2-Mediated Melanoma Invasion and Metastasis, JNCI: Journal of the National Cancer Institute 102, 2010, 1148-1159. Amna T., Park K.M., Cho I.-K., Choi T.J., Lee S.S., Seo K.-S., Hwang I., Substantial Effect of Melanin Influencing Factors on In vitro Melanogenesis in Muzzle Melanocytes of Differently Colored Hanwoo, Asian Australas. J. Anim. Sci 25, 2012, 1029-1037. Armstrong C.A., Murray N., Kennedy M., Koppula S.V., Tara D., Ansel J.C., Melanoma-Derived Interleukin 6 Inhibits In Vivo Melanoma Growth. Journal of Investigative Dermatology 102, 1994, 278-284. Aubrecht J., Goad M.E.P., Czopik A.K., Lerner C.P., Johnson K.A., Simpson E.M., Schiestl R.H., A high G418-resistant neo R transgenic mouse and mouse embryonic fibroblast (MEF) feeder layers for cytotoxicity and gene targeting in vivo and in vitro, Drug and Chemical Toxicology 34, 2011, 433-439. Belsham D.D., Cai F., Cui H., Smukler S.R., Salapatek A.M.F., Shkreta L., Generation of a Phenotypic Array of Hypothalamic Neuronal Cell Models to Study Complex Neuroendocrine Disorders, Endocrinology 145, 2004, 393-400. Bidou L., Bugaud O., Belakhov V., Baasov T., Namy O., Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells, RNA Biology 14, 2017, 378-388. Boukamp P., Stanbridge E.J., Foo D.Y., Cerutti P.A., Fusenig N.E., c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy, Cancer Res. 50, 1990, 2840-2847. Chapman S.W.K., Metzger N., Grest P., Feige K., von Rechenberg B., Auer J.A., Hottiger M.O., Isolation, establishment, and characterization of ex vivo equine melanoma cell cultures, In Vitro Cell.Dev.Biol.- Animal 45, 2009, 152-162. Davies J., Jimenez A., A New Selective Agent for Eukaryotic Cloning Vectors, The American Journal of Tropical Medicine and Hygiene 29, 1980, 1089-1092. De Luca M., D’Anna F., Bondanza S., Franzi A.T., Cancedda R., Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis, The Journal of Cell Biology 107, 1988, 1919-1926. Di Palma T., Filippone M.G., Pierantoni G.M., Fusco A., Soddu S., Zannini M., Pax8 has a critical role in epithelial cell survival and proliferation, Cell Death Dis 4, 2013, e729-e729. Floquet C., Deforges J., Rousset J.-P., Bidou L., Rescue of non-sense mutated p53 tumor suppressor gene by aminoglycosides, Nucleic Acids Research 39, 2011, 3350-3362. Fountain J.W., Lockwood W.K., Collins F.S., Transfection of primary human skin fibroblasts by electroporation, Gene 68, 1988, 167-172. Gledhill K., Guo Z., Umegaki-Arao N., Higgins C.A., Itoh M., Christiano A.M., Melanin Transfer in Human 3D Skin Equivalents Generated Exclusively from Induced Pluripotent Stem Cells, PLoS ONE 10, 2015, e0136713. Halaban R., Culture of Melanocytes from Normal, Benign and Malignant Lesions, in: Pfragner R., Freshney R.I. (Eds.), Culture of Human Tumor Cells, John Wiley & Sons, Inc., Hoboken, NJ, USA, 2005, 289-318.

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Cardiac fibrosis, its causes and consequences

Mateusz Bartkowiak1,2, Krzysztof Bartkowiak2, Ewa Jankowska-Steifer*3 1 Department of History of Medicine, Medical University of Warsaw 2 Students Science Association HESA – Department of Histology and Embryology, Medical University of Warsaw 3 Department of Histology and Embryology, Medical University of Warsaw *Corresponding author: Ewa Jankowska-Steifer, Department of Histology and Embryology, Medical University of Warsaw, 5 Chałubińskiego str., 02-004 Warsaw, e-mail: [email protected] ABSTRACT Cardiac fibrosis (CF) is a condition characterized by changes in the heart collagen network. It can be caused by different pathological states such as cardiac ischemia, hypertension, heart failure, systemic diseases. These conditions contribute to over the 30% of worldwide deaths. Numerous cell types are involved in CF. However, the main role in the production of the extracellular matrix in CF is played by fibroblasts and myofibroblasts, whose functions are regulated by transforming growth factor β (TGF-β), interleukin-10 (IL-10), rennin-angiotensin-aldosterone (RAA) pathway and numerous miRs, especially miR-26a. Other types of cells, such as macrophages, vascular endothelial cells or pericytes also have an impact on CF. All of them play a regulatory role in process of CF. Cardiac fibrosis may contribute to the development of heart dysfunction, arrhythmias or to the progression of heart failure. It can be assessed by biopsy, blood biomarkers evaluation or imaging tests. Keywords: cardiac fibrosis, myofibroblasts, miR-26a INTRODUCTION Fibrotic diseases cause more than 800 000 deaths annually in worldwide and one of the most common condition connected with fibrosis is CF. Cardiac fibrosis (CF) is a pathology characterized by changes in the heart collagen network, in which an inappropriate balance of extracellular matrix (ECM) production and degradation occurs. It is caused by different pathological states such as cardiac ischemia, hypertension, heart failure, systemic diseases and others. These pathological condition contribute to more than 30% deaths in worldwide. Cardiac fibrosis may contribute to the development of heart dysfunction, arrhythmias or the progression of heart failure (Hinderer and Schenke-Layland, 2019). Fibrosis in the heart can by classified into different types, depending on its cause and pathoanatomy. Reactive fibrosis (RF) is caused by inappropriate and long-term stimulation by the inflammatory cells, and/or such factors as paracrine signals or oxidative stress. In this type of fibrosis, the increase in collagen synthesis without large, widespread effects on the cardiac cells viability occurs. Collagen deposits are observed interstitially or perivascularly. Interstitial fibrosis is linked with a diffused spread of the extracellular collagen network without cardiomyocytes’ necrosis, and it is also called diffuse fibrosis. If the increase of collagen network in the ECM can be observed near cardiac micro- vasculature, then this type of fibrosis is called perivascular. The pathological changes

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Cardiac fibrosis, its causes and consequences in RF can probably be reversible if appropriate treatment were to be administered. Reactive fibrosis is characteristic especially in the elderly and in some clinical conditions including aortic stenosis, cardiomyopathies, coronary heart disease without infarction, and heart failure. Multiple conditions, such as endothelial and coronary microvascular dysfunctions, age-dependent pathologies like hypertension, diabetes, autoimmune rheumatic disease as well as kidney diseases can also contribute to the development of RF (Epelman et al., 2014; Frangogiannis, 2019; Tian et al., 2017). Replacement fibrosis also known as scar fibrosis or reparative fibrosis is a type of fibrosis which occurs in places where cardiomyocytes are removed after necrosis. Replacement fibrosis is probably irreversible, and as suggested by some Authors, prevents muscle rupture after myocardial infarction. Reactive and replacement fibroses can coexist in numerous patients (de Boer et al., 2019; Epelman et al., 2014). Infiltrative interstitial fibrosis (IIF) is a third type of fibrosis. It has been seen in such situations as amyloidosis or Anderson-Fabry disease. These conditions are characterized by a progressive deposition of insoluble proteins or glycosphingolipids in the heart which contributes to an induction of fibrosis. This type of fibrosis also occurs in the right ventricle of systemic sclerosis-associated pulmonary arterial hypertension, where inflammatory cells infiltrate the heart and promote fibrosis (Tian et al., 2017). Furthermore, some types of CF such as endomyocardial fibrosis (EF), also called Davies disease, occur endemically. This type of fibrosis is characterized by fibrotic thickening of the endocardium and myocardium in the left and right ventricle. It is an important cause of heart failure, occurring in tropical and subtropical regions of developing countries. The etiology and pathogenesis of this type of fibrosis is still unknown. However, pathological analysis shows that fibrosis extends from the apex to the mitral valve and outflow tract from the left ventricle. It can be also observed in papillary muscles and chordae tendineae. Microscopically, deposits of a dense fibrous tissue in the subendocardium with superimposed thrombosis and advanced stages of the calcification are observed. The treatment of EF is only symptomatic. The prognosis in Davies disease is poor; one-third to half of patients with this disease in the advanced stage die within two years (Duraes et al., 2019; Frangogiannis, 2019). Heart failure and the fibrosis Cardiac interstitial fibrosis is one of the frequent findings in patients with heart failure (HF). This type of fibrosis contributes to left ventricular dysfunction and consequently to the development of a HF with preserved or reduced ejection fraction. The increased production of collagen fibers in the ECM reduces the diastolic function of the heart. An increased fibrosis near blood vessels in the heart also reduces coronary flow. Pathology in interstitial fibrosis depends on the etiology of the HF. For example, in a pressure- overloaded related HF, the production of type I collagen is higher than that of type III collagen. These changes contribute to increased myocardial stiffness. Cardiac fibroblasts respond to mechanical changes in the environment through specific adhesion proteins and mechanosensitive signaling pathway. Increased myocardial stiffness in HF contributes to the induction of active myofibroblasts differentiation. These cells produce proteins involved in the development and regulation of fibrosis. Increased stiffness

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Mateusz Bartkowiak, Krzysztof Bartkowiak, Ewa Jankowska-Steifer of the myocardial environment also contributes to the inappropriate contraction and relaxation of cardiomyocytes which may lead to a hypertrophy of these cells. In some clinical situations, interstitial fibrosis may impact the outcome in patients with HF, for example by increasing mortality after invasive procedures in some cardiac events or aortic valve replacement (Duraes et al., 2019; Gonzales et al., 2018; Hinderer and Schenke-Layland, 2019). FIBROBLASTS AND MYOFIBROBLASTS AS A MAIN CELL TYPES INVOLVED IN THE CARDIAC FIBROSIS Fibroblasts form one of the largest stromal cells population in the heart. Their crucial function is to produce and to control the ECM turnover and to maintenance of tissue structure. Based only on the expression of the single cell surface antigen, identification of these cells is problematic since the unique, fibroblast-specific marker is not available. Markers of fibroblasts (like fibroblast specific protein, FSP-1) are also expressed in other cell types (e.g. vascular cells or macrophages). The identification of cardiac fibroblasts often relies on their morphological features, function and typical localization in the heart tissue (Kong et al., 2013). Cardiac fibroblasts can undergo a transdifferentiation process, which results in the formation of myofibroblasts. These cells are present in the heart in normal and pathological conditions (e.g. myocarditis, alcoholic cardiomyopathy, myocardial infarction and pressure or volume overload). They produce ECM proteins, like fibroblasts. α-smooth muscle actin (α-SMA) is expressed in these cells, and it serves as a useful marker. In the early stages of transdifferentiation of fibroblasts into myofibroblasts α-SMA is not produced. Therefore, at these stages of differentiation such cells are referred as proto-myofibroblasts (Hinz, 2010). Recent studies suggest that the population of myofibroblasts comes from cardiac resident fibroblasts, but some investigators propose that these cells have endothelial, or hematogenic origin. Moreover, other authors postulate that myofibroblasts can also differentiate from pericytes. The origin of myofibroblasts in cardiac tissue depends on a mechanism leading to the heart dysfunction. In the infarcted state, cardiomyocytes die. However, since cardiac resident fibroblasts are less susceptible to the lack of oxygen and nutrition supply, they can survive. If the ischemia of the heart tissue is prolonged, fibroblasts also die, and myofibroblasts in the ischemic region come mainly from neighboring interstitial cells of non-infarcted areas. In myocarditis, many cytokines which have a chemoactive function and recruit circulating fibroblast progenitors are produced (Frangogiannis, 2019). Activation of the myofibroblast population formation depends on many cytokines and other proteins present in the ECM. TGF-β and IL-10 which are secreted by macrophages, mast cells and infiltrating lymphocytes play a crucial role in this process. Moreover, stimulation can also come from cardiomyocytes or vascular cells in the injured area. The transdifferentiation of cardiac resident fibroblasts into myofibroblasts can be also mediated by the RAA pathway (van den Borne, 2009).

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Therefore, cardiac resident fibroblasts are a crucial cell population, which can be activated in many pathways in CF. Pharmacological therapies focused on the inhibition of these mechanisms are often useful in the limitation of CF (Frangogiannis, 2019). ROLE OF MACROPHAGES IN CARDIAC FIBROSIS Immune cells, such as macrophages, are also involved in the regulation of CF. In normal condition, a small population of resident macrophages is present in the heart. They play a role in the regulation of cardiac homeostasis (e.g. facilitating atrio – ventricular conduction). These macrophages are derived from embryonic yolk sac (Epelman et al., 2014). In pathological states, cardiac macrophages are enriched by a population of monocyte-derived macrophages. Macrophages play various roles in the injured heart, including the regulation of inflammation, fibrosis, remodeling of the ECM, regeneration or angiogenesis. Activated macrophages can regulate the fibrosis process, since they are the main producers of the profibrotic cytokines and growth factors such as TGF-β, platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), fibroblast growth factors (FGFs) as well as proteases and other ECM proteins (Frangogiannis, 2019). Fibrogenic phenotype in cardiac macrophages is stimulated in a various ways. Firstly, in infarcted hearts, the damage-associated molecular patterns (DAMPs) activate the toll-like receptor (TLR) signaling pathway that leads to an inflammatory reaction which contributes to the activation of fibroblasts (Wang et al., 2017). Secondly, such molecules as angiotensin II and aldosterone can stimulate macrophages to produce profibrotic factors. Moreover, pro-inflammatory cytokines like IL-1, tumor necrosis factor-α (TNF-α) can promote genes transcription of the TGF-β superfamily members, and by this activate the fibroblast and fibrosis process (Huynh et al., 2002). ROLE OF VASCULAR ENDOTHELIAL CELLS IN CARDIAC FIBROSIS Vascular endothelial cells (VECs) contribute to the activation of different cells to pro- or anti-fibrotic pathways in various ways. It has been demonstrated that in infarcted and pressure-overloaded hearts, VECs produce adhesion molecules, such as inter- cellular adhesion molecule 1 (ICAM-1), which are able to stimulate macrophages and inflammatory cells to the production of factors leading to fibrosis. Furthermore, VECs themselves are one of the main producers of pro-fibrotic mediators like TGF-β1, FGFs, or endothelin (ET)-1. Vascular endothelial cells secrete pro-inflammatory cytokines and chemokines which also contribute to the development of the leukocytes’ profibrotic response (Adiarto et al., 2012; Frangogiannis, 2019). In addition, endothelial cells are able to undergo an endothelial to mesenchymal transition by means of an activated canonical Wnt pathway. Through this phenomenon, they contribute to an increase in the number of cardiac fibroblasts, which are engaged in fibrosis, scar formation, and neovascularization (Aisagbonhi et al., 2011). Vascular endothelial cells also function as negative regulators of CF deactivating of the profibrotic cardiac fibroblasts. Hypoxia-inducible factor-1 (HIF1) produced by VECs suppresses the TGF-β pathway and reduces fibrosis (Wei et al., 2013). It was also shown, that VECs are able to produce the CXC chemokine, Interferon-γ-inducible protein 10 (IP)-10/CXCL10, which inhibits the migration of fibroblasts in the infarcted myocardium. Therefore VECs regulate the pro- and anti-fibrotic pathways in various ways (Frangogiannis, 2019).

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ROLE OF THE PERICYTES IN CARDIAC FIBROSIS Pericytes constitute 5% of the total non-myocyte population in the heart. The cardiac pericytes are derived from mesenchymal angioblasts. Pericytes sit on abluminal side of the capillaries, or as adventitial pericytes in arterioles or venules. Pericytes are involved in numerous functions such as perfusion, vascular integrity, angiogenesis, inflammation, regeneration as well as fibrosis. In pathological conditions they can secrete inflammatory mediators and fibroblast-activating growth factors. Pericytes are able to separate from the VECs, migrate and transdifferentiate into myofibroblasts-like cells. Therefore, they enrich the population of active myofibroblasts, which produce components of the ECM. It was shown that inhibition of the proliferation of pericytes and their contribution to the ECM causes a reduction of fibrosis and facilitates healing (Alex and Frangogiannis, 2019; Frangogiannis, 2019). ROLE OF MIRNAS IN CARDIAC FIBROSIS MicroRNAs (miRNAs; miRs) are small, non-coding RNAs, which negatively regulate gene expression by translation inhibition or mRNA degradation. It was shown that some miRNAs can regulate different pathways involved in the fibrosis, playing pro- or anti-fibrotic role (Vegter et al., 2016). Many studies have been conducted, concerning the role of miR-21, miRs-29 family and miR-26a on CF. miR-21 has a critical pro-fibrotic effect. Its overexpression leads to increased CF caused by the modulation of endothelial to mesenchymal transition (EMT), the process which is regulated by TGF- . MiR-21 regulates the TGF- receptor and TGF- pathway. It has been shown that miR-21 promotes fibrotic lung diseases by targeting Smad7 (an inhibitory Smad) to enhance the TGF-β signaling pathway (Kang, 2017). The activity of certain other miRNAs leads to the inhibition of fibrosis. The miR-29 family is involved in regulation of fibrosis by control of such proteins as fibrillin, elastin or collagens as well as TGF- /Smad3 signaling pathway. In vitro overexpression of miR-29 contributes to the reduction of CF, and knockdown of miR-29 causes the opposite effect. miR-26a also has an anti-fibrotic effect. It has been shown that fibrosis of different organs (e.g. heart, lung, liver and kidney) is accompanied by a decreased expression of this miR. Contrary, its overexpression depresses CF and insulin resistance (Wang et al., 2019). The targets of miR-26a are, among others, GSK-3β (glycogen synthase kinase 3), PTEN (phosphatase and tensin homolog), CTGF (connective tissue growth factor – a major TGF-β1 signaling effector), collagen I, and FoxO1 (Forkhead box protein O1) (tab. 1) (Xin et al., 2018). Most of the information about the inhibitory role of miR-26a comes from studies on idiopathic pulmonary fibrosis (IPF). miR-26a directly targets Smad4 and by this inhibits the nuclear translocation of p-Smad3, and further represses TGF-β1-induced fibrogenesis (Liang et al., 2014). In IPF miR-26a also targets HMGA2 (high-mobility group protein A2), a critical factor required for TGF-β1-induced EMT (Liang et al., 2014). In CF, miR-26a affects type I collagen and CTGF expression (Wei et al. 2013). In myocardial infarction, miR-26a targets the ataxia-telangiectasia mutated (ATM) protecting the heart against cardiac apoptosis and reducing expression of type I collagen and CTGF (Chiang et al., 2020). It has also been shown that miR-26a regulates cardiac fibroblasts collagen gene expression by targeting an unc-51 like autophagy

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Cardiac fibrosis, its causes and consequences activating kinase 1 (ULK1) – a key component in autophagy pathway (Zheng et al., 2018). Forkhead box protein O1 (FoxO1) is another important factor that contributes to CF. Its activation is regulated by TGF-β1 and plays a critical role in cardiac fibroblast differentiation into cardiac myofibroblasts. FoxO1 down-regulation by miR-26a decreases the cytostatic effects of TGF-β1 in cardiac fibroblasts (Vivar et al., 2016). DIAGNOSIS AND MEASUREMENT OF CARDIAC FIBROSIS The gold standard of diagnosing and measuring CF in humans is to take the tissue sample during cardiac myectomy, open heart surgery or catheter-based endocardial biopsy. These procedures have a lot of limitations including non-representative sampling, only local and small tissue fragments assessment, and they are invasive (de Boer et al., 2019; Espeland et al., 2018). It was shown that the levels of some blood biomarkers reflect cellular and molecular changes related to the quantity of fibrotic tissue. Such biomarkers as procollagen type I and procollagen type III N-terminal propeptides, or glycoproteins that modulate the collagen production or maturation (e.g. periostin, mimecan, monocyte chemoattractant protein-1, as well as galectin-3) are used for assessment of the fibrosis level. Several matrix metalloproteinases (MMP-3, MMP-9, MMP-11 and MMP-12) or their tissue inhibitors (TIMP-1, TIMP-3) can be also used as markers of fibrosis (de Boer et al., 2019; Espeland et al., 2018). OTHER BIOMARKERS OF CF ARE MICRORNAS Cardiac fibrosis can be also assessed by visualization techniques such as echocardiography with a specific mode which evaluates tissue velocity and global longitudinal systolic strain. However, the best way to visualize fibrosis in the heart is magnetic resonance imaging (MRI). In this technique it is possible to evaluate the scar tissue, focal reparative fibrosis, interstitial edema, protein degradation and aggregation, lipid accumulation as well as other parameters. This technique is more accurate and reproducible than the echocardiography (de Boer et al., 2019; Espeland et al., 2018). CONCLUSION Despite the current knowledge on the mechanisms of cardiac fibrosis, we still cannot counteract this phenomenon. Careful understanding of the signaling pathways between the cells involved in fibrosis, including the mechanisms of regulation through miRNA- mediated RNA interference, may improve diagnostic methods and enable potential antifibrotic treatments.

Table 1. Anti-fibrotic activity of miR-26a TARGETS TYPE OF FIBROSIS/ CELL REFERENCES

Smad4 Pulmonary fibrosis (Liang et al., 2014; Zhang et Hypertensive myocardial al., 2020) fibrosis

CTGF Pulmonary fibrosis (Koga et al., 2015; Wei et al., Hypertensive myocardial 2013; Zhang et al., 2020) fibrosis

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HMGA2 Pulmonary fibrosis (Liang et al., 2014)

ULK1 Cultured primary cardiac (Zheng et al., 2018) fibroblasts

FoxO1 Uremic cardiomyopathy (Wang et al., 2019)

PTEN Myocardial fibrosis (Zhang et al., 2018)

ATM Myocardial infartion (Chaing et al., 2020)

ACKNOWLEDGMENT This paper was funded by the ERA-CVD Research Program which is a translational R&D program jointly funded by national funding organizations within the framework of the ERA-NET ERA-CVD. Funding agency: NCBR, Poland, grant number: ERA- CVD/LyMitDis/1/2017. REFERENCES Adiarto S., Heiden S., Vignon-Zellweger N., Nakayama K., Yagi K., Yanagisawa M. et al., ET-1 from endothelial cells is required for complete angiotensin II-induced cardiac fibrosis and hypertrophy, In: Life sciences, 2012; 91(13-14): 651-657. Aisagbonhi O., Rai M., Ryzhov S., Atria N., Feoktistov I..Hatzopoulos A.K., Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition, In: Disease models & mechanisms, 2011; 4(4): 469. Alex L., Frangogiannis N.G., Pericytes in the infarcted heart, In: Vascular Biology, 2019; 1(1): H23. Chiang M.-H., Liang C.-J., Lin L.-C., Yang Y.-F., Huang C.-C., Chen Y.-H. et al., miR-26a attenuates cardiac apoptosis and fibrosis by targeting ataxia – telangiectasia mutated in myocardial infarction, In: 2020; 235(9): 6085-6102. de Boer R.A., De Keulenaer G., Bauersachs J., Brutsaert D., Cleland J.G., Diez J. et al., Towards better definition, quantification and treatment of fibrosis in heart failure. A scientific roadmap by the Committee of Translational Research of the Heart Failure Association (HFA) of the European Society of Cardiology, In: Eur J Heart Fail, 2019; 21(3): 272-285. Duraes A.R., de Souza Lima Bitar Y., Roever L., Neto M.G., Endomyocardial fibrosis: past, present, and future, In: Heart failure reviews, 2019. Epelman S., Lavine K.J., Beaudin A.E., Sojka D.K., Carrero J.A., Calderon B. et al., Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation, In: Immunity, 2014; 40(1): 91-104. Espeland T., Lunde I.G., B H.A., Gullestad L., Aakhus S., Myocardial fibrosis, In: Tidsskr Nor Laegeforen. 2018; 138(16). Frangogiannis N.G., Cardiac fibrosis: Cell biological mechanisms, molecular pathways and therapeutic opportunities, In: Molecular aspects of medicine, 2019; 65, 70-99. Gonzalez A., Schelbert E.B., Diez J..Butler J., Myocardial interstitial fibrosis in heart failure: biological and translational perspectives, In: J Am Coll Cardiol. 2018; 71(15): 1696-1706. Hinderer S., Schenke-Layland K., Cardiac fibrosis – A short review of causes and therapeutic strategies, In: Advanced Drug Delivery Reviews, 2019; 146, 77-82. Hinz B., The myofibroblast: paradigm for a mechanically active cell, In: Journal of biomechanics. 2010; 43(1): 146-155.

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Huynh M.L., Fadok V.A..Henson P.M., Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-beta1 secretion and the resolution of inflammation, In: J Clin Invest. 2002; 109(1): 41-50. Kang H., Role of microRNAs in TGF-β signaling pathway-mediated pulmonary fibrosis, In: International journal of molecular sciences. 2017; 18(12). Koga K., Yokoi H., Mori K., Kasahara M., Kuwabara T., Imamaki H. et al., MicroRNA-26a inhibits TGF-β-induced extracellular matrix protein expression in podocytes by targeting CTGF and is downregulated in diabetic nephropathy, In: Diabetologia. 2015; 58(9): 2169-2180. Kong P., Christia P., Saxena A., Su Y., Frangogiannis N.G., Lack of specificity of fibroblast-specific protein 1 in cardiac remodeling and fibrosis, In: 2013; 305(9): H1363-H1372. Liang H., Gu Y., Li T., Zhang Y., Huangfu L., Hu M. et al., Integrated analyses identify the involvement of microRNA-26a in epithelial-mesenchymal transition during idiopathic pulmonary fibrosis, In: Cell Death Dis. 2014; 5(5): e1238-e1238. Liang H., Xu C., Pan Z., Zhang Y., Xu Z., Chen Y. et al., The antifibrotic effects and mechanisms of microRNA-26a action in idiopathic pulmonary fibrosis, In: Mol Ther. 2014; 22(6): 1122-1133. Tian J., An X., Niu L., Myocardial fibrosis in congenital and pediatric heart disease, In: Exp Ther Med. 2017; 13(5): 1660-1664. van den Borne S.W., Isobe S., Zandbergen H.R., Li P., Petrov A., Wong N.D. et al., Molecular imaging for efficacy of pharmacologic intervention in myocardial remodeling, In: JACC. Cardiovascular imaging. 2009; 2(2): 187-198. Vegter E.L., van der Meer P., de Windt L.J., Pinto Y.M..Voors A.A., MicroRNAs in heart failure: from biomarker to target for therapy, In: Eur J Heart Fail. 2016; 18(5): 457-468. Vivar R., Humeres C., Muñoz C., Boza P., Bolivar S., Tapia F. et al., FoxO1 mediates TGF-beta1- dependent cardiac myofibroblast differentiation, In: Biochimica et Biophysica Acta (BBA) – Molecular Cell Research. 2016; 1863(1): 128-138. Wang B., Zhang A., Wang H., Klein J. D., Tan L., Wang Z.-M. et al., miR-26a limits muscle wasting and cardiac fibrosis through exosome-mediated microRNA transfer in chronic kidney disease, In: Theranostics. 2019; 9(7): 1864-1877. Wang J.-W., Fontes M.S.C., Wang X., Chong S.Y., Kessler E.L., Zhang Y.-N. et al., Leukocytic toll-like receptor 2 deficiency preserves cardiac function and reduces fibrosis in sustained pressure overload, In: Sci Rep. 2017; 7(1): 9193. Wei C., Kim I.-K., Kumar S., Jayasinghe S., Hong N., Castoldi G. et al., NF-κB mediated miR-26a regulation in cardiac fibrosis, In: Journal of Cellular Physiology, 2013; 228(7): 1433-1442. Wei H., Bedja D., Koitabashi N., Xing D., Chen J., Fox-Talbot K. et al., Endothelial expression of hypoxia-inducible factor 1 protects the murine heart and aorta from pressure overload by suppression of TGF-β signaling, In: 2012; 109(14): E841-E850. Xin Z., Ma Z., Hu W., Jiang S., Yang Z., Li T. et al., FOXO1/3: Potential suppressors of fibrosis, In: Ageing Research Reviews, 2018; 41(42-52. Zhang S..Cui R., The targeted regulation of miR-26a on PTEN-PI3K/AKT signaling pathway in myocardial fibrosis after myocardial infarction, In: European review for medical and pharmacological sciences, 2018; 22(2): 523-531. Zhang W., Wang Q., Feng Y., Chen X., Yang L., Xu M. et al., MicroRNA‐26a protects the heart against hypertension‐induced myocardial fibrosis, In: 2020; 9(18): e017970. Zheng L., Lin S., Lv C., MiR-26a-5p regulates cardiac fibroblasts collagen expression by targeting ULK1, In: Sci Rep. 2018; 8(1): 2104.

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Review: Role of pregnancy in eye functioning

Małgorzata Kowalik-Jagodzińska*¹, Adam Jagodziński² ¹ Department and Clinic of Ophthalmology, Wroclaw Medical University ² 1st Department and Clinic of Gynaecology and Obstetrics, Wroclaw Medical University *Corresponding author: Małgorzata Kowalik-Jagodzińska, Department and Clinic of Ophthalmology, Wroclaw Medical University, e-mail: [email protected] ABSTRACT Pregnancy causes many changes in all systems of the woman's body. The visual system is also affected. A wide range of functional and structural changes can occur. They are physiological, transient and usually do not indicate disease. To begin with, arising level of sex hormones leads to changes in body water metabolism. Fluid retention causes corneal edema what generates the corneal refractive index changes. Another sign of this phenomenon is an arrising hydration of the lens and a significant increase in choroid thickness observed during pregnancy compared to nonpregnant women. Furthermore, gestation can lead to poorer lacrimation. Changes in intraocular pressure may be a specific adjustment for natural delivery, preventing too high rise of this parameter during second phase of labour. Maternal diseases complicating pregnancy may induce visual sight-threatening effects. Sometimes preexisting ocular disorders can worsen during pregnancy. Diseases affecting a visual organ may manifest in cases of pre/gestational diabetes mellitus, chronic/pregnancy induced hypertention, coagulation disorders as soon as obstetric emergencies. Moreover visual symptoms can have extraocular background such as vascular events. On the other hand pregnancy may cause remission of some inflammatory or autoimmuno- logic ocular disorders such as uveitis and sclerosis multiplex. The aim of the review is to summarise actual knowledge about pregnancy reflected in ophthalmology. INTRODUCTION Pregnancy leads to plenty of changes in body functioning. All of them have hormonal background as metabolic adjustment for whole period of pregnancy and for natural delivery. Some specific visual symptoms may occur for that reason it is essential to distinguish physiological reactions from pathologies. Changes that take place during physiological pregnancy prepare the mother for labour and delivery. Rising level of oestradiol modulate the synthesis of nitric oxide and leads to peripheral vasodilatation in early pregnancy. Observed fall in systemic vascular resistance generate an increase in cardiac output due to compensation mechanism. In the end of second trimester it reaches the highest level about 40%. Renal sodium and water retention as a result of a renin-angiotensin-aldosterone system activation are characteristic for pregnancy. The increase in extracellular and also plasma volume is necessary to keep uteroplacental perfusion on a sufficient level for a developing fetus. Human chorionic gonadotropin produced during pregnancy by a trophoblast has a role in upregulation of a vasopressin secretion and leads to decreased plasma osmolarity level. Body water metabolism changes may generate refractive error fluctuances, differences in corneal hydration, anterior chamber plasticity modivication, lens abnormalities and dry eye syndrome. The article is focused mainly on physiological pregnancy with following natural consequences noticed in the eye or visual anomalies independent from eye functioning.

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Furthermore, some ocular emergencies typical for obstetric complications and preexisting ocular diseases deteriorating during pregnancy are mentioned. The aim of the review is to summarise actual knowledge about pregnancy reflected in ophthalmology. DRY EYE SYNDROME Tear film consists of three main layers: aqueous, mucin and phospholipid layer. Aqueous layer component is produced mainly by lacrimal gland and by accessorial glands. Its role is to moisturize the surface of the eye, wash out wastes and bacteria. In addition it consists of nutritional elements for cornea. Mucin is secreted by goblet cells situated in conjunctiva and it is responsible for stabilization of the aqueous against hydrophobic epithelium. Last but not least phase is phospholipd one which is secreted by Meibomian glands. This phase heightens vaporising temperature of tears to prolonged corneal and conjuctival moisturisation and keeps higher surface tension. Any disturbances in balance between tear film phases may lead to specific uncomfortable symptoms such as itching, irritation, foreign body sensation, blurred vision or increased tearing after exposition, for example to wind. Moreover this syndrome is connected with higher susceptibility to infections. During pregnancy tear film physiology is affected. It is said that arising levels of prolactin, epidermal growth factor, transforming growth factor beta-1 lead to acinar cells' destruction and following reduction in water phase secretion (Yenerel and Küçümen, 2015)⁠. Dryness is exacerbated by dehydratation that is why fluid intake should be at least 2,5 liters daily. Symptoms are troublesome especially in third trimester but in cases with tiring nausea and vomiting they may be noticeable even during first weeks of pregnancy. Hormonal changes may also affect oil phase production because of their influence on Meibomian glands function. As a result an instability of tear film is observed. Performing Schirmer test and break-up-time test among pregnant women reveals significant decrease of values during third trimester in comparison to second trimester and post-partum period (Nkiru Z et al., 2019)⁠. Although tear production during pregnancy decreases, it was detected that tear osmolarity also diminishes. This correlation may be protective for eye surface (Duran and Güngör, 2019)⁠. INTRAOCULAR PRESSURE Intraocular pressure (IOP) is dependent on several factors such as aqueous production, outflow and anatomy of the irydocorneal angle. The fluid is secreted by ciliary body and is responsible to keep correct tension and shape of the eye bulb. Furthermore this medium provides nutritional substances to non-vascularized structures such as lens and cornea. Trabecular route is the main route of fluid outflow and circa 30% of aqueous humour leaves the eye by uveoscleral route. Conventional route is dependent on pressure in episcleral veins. Decreased IOP among pregnant women is detected. Medium drop of IOP hesitates between 10-25% and is higher in women with preexisting ocular hypertension (Yenerel and Küçümen, 2015; Chawla et al., 2013)⁠. According to the course of pregnancy it is significant mostly between 12th and 18th week of gestation and lasts for several months

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Małgorzata Kowalik-Jagodzińska, Adam Jagodziński after delivery. Some mechanisms are suspected to be responsible for these fluctuances. Increased uveoscleral outflow has possibly the major meaning. Due to developing fetus and hormone changes during pregnancy vein pressure in the lower part of the body is higher than in the upper part. As a result episcleral venous pressure is decreased what facilitates uveoscleral outflow. General acidosis, low scleral rigidity and corneal edema may also be favourable to IOP fall (Naderan, 2018; Efe et al., 2012)⁠. Changes in anterior chamber and irydocorneal angle measurements are also confirmed (Taradaj et al., 2018)⁠. Anterior chamber becomes deeper and it also may play a role in IOP drop but the differences between pregnant and non-pregnant women are not significant. Changes in IOP may be a specific adjustment for natural delivery preventing too high rise of this parameter during second phase of labour. REFRACTION, CORNEA AND LENS, ANTERIOR CHAMBER Physiological reactions noticed in eye structures during pregnancy may lead to different refraction in comparison to the pregestational period. Commonly simple myopia is observed (Nkiru et al., 2018)⁠. However simple myopic astigmatism, compound myopic astigmatism, simple hypermetropia and hypermetropic astigmatism are noticed. Decreased visual acuity (VA) may interfere with everyday functioning of pregnant women and sometimes could be an origin of fear. Deterioration of visual acuity is transient and refraction returns to baseline after delivery. The knowledge about physiological changes which occur in the eyes during uncomplicated pregnancy allows physicians and obstetricians distinguishing normal reactions form pathologies and ocular emergencies. The most typical causes of refraction error are changes in central corneal thickness (CCT), its curvature and lens' structure abnormalities with accomodative defects. On the basis of metaanalysis (Wang et al., 2017)⁠ it is a common phenomenon for second and third trimesters that CCT values increases. Corneal measurements as soon as IOP are affected by hormonal changes. Both of them are dependent from raising levels of progesterone, oestrogen, relaxin and β-human chorionic gonadotropin. The most significant raise in CCT value is described during second and third trimesters as a result of fluid collection. Although most of the researches reveals this tendency not all of them confirm its significance (Örnek, Özcan Dağ and Örnek, 2017)⁠. Fluid retension causes corneal edema that is why the corneal refractive index changes. Furthermore change in corneal curvature and decreased sensitivity are detected. In spite of the fact that almost all of the corneal physiologic reactions during pregnancy are temporary there are some cases of permanent corneal pathologies which can occur. For instance, if the shape of the cornea extremely changes, it could lead to keratoconus formation. All of the mentioned corneal reactions together with poor lacrimation may cause contact lens intolerance (Naderan, 2018)⁠. According to corneal anomalies the Krukenberg's spindle is commonly seen but is not accompanied by rising IOP because of physiological drop of this parameter.

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Refraction as soon as accomodation may be affected because of lens hydration differences. Lower autofluorescence of the lens during pregnancy as a proof of liquid retension was confirmed (Beneyto and Pérez, 2006)⁠ what could lead to cataract formation or exacerbation of preexisting lens opacities. In addition lens curvature also increases contributing to myopic shift. The following phenomenon connected with lens function is accomodative absolute loss or weakness which could be present even during breastfeeding period (Chawla et al., 2013)⁠. Postponing in prescribing new contact lenses or spectacles recipes is advised due to transient character of refraction change. Refractory surgery is contraindicated during pregnancy and post-partum period. CHOROIDAL THICKNESS Cardiovascular adjustment to pregnancy consists in increase in cardiac output, heart rate and blood volume, decrease in vascular resistance and stimulation of angiotensin- renin-aldosterone system. Choroidea is the most vascularised tissue in the organism that is why these phenomena could be judged during its examination. Contemporary ophthalmology allows observing thickness of the choroidea during natural course of pregnancy by using safe and reproducible methods such as optic coherent tomography. It was observed in several researches that there is significant increase in choroid thickness during pregnancy compared to its thickness among nonpregnant women (Roskal-Wałek et al., 2017; Dadaci et al., 2015)⁠. The most significant change is noticed for the first trimester and is decreasing during following trimesters. Subfoveal region is the main point of interest (Kara et al., 2014)⁠. Although the meaning of choroid thickening is still unclear and demands further investigation, it is valuable to better understand the ocular pathologies such as central serous chorioretinopathy. VISUAL FIELD DEFECTS Pregnancy causes typical visual symptoms not only due to its influence on eye physiology but also on affecting the visual route. The most common symptom is restriction of visual field presented as bitemporal defect which is a sign of arising pressure on the optic chiasm (Brewington et al., 1974)⁠. The pressure is caused by natural enlargement of pituitary gland in reaction to pregnant state and is physiologically reversible after pregnancy and breastfeeding periods. Otherwise if the defect is still detectable or is getting worse, it may be connected with hormone-dependent tumors arising or growing during pregnancy such as pituitary adenomas. EYELIDS AND CONJUNCTIVA Increased stimulation of melanocytes as a result of arising concentration of melanocyte- stimulating hormone leads to darkening of the skin especially in sun-exposed areas (Jadotte and Schwartz, 2010)⁠. Chloasma is one of the well known signs of pregnancy and may be presented on cheeks and spreads to the eyelids. In addition unilateral ptosis is usually observed due to liquid retention in levator aponeurosis. Acoording to conjunctival changes growing granularity of the vessels because of decreased blood flow rate could be presented.

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OCULAR EMERGENCIES AND PREEXISTING OCULAR DISEASES DURING PREGNANCY Ophthalmic emergencies observed during the course of pregnancy are mainly connected with complicated pregnancies. Pregnancy induced hypertension, chronic hypertension, gestational and pre-gestational diabetes mellitus, coagulation disorders as soon as obstetric emergencies like pre-eclampsia, eclampsia, disseminated intravascular coagulation may lead to vision- and life-threatening states. Moreover pregnant state could change the course of ocular inflammatory diseases, lead to ocular diseases like central chorioretinopathy and progression of preexisting tumors (pituitary adenomas, meningiomas, uveal melanomas). Diabetes mellitus type I and II are one of the most important challenges for contemporary obstetrics starting from pre-conceptional period. For ophthalmologists the proper care of pregnant women with preexisting diabetes mellitus is essential for keeping sight. Pregnant state is known as a risk factor of diabetic rethinopathy's progression (Morrison et al., 2016; Rahman et al., 2007)⁠⁠⁠. The progression is a result of higher VEGF concentration due to arising progesterone level (Swiatek-De Lange et al., 2007)⁠. The importance of the progression is dependent on a degree of retinopathy before gestation, glycemic control and comorbidities. Diabetic macular edema could also increase. On the contrary, it is non common to develope diabetic retinopathy during gestational diabetes (Yenerel and Küçümen, 2015)⁠. In addition chronic hypertension in association with diabetes may accelerate the progression. Ocular treatment possibilities are limited in relation to fetal safety. Especially safety of anty-VEGF agents is uncertain (Sullivan et al., 2014). Coagulation disorders, for instance antyphospholipid syndrome or DIC as a result of eclampsia/pre-eclamspia, placental abruption or amniotic fluid embolism may lead to ocular occlusive disorders. Purtcher-like retinopathy is a transient manifestation seen in posterior pole (Naderan, 2018).⁠ It is manifestated after labour as bilaterial vision loss with cotton-wool spots with or without retinal haemorrhage. Retinal arteries or veins occlusion is less commonly detected. The most typical complaint during eclampsia/ preeclampsia is blurred Visio (Abu Samra, 2013).⁠ Moreover symptoms such as photopsia, visual field defects or even complete blindness may occur as a consequence of cortex, retina or optic nerve affection. During examination narrowed vessels are detected so the retinopathy is similar to hypertensive one. Serous retinal detachment is not commonly observed but possible due to choroidal ischemia (Lee et al., 2019). Idiopatic chorioidopathies like choroidal neovascularisation or punctate inner chorioretinopathy was described (De Silva et al., 2016)⁠⁠⁠. Another example of sight affecting complications is Shehan's syndrome which is a result of pituitary infarction due to massive haemorrhages during delivery (Naderan, 2018)⁠. The symptoms of this syndrome are headache, vision or visual field defect, diplopia and ophthalmoplegia. As a consequence of compression on cavernous sinus ptosis, mydriasis, lateral-inferior deviation of the globe are revealed. Moreover Horner syndrome may occur by affecting sympathetic nerves.

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Quick weight gaining could lead to idiopatic intracranial hypertension with headache, nausea, vomiting, retrobulbar pain and visual abnormalities like scotomata, photopsias, diplopia and tinnitus. During examination optic edema is observed. ⁠ Pregnancy causes decreased activity of immunological system what generates usually remission of ocular inflammatory diseases and autoimmunological disorders such as uveitis or sclerosis multiplex. Contrary to remission Grave's disease typically worsen during first trimester and after delivery. On the other hand the immunosupression is connected with higher susceptibility to reactivation of latent infections. The most typical infective disease is toxoplasmosis which is sight-threatening and also dangerous for the fetus. Due to IOP drop during pregnancy preexisting glaucoma is better controlled or even improve. According to treatment, laser trabeculoplasty are recommended rather than using medications which could be teratogenic. SUMMARY During uncomplicated pregnancy changes in all body systems may be observed. They are physiological, transient and usually do not indicate disease. Due to specyfic hormonal adjustment causing eye functioning and structural differences as a result of body water metabolism modification. For instance poor lacrimation, decrease in intraocular pressure, new refractive error, corneal oedema, thickening of the choroidea could be detected. Moreover some neuroophtalmic and skin signs are commonly seen. On the contrary to physiological changes, obstetric emergencies could lead to sight- threatening complications and the course of pregnancy may deteriorate preexisting ocular diseases. REFERENCES Abu Samra K., The eye and visual system in the preeclampsia/eclampsia syndrome: What to expect?, In: Saudi Journal of Ophthalmology, Saudi J Ophthalmol, 2013; pp. 51-53, doi: 10.1016/j.sjopt.2012.04.003. Beneyto P., Pérez T.M., Study of lens autofluorescence by fluorophotometry in pregnancy. Experimental Eye Research, 2006; Academic Press, 82(4), pp. 583-587, doi: 10.1016/j.exer.2005.08.018. Brewington T.E. et al., The effect of pregnancy on the peripheral visual field, Journal of the National Medical Association, National Medical Association, 1974; 66(4), p. 330. Chawla S. et al., Ophthalmic considerations in pregnancy, Medical Journal Armed Forces India, Medical Journal Armed Forces India, 2013), pp. 278-284, doi: 10.1016/j.mjafi.2013.03.006. Dadaci Z. et al., Changes in choroidal thickness during pregnancy detected by enhanced depth imaging optical coherence tomography, British Journal of Ophthalmology. BMJ Publishing Group, 99(9), 2015; pp. 1255-1259. doi: 10.1136/bjophthalmol-2014-306343. Duran M., Güngör İ., The effect of pregnancy on tear osmolarity, Contact Lens and Anterior Eye. Elsevier B.V., 42(2), 2019; pp. 196-199, doi: 10.1016/j.clae.2018.10.007. Efe Y.K. et al., The course of corneal and intraocular pressure changes during pregnancy, Canadian journal of ophthalmology, Journal canadien d’ophtalmologie, Elsevier B.V., 2012; 47(2), pp. 150-4, doi: 10.1016/j.jcjo.2012.01.004. Jadotte Y.T., Schwartz R.A., Melasma: Insights and perspectives, Acta Dermatovenerologica Croatica, 18(2), 2010; pp. 124-129, Available at: https://pubmed.ncbi.nlm.nih.gov/20624364/ (Accessed: 12 June 2020).

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Kara N. et al., Evaluation of subfoveal choroidal thickness in pregnant women using enhanced depth imaging optical coherence tomography, Current Eye Research. Informa Healthcare, 2014; 39(6), pp. 642-647, doi: 10.3109/02713683.2013.855236. Lee C.S. et al., Serous retinal detachment in preeclampsia and malignant hypertension, Eye (Basingstoke), Nature Publishing Group, 33(11), 2014; pp. 1707-1714, doi: 10.1038/s41433-019-0461-8. Morrison J.L. et al., Diabetic retinopathy in pregnancy: a review, Clinical and Experimental Ophthalmology, Blackwell Publishing, 2016; pp. 321-334. doi: 10.1111/ceo.12760. Naderan M., Ocular changes during pregnancy, Journal of Current Ophthalmology, Iranian Society of Ophthalmology, 2018; pp. 202-210. doi: 10.1016/j.joco.2017.11.012. Nkiru Z.N. et al., Visual acuity and refractive changes among pregnant women in Enugu, Southeast Nigeria, Journal of family medicine and primary care, Wolters Kluwer – Medknow Publications, 7(5), 2018; pp. 1037-1041. doi: 10.4103/jfmpc.jfmpc_335_17. Nkiru Z.N. et al., Dry eye disease: A longitudinal study among pregnant women in Enugu, south east, Nigeria, Ocular Surface. Elsevier Inc., 17(3), 2019; pp. 458-463, doi: 10.1016/j.jtos.2019.05.001. Örnek N., Özcan Dağ Z., Örnek K., Corneal Endothelial Cell Density and Morphology in Different Trimesters of Pregnancy, Eye and Contact Lens. Lippincott Williams and Wilkins, 2017; 44 Suppl 1, doi: 10.1097/ICL.0000000000000354. Rahman W. et al., Progression of retinopathy during pregnancy in type 1 diabetes mellitus, Clinical and Experimental Ophthalmology, 2007; 35(3), pp. 231-236, doi: 10.1111/j.1442-9071.2006.01413.x. Roskal-Wałek J. et al., Choroidal Thickness in Women with Uncomplicated Pregnancy: Literature Review, BioMed Research International, 2017, doi: 10.1155/2017/5694235. De Silva S.R. et al., Idiopathic choroidal neovascularisation in pregnancy: Treatment options and a successful outcome, BMJ Case Reports, BMJ Publishing Group, 2016, doi: 10.1136/bcr-2016-215787. Sullivan L. et al., Intravitreal bevacizumab injection in unrecognised early pregnancy, Eye (Basingstoke), Nature Publishing Group, 28(4), 2014; pp. 492-494, doi: 10.1038/eye.2013.311. Swiatek-De Lange M. et al., Membrane-initiated effects of progesterone on calcium dependent signaling and activation of VEGF gene expression in retinal glial cells, GLIA. Glia, 2007; 55(10), pp. 1061-1073. doi: 10.1002/glia.20523. Taradaj K. et al., Changes in the parameters of the anterior segment of the eye in pregnant women - literature review, Ginekologia Polska, Via Medica, 2018; pp. 169-173, doi: 10.5603/GP.a2018.0028. Wang C. et al., Changes in intraocular pressure and central corneal thickness during pregnancy: A systematic review and Meta-analysis’, International Journal of Ophthalmology, International Journal of Ophthalmology (c/o Editorial Office), 10(10), 2018; pp. 1573-1579, doi: 10.18240/ijo.2017.10.15. Yenerel N.M., Kücümen R.B., Pregnancy and the eye, Turk Oftalmoloiji Dergisi, Turkish Ophthalmology Society, 2015; pp. 213-219, doi: 10.4274/tjo.43815.

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Angiogenesis and pulmonary vascularisation disorders in lung hypoplasia in the course of Congenital Diaphragmatic Hernia

Agata Cyran1, Dorota M. Radomska-Leśniewska2 Corresponding Author 1 Department of Pathology, Medical University of Warsaw 2 Department of Histology and Embryology, Medical University of Warsaw Chalubinskiego str. 5; 02-004 Warsaw Email: [email protected] ABSTRACT Congenital diaphragmatic hernia (CDH) is a rare and severe congenital anomaly, characterized by defective formation and/or muscularization of the diaphragm with displacement of abdominal contents into the chest cavity. CDH is almost invariably accompanied by lung hypoplasia and often persistent pulmonary hypertension (PPH). The lungs vasculature in patients with CDH show signs of immaturity in relation to fetal age such as: reduced pulmonary vessels number and size, complexity of proximal pulmonary artery branching and paucity of microvascular or capillary networks. Disturbed balance between proangiogenic and antiangiogenic factors and its altered expression has been recently described in CDH. Dysfunctional expression of hypoxia-inducible transcription factor 2 α, a decreased level of vascular endothelial growth factor (VEGF) and VEGFR2 receptor, accompanied by reduced expression of other proangiogenic factors, such as Slit 3 and their receptors Robo and eNOS was observed in CDH patients. Increased metalloproteinases expression and reduced expression of their inhibitors appear to correlate with vascular disorders in CDH. Elevated transforming growth factor-beta (TGFβ-1) level in CDH lung tissue may participate in suppression of lung growth. Angiogenesis disorders are one of the causes of lung hypoplasia and may impair normal lungs development in CDH patients. PPH in the course of CDH is characterized by disturbed development, structure, tone and reactivity of pulmonary vessels, and hypertensive vascular remodelling. Its structural abnormalities leads to size reduction of the vessels and predisposes to vasoconstriction and lowering vasodilatation response. These structural changes are partly dependent on endothelial cell (EC) dysfunction, which produces vasoactive mediators at the improper level. Impro- vement of pulmonary parameters in hypoplasia may be achieved by the use medicines, which positively influenced the expression of VEGF and other angiogenic factors INTRODUCTION Congenital diaphragmatic hernia (CDH), a birth defect affecting 1 in 2500 live born infants, characterized by anatomic abnormalities which are: replacement of abdominal viscera into the thoracic cavity through the defect in the diaphragm and abnormal lung development including lung hypoplasia and pulmonary vascular abnormalities (Lath, 2011; Laudy, 2000). Pulmonary hypoplasia, an element of CDH, moderately defined disorder of under- developed lungs preventing from reaching anatomical mature size. This immaturity is due to reduce number of bronchiolar branching, decrease number of blood vessels per unit of lung tissue and impair cartilage development and acinar complexity. Prevalence of pulmonary hypoplasia in the population is estimated at 14 per 10000 of all births (Knox, 1986) and its perinatal mortality is high, ranges from 47 to 75% (Winn, 2000; Delgado-Peña, 2015). According to Winn et al. the incidence of pulmonary hypoplasia among children born between 15 and 28 weeks’ gestation due to preterm rupture of the amniotic membranes without others anomalies was 12,9%.

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Postero-lateral hernia (Bochdalek hernia) is the most common type (approximately 75%) situated on the left side (85%). Less common type are anterior (Morgagni 23-28%) and central located (approximately 7%) (Chandrasekharan, 2017). Because the etiology of CDH is moderately known, first it was considered that lung hypoplasia is secondary to compression of the lung. This compression due to presence of the abdominal organs in thoracic cavity arrest lung development. However, a second hypothesis was put forward: lung hypoplasia is primary and leads to disturbances is diaphragmatic development. Simultaneous development of the airway and blood vessels determines normal lung development. According to Nakamura’s study (Nakamura, 1992), severity of damage depends on prenatal stage lung development. Lung injury before canalicular period (before 16 weeks of gestation) causes reduced bronchiolar branching, impaired cartilage development, acinar complexity and maturation and decreased number of blood vessels. Incorrect vasculogenesis corresponds to decrease total number of blood vessels, number of vessels per unit of lung tissue and increase thickness of muscle layer. These changes are observed in pulmonary hypoplasia (Laudy, 2000). In the developing fetus, new blood vessels are formed in the first stage by vasculogenesis, followed by vasculogenesis and angiogenesis. Vasculogenesis refers to the process beginning with angioblast forming a primitive vascular network. Angiogenesis is a physiological process involving the growth of new blood vessels from preexisting vessels (Demir, 2007) and it is considered to play a central role in human tissue development and regeneration. Since vascular development parallels that of airways hypoplasia, underdevelopment of the pulmonary vascular bed is the feature of hypoplastic lung (Boucherat, 2014) SEARCH STRATEGY AND SELECTION CRITERIA Research strategy was focused on providing current data on angiogenesis and vascular disorders in lung hypoplasia in the course of the CDH. The terms like: "congenital diaphragmatic hernia", "lung hypoplasia", "lung development", "lung embryology", "angiogenesis", "vasculogenesis", "hypoxia-inducible transcription factor 2 α", "vascular endothelial growth factor", "vascular endothelial growth factor receptor", "endothelial nitric oxide synthase", "Slit 3 and Robo receptors", "vascular remodelling" and "persistent pulmonary hypertension", "expression of metalloproteinases" as well as "tissue inhibitors of metalloproteinase", "hypermuscularisation" and "neomuscu- larisation" were searched in PubMed, ResearchGate and Google Scholar database. Scientific articles from 1962 to 2019 and relevant papers were reviewed. Search results were limited by the association of angiogenesis and vascularization disorders in CDH. REVIEW PRENATAL LUNG DEVELOPMENT Normal prenatal lung development is divided into four stages. Depending on the week of gestation its include embryonic period (up to 6 weeks of gestation), pseudoglandular period (weeks 6 to 16), canalicular period (weeks 16 to 24) and saccular period (weeks 24 to 40).

150 Angiogenesis and pulmonary vascularisation disorders in lung hypoplasia in the course of Congenital Diaphragmatic Hernia

According to two-hit hypothesis based on the rat nitrofen model, the initial hit is the bilateral lung hypoplasia and the second one is physical compression of the lungs by abdominal organs. The first hit appears during organogenesis (Keijzer, 2000). The first period of organogenesis of the lungs is to appear two ventral buds of the oesophagus. This buds at the end of the 6th week become tubes with lobar and segmental branching (Mullassery, 2015). Branching pattern is initiated by signals from mesenchyme to the buds, without them this branching is impossible (Alescio, 1962). Disturbance in lung bud development can lead to impairment of post hepatic mesenchymal plate. At the same times vasculogenesis take place. The presence of primitive endothelial plexus surrounding the newly formed airway at 9.5 day was described (Parera, 2005) at the very beginning of lung development. Vascular connection between the heart and the lung has been described as early as 34 day gestation in human (Peng, 2013). At the end of this period 2 pulmonary arteries become a vascular plexus origin. The pseudoglandular period is the time of repeatable branching of the lung buds (“branching morphogenesis”). The airway epithelium starts to grow and differentiates from proximal to distal end. This is the time when neuroendocrine, goblet and ciliated cells are present; cartilage and smooth muscle cells appear. At the end of this period airways are formed, vascular development is completed: pre-acinar pulmonary vessels are present in each segment. Lung injury before canalicular period (before 16 weeks of gestation) causes reduced bronchiolar branching, impaired cartilage development, acinar complexity and maturation and decreased number of blood vessels. Incorrect vasculogenesis corresponds to decrease total number of blood vessels, number of vessels per unit of lung tissue and increase thickness of muscle layer. In next stage-canalicular period-is observed formation of air-blood barrier, respiratory bronchioles which branch into alveolar ducts and surfactant secretion. At this stage, epithelial cells differentiate into pneumocytes type 1 and type 2 which are involved in the surfactant production. Surfactant starts to be secreted to the airway lumen from about 30 weeks of gestation. Cell specialization is a prerequisite for lung development. The pulmonary vascular development includes vessel proliferation and becoming capillary network surrounding alveoli. During this period and the next one, vessels length and diameter growths. Saccular period is the last stage while terminal saccules is born. At the end of this stage all generations of respiratory branches are completed. Another important element that prepares the fetus for birth is increasing parenchyma amount (Mullassery, 2015; Smith, 2010) ANGIOGENESIS DISTURBANCES IN LUNG HYPOPLASIA Angiogenesis, a new blood vessels formations from preexisting one, is an important process to maintain homeostasis of the organism (Carmeliet, 2011).

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In order to gas exchange between blood and the growing airways, microcirculation is adapted along with the expansion of the airways. The formation of a new blood vessels primarily occurs through sprouting of a new capillaries from preexisting vessels at the growing tips of branching epithelium, a process called distal angiogenesis (Parera, 2005). The main factor inducing angiogenesis is hypoxia. Among the factors involved in angiogenesis are such as: hypoxia inducible factors (HIFs), vascular endothelial growth factor (VEGF), metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMP), platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGFβ), angiopoietins (Angs), Slit proteins, Robo receptors. Disorders of expression and functioning of these factors may contribute to new vessel formation disturbances. HYPOXIA INDUCIBLE FACTORS HIFs are the key component of the cellular response to hypoxia. HIFs are heterodimer transcription factors composed of two subunits: a stable HIF1β and one of the three oxygen sensitive subunits HIF1α, HIF2α or HIF3α (Fong, 2008). HIF-1, when stabilized by hypoxic conditions, upregulates several genes to promote survival in low- oxygen conditions. It increases the expression of proangiogenic proteins such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and endothelial nitric oxide synthase (eNOS) and diminishes the expression of angiogenesis inhibitors. Physiologically HIF1α expression is localized in endothelial cells of internal membrane and in the middle membrane of the vessel wall. De Rooij and coworkers have shown that HIF1α was expressed only in middle membrane in CDH patients (de Rooij, 2004). Moreover, in human pulmonary artery fibroblasts (PAF) preincubated with a specific p38 MAPK inhibitor (SB203580) and grown in condition of acute hypoxia HIF1α expression was decreased suggesting that HIF1α may be downstream effector of p38 MAPK signaling (Mortimer, 2006, Pak, 2007). Furthermore high expression of HIF2α was demonstrated in CDH patients in comparison to healthy individuals (van der Horst, 2011). This expression correlated with severity of persistent pulmonary hypertension (PPH). Huang et al. revealed that, the expression of HIF2α of healthy neonates born after 37 weeks was localized in distal epithelial cells, alveolar epithelial cells and alveolar type II cells while in CDH patients HIF2α was present in epithelial cells and endothelial cells (Huang, 2018). Authors suggested that hypoxia and HIF may be involved in pathogenesis of pulmonary hypertension. Prolonged exposure to hypoxia can lead to pulmonary artery hypertension (Pak, 2007). VASCULAR ENDOTHELIAL GROWTH FACTOR VEGF is the strongest angiogenesis stimulator. It was descripted over 30 years ago as vascular permeability factor secreted by tumor cells (Senger, 1986) and since then several VEGF splice variants have been identified (Ferrara, 2003). The most abundant VEGF protein isoform is VEGFA displaying pleiotropic functions. It influences endothelial cells (ECs) by increase their proliferation, migration, chemotaxis and survival (Voelkel, 2014). During development it induces/stimulates embryogenesis, placental development and organogenesis. It has proinflammatory activity.

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VEGFA activates tyrosine-kinase receptors (mainly VEGFR-1, VEGFR-2) localized among others cells such as: endothelial, monocytes and cancer cells. VEGFR-2 activates downstream signaling pathways such Erk, Act, eNOS which are necessary for early angiogenesis (Koch, 2012). The majority of studies showed a decreased level of VEGF and its VEGFR2 receptor in CDH. However Patel et al. study showing the relationship between plasma level of VEGF and placental growth factor (PLGF), which is the members of the VEGF family, produced different results (Patel, 2014). Plasma VEGF was higher and PLGF was lower in CDH neonates compared to existing normative data. VEGF:PLGF ratio correlated with CDH severity as a pulmonary vascular disease. Studies on animal model revealed that administration of teratogenic herbicide – nitrofen to the rats dams causes a decreased level of VEGF and its receptors in fetuses and lead to hypoplasia of their pulmonary vessels. This is the reason of impairment of lung development following by lung hypoplasia and pulmonary hypertension (Sbragia, 2014; Schmidt, 2012). Compernolle and coworkers published that HIF2 and VEGF inhibition attenuated fetal lung maturation and simultaneously treatment with VEGF prevented fetal respiratory distress in premature mice, which may be due to lack of surfactant. Authors explained that VEGF participates in lung development and surfactant production, and induces glycogen conversion to surfactant in pneumocytes II (Compernolle, 2002). Impaired angiogenesis and pulmonary vascular development participate in development of hypoplasia. VEGF is released by the interstitial lung cells and the apex of the growing airways (Healy, 2000). The classic signaling pathway of VEGF affecting angiogenesis involves the stimulation of eNOS by VEGF, which leads to release of NO and feedback stimulation of cGMP which positively regulate angiogenesis. During CDH the eNOS and cGMP level are lowered (Luong, 2011; Michelakis, 2003). Sildenafil, used to therapy of primary pulmonary hypertension, inhibits the action of cGMP phosphodiesterase (PDE 5), which decompose cGMP to GMP (Aman, 2016; Michelakis, 2003). In CDH increasing activity of PDE5 decreases expression of VEGF. Studies by Russou et al. proved the efficacy of sildenafil in CDH (Russou, 2016). In this study growth of the vascular network induced improvement of pulmonary parameters, including maturation of alveoli and their function. Park et al. investigation suggested that inhibition by sildenafil PDE5 may lead to HIF activation concluded that production o VEGF can be regulated by activation of HIF via cGMP (Park, 2013). SLIT PROTEINS AND ROBO RECEPTORS Slit proteins (1-3) and their Rounabout (Robo) receptors are involved in the processes of growth of neurons; diaphragm, kidneys, heart, lungs development and angiogenesis (Zhang, 2009). ECs express Robo 1-4 receptors on their surface, produced and released Slit 3 into extracellular matrix (ECM). A major role in angiogenesis plays molecule Slit 3 and their partner Robo 4 receptor (Paul, 2013). Slit 3 stimulates of EC proliferation, migration, chemotaxis, induces formation of vascular network (in vitro study), and buds of new vessels. Zhank et al. showed that Slit-/- mice exhibit disturbed angiogenesis during embryogenesis (Zhank, 2009). Furthermore SLIT 3 and ROBO mutants have kidney, heart and lung defects (Robo 1) and develop CDH (Dickinson,

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2010). Robo 4 is involved, among others, in regulating angiogenesis during vascular development (Dai, 2019). Human placental multipotent mesenchymal stromal cell (hPMSC)-derived Slit 2 and its receptor Robo 4 modulated EC migration, tube formation regulating angiogenesis in placenta (Chen, 2016). METALLOPROTEINASES AND TISSUE INHIBITORS OF METALLOPROTEINASE Degradation of basement membrane of the maternal vessel as well as surrounding ECM is essential step of new blood vessels formation mediated by proteolytic activity of MMPs and plasmin (Bellon, 2004). MMPs and tissue inhibitors TIMPs are involved in the balance between deposition and turnover of ECM. In CDH lungs the expression of MMP-1 increases significantly in capillaries and fibroblasts, while expression of TIMP-2 decreases (Masumoto, 2006) indicating a disturbance in the balance between MMPs and TIMPs. Besides MMP-2 and -9 expression area was elevated in arterioles of CDH lungs. Disorders in the concentration of MMP-9 and its inhibitor, TIMP-1, was found in rat lung tissue. Increase of MMP-9 activity due to the absence of TIMP-1 in fetal rats with nitrofen-induced CDH, a model of respiratory disorders, was presented by Tekawa et al. (Tekawa, 2003). Wild et al. studies performed on CDH rat model showed induction of elastolytic and MMP activities as well as an elevation of epidermal growth factor (EGF) and osteo- pontin deposition in the abnormal lung vasculature (Wild, 2017). These changes may be important in explaining the reason of disturbances in interstitium and pulmonary arteries found in CDH (Masumoto, 2006). ANGIOPOIETINS Angs are the specific growth factors for endothelial cells that act in a late phase of angiogenesis. They bind tyrosine kinase receptors called TIE 1 and TIE 2. Ang 1 and Ang 2 act competitively in relation to the TIE 2 receptor. Ang 2 can block the function of Ang 1 a factor displaying strongly proangiogenic effect. The action of Ang 2 is depended on concentration of VEGF in the environment. In the absence or low concentration of VEGF Ang 2 exerts antiangiogenic action (Fong, 2008). Borcherat et al. found that the concentration of Ang-1 and TIE 2 did not differ from normal, but the concentration of Ang 2 and VEGF was higher in the hypoplastic lung of the human fetus (in post mortem tissue) during pregnancy (Borcherat, 2010). Biological effects of VEGF and angiopoietins are mediated by eNOS (Sluiter, 2012; Michelakis, 2003). The eNOS level was more decreased in hypoplastic lungs in the course of CDH then in the lung with hypoplasia due to other reason (Borcherat, 2010). Boucherat concluded that defectiveness of angiogenesis in hypoplastic lungs despite increase level of VEGF and Ang 2 production, might result from insufficient eNOS expression. PLATELET-DERIVED GROWTH FACTOR AND TRANSFORMING GROWTH FACTOR- BETA Factors that stimulate late phase of angiogenesis include mitogen and chemoattractant of pericytes – PDGF and inducer of pericyte differentiation – TGFβ. TGFβ is a key player in lung development and repair (Vuckovic, 2016). Increased expression of TGFβ-1 in the lung of CDH rat model was presented by Xu and coworkers (Xu,

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2009). Authors suggest that elevated TGFβ-1 level in lung tissue may participate in suppression of lung growth and development. Prenatal treatment with Tetrandrine can improve the growth of the lung of the nitrofen-induced CDH fetuses and its mechanism seems to be involved in downregulating the expression of TGFβ-1. Vuckovic evaluated pulmonary expression of TGF-signaling components, downstream effectors, and extracellular matrix targets in CDH neonates who died between birth and the first few weeks of life after prenatal conservative management or tracheal occlusion (TO), and in rabbit pups that were prenatally randomized for surgical CDH and TO vs. sham operation. They found that prenatal tracheal occlusion increases TGF-/Rho kinase pathway, myofibroblast differentiation, and matrix deposition in neonatal rabbit and human CDH lungs. The upregulated protein and gene expression of EGF and TGFα involved in the fetal lung growth and development, in the proximal airways in the CDH hypoplastic lung was reported by Guarino et al. Authors suggest persistence of fetal stage of pulmonary airway development in CDH is due to disturbances of EGF and TGFα expression in lung (Guarino, 2004). VASCULAR DISORDERS IN CDH Lungs of CDH patients display vascular abnormalities classifying CDH to a vascular disease (Sluiter, 2012). There is a smaller number of vessels and their lower density in the hypoplastic lungs. The degree of proximal ramification is lower and the network of capillaries characteristic for the alveolarization of distal parts of the lungs for normal age is poorer. The size of the vessels are smaller (means smaller lumen). High mortality and morbidity rates in CDH are mostly due to the concomitant pulmonary hypertension (PH) (Mous, 2018). PH associated with CDH is characterized by disrupted development and structure of pulmonary vessels, disturbed reactivity of the vessels, disorderly postnatal reconstruction of vessels. Structural abnormalities of the vessels start to develop when the lung is very immature (Mous, 2018). Vascular disorders are mainly caused by hypertrophy of the muscle layer, especially of small arterioles, but also of medium and large arteries. It was reported to be much thicker in the pulmonary arteries of newborns and stillborns with CDH. Excessive muscularization occurs early in gestation (Sluiter, 2013). Furthermore, the presence of a layer of smooth muscle cells in capillaries called neomuscularization is a characteristic feature of vascularization in CDH. Under normal conditions in capillaries such cells are not present (Kool, 2014; Sluiter, 2011; Rottier, 2005). Neomuscularization is considered to be the results of abnormal differentiation of pericites precursor cells (Mous, 2018; Armulik, 2011) and interstitial cell into smooth muscle cells (Sluiter, 2012). Other cells such circulating progenitor cells, residual vascular progenitor cells, activated fibroblasts and transdifferentiated to mesenchymal cells EC can also participate in this reconstruction/remodeling (Davie, 2004). In the persistent pulmonary hypertension of newborns, a hypothesis was formulated that the transformation of smooth muscle cells in the fetus to mature cells is disturbed. Muscularization of immature pulmonary vessels leads to migration of fibroblasts from the adventitia and smooth muscle cells of the middle membrane, which with increased production

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Agata Cyran, Dorota M. Radomska-Leśniewska of ECM proteins makes the vessel wall in incompletely dilated state (Stenmark, 1993). Moreover, adventitia is also, just like the media, much thicker in the pulmonary arteries in CDH. Physiologically, a decrease in adventitia layer is observed after birth, as a remodeling disorders. Sehata studies show this structural remodeling impairment in CDH infants in adventitial layer was significantly thicker than in controls (Sehata, 1999). Structural changes of the vessels are partly dependent on endothelial cells dysfunction. Altered expression of vasoactive factors, released by endothelial cells may cause unpredictable vascular reactivity of vasculature in CDH. In humans, an increase in: endothelin receptor A (ETA) and endothelin receptor B (ETB) levels has been reported in the PH (de Lagausie, 2005]). High levels of endothelin 1 (ET-1) in plasma correlated with the severity of pulmonary hypertension disease in this population (Keller, 2010). Studies on the level of endothelial NO synthase (eNOS) differed between researchers in human studies from the decrease in eNOS (North, 1995), lack of difference or even increase in eNOS (Sehata, 2006). Sonic Hedgehog (SHH) signaling take a part in development of respiratory bronchioles and thinning of the interstitium. Normally SHH shows the highest concentration at 16-18 weeks of fetal life and disappears at 35. In CDH, decreased SHH expression was detected in early stages of development and the maximum expression was in 21-34 weeks of fetal life, i. e. it was not continued until 35 weeks. In addition, SHH expression is delayed in CDH, suggesting that its disturbances may contribute to the formation of structural and pulmonary function disorders (Unger, 2003) CONCLUSION Congenital diaphragmatic hernia (CDH) is a birth defect characterized by anatomic abnormalities including lung hypoplasia and pulmonary vascular abnormalities. Lung hypoplasia as an element of CDH is still poorly known. One of the causes of lung hypoplasia during embryonic development is abnormal angiogenesis. Angiogenesis disorders in CDH are mainly caused by altered expression of VEGF and its receptors. They are accompanied by disturbances in the expression of other factors modulating angiogenesis such as: Slit3 and their receptors Robo, eNOS, cGMP, MMPs, TIMPs , Angs or TGFβ. Improvement of pulmonary parameters in hypoplasia may be achieved by the use medicines, which positively influenced the expression of VEGF and other angiogenic factors. REFERENCES Alescio T., Cassini A., Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium, Journal of Experimental Zoology, 1962; 150:83-94. Aman J., Bogaard H.J., Noordegraaf A.V., Why vessels do matter in pulmonary disease, Thorax. 2016; 71:767-769. Armulik A., Genové G., Betsholtz C., Pericytes: developmental, physiological, and pathological perspectives, problems, and promises, Dev Cell. 2011; 21:193-215. Bellon G., Martiny L., Robinet A., Matrix metalloproteinases and matrikines in angiogenesis, Crit Rev Oncol Hematol. 2004; 49:203-20.

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North A.J, Moya F.R., Mysore M.R., Thomas V.L., Wells L.B., Wu L.C. et al., Pulmonary endothelial nitric oxide synthase gene expression is decreased in a rat model of congenital diaphragmatic hernia, Am J Respir Cell Mol Biol. 1995; 13: 676-682. Pak O., Aldashev A., Welsh D., Peacock A., The effects of hypoxia on the cells of the pulmonary vasculature, Eur Respir J. 2007; 30: 364-372. Parera M.C., van Dooren M., van Kempen M., de Krijger R., Grosveld F., Tibboel D. et al., Distal angiogenesis: a new concept for lung vascular morphogenesis, Am J Physiol Lung Cell Mol Physiol. 2005; 288: L141-9. Park H.S, Park J.W, Kim H.J, Lee H.J., Kim B., Chun Y.S., Sildenafil alleviates bronchopulmonary dysplasia inneonatal rats by activating the hypoxia-inducible factor signaling pathway, Am J Respir Cell Mol Biol. 2013;48:105-13. Patel N., Moenkemeyer F., Germano F., Cheung M.M.H., Plasma vascular endothelial growth factor A and placental growth factor: novel biomarkers of pulmonary hypertension in congenital diaphragmatic hernia, Am J Physiol Cell Mol Physiol. 2014; 308: L378-L383. Paul J.D., Coulombe K.L.K., Toth P.T., Zhang Y.M., Marsboom G., Bindokas V.P. et al., SLIT3-ROBO4 activation promotes vascular network formation in human engineered tissue and angiogenesis in vivo, J Mol Cell Cardiol. 2013; 64: 124-131. Peng T., Morrisey E.E., Development of the pulmonary vasculature: Current understanding and concepts for the future, Pulm Circ. 2013;3(1):176-178. de Rooij J.D., Hosgor M., Ijzendoorn Y., Rottier R., Groenman F.A., Tibboel D. et al., Expression of angiogenesis-related factors in lungs of patients with congenital diaphragmatic hernia and pulmonary hypoplasia of other causes, Pediatr Dev Pathol. 2004 ;7(5):468-477. Rottier R., Tibboel D., Fetal lung and diaphragm development in congenital diaphragmatic hernia, Semin Perinatol. 2005; 29: 86-93. Russo F.M., Toelen J., Eastwood M.P., Jimenez J., Miyague A.H., Vande Velde G. et al., Transplacental sildenafil rescues lung abnormalities in the rabbit model of diaphragmatic hernia, Thorax. 2016; 71 (6): 517-25. Sbragia L., Nassr A.C., Gonçalves F.L., Schmidt A.F., Zuliani C.C., Garcia P.V. et al., VEGF receptor expression decreases during lung development in congenital diaphragmatic hernia induced by nitrofen, Braz J Med Biol Res. 2014;47(2):171-8. Schmidt A.F., Gonçalves F.L., Regis A.C., Gallindo R.M., Sbragia L., Prenatal retinoic acid improves lung vascularization and VEGF expression in CDH rat, Am J Obstet Gynecol. 2012 ;207(1):76.e25-32. doi: 10.1016/j.ajog.2012.04.025. Epub 2012 Apr 30. Senger D.R., Perruzzi C.A., Feder J., Dvorak H.F., A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines, Cancer Res. 1986;46:5629-5632. Shehata S.M., Tibboel D., Sharma H.S., Mooi W.J., Impaired structural remodelling of pulmonary arteries in newborns with congenital diaphragmatic hernia: a histological study of 29 cases, J Pathol. 1999;189(1):112-118. Shehata S.M., Sharma H.S., Mooi W.J., Tibboel D., Pulmonary hypertension in human newborns with congenital diaphragmatic hernia is associated with decreased vascular expression of nitric-oxide synthase, Cell Biochem Biophys. 2006; 44: 147-155. Sluiter I., Reiss I., Kraemer U., de Krijger R., Tibboel D., Robbert J Rottier R.J., Vascular abnormalities in human newborns with pulmonary hypertension, Expert Rev Respir Med. 2011; 5: 245-256. Sluiter I., Veenma D., van Loenhout R., Rottier R., de Klein A., Keijzer R. et al., Etiological and pathogenic factors in congenital diaphragmatic hernia, Eur J Pediatr Surg. 2012; 22: 345-354.

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Sluiter I., van der Horst I., van der Voorn P., Boerema-de Munck A., Buscop-van Kempen M., de Krijger R. et al., Premature differentiation of vascular smooth muscle cells in human congenital diaphragmatic hernia, Exp Mol Pathol. 2013; 94: 195-202. Smith L.J., McKay K.O., van Asperen P.P., Selvadurai H., Fitzgerald D.A., Normal Development of the Lung and Premature Birth, Paediatr Respir Rev. 2010;11(3):135-42. Spees J.L., Whitney M.J., Sullivan D.E., Lasky J.A., Laboy M., Ylostalo J. et al., Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension, FASEB J. 2008;22(4):1226-1236. Stenmark K.R., Badesch D.B., Dempsey E.C. et al., Regulation of pulmonary vascular wall cell growth, Eur Respir Rev. 1993;3:629-637. Tatekawa Y., Kanehiro H., Hisanaga M., Nakajima Y., Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1: expression in the lung of fetal rats with nitrofen-induced diaphragmatic hernia, Pediatr Surg Int. 2003 ;19(1-2):25-8. Unger S., Copland I., Tibboel D., Post M., Down-regulation of sonic hedgehog expression in pulmonary hypoplasia is associated with congenital diaphragmatic hernia, Am J Pathol. 2003;162(2):547-555. Wild B., St-Pierre M.E., Langlois S., Cowan K.N., Elastase and matrix metalloproteinase activities are associated with pulmonary vascular disease in the nitrofen rat model of congenital diaphragmatic hernia, J Pediatr Surg. 2017;52(5):693-701. Winn H.N., Chen M., Amon E., Leet T.L., Shumway J.B., Mostello D., Neonatal pulmonary hypoplasia and perinatal mortality in patients with midtrimester rupture of amniotic membranes – A critical analysis, American Journal of Obstetrics and Gynecology, 2000, 182: 1638-1644. van der Horst I.W., Rajatapiti P., van der Voorn P., Francien H van Nederveen F.H., Tibboel D.l, Rottier, R. et al., Expression of hypoxia-inducible factors, regulators, and target genes in congenital diaphragmatic hernia patients, Pediatr Dev Pathol. 2011; 14: 384-390. Voekel N.F., Gomez-Arroyo J., The role of vascular endothelial growth factor in pulmonary arterial hypertension. The angiogenesis paradox, Am J Respir Cell Mol Biol. 2014; 51(4): 474-84. Vuckovic A., Herber-Jonat S., Flemmer A.W., Ruehl I.M., Votino C., Segers V. et al., Increased TGF-: a drawback of tracheal occlusion inhuman and experimental congenital diaphragmatic hernia?, Am JPhysiol Lung Cell Mol Physiol. 2016; 310: L311-L327. Xu C., Liu W., Chen Z., Wang Y., Xiong Z., Ji Y., Effect of prenatal tetrandrine administration on transforming growth factor-beta1 level in the lung of nitrofen-induced congenital diaphragmatic hernia rat model, J Pediatr Surg. 2009;44(8):1611-20. Zhang B., Dietrich U.M., Geng J.G., Bicknell R., Esko J.D.Wang L., Repulsive axon guidance molecule Slit3 is a novel angiogenic factor, Blood. 2009, 114, 4300-9.

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The role of melatonin and noradrenaline interaction in REM sleep phase modulation

Wojciech Szlasa1*, Miriam Lang1, Michał Gebuza1, Kacper Turek1, Nina Rembiałkowska2, Julita Kulbacka2 Faculty of Medicine, Wroclaw Medical University, Poland Department of Molecular and Cellular Biology, Faculty of Pharmacy, Borowska 211, 50-556 Wrocław Wroclaw Medical University, Poland *Corresponding author: [email protected], Faculty of Medicine, Wroclaw Medical University, Poland ABSTRACT Melatonin and noradrenaline are the hormones responsible for the regulation of the sleep. The hormones’ interplay occurs on three levels – neuronal, physiological, and biochemical. REM-ON and REM-OFF neurons remain under the regulation by noradrenaline. Moreover, the increased concentration of noradrenaline in the brain tissue, by the upregulation of the hormone’s transporters, leads to the increased excitability of the brain, the downregulation of melatonin biosynthesis, and eventually to the lack of sleep. The biosynthesis pathways of both hormones involve the same enzyme – Monoamine oxidase (MAO), thus their interaction might appear on the biochemical level. The dysregulation of melatonin and noradrenaline concentration has an impact on the progression and development of severe physiological complications, such as diabetes or even cancer. The review focuses on the detailed description of metabolic pathways where melatonin and noradrenaline can interact and cause the modulation of the sleep – in particular REM sleep phase. In the end, all the data is connected with the clinical disorders concerning the melatonin-noradrenaline equilibrium dysregulation. INTRODUCTION In the late 20th century researchers established that the changes in the noradrenaline levels in the pineal gland affect the melatonin production by that organ and thus affects the REM sleep phase (Mitchell, 2010). Aside from various functions of the hormones, both are involved in the sleep regulation (Brown, 2012). There are several levels on which the hormones interact. The first one is connected with the neuronal regulation of melatonin biosynthesis. The second is connected with the increased excitability of the brain by the elevated noradrenaline outflow from the neurons to the extracellular space, leading to the reduced sleep. The third one is related to the biochemical modulation of melatonin biosynthesis by the changes in the relative amounts of substrates and enzymes in the melatonin and noradrenaline biosynthesis pathways. All three levels will be described in the presented review. Melatonin is a monoamine hormone biosynthesized in the pineal gland. This compound is responsible for various biological processes among which the most important is sleep regulation (Tordjman, 2017). The hormone is being released from the pineal gland with respect to the circadian clock. Suprachiasmatic nucleus (SCN) is being the main regulator of pineal function, which acts via the melatonin receptors (MT1 and MT2). Namely, when the proper amount of the hormone is being released by the gland, high-affinity receptors (MT1 and MT2), localized on the SCN, decrease the stimulation of the pineal gland, leading to the attenuation of the hormone’s release (Doghramji, 2007). Melatonin induces the sleep, by introducing hypothermia or by the inhibition

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Wojciech Szlasa, Miriam Lang, Michał Gebuza, Kacper Turek, Nina Rembiałkowska, Julita Kulbacka of wakefulness structures and the activation of central GABAergic neurons (Lubo- shizsky, 1998). By the attenuation of the brain excitability, the hormone affects the sleep-inducing areas. Conversely, noradrenaline (norepinephrine) is responsible for maintaining the proper general excitation of the central nervous system (O’Donnell, 2012). Namely, the organism becomes more energetic and unable to relax, which is necessary in falling asleep. Aside from the central nervous system, this hormone leads to an increase in blood pressure and the transmission in the sympathetic neurons of the peripheral autonomous nervous system (Drouin, 2016). The mechanism of noradrenaline action in the sleep regulation involves mostly the REM-OFF neurons, which switch off the effective falling asleep. However, it is more complex, thus is being discussed in the following paragraphs. The model of conjugated metabolism of sleep-regulating hormones – noradrenaline and melatonin, is complicated. Few parameters have to be taken into account when considering the problem. First, there has to be a metabolic node, which gathers the metabolism of both analyzed hormones. Indeed, monoamine oxidase (MAO) is a substrate-non-specific enzyme, involved in the metabolism of both melatonin and noradrenaline (Lin, 2013). Due to the fact, that both compounds undergo metabolism by the same enzyme, the increase in the concentration of one hormone affects the metabolism rate of the other (Mitchell, 2010). To make it happen, both hormones have to be present at the same time inside the cell (Chuluyan, 1991). Moreover, to compete for the binding site of the enzyme, both of the substrates should have a similar affinity to MAO. In case of MAO, there are two isoforms of the enzyme. MAO-A has a high affinity to tyramine, noradrenaline, serotonin and dopamine. On the other hand, MAO- B is involved in the dopamine and tyramine metabolism (Finberg, 2016). The model of conjugated metabolism of both hormones could be analyzed from two perspectives. In physiological conditions, the interactions occur inside pineal gland cells, which leads to an increase in melatonin biosynthesis (Axelrod, 1969). Conversely, during melatonin-mediated insomnia treatment, the interactions take place in locus coeruleus (Chen, 2003). In this case, the pharmacologic elevation of melatonin plasma level leads to the decrease in noradrenaline biosynthesis and therefore to the relieve of insomnia symptoms (Jameie, 2019). As an antioxidant, the exogenous melatonin inhibits mutagenesis and the development of tumors by the cleavage of reactive oxygen species (Anisimov, 2003). Moreover, it affects the longevity of the organism. In contrast, the endogenous hormone secreted by the pineal gland, acts more locally on the brain tissue, due to the fact, that smaller amounts of the hormone enter the blood flow. SEARCH STRATEGY AND SELECTION CRITERIA This study aimed to provide the molecular description of conjugated metabolism of noradrenaline and melatonin. To achieve this, the authors reviewed the literature indexed in PubMed and Google Scholar databases by entering: Monoamine connection, noradrenaline and melatonin conjugated metabolism, melatonin regulates noradrenaline level, noradrenaline regulates melatonin biosynthesis. The researchers considered only English-language articles. However, due to the scarce number of papers concerning the analyzed problem, the authors have also analyzed older articles with high impact in the field.

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REVIEW OF HORMONES CONNECTION IN SLEEP REGULATION ANATOMICAL BASES OF INTERACTIONS BETWEEN THE SLEEP-REGULATING HORMONES BRAIN REGIONS RESPONSIBLE FOR REM SLEEP Based on the extensive research, scientists established that noradrenaline plays a crucial role in the rapid eye movement phase (REM phase) regulation. Over the last quarter of the 20th century, scientists identified specific brain regions, responsible for rapid eye movement control and tried to understand the physiological mechanisms responsible for the phenomenon (Maquet, 1996). There were proposed several REM sleep regulation models. The most physiologically-accepted one was proposed in 2002 by Mallick et al. (Mallick, 2002 a). In the experiments, the brain was lesioned or transected to analyze the electrophysiological functions of the specific regions of the brain, such as locus coeruleus. Mallick et al. showed that the structures within the pons were active during REM sleep phase (Mallick, 2002 a). The observation was in acceptance with other lesion studies, which suggested that locus coeruleus (LC) was the region of interest (Porkka-Heiskanen, 1995). LC is the primary norepinephrine producing site of the brain (Aston-Jones, 2005). Studies in which areas of the LC were locally cooled or lesioned by 6-hydroxydopamine revealed that the principal was the specifically REM-related site of LC (Brown, 2012). NEURONS RESPONSIBLE FOR REM PHASE INDUCTION LC neurons prevent REM sleep induction and remain active during REM sleep phase (Choudhary, 2014). Since LC-neurons are noradrenergic, the researchers concluded that norepinephrine might be significant for REM sleep regulation. Hence LC-neurons, which are not active during REM sleep phase were classified as "REM-OFF" neurons (Aston-Jones, 1981). Conversely, ventral to the LC, there were observed neurons active exclusively during REM sleep which were classified as "REM-ON". Based on NE agonist and antagonist studies REM-ON neurons were assigned to be cholinergic (Luebke, 1992; Mallick, 2002 a). Since acetylcholine is not an inhibitory neurotransmitter, Mallick et al. proposed that the inhibition of REM-OFF neurons is the result of GABA-ergic interneurons. Indeed, it was experimentally proven that GABA levels in the LC increased during REM sleep and GABA-ergic neutrons remained active (Mallick, 2002 a). Further, the influence of waking areas of the brain was studied. Researchers found out that reticular wake- inducing area activates LC-REM-OFF and inhibits the REM-ON neurons (Pal, 2007). GABA MEDIATED MODEL OF REM SLEEP REGULATION GABA mediated model was mostly derived by Mallick et al. According to the model, in the standby physiological mode, the wake centre keeps REM-OFF neurons active and REM-ON neurons inactive (Mallick, 2001). Later it was discovered that the neuronal input reaches the REM-related neurons from the reticular part of the substantia nigra (SN) (Pal, 2009). Namely, the GABA level increases in the LC causing the LC-REM-OFF neurons to decrease their activity (Kaur, 2001). The attenuation of REM-OFF neurons activity resulted in cessation of the REM-ON derived inhibitory

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Wojciech Szlasa, Miriam Lang, Michał Gebuza, Kacper Turek, Nina Rembiałkowska, Julita Kulbacka effects. Namely, pre-REM sleep phase occurred after REM-ON neurons became active (Pal, 2007). Inhibition of the vast majority of REM-OFF neurons by high GABA level leads to the constant activation of REM-ON neurons. The whole sequence of neuronal events results in REM sleep induction (Choudhary, 2014). PATHOLOGY OF MELATONIN LEVEL DEPRIVATION Melatonin concentration is related to the age of the patient by the activity of the pineal gland – the melatonin producing organ. The size of the pineal gland increases from birth until the second year of life and since then remains constant up to 20th year of life. Afterwards, change in shape occurs (Sumida, 1996). The shape of pineal gland depends on age – in young adults it usually remains pea-shaped and in elderly – irregular or fusiform (Afroz, 2016). Several physiological or pathological conditions, like obesity or primary insomnia, influence the volume of the gland. The phenotype varies with health status and environmental factors (Tan, 2018). The pineal gland is histologically composed of pinealocytes, microglia and astrocytes, covered with the pia mater capsule. The fibrous tissue from the pia mater forms septa which divide the gland into lobules (Tomonari, 2012; Gheban, 2019). The microscopic structure differs significantly between young and older individuals. Namely, the septa are more developed in the elderly age (Arunkumar, 2015). In infants, the pineal gland is relatively bigger and cells from alveoli. The fragile structure of the organ leads to the increased susceptibility to injury, resulting in cystic, hemorrhagic and necrotic changes (Laure-Kamionowska, 2003). Ageing induces the accumulation of pineal sand. The calcification of the pineal gland results in the formation of acervuli - deposits of calcium phosphate, calcium carbonate, magnesium and ammonium phosphates (Zimmerman, 1982; Vigh, 1989). The process occurs without significant differences between countries, regions, gender, races and concerns the vast majority of people all over the world. The incidence of the visible pineal gland calcification (PGC) increases with age – 2% in 0-9 yr., 32% in 10-19 yr., 53% in 20-29 yr. and 83% in over-30 yr. (Tan, 2018). PGC reduces the melatonin level in cerebrospinal fluid. In the mechanism of redox equilibrium dysregulation, it results in circadian rhythm disturbances, insomnia and migraine (Pandi-Perumal, 2006). Few hypotheses are explaining the pathological mechanism of PGC. The first one states that calcium deposits accumulate in the connective tissue due to the pathologic infiltration of mast cells. The cells induce the calcification process by the release of biologically active substances (Maślińska, 2010). The second hypothesis includes the excretion of polypeptides by pinealocytes into the extracellular space. The calcium layers are gradually deposited on the surface of the protein concretions and forming acervuli (Tan, 2018). Different PGC mechanism involves the accumulation of hydroxyapatite crystals by the pinealocytes, due to the Ca2+-ATPase disorder (Krstić, 1986). The number of calcium layers of the pineal sand increases with age (Schmid, 1995). Addi- tional studies proved that pineal calcification is related to the progression of neuro- degeneration, especially in Alzheimer’s disease (Mahlberg, 2008). The age-dependent progressive loss of pinealocytes with the simultaneous increase in the number of neuroglial cells was observed in humans (Afroz, 2016). The degene-

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The role of melatonin and noradrenaline interaction in REM sleep phase modulation ration leads not only to the decrease in the number of pinealocytes but also in the general metabolism rate of the organ (Afroz, 2016). With the pathological changes in the pineal gland, melatonin biosynthesis becomes insufficient. BIOCHEMICAL BASES OF CONJUGATED NE AND MELATONIN METABOLISM ENZYMATIC CHANGES IN DEPRIVED REM SLEEP Melatonin and noradrenalin might also interact on the metabolic level of their biosynthesis and degradation. The key enzyme, involved in the crosstalk between the hormones is the Monoamine oxidase, represented by two isoforms – A and B. MAO-A and MAO-B are differently affected by REM sleep deprivation. Isoforms of MAO differ in response to the pathology, namely sleep deprivation more rapidly affects MAO-A (after 1 day) than further MAO-B (after 2 days of sleep deprivation) (Thakkar, 1993). There is an inconsistency in the available literature concerning the molecular changes after sleep deprivation. Some authors support the lack of brain degeneration after sleep deprivation (Cirelli, 1999; Gopalakrishnan, 2004). Conversely, the others support the decrease in melatonin production caused by ROS stress (Zang, 1998; Bandyopadhyay, 2006; Bonnefont-Rousselot, 2011). Tyrosine hydroxylase (TH) increases the activity of MAO in response to sleep disruption (Basheer, 1998). On the molecular level, TH is activated by phosphorylation. ROS accumulation leads to the upregulation of protein kinases (i.e. PKA) and downregulation of protein phosphatases (i.e. PP2A) (Raghuraman, 2009). Moreover, the level of norepinephrine transporters’ mRNA increases, leading to the elevated outflow of NE from the hormone-producing cells (Basheer, 1998). Free NE diffuses throughout the brain – in particular to the pineal gland (de Iraldi, 1966), leading to the enhanced brain stress-activation (Farley, 1977). The upregulation of NE transporter disrupts the NE equilibrium in the locus coeruleus. The product of the TH-catalyzed reaction diminishes, leading to the simultaneous lowering of the [NA]/[Dopamine] intracellular ratio. When the current [product]/[substrate] ratio remains lower than the constant of the reaction (Keq), the chemical process proceeds, resulting in the enhanced NE biosynthesis (Atkins, 2010). COUPLING OF NE AND MELATONIN METABOLIC PATHWAYS MAO is a mitochondrial enzyme that occurs in two isoforms in the human body. Each isoform has different properties, such as affinity to serotonin and melatonin or catalytic rate (Youdim, 2009). High cell membrane permeability for melatonin and noradrenaline, as well as the crosstalk between 5-Hydroxytryptophan and NE neuronal pathways in the brain (Ungerstedt, 1971), enables both hormones to flow into the cell and compete for the active site of MAO. The destruction of pinealocytes lead to the depletion of melatonin biosynthesis and consequently to the low [melatonin]/[serotonin] ratio (Rozencwaig, 1987). The accumulated excess of serotonin has to be excreted, thus it is being metabolized to 5-hydroxyindoliloacetate acid (5-HIAA) by monoamine oxidase (MAO) (Héry, 1972). The involvement of MAO in the serotonin metabolism decreases the number of free

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Wojciech Szlasa, Miriam Lang, Michał Gebuza, Kacper Turek, Nina Rembiałkowska, Julita Kulbacka units of the enzyme. Without the freely accessible enzyme, the conversion of dopamine into homovanilic acid (HVA) does not occur or occurs with the use of the other enzyme – COMT. When the excess of serotonin has to catabolized, the vast majority of MAO is involved in the 5-HIAA production. The physiological condition leads to the redirection of dopamine to noradrenaline synthesis, which is catalyzed by dopamine beta-hydroxylase (fig. 1) (Rubí, 2010). Aside from the increase in noradrenaline biosynthesis, the stimulation of tryptophan conversion into 5-hydroxytryptophan occurs, leading to further substrates flow in presented metabolic pathways (Ariëns Kappers, 1978).

Figure 1. Noradrenaline and melatonin biosynthesis pathways during the impaired melatonin biosynthesis and the increased noradrenaline production. 1 – the pineal calcification leads to the decreased melatonin biosynthesis and its concentration; 2 – the excess of serotonin is being metabolized by MAO to the 5-HIAA; 3 – dopamine is not catabolized to homovanillic acid; 4 – dopamine metabolism is shifted towards noradrenaline biosynthesis

CLINICAL SIGNIFICANCE OF THE MELATONIN LEVEL DECREASE Several clinical complications relate to the dysregulation of melatonin to noradrenaline systemic ratio. The increased concentration of noradrenaline in the brain leads to the stimulation of noradrenergic REM-OFF neurons. The neurons prevent the REM sleep phase to occur via the GABA-mediated interneuron stimulation of REM-ON neurons. Besides, norepinephrine also leads to the general psychical excitation, simultaneously loosing regeneration and relaxation (O’Donnell, 2012).

Noradrenaline prevents entering into REM sleep phase via binding to the α1 adrenergic receptors (Mallick, 2002 b). The agonist raises the IP3 level in the cytoplasm, resulting in an elevation of Ca2+ concentration. Calcium ions bind to calmodulin, which activates calcineurin (protein phosphatase). This promotes Na+/K+ pump dephosphorylation and consequently enhances the activity of the transporter. The presented pathway increases the excitability of the neurons, therefore changing the behavior (Mallick, 2002 b). Increased aggressiveness, sexual disorders, increased excitability and loss in memory consolidation are thought to be some of the results of REM sleep deprivation (Vogel,

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+ + 1975). Stimulation of α1A adrenergic receptors enhances Na /K ATPase activity, leading to increased neural excitability (Mallick, 2002 b). Interestingly, REM sleep deprivation also decreases cell membrane fluidity (Nath Mallick, 1995). This affects membrane-bound enzymes activity, by preventing them from moving to the lipid rafts (Devaux, 2004; Gomes, 2019). For instance, the changes in receptor densities are responsible for synaptic signal transmission alternations (Nath Mallick, 1995). However, GPI-linked enzymes, like acetylcholinesterase, are affected by the changes in the membrane fluidity as well. Indeed, it was observed that acetylcholinesterase, increase its activity during deprived sleep (Thakkar, 1991). Thus the decrease of acetylcholine content results in increased heart rate and energy expenditure (Sakmann, 1983). Melatonin is particularly important for sleep initiation (Cajochen, 2003). People with low levels of this hormone most often have trouble with falling asleep as well as often suffer different sleep disorders, such as: • circadian rhythm sleep disorders – characterized by abnormal daily rhythm. People suffering from this type of disorder sleep appropriate amount of time, but during the daytime (Turek, 2004); • dyssomnias – a disorder characterized by abnormal quality, amount and duration of sleep. Patients suffering from dyssomnia find it difficult to fall and remaining asleep (Thorpy, 1988); • parasomnias – a disorder characterized by the occurrence of abnormal events during sleep, due to inappropriately timed activation of physiological systems. Symptoms include nightmare disorder, sleep terror disorder, and sleepwalking disorder (Xie, 2017). DECREASED LEVEL OF MELATONIN REDUCES THE EFFICIENCY OF SLEEP The general effect of sleep deprivation is based on the NA excitation of the nervous system. It is being manifested through irritability, cognitive impairment, depression, impulsive behavior, hallucinations paranoia or suicidal thoughts (Pilcher, 1996) (Killgore, 2010). Improper sleep regulation results in abnormal muscle movement, tremors, increased reaction time and decreased accuracy and precision of the moves. A lack of REM sleep also increases the risk of heart attack, impairment of the immune system and growth suppression in adolescence (Moldofsky, 1989; Takase, 2004). Moreover, the risk of precocious death is elevated in sleep deficiency. 27 independent cohort samples showed, that sleeping for less than 6-8 hours during the night increases the risk of premature death by 12% (Cappuccio, 2010). MELATONIN AND DIABETES TYPE 2 A low level of melatonin secretion or mutations in melatonin receptors also poses an noticeable influence on the development of diabetes type-2 (McMullan, 2013). Via interaction of melatonin and its receptor, Gi protein is being activated. This result in suppression of cAMP production and therefore leads to the insulin release. Different 2+ pathway activates Gq protein, which via IP3 and phospholipase C increases Ca cyto- plasmatic concentration (Peschke, 2008). Curiously, too high level of melatonin may lead to hyperglycemia and increase the risk of developing diabetes type-2 (Tuomi, 2016).

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RESTORING THE NORMAL LEVEL OF MELATONIN – THERAPY APPROACH A supplementation of melatonin proved its biological effects in various studies(Lahiri, 2004). Some of them even state that supplementation reduces levels of amyloid-β peptides in the cerebral cortex and might be useful in Alzheimer’s disease prevention (Feng, 2006). Studies performed on insomniac patients (group over 50 years old), revealed that oral supplementation of 0.3 gram melatonin, 30 minutes before bedtime has the biggest positive impact on sleep efficiency (Zhdanova, 2001). Animal (rats) studies showed that administration of 0.4 µg of melatonin per mL of water to older rats for 10 weeks resulted in the lowering of insulin, leptin and retroperitoneal fat levels into the concentration typical for younger organisms (Rasmussen, 1999). Since an increased visceral fat is associated with increased insulin resistance, diabetes, and cardiovascular disease, these results suggest, that an appropriate melatonin supplementation may potentially provide prophylaxis or therapy for some prominent pathologies associated with ageing (Rasmussen, 1999). MELATONIN SUPPLEMENTATION DECREASES THE LEVEL OF PLASMA LEPTIN, PLASMA INSULIN AND RETROPERITONEAL FAT. With age, the levels of plasma insulin, leptin and the amount of visceral fat raises (Rasmussen, 1999). Melatonin deficiency affects cholesterol, adiponectin, glucose and triglycerides metabolism over mammals as well (Ríos-Lugo, 2010). Animal studies proved, that these changes could be prevented by melatonin supplementation. Protective effects on fat-connected disorders, such as non-alcoholic fatty liver disease, are the result of antioxidant actions of melatonin (Pan, 2006). MELATONIN INHIBITION OF CANCER GROWTH IN VIVO Melatonin inhibits the growth of cancer in vivo and results in suppression of tumour fatty acid metabolism (Fenselau, 1976). Via melatonin receptor, the cAMP levels lower. Afterwards, the inhibition of fatty acids transport to the cells occurs. This results in the suppression of linoleic acid biotransformation into 13-HODE (mitogenic signaling molecule), which results in cancer growth inhibition (Fenselau, 1976). Numerous experimental in vitro studies indicate that melatonin primarily slows down cancer promotion and progression through suppression of mitogens, hormones and growth factors such as EGF, estradiol, and prolactin (Blask, 1999). CONCLUSIONS The available data indicated that norepinephrine and melatonin interact with each other on various levels in the sleep modulation. The interactions occur on the physiological, anatomical and biochemical levels. The dysregulation of melatonin to noradrenaline ratio leads to the severe clinical complications, like cancer growth or diabetes. The understanding of their conjugated metabolism enables the clinicians to avoid severe complications and open new ways of hormonal disorders treatment. FINANCING The work was financed from the subsidy of the Department of Molecular and Cellular Biology Np.:SUB.D260.20.009 and sponsorship account of the same Department No. 846 183.

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Emilia Królewicz*, Irena Kustrzeba-Wójcicka Department of Medical Biochemistry, Wroclaw Medical University, Chałubinskiego 10, 50-368 Wrocław, Poland *Corresponding author: Emilia Królewicz, PhD, Department of Medical Biochemistry, Wroclaw Medical University, e-mail: [email protected] ABSTRACT Annexin 5 (An-5) is a cytoplasmic protein present in many tissues of the human body and connected with regulation of the dynamics of biological membranes. It is involved in mechanisms of cell proliferation, apoptosis, blood coagulation, immunology reaction, signal transduction and maintenance of calcium homeostasis. It has long been known as a good tool in research and diagnostic. Scientific studies in the field of medicine and related sciences prove that Annexin 5 is considered as a multifunctional marker and molecular imaging agent. It has been investigated, that the anticoagulant activity of An-5 plays a role in preventing thrombotic processes that occur on placental villi in contact with maternal blood. This knowledge is crucial for diagnostics and research in the field of hematology and gynecology. Recent studies showed also that Annexin 5 is internalized into the cytosol by stressed cardiomyocytes, myoblasts and apoptotic neurons. Investigating this phenomenon contributes to a better understanding of the pathomechanism of cardiological diseases and development of the inflammation process. Until now, An-5 is also commonly used as a marker of early phase of cell apoptosis, but is not used as a marker for allergy diagnosis. The present paper describes a novel basophil activation test (BAT) employing An-5 as an effective marker for moulds allergy recognition. Using this protocol in diagnosing allergy to Alternaria alternata, allows to discriminate between allergic patients and healthy volunteers. High sensitivity (90-100%) and up to 100% specificity in conducted studies suggest that BAT with An-5 should be considered as a promising test for basophil activation and a costs-saving alternative to currently used BATs. Further research is needed for better validation and standardization of this assy. INTRODUCTION Annexins are a group of proteins known for over 40 years and occurring mainly in the world of eukaryotes. They are proteins with a molecular weight in the range of 35-40 kDa and have the ability to bind calcium ions Ca2+ and phospholipids present in cell membranes. The annexin name derives from a Greek word "synexis" which means "a meeting". The name "synexin" was created by Carl Creutz (Creutz et al., 1979). In 1979 the Researcher worked on the adrenal medullary protein – a receptor for calcium ions in the process of exocytosis. About 10 years later Geisow (Geisow et al., 1987) and coworkers, who continued research initiated by Creutz, used the generic term "annexin family" in relation to the group as a whole. The name "annexin" is considered to derive from the Greek word "annex", meaning "to bring / hold together", and indicates the function of providing a compact and stable form to biological structures. Annexins are usually cytosolic proteins with a soluble and a stable form. Annexins are reversibly associated with components of the cytoskeleton or proteins that mediate interactions between a cell and an extracellular matrix (Mirsaeidi et al., 2016). Nowadays more than 100 annexin family members have been discovered in many species (Gerke, Moss, 2002). Annexins present in vertebrates were classified in the annexin "A" family, while the descriptor "B" denotes their presence in Invertebrates and "C" – in Mycetozoa and Fungi. In addition, "D" denotes annexins present in Plants

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Emilia Królewicz, Irena Kustrzeba-Wójcicka and "E" – in Protista (Einarsson et al., 2016; Lizarbe et al., 2013). Bioinformatic analysis of bacterial genomic sequences also revealed the presence of annexin-like proteins – about thirty annexin homologues, including single-domain and multi-domain annexins, were identified in seventeen bacterial species (Kodavali et al., 2014; Morgan et al., 2006). The sequences of bacterial annexins exhibited a remote but statistically significant similarity to sequence profiles identified in eukaryotic organisms (Kodavali et al., 2014). Thirteen proteins have been identified in humans and conventionally named as annexins A1-13 (ANXA1-ANXA13), leaving ANXA12 unassigned in the official nomenclature. Table 1. presents the human annexins nomenclature according to The HUGO Gene Nomenclature Committee database (Website of Human Gene Nomenclature Committee. Available at: www.gene.ucl.ac.uk/nomenclature) and in The UniProt Knowledgebase (Website of The UniProt Knowledgebase. Available at: www.uniprot.org). Human Annexin 5, labelled as ANXA5 in this nomenclature and widely used as An-5 in the literature, is of particular interest as a multifunctional protein with a significant diagnostic potential.

Table 1. Human annexins nomenclature Approved Approved name Previous symbols Molecular weight Reference symbol [kDa] ANXA1 annexin A1 ANX1, LPC1 38.714 (Einarsson et al., 2016) ANXA2 annexin A2 ANX2, ANX2L4, 38.604 (Fal et al., 2003; CAL1H, LPC2D Faust et al., 2009) ANXA3 annexin A3 ANX3 36.375 (Floyd et al., 2000) ANXA4 annexin A4 ANX4 35.883 (Gauer et al., 2013) ANXA5 annexin A5 ENX2, ANX5 35.937 (Geisow et al., 1987) ANXA6 annexin A6 ANX6 75.873 (Gerke, Moss, 2002) ANXA7 annexin A7 ANX7 52.739 (González-Muñoz et al., 2008) ANXA8 annexin A8 ANX8 36.881 (Hayes et al., 2004) ANXA8L1 annexin A8 like 1 ANXA8L2 36.879 (Hayes et al., 2004) ANXA9 annexin A9 ANX31 38.364 (Hoetzenecker et al., 2005) ANXA10 annexin A10 37.278 (Huber et al., 1990) ANXA11 annexin A11 ANX11 54.390 (Jones et al., 2011) ANXA13 annexin A13 ANX13 35.415 (Katsuta et al., 2006)

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According to the information from the HUGO Gene Nomenclature Committee database (Website of Human Gene Nomenclature Committee. The official nomenclature source of the Human Gene Organization (HUGO) for human genes and their cognate orthologs. Available at: www.gene.ucl.ac.uk/nomenclature) and from the UniProt Knowledgebase (Website of The UniProt Knowledgebase. Available at: www.uniprot.org). Twelve human annexin genes range in size from 15 kb to 96 kb and are present in the genome on eight chromosomes: 1, 2, 4, 5, 8, 9, 10, and 15 (Hayes & Moss, 2004). Annexins consist of a unique amino-terminal domain followed by a homologous C-terminal core domain containing the calcium-dependent phospholipid-binding sites. The C-terminal domain usually comprised 60-70 amino acid repeats, known as annexin repeats or an endonexin fold. The four annexin repeats form a highly alpha-helical, tightly packed disc known as the annexin domain, which binds to phospholipids in the membrane in a calcium-dependent manner. SEARCH STRATEGY AND SELECTION CRITERIA The aim of this work was to present a literature review about Annexin 5 as a multifunctional protein in biomedical research. Particular attention has been paid to Annexin 5 as a protein that may be considered as a basophil activation marker in diagnosis of allergic diseases. To prepare a paper, following data bases were used: PubMed (NCBI), Google Scholar, Web of Science, Scopus, HUGO Gene Nomenclature Committee database, UniProt Knowledgebase. We searched through primary original articles and reviews published from 1987 until 2018. ANNEXIN 5 AS A PROTEIN Annexin 5 was first isolated in late 1970s from aqueous extracts of human placenta by gelfiltration (Bohn, Kraus, 1979). Around 1985, the protein was discovered independently in homogenate of human umbilical cord arteries and named vascular anticoagulant protein alpha (VAC-α) because of its ability to inhibit blood coagulation (Reutelingsperger et al., 1985). At present, a recombinant form of An-5 can be produced by the expression of its complementary DNA in Escherichia coli. This allows to employ An-5 worldwide for various biochemical experiments. Annexin 5 is a 35.7 kDa protein whose tertiary structure consists four domains of similar structure. The molecule consists of 319 amino acids and has dimensions of 64 x 40 x 30 Å3. Each domain is built of five α-helices wound into a right – handed superhelix yielding a globular structure of 18 Å diameter (Huber et al., 1990; Sopkova- De Oliveira Santos et al., 2001). This arrangement makes the molecule is slightly curved and convex in shape (fig. 1). The Ca2+ and phosphatidylserine binding sites are located at the convex, membrane-facing side of the protein. A short amino-terminal tail is located at the concave side of the molecule. X-ray crystallography studies have revealed that the domains within An-5 interact with each other in a non-covalent manner (Van Genderen et al., 2008). The interdomain interactions result in the formation of two tightly associated modules (Richter et al., 2005). The first module

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Emilia Królewicz, Irena Kustrzeba-Wójcicka consists of domains I and IV and the second one of domains II and III (fig. 1). The interaction between domain I and IV is mediated non-covalently by the amino-terminal tail. Domain II and III are covalently linked via a short inter-helical turn. It is assumed that An-5 trimer is the basic building block in the crystal lattice (Gauer et al., 2013).

Figure 1. The crystal lattice of Annexin 5

Annexin 5 belongs to the family of cytoplasmic proteins and is found in many tissues of the body, including kidney epithelial cells, myocardium, skin, skeletal muscles as well as on the surface of some blood cells including thrombocytes, erythrocytes, lymphocytes and macrophages (Boersma et al., 2005; Gerke, Moss, 2002). Although physiological significance of An-5 is not fully elucidated, several of its properties are well known (tab. 2).

Table 2. Biochemical functions of Annexin 5 Function Reference anticoagulant activity (Kuypers et al., 2007) regulatory functions for apoptosis (Williamson, Schlegel, 2002) regulatory functions for inflammation (Tritsch et al., 2013) inhibition of microparticle generation (Markoff et al., 2007) regulation of proliferation (Li et al., 2010) regulation of signal transduction (Jones, Howl, 2011) inhibition of phospholipase A2 (Buckland, Wilton, 1998) regulation of calcium homeostasis (Mirsaeidi et al., 2016)

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As early as in the 1990s, An-5 was shown to bind phosphatidylserine (PS) on the surface of activated platelets in a calcium-dependent manner. As so, An-5 competitively inhibits prothrombin binding. It was assumed that An-5 changes it’s conformation when binds to a PS-expressing membrane (Schutters, Reutelingsperger, 2010; Meers, Mealy, 1993) and forms a membrane-bound two-dimensional crystal lattice. Since the 1990, diagnostic methods for detecting PS presence on the external surface of the cell have been developed (Ravanat et al., 1992). Externalization of PS can be demonstrated in vitro using fluorescently labeled An-5, or in vivo employing radiolabeled An-5 in animal models and in humans (Van Genderen et al., 2008; Faust et al., 2009; Lingao et al., 2018). It has been found that a recombinant An-5 has better PS binding affinity than a natural one (Schutters, Reutelingsperger, 2010). However, electron crystallography studies indicated that the membrane-bound form of An-5 monomer has nearly the same structure as the soluble form (Oling et al., 2000). The calcium ions concentration [Ca2+] which is approximately 1 mmol/L favors a rapid binding of An-5 to PS expressed on the cell surface. Andree and coworkers demonstrated that binding of An-5 to PS occurs when the membrane contains a low mole fraction of PS of about 0.22-1.5 mmol/L (Andree et al., 1990). Interestingly, the An-5 binding to phospholipids is not only determined by PS content but also by the presence of phosphatidylethanolamine (PEA). Flow cytometry measurements showed that the minimal mole fraction PS necessary for An-5 binding decreases in the presence of PEA (Stuart et al., 1998). It is also worth mentioning that zinc ions [Zn2+] at concentration of 12-16 µmol/L probably influence the An-5 binding to PS by increasing affinity between An-5 and PS (Rink, Kirchner, 2000). One can therefore assume that An-5 – PS binding depends on the environment in a cell membrane. Although An-5 releasing mechanism is still unexplained, its purpose is known. The protein is localized on the apical surface of all syncytiotrophoblasts and its concentration in blood plasma remains low and constant throughout pregnancy and postpartum. Masuda (Masuda et al., 2004) and coworkers hypothesized that these biochemical conditions in pregnancy suggest that An-5 prevents clotting in the placenta. In addition, it was suggested that An-5 plays a role in extracellular placental compartment in contact with maternal blood (Masuda et al., 2004). Monceau (Monceau et al., 2004) et al. demonstrated that An-5 is externalized at a very early stage of apoptosis and could have a proapoptotic effect in cardiomyocytes. Furthermore, An-5 induces the internalization of tissue factor expressed on the surface of apoptotic THP-1 macrophages (Ravassa et al., 2005). CLINICAL SIGNIFICANCE OF APOPTOSIS IN THE DIAGNOSIS AND THERAPY OF ALLERGIC DISEASES. ANNEXIN 5 IN APOPTOSIS Apoptosis is a tightly regulated cellular process that plays an essential role in the cancer biology and immune response. Apoptosis as ordered and controlled cell death is an equally physiological phenomenon as the aging of the organism as a whole. In contrast, disturbances in the process of apoptosis play a key role in the pathogenesis of many diseases. In some groups of diseases, such as degenerative diseases, the reason is too much apoptosis, while in others, for example in cancer, the cause is insufficient apoptosis. Apoptosis is a very complicated and multidirectional process. A disorder may occur at any stage of the controlled cell death, which can change the course of the disease and manifest symptoms and cause a changed response to treatment. However,

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Emilia Królewicz, Irena Kustrzeba-Wójcicka following the process of apoptosis, its severity and course can provide valuable information about the progress and course of the disease, as well as the effectiveness of the treatment. Therefore, interest in apoptosis is enormous, and the number of publications associated with this issue has been rapidly increasing in recent years, primarily in relation to oncology, as well as virology, parasitology and broadly understood degenerative diseases. In allergic diseases, apoptosis attracts less attention in comparison to other areas of medicine, although from a theoretical point of view the issue is very promising. Even if research limited only to eosinophil, mastocyte and basophil as the key cells of allergic inflammation, learning the mechanisms of induction and the course of apoptosis occurring in them would create completely new diagnostic and therapeutic possibilities. The disruption of apoptotic processes in allergic diseases has been postulated for a long time. So far, the most interest is caused by eosinophils. The first works on the impact of impaired eosinophil apoptosis on the course of asthma appeared at the turn of the century (Brutsche et al., 2001; Fal et al., 2003; Vignola et al., 1999; Woolley et al., 1996). However, this knowledge does not translate into therapeutic use because there are no preparations with satisfactory cellular specificity and safety profile. This applies primarily to corticosteroids, which have long been known to have the ability to induce apoptosis. There are also numerous works on the role of apoptosis in allergic dermatitis (Katsuta et al., 2006; Meyer et al., 2010; Rebane et al., 2012; Trautmann et al., 2001), but this research also does not translate into practical application, although it is known that many drugs affect the apoptosis of cells involved in the pathomechanism of this disease entity (Bhol, Schechter, 2005; Hoetzenecker et al., 2005). Treatment in diseases with excessive eosinophilic activity should be focused on induction of their apoptosis. Theoretically, such a procedure is possible. In 2001, Ruth Belostotsky and Haya Lorberboum-Galski presented a very interesting paper (Belostotsky, Lorberboum-Galski, 2001) showing a possibility of inducing apoptosis on cell lines from different species. The researchers used a chimeric protein called Fcɛ- Bak/Bax, a ligand combination for FcɛRI and pro-apoptotic proteins Bak and Bax (Belostotsky, Lorberboum-Galski, 2001). After internalization of the receptor with the attached Fcɛ Bak/Bax, target cells demonstrated a massive apoptosis, but not degranulation. In the experiment, selective elimination of mast cells and basophils (as cells possessing sufficient FcɛRI density) was demonstrated. Belostotsky and Lorberboum- Galski suggested, that Fcɛ-Bak/Bax chimeric proteins may prove to be powerful agents for the treatment of severe forms of allergy and asthma. It may be possible due to the strong and specific apoptotic effect of these chimeras on the target FcɛRI-positive cells, without triggering degranulation and the immune response. At the beginning of the 21st century, the Siglec-8 protein (sialic acid-binding Ig-like lectin 8) was of particular interest (Floyd et al., 2000). This protein selectively occurs on human eosinophils, and to a lesser extent also on mastocytes and basophils. Siglec-8 inhibits IgE-dependent release of histamine from mast cells and induces apoptosis of eosinophils by activating caspases. However, the potential for the modification of Siglec-8's has not been implemented into practice.

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Even less is known about the possibility of monitoring the course of apoptosis for diagnostic purposes. One of the practical methods for detecting apoptosis in cells is to detect PS presence on their surface (Xiong C et al., 2011; Zhou et al., 2016). In non- apoptotic cells, PS is found almost exclusively on the cytoplasmic side of cell membrane, thus demonstrating its presence on the apical cell surface is a relatively sensitive and specific method to demonstrate the cell apoptosis. A very important advantage of this method is the possibility of using it both in vitro and in vivo. This allows tracking the severity of apoptosis. ANNEXIN 5 IN BAT Basophil activation test (BAT) was carried out for the first time in 1994 by Sainte- Laudy (Sainte-Laudy et al., 1994) and coworkers. The assay was based on the cytometric measurement of the CD63 expression on the basophil surface after stimulation by allergens and anti-IgE. It is known that An-5 is commonly used as a marker for early phase of cell apoptosis, but is not used as a marker for basophil activation. Modern techniques of apoptosis detection are based on the detection of biochemical and morphological changes in the cell. This includes the disappearance of asymmetry of the cell membrane in terms of lipid distribution in the external and internal layer, without compromising the integrity of its structure. Externalization of PS can be monitored using An-5 linked with fluorescein isothiocyanate (FITC), resulting in green fluorescence in flow cytometry imaging. In 2010, Jean Sainte-Laudy and Catherine Ouk (Sainte-Laudy, Ouk, 2010) presented a paper in which they activated human basophils with commonly used stimulating factors: fMLP, anti-IgE and wasp venom. They labeled basophils with common antibodies associated with various fluorochromes, including anti-CCR3, anti- CD63, anti-CD203c, anti-CD11b and An-5. The results of cytometric tests confirmed change of conformation of the basophil cell membrane resulting from their activation with specific and non-specific stimuli(Sainte-Laudy, Ouk, 2010). This experiment encouraged us to verify if An-5 and the expression of PS as its ligand can be used to construct an effective BAT in allegy diagnostics. In 2018 Królewicz (Królewicz et al., 2018) presented the BAT protocol employing An- 5 to demonstrate basophil activation in Alternaria alternata allergy. The study was performed on 65 individuals of whom 32 were sensitized to Alternaria i.e. presented clinical symptoms if exposed to the culprit allergen and demonstrated positive skin prick-test. The remaining 33 allergy-free participants served as controls. In each investigated person the An-5 binding test was performed using three decreasing concentrations of Alternaria allergenic extract: 100 SBU/ml, 10 SBU/ml, and 1 SBU/ml, respectively. Analysis of ROC curves yielded a threshold value of 4.95% activated basophils when Alternaria alternata – sensitized patients were studied with 100 SBU/ml mould extract. A slightly lower test efficiency was demonstrated both for the intermediate concentration of 10 SBU/ml and for the lowest one of 1 SBU/ml allergen extracts. Statistically significant differences were found in the cut-off value expressed in % for all three concentrations (for all tested samples p < 0.05).

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We have demonstrated 90-100% sensitivity and up to 100% specificity in diagnosing allergy to A.alternata. Two similar BAT experiments with house dust mite and rye allergens have been carried out so far at the Medical University of Wrocław (Barg, 2015; Skotny, 2016). Both studies have shown high sensitivity and specificity of BAT using An-5 in activating basophils under the influence of specific allergens in allergic individuals. The results of research by Królewicz (Królewicz et al., 2018) et al. prove that the sensitivity and specificity of the BAT protocol using Annexin 5 are similar to other BAT tests in which commonly known markers for basophil activation are used, among which the CD63 antigen is the most effective and considered the "gold standard" (Kim et al., 2016; Ocmant et al., 2007; Santos et al., 2016; Sanz et al., 2001; Sudheer et al., 2005). Kim (Kim et al., 2016) and coworkers conducted basophil activation tests in a group of atopic and non-atopic patients for autoimmune urticaria diagnosis. To improve basophils identification they used CCR3 vs. CD123 and CD63 vs. CD203c gating strategy in experiments with cells primed or unprimed with IL-3. The researchers recommended a combination of CD123 and CCR3 markers for basophil identification, while CD123 alone may be used as an alternative. Kim et al. have proven that three activation markers on the atopic donor basophils demonstrated 100% test sensitivity: CD203c on unprimed basophils, and CD63+ CD203+ or CD63 alone on primed basophils. The study revealed that CD203c alone or in combination with CD63 is a necessary activation marker. This finding that is consistent with those of previous reports that favor CD203c over CD63 (Ocmant et al., 2007; Sturm et al., 2010; Yasnowsky et al., 2006). It is worth noting that many attempts have been made to show whether CD203c is a better marker compared to CD63 that did not produce conclusive results. It seems that the results depend on how the BAT test procedure is carried out, time and conditions of incubation of samples, type and purity of allergen used and reaction mechanism. Not all scientists use standardized and calibrated allergens for in vitro tests in BAT protocols. Some researchers utilize allergen extracts for skin prick tests and achieve satisfactory results (González-Muñoz et al., 2008). In the case of Hymenoptera venom allergy, the use of the CD203c antigen was slightly more effective as compared to CD63. The test sensitivity was 85% and slightly below 80%, respectively (Sturm et al., 2010). Interesting findings come from Abuaf (Abuaf et al., 2012) and coworkers, who studied influence of non-steroidal anti-inflammatory drugs on the activation of basophils and mastocytes. They demonstrated that the sensitivity of BAT test with acetylsalicylic acid for the CD63 marker was 37%, and the specificity was 90%. Abuaf (Abuaf et al., 2012) and colleagues demonstrated that CD63 was at least 5 times more sensitive than CD203c (P < 0.0001). Nevertheless, Kim (Kim et al., 2016) proved that CD203c should be measured on unprimed basophils. If basophils were primed, CD63 must be included as an activation markers (i.e. either CD63+ or CD203c+CD63+). Therefore, the basophils of non-atopic donors, even after being primed, are not comparable to the primed basophils of atopic donors. Furthermore, unprimed basophils of atopic donors are not comparable to primed basophils of non- atopic donors (Kim et al., 2016).

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The results of BATs to peanuts conducted by Santos (Santos et al., 2016) and colleagues were quite surprising. It was found that different strategies for basophil gating significantly affect sensitivity and specificity. In the case of CD123+/HLADR− gating strategy the sensitivity and specificity reached 88% and 94%, while in the experiment according to the CD203c+/CD123+/HLADR- protocol the sensitivity and specificity were found to be 98% and 96% (Santos et al., 2016). Sanz and Sanchez (Sanz et al., 2001) determined by flow cytometry the percentage of activated basophils after in vitro stimulation by Dermatophagoides pteronyssinus and Lolium perenne allergens and expressing the CD63 marker. It was demonstrated that BAT is a highly reliable technique in the diagnosis of allergy to inhalant allergens. The sensitivity of the BAT was 93.3%, and its specificity 98.4% (Sanz et al., 2001). Ocmant (Ocmant et al., 2007) worked on a comparison of CD63 and CD203c expression using BAT in a model of cat allergy and to determine optimal experimental conditions for both markers. Sensitivity and specificity in both protocols was found to be 100% (Ocmant et al., 2007). Sudheer (Sudheer et al., 2005) and his coworkers from United Kingdom proved that the sensitivity of basophil activation test with CD63 and CD203c for the muscle relaxants was found to be 79% and 36%, respectively and the specificity was found to be 100% (Sudheer et al., 2005). CONCLUSIONS In this paper the chemical structure and biochemical functions of Annexin 5 were presented. This protein is involved in many different processes in human organisms and thus it is possible to use An-5 as a multifunctional marker in various biomedical research. The use of Annexin 5 in allergology research has been of particular interest in recent years. The results presented in the paper proved that An-5 is an effective marker of basophil activation. The sensitivity and specificity calculated in the experiment with extract of A.alternata allergens are in the range of 90-100%. This fact may contribute to the improvement of the diagnostic value of basophil activation tests, which at present are conducted with antigens such as CD63, CD203c and others. The use of An-5 as a new marker for BAT testing would significantly reduce the costs of performing cytometric tests, which have the disadvantage of a high price. This would create an opportunity to introduce basophil activation tests for allergy diagnostics, but would require further standardization and validation. The experiments discussed in this work, initiated by Jean Saint-Laudy and Catherine Ouk and then developed by scientists from Wroclaw Medical University are a novelty in allergy diagnostics. Even though the biological role of An-5 in the process of apoptosis as programmed cell death is known, the number of literature reports on its function in the process of basophil apoptosis caused by stimulation of various allergens in vitro is clearly insufficient. At present, there is only a pilot study evaluating the effectiveness of An-5 compared to the markers commonly used in BAT. It is therefore necessary to carry out further studies directly comparing the effectiveness of the BAT protocol with An-5 and other basophil activation markers in use.

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CONFLICT OF INTEREST The contribution of the authors in the order from affiliation. Authors Emilia Królewicz and Irena Kustrzeba-Wójcicka declare that they have no conflict of interest. ABBREVIATIONS anti-CCR3 – anti-Cys-Cys chemokine Receptor 3 anti-IgE – anti-Immunoglobulin E An-5 – Annexin 5 BAT– Basophil Activation Test CD – Cluster of Differentiation FITC – Fluorescein IsoThioCyanate PE – PhycoErythrin PEA – PhosphatidylEthanolAmine PS – PhosphatidylSerine ROC curve – Receiver Operating Characteristic curve SBU – Standard Biological Units THP-1 macrophages – Tamm-Horsfall Protein-1 macrophages VAC-α – Vascular AntiCoagulant protein α REFERENCES Abuaf N., Rostane H., Barbara J., Toly-Ndour C., Gaouar H., Mathelier-Fusade P. et al., Comparison of CD63 Upregulation Induced by NSAIDs on Basophils and Monocytes in Patients with NSAID Hypersensitivity, In: J Allergy (Cairo), 2012; 2012:580873, https://doi.org/10.1155/2012/580873. Andree H.A., Reutelingsperger C.P., Hauptmann R., Hemker H.C., Hermens W.T., Willems G.M., Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers, In: J Biol Chem. 1990; 265(9): 4923-8. Barg W., Annexin-V binding assay as a marker for the activation of a basophil induced by specific allergens, 2015; DSc thesis, Wroclaw Medical University, (Poland); ISBN: 978-83-7055-471-2. Belostotsky R., Lorberboum-Galski H., Apoptosis-inducing human-origin Fcepsilon-Bak chimeric proteins for targeted elimination of mast cells and basophils: a new approach for allergy treatment, In: J Immunol. 2001; 167(8): 4719-28, https://doi.org/ 10.4049/jimmunol.167.8.4719. Bhol K.C., Schechter P.J., Topical nanocrystalline silver cream suppresses inflammatory cytokines and induces apoptosis of inflammatory cells in a murine model of allergic contact dermatitis, In: Br J Dermatol. 2005; 152(6): 1235-42, https://doi.org/10.1111/j.1365-2133.2005.06575.x. Boersma H.H., Kietselaer B.L., Stolk L.M., Bennaghmouch A., Hofstra L., Narula J. et al., Past, present, and future of annexin A5: from protein discovery to clinical applications, In: J Nucl Med. 2005; 46(12): 2035-50, https://doi.org/46/12/2035 [pii]. Bohn H., Kraus W., Isolation and characterization of a new placenta specific protein (PP10) (author’s transl), In: Arch Gynecol. 1979; 227(2): 125-34, https://doi.org/10.1007/BF02103286.

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Oling F., Santos J.S.O., Govorukhina N., Mazères-Dubut C., Bergsma-Schutter W., Oostergetel G. et al., Structure of Membrane-bound Annexin A5 Trimers: A Hybrid Cryo-EM - X-ray Crystallography Study, In: J Mol Biol. 2000; 304(4): 561-73, https://doi.org/10.1006/JMBI.2000.4183. Ravanat C., Archipoff G., Beretz A., Freund G., Cazenave J.P., Freyssinet J.M. et al., Use of annexin-V to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic balance by endothelial cells, In: Biochem J. 2009; 282( Pt 1): 7-13, https://doi.org/10.1042/bj2820007. Ravassa S., Bennaghmouch A., Kenis H., Lindhout T., Hackeng T., Narula J. et al., Annexin A5 down- regulates surface expression of tissue factor: A novel mechanism of regulating the membrane receptor repertoir, In: J Biol Chem. 2005; 280(7): 6028-35, https://doi.org/10.1074/jbc.M41171020. Rebane A., Zimmermann M., Aab A., Baurecht H., Koreck A., Karelson M. et al., Mechanisms of IFN-γ- induced apoptosis of human skin keratinocytes in patients with atopic dermatitis, In: J Allergy Clin Immunol. 2012; 129(5): 1297-306, https://doi.org/10.1016/j.jaci.2012.02.020. Reutelingsperger C.P.M., Hornstra G., Hemker H.C., Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord, In: Eur J Biochem. 1985; 151(3): 625-9, https://doi.org/10.1111/j.1432-1033.1985.tb09150.x. Richter R.P., Him J.L.K., Tessier B., Tessier C., Brisson A.R., On the kinetics of adsorption and two- dimensional self-assembly of annexin A5 on supported lipid bilayers, In: Biophys J. 2005; 89(5): 3372- 85, https://doi.org/10.1529/biophysj.105.064337 Rink L., Kirchner H., Zinc-Altered Immune Function and Cytokine Production, In: J Nutr. 2000; 130(5 S Suppl): 1407S-11S, https://doi.org/10.1093/jn/130.5.1407S. Sainte-Laudy J., Vallon C., Guérin J.C., Analysis of membrane expression of the CD63 human basophil activation marker. Applications to allergologic diagnosis, In: Allerg Immunol (Paris). [French]. 1994; 26(6): 211-4, https://pubmed.ncbi.nlm.nih.gov/7524523. Sainte-Laudy J., Ouk C., Use of lipid rafting for the analysis of human basophil activation by flow cytometry, In: Inflamm Res. 2010; 59(Suppl 2): S193-5, https://doi.org/10.1007/s00011-009-0126-3. Santos A.F., Bécares N., Stephens A., Turcanu V., Lack G., The expression of CD123 can decrease with basophil activation: Implications for the gating strategy of the basophil activation test, In: Clin Transl Allergy, 2016: 6-11, https://doi.org/10.1186/s13601-016-0100-4. Sanz M.L., Sánchez G., Gamboa P.M., Vila L., Uasuf C., Chazot M. et al., Allergen-induced basophil activation: CD63 cell expression detected by flow cytometry in patients allergic to Dermatophagoides pteronyssinus and Lolium perenne, In: Clin Exp Allergy. 2001; 31(7): 1007-13, https://doi.org/10.1046/j.1365-2222.2001.01122.x. Schutters K., Reutelingsperger C., Phosphatidylserine targeting for diagnosis and treatment of human diseases, In: Apoptosis. 2010; 15(9): 1072-82, https://doi.org/10.1007/s10495-010-0503-y. Skotny A., Annexin-V Binding Assay on the basophil surface in the diagnosis of rye allergy, 2016; PhD Dissertation, Wroclaw Medical University, (Poland). Sopkova-De Oliveira Santos J., Vincent M., Tabaries S., Chevalier A., Kerbœuf D., Russo-Marie F. et al., Annexin A5 D226K structure and dynamics: Identification of a molecular switch for the large-scale conformational change of domain III, In: FEBS Lett. 2001; 493(2-3): 122-8, https://doi.org/10.1016/S0014-5793(01)02285-2. Stuart M.C.A., Reutelingsperger C.P.M., Frederik P.M., Binding of annexin V to bilayers with various phospholipid compositions using glass beads in a flow cytometer, In: Cytometry, 1998; 33(4): 414-9, https://pubmed.ncbi.nlm.nih.gov/9845435. Sturm E.M., Kranzelbinder B., Heinemann A., Groselj-Strele A., Aberer W., Sturm G.J., CD203c-based basophil activation test in allergy diagnosis: Characteristics and differences to CD63 upregulation, In: Cytometry B Clin Cytom. 2010; 78(5): 308-18, https://doi.org/10.1002/cyto.b.20526.

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Sudheer P.S., Hall J.E., Read G.F., Rowbottom A.W., Williams P.E., Flow cytometric investigation of peri-anaesthetic anaphylaxis using CD63 and CD203c, In: Anaesthesia. 2005; 60(3): 251-6, https://doi.org/10.1111/j.1365-2044.2004.04086.x. Trautmann A., Akdis M., Klunker S., Blaser K., Akdis C.A., Role of apoptosis in atopic dermatitis, In: Int Arch Allergy Immunol. 2001; 124(1-3): 230-2, https://doi.org/10.1159/000053720. Tritsch E., Mallat Y., Lefebvre F., Diguet N., Escoubet B., Blanc J. et al., An SRF/miR-1 axis regulates NCX1 and Annexin A5 protein levels in the normal and failing heart, In: Cardiovasc Res. 2013; 98(3): 372-80, https://doi.org/10.1093/cvr/cvt042. Van Genderen H.O., Kenis H., Hofstra L., Narula J., Reutelingsperger C.P.M., Extracellular annexin A5: Functions of phosphatidylserine-binding and two-dimensional crystallization, In: Biochim Biophys Acta. 2008; 1783(6): 953-63, https://doi.org/10.1016/j.bbamcr.2008.01.030. Vignola A.M., Chanez P., Chiappara G., Siena L., Merendino A., Reina C. et al., Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis, In: J Allergy Clin Immunol. 1999; 103(4): 563-73, https://doi.org/10.1016/s0091-6749(99)70225-3. Website of Human Gene Nomenclature Committee. The official nomenclature source of the Human Gene Organization (HUGO) for human genes and their cognate orthologs. Available at: www.gene.ucl.ac.uk/nomenclature [accessed: 12.06.2020 ]. Website of The UniProt Knowledgebase. Available at: www.uniprot.org [accessed: 12.06.2020]. Williamson P., Schlegel R.A., Transbilayer phospholipid movement and the clearance of apoptotic cells, In: Biochim Biophys Acta. 2002; 1585(2-3): 53-63, https://doi.org/ 10.1016/s1388-1981(02)00324-4. Woolley K.L., Gibson P.G., Carty K., Wilson A.J., Twaddell S.H., Woolley M.J. et al., Eosinophil apoptosis and the resolution of airway inflammation in asthma, In: Am J Respir Crit Care Med. 1996; 154(1): 237-43, https://doi.org/10.1164/ajrccm.154.1.8680686. Xiong C., Brewer K., Song S., Zhang R., Lu W., Wen X.L.C. et al., Peptide-Based Imaging Agents Targeting Phosphatidylserine for the Detection of Apoptosis, In: J Med Chem. 2011; 54(6), 1825-35, https://doi.org/DOI: 10.1021/jm101477d. Yasnowsky K.M., Dreskin S.C., Efaw B., Schoen D., Vedanthan P.K., Alam R. et al., Chronic urticaria sera increase basophil CD203c expression, In: J Allergy Clin Immunol. 2006; 117(6): 1430-4, https://doi.org/10.1016/j.jaci.2006.02.016. Zhou H., Wang Q., Yuan D., Wang J., Huang Y., Wu H. et al., Early apoptosis real-time detection by label-free SERS based on externalized phosphatidylserine, In: Analyst. 2016; 141(14): 4293-8, https://doi.org/10.1039/c6an00606j.

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Body donation as the basis for advance in medicine in past and today – meaning, principles, controversies

Jędrzej Siuta1*, Zygmunt Domagała2, Dominika Pluta3, Miron Tokarski3, Dariusz Iwan4, Tomasz Jurek1, Tadeusz Dobosz3 1 Department of Forensic Medicine, Wroclaw Medical University, ul. J. Mikulicza- Radeckiego 4, 50-345 Wrocław Poland; 2 Department of Anatomy, Wroclaw Medical University, ul. Chałubińskiego 6a, 50-368 Wrocław Poland; 3 Department of Molecular Techniques, Wroclaw Medical University, ul. M. Curie-Skłodowskiej 52, 50-369 Wrocław Poland; 4 Museum and Institute of Zoology, Polish Academy of Sciences, ul. Wilcza 64, 00-679 Warszawa Poland. *Corresponding author: Jędrzej Siuta, Department of Forensic Medicine, Wroclaw Medical University, e-mail: [email protected] ABSTRACT It is impossible to imagine the development of medicine without scientific research and educational courses carried out with the use of human corpses. This applies to both traditional medical sciences such as anatomy, pathological anatomy and forensic medicine, as well as modern biomedical research. Although this statement seems obvious, since ancient times up to the present, both cultural and religious aspects, and legal acts based on them, effectively limited the use of corpses for scientific research and teaching purposes. Currently, the main source of acquiring corpses by scientific medical institutions in Poland is body donation, both by authorised entities and the deceased persons themselves, by expressing voluntary will to donate the body before dying. The rules of dealing with human corpses, including the possibilities and conditions of donating bodies for scientific purposes, are strictly defined by legal acts. However, they still raise some controversy among the considerations of ethicists and theologians. The development of programmes of conscious body donation carried out by various universities had a positive impact on the increase in the number of bodies acquired. Nevertheless, the number of corpses at the disposal of medical universities is insufficient. That is why it is extremely important to know the provisions and procedures regarding the principles of handing over the body and to promote the idea of body donation in society by the medical community. INTRODUCTION It is difficult to imagine good medical practice based only on knowledge gained from textbooks. Such knowledge is of course extremely important, but without proper practice it does not bring the expected results. Each future physician should learn the secrets of the structure of the human organism, which are best explored in anatomy lessons in the university dissecting rooms. The development of modern technologies, enabling the use of computerized phantoms, is not able to replace traditional lessons enabling contact with the real human body. Acquiring valuable knowledge in this way would be impossible if people did not sacrifice their bodies for didactic purposes. Nevertheless, body donation was subject to strict legal restrictions from the very beginning of medicine as a modern science. Depending on the era and the evolving worldview of the society, the regulations in force in Poland became more or less restrictive. Similarly, the awareness of people whose open approach to the issue of donation broadens the perspectives of medicine is changing (Estai, 2016; Pilonis, 2012 online; www.rynekzdrowia.pl online, 2019).

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SEARCH STRATEGY AND SELECTION CRITERIA A comprehensive search of medical, legal (including legal acts), sociological, historical and other Polish and worldwide literature, including internet sources, was carried out. The historical and law aspects of body donation are described since the time of creation of the Second Polish Republic (1918) up to the present. The work also contains a description of ethical and religious aspects of body donation, as well as so-called unusual practices associated with donated bodies. REWIEV BODY DONATION – LEGAL ASPECTS IN THE PAST AND TODAY DONATION IN THE PAST The history of body donation for scientific purposes is relatively short. In most of the ancient cultures any postmortem surgery of the body was forbidden due to religious and moral canons. This included, with small exceptions, i.a. Greece, Rome, India or even Egypt (where only closed caste of priests learned anatomy for mummification purposes) (Gulczyński, 2011; Szumowski, 1961; Bilikiewicz, 1978). For this reason, ancient anatomists practiced primarily on corpses of animals, as well as on living soldiers who suffered extensive wounds in battles (Bilikiewicz, 1978). The bodies of convicts, which were exposed to public view after death, were also examined. This type of "donation" (bodies obtained from convicts), though not voluntary, remained the most popular source of acquiring bodies for scientific purposes up to modern times (Bilikiewicz, 1978; BBC online, 2019). In the Alexandrian school, the so-called Vivisections – autopsies started while the "patient" was still alive (Bilikieiwcz, 1978). The first, systematic and typical autopsy of human body was performing in ca. 1300 in Bologna by Mundino de Lucci. During this period, performing an autopsy by a University required special permission from the reigning monarch or pope (the ordinance of Frederick II of 1238 allows the performance of one autopsy over the period of five years, and the first autopsy carried out in Montpellier with the permission of the Pope took place in 1376). Only Vesalius (Andreas Vesalius), thanks to numerous post-mortem examinations carried out on corpses (illegally) stolen from the cemetery, showed irregularities in the science of anatomy based on animal autopsies by finding over 200 mistakes in the works of the so-far regarded as the almost infallible Galen (Bilikiewicz, 1978; Szumowski 1961; Gulczyński, 2011). In the following years, there was a slight increase in the number of scientific autopsies carried out at universities. However, they were relatively rare, and students who wanted to practice anatomy had to acquire bodies illegally. To some extent, this has remained that way to this day, where the first year students often still have to buy natural human skull for anatomy lessons from gravediggers, which of course is a crime (Bilikiewicz, 1978; Wyborcza online, 2018). In Poland, one of the first surviving protocols of autopsy concerns the autopsy of King Stefan Batory, conducted by Mikołaj Bucella and Jakub Gosławski. The first purely scientific, anatomical and public autopsy was performed in 1613 by Joachim Oelhafius. At that time it was Gdańsk and Toruń, that were at the forefront of the science of anatomy. In other parts of Poland, although anatomy lessons or even publishing anatomical works were also taught, the autopsies for didactic

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Body donation as the basis for advance in medicine in past and today – meaning, principles, controversies purposes began to be carried out mostly at the end of the 18th century (Moraczewski, 1842; Bilikiewicz 1978). Establishing an anatomical theatre in Warszawa in 1777 caused protests of the outraged population, from whom the doctor conducting autopsies with the students had to run away. At the beginning of its operation, the anatomical theatre, run from approx. 1780 as part of the Collegium Medicum in Kraków by Rafał Czerwiakowski, had to be guarded. At that time, bodies were obtained from hospitals and bought (legally, according to the "records of expenditure on the anatomy school" in Kraków run by Wincenty Szaster) from gravediggers. Bodies of convicts were also obtained. Specimens with preserved fragments of the human body for scientific purposes were also produced (Bilikiewicz, 1978; Giżewski, 1978; Leociak, 2005; Moraczewski, 1842). In the 19th century, there was a significant development in the field of learning anatomy on the basis of human corpses and preparations obtained from them. At that time anatomical museums were created (with exhibits in the form of preparations made from human organs), from which some of the exhibits have survived to this day (Pluta, 2016). In the reborn Poland, the issue of body donation for scientific purposes was initially regulated by the Circular of the Minister of Public Health dated 29 January 1923 regarding the introduction of human corpses to university institutions for scientific purposes. It stated, among others, that bodies from hospitals, anatomy laboratories, prisons, nursing homes, foundlings houses or "other similar institutions" may be handed over to anatomy facilities of state universities for scientific purposes if the family of the deceased did not request to bury their body within 48 hours of death. The circular also specifies the principles of transport and preservation of the bodies. The previous Basic Sanitary Act of 19th July 1919 (Journal of Laws – Dz. PR. P.P. 1919 no. 63 item 371) did not directly define the principles of body donation. The next Act, which further regulated the rules of donation, was the Act of 17 March 1932 on Burials and Determining the Cause of Death (Journal of Laws – Dz. U. of 1932, no. 53, item 359). The Act stated that "Poviat authorities of general administration may hand over bodies that were not buried by the family to university units for scientific purposes", whereas the family, as in the Act currently in use, was considered to be a spouse, ascendants, descendants and lateral relatives up to the fourth degree of consanguinity as well as first-degree relatives by marriage in direct line. In some cases, the appropriate military and religious authorities were also considered to be "families" within the meaning of the above provision. It was also required – as it is now – to enter the declaration and cause of death into the death certificate. The Act was in force until 1959, when it was replaced by the Act on Cemeteries and Burials (Journal of Laws – Dz.U. of 1959, no. 11, item 62, as amended). DONATION IN POLAND TODAY Nowadays, the rules of dealing with human corpses, including the possibilities and conditions of donating bodies for scientific purposes, are strictly defined by legal acts. The basic one is the Act on Cemeteries and Burials (Journal of Laws – Dz.U. of 1959, no. 11, item 62, as amended), which defines, among others: the functioning of cemeteries, rules of transport and storage of bodies, rules of issuing a death certificate, rules of exhumation, and – most importantly in the context of this paper – principles of donation for scientific purposes and persons authorised to bury bodies.

189 Jędrzej Siuta, Zygmunt Domagała, Dominika Pluta, Miron Tokarski, Dariusz Iwan, Tomasz Jurek, Tadeusz Dobosz

Persons authorised to bury bodies according to the abovementioned acts are: the spouse of the deceased, ascendants and descendants as well as lateral relatives up to the fourth degree of consanguinity and first-degree relatives by marriage in a direct line. Relatives by marriage are the spouse's family, i.e. people who are not biologically related. In addition, the right to burial is in some cases granted to military and state authorities as well as social institutions and organisations, and "to any person who undertakes to do so", even if completely unrelated to the deceased. The list of people who potentially have the right to bury is really extensive, and in accordance with Art. 10.2 of the cited Act, only bodies that were not buried by the abovementioned entities (persons or institutions) may be handed over to a "public medical university or public university conducting teaching and research activities in the field of medical sciences" for scientific purposes. Therefore, it is not a matter that can be resolved easily and "automatically", and thus this procedure is not implemented by local authorities and universities often enough. The situation is much simpler when the deceased issued a written statement on the will to donate his/her body to an appropriate university before his/her death (Art. 10.6 of the cited Act) - then the will of the deceased is more important than the right or will of other people. Unfortunately, the lack of awareness of the importance of scientific work on human corpses for educational purposes and knowledge development, the lack of knowledge of legal regulations in society as well as cultural and religious barriers make this form of donation occur much too rarely (Klinger, 2017). The act also specifies, among others, the need to enter – before handing the body over to the university – the full cause of death of the deceased into the death certificate "containing an endorsement of the registration of death from the civil registry office" and the need for the university to bear the costs of body transport. The other legal acts related to and resulting from the cited Act on Cemeteries and Burial, specifying the rules of dealing with human corpses, are regulations of competent ministers, such as: Ordinance of the Minister of Health of 7th December 2001 on dealing with human bodies and remains (Journal of Laws – Dz. U. of 2001, no. 153, item 1783, as amended), Regulation of the Minister of Justice of 7th July 2010 on dealing with corpses of persons deprived of liberty who died in prisons and detention centers (Journal of Laws – Dz. U. of 2010, no. 123, item 839). Regulation of the Minister of Internal Affairs of 22nd January 2015 on dealing with corpses of foreigners placed in a guarded centre or custody for foreigners (Journal of Laws – Dz. U. of 2015, item 179), or finally the Regulation of the Minister of Health of 30th July 2009 on the procedure and conditions for the donation of bodies for scientific purposes (Journal of Laws – Dz. U. of 2009, no. 129, item 1067). Important additional information contained in the cited regulations include the possibility of transferring the body of a foreigner who died in a guarded center or detention centre for foreigners if the body is not picked up by the entitled persons within 14 days from the date of death and the body of a person deprived of liberty who died in a prison or detention center for whom the entitled persons do not express the will to bury their body within 72 hours of death. The process of donating bodies for scientific purposes must be carried out in accordance with other provisions of the cited acts and "in a way that ensures dignity due to the deceased". The regulations do not specify the time for which a university can store the body received by way of donation – if the person who

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Body donation as the basis for advance in medicine in past and today – meaning, principles, controversies donated his or her body to a university before death does not stipulate otherwise, the body is donated to the university "forever". The purpose of using the body by a university must be "scientific" – it means that in Poland it is not possible to use bodies for commercial purposes. It should be noted that any deviation from the abovementioned provisions can be treated as a crime – i.a. insulting the corpse which is described in Art. 262 of the Criminal Code (Journal of Laws – Dz. U. of 1997 no. 88 item 553, as amended). This also applies to accepting any material benefit for the donation of bodies for scientific purposes which is equivalent to the trade of human bodies or remains, which is prohibited in Poland. In addition, the family of a person whose body was donated for scientific purposes usually loses the right to funeral grant, which is motivated by the fact that they do not deal with the organisation of the funeral and thus do not bear its costs. In practice, however, it is possible to get a funeral grant in such a situation, for example for the so-called symbolic burial which does not require the physical burial of the deceased. According to the articles in the popular press, this issue is controversial for potential body donors and their families – these controversies, however, most often result from the lack of information in the society (Bodył, 2016). Another issue that is often confused – as results from the authors' observations and research of various online forums – is mistaking the notion of body donation for scientific purposes for the lack of objection to postmortem organ transplants. It should be clearly stated that these are entirely different issues, regulated by separate legal acts (Journal of Laws – Dz.U.2017.1000). A positive trend in the field of donating bodies for scientific purposes, observed in recent years, is the development of programmes of conscious body donation by medical universities. The first such programme was launched in 2003 by the Medical University of Silesia in Katowice. Currently, it is also run by numerous other medical universities or universities with medical faculties, such as: Jan Kochanowski University in Kielce, Jagiellonian University Medical College in Kraków, University of Warmia and Mazury in Olsztyn, Medical University of Lublin, Medical University in Białystok, Wrocław Medical University and others. Each university has its own rules of the donation programme, however, the differences between the programmes are rather small. By taking part in the programme of conscious donation, a given person donates his or her body to a specific university by filling out an appropriate certificate while alive. In this situation, in accordance with the regulations, the donor may sometimes state additional wishes in the act of body donation, e.g. regarding the place of burial of the body after it is used by a university or placing the donor's personal information on a comme- morative plaque (SUM in Katowice online, 2020; CMUM in Kielce online, 2020; CMUJ in Kraków online, 2020; UMW in Olsztyn online, 2020; UM in Lublin online, 2020; UM n Białystok online, 2020; UM in Wrocław online 2020). It should be noted that only a public university can participate in the process of body donation (which results from Art. 10.2 of the Act on Cemeteries and Burial (Journal of Laws – Dz.U. 1959, nr 11, poz. 62 as amended art. 2)). This provision completely marginalises the ability of private medical universities to practice anatomy. The above programme had a very positive impact on the availability of bodies for students, which is why it became popular among medical universities. The factor that has a positive influence on the number of body donors is also the official position of the Catholic church (which is

191 Jędrzej Siuta, Zygmunt Domagała, Dominika Pluta, Miron Tokarski, Dariusz Iwan, Tomasz Jurek, Tadeusz Dobosz the largest religious association in Poland), which fully allows the donation of bodies and their autopsy for scientific purposes. In addition, it is a saint of the Catholic church, St. Franciszek Salezy, that is sometimes mentioned as one of the first people in history who were a potential donor of their own body for scientific purposes (Morciniec, 2009; SUM online, 2018). BODY DONATION – RELIGIOUS, ETHICAL AND LEGAL ASPECTS. Human corpses are material remains of a person, the visible form in which a given individual appeared during his or her lifetime. It is worth noting that most ethical and moral standards derive from some sort of a chain of our affiliation to the Homo sapiens species. This affiliation does not necessarily have to end at the moment of death of a given person but usually also continues after death, as long as the corpse is suffi- ciently intact. Piotr Morciniec states that this affiliation constitutes certain moral laws that are inalienable for men also after death, thus constituting a kind of "species dignity" which concerns a dead body (Morciniec, 2009). Although, as Morciniec himself claims, ontologically corpses are neither a person nor a living creature – even when not all cells that build the body have died – but more of a material object, unlike things they may not only be damaged but also injured. This is particularly important if bodies are dismembered and exhibited, and thus ridiculed. Such activities are directly prohibites because people who are still alive have direct moral obligation towards dead people. Thus, it is morally necessary to treat the body of the deceased with respect (Morciniec, 2009; Birnbacher, 1998). On the other hand, F.A. Ingelheim postulates in his paper a principle derived from the above beliefs, the retaining of which is necessary in the case of medical examinations on bodies: "during the examination of corpses, the rules of medicine and medical ethics must be followed as closely as while treating living patients. There is the same obligation to protect the interests of the dead as the interests of the living" (Ingelheim, 1981). Similarly to the general ethical norms, the church doctrine discusses the matter of handling human body after death. Justified by the anthropological foundation of "God's image and likeness to God", the belief in the existence of a particle of the Creator in every body brings with it a modification of reference to this body also after the death of a human being. That is why it should not be accepted to treat the human corpse in a way that offends the dignity of God's creation or as useless remains of the body after death. As in the case of general moral norms, also in theological tradition a consistent reference to the theology of redemption and resurrection is clearly indicated as a justification for treating bodies of deceased persons with respect (Katechizm KK., 1994). Nevertheless, further consideration of this issue leads to the conclusion that the Christian resurrection is based on the belief in the resurrection of a transformed body and not literal resurrection of a cadaver to life. Historically, the belief in the need to treat the body with dignity and respect is a common denominator of different eras and religious traditions. Of course it should be remembered that these attitudes were not always respected. This problem will be discussed in more detail in the next chapter. As it has been already mentioned, unworthy treatment of human corpses consists primarily in the violation of the dignity of the deceased, which is thereby equivalent to the dignity of the person who was the body. This attitude gives rise to a very important ethical context that human corpses are

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Body donation as the basis for advance in medicine in past and today – meaning, principles, controversies inseparable from the human being who was previously an individual, and the disruption of this relationship inevitably leads to a refusal to respect human remains. This does not mean, however, that the position of the Church in this matter remained unchanged. Initially, for a long time in the ethical-moral catholic doctrine prevailed the position derived from a form of pietism – respect for the human body after death and preserving the memory of the deceased. Currently, these features also include the principle of solidarity, which is about helping the sick and suffering through the use of corpses for scientific research. Therefore, instead of the categorical opposition to violating the integrity of the human body after death, today the Church permits such activities as far as they bring help to one's neighbor (Conte a Coronata, 1951; Katechizm KK, 1994; Morciniec, 2009). In addition, it is also required that the scientific unit, after the time specified in the act of donation, organises a Catholic funeral, in which the family of the deceased may attend. Catholic ethicists point out, however, that it is unethical to accept that institutions or persons obliged to make funerals decide on handing over the body for scientific or didactic purposes on their own (Majer, 2016). Currently, both from the ethical and moral point of view as well as considering the formal and legal requirements for conducting scientific research on human corpses or using organs and tissues for didactic purposes, it is believed that such studies are allowed only with an ante-mortem consent of the person concerned. Such consent must be informed and expressed explicitly in a written declaration of will, which is the first and at the same time necessary condition for such activities to be ethically and legally accepted. It is not possible to use an implied consent (based on lack of objection) under the provisions of the Act on the collection, storage and transplantation of cells, tissues and organs in order to use parts of the body for scientific or didactic purposes (Nesterowicz, 2009; Żelichowski, 2007; Journal of Laws – Dz.U.2017.1000). An issue necessary to consider is the potential objection of the relatives of the deceased to donate the body. It seems that the objection should be respected only in cases that leave no doubt – when the planned studies could offend the dignity of the human body or due to changes in the values of the donor which were not included in the donor's declaration of will (Nesterowicz, 2009; Żelichowski, 2007; Journal of Laws – Dz.U.2017.1000). However, some authors believes that "one cannot conclude a priori that handing over human corpses for research purposes is against the personal well- being - the cult of a loved one" and the act of donation drawn up on the basis of Art. 10 sec. 5 of the Act of 1959 on Cemeteries and Burials (Journal of Laws – Dz.U. 1959, nr 11, poz. 62 with later change) is a legally permissible restriction of personal rights of the relatives of the deceased, who cannot oppose to the deceased (Krekora-Zając, 2017). The explanation of such position was supposed to be the notion of a noble purpose – consistent with the general welfare of mankind. The author even goes to accept the existence of an obligation of relatives to hand the body over to a scientific unit in accordance with the act of donation and the right of this unit to a contractual claim in the event of not meeting such obligation (Krekora-Zając, 2017). Some scientists believe that the current or amended legal provisions limit the progress of medical science due to the inability to obtain biological material from corpses which

193 Jędrzej Siuta, Zygmunt Domagała, Dominika Pluta, Miron Tokarski, Dariusz Iwan, Tomasz Jurek, Tadeusz Dobosz is extremely important from their point of view. It is impossible to create extensive anatomical collections and conduct multiple scientific research using the organs or tissues of the dead without their prior consent (Nesterowicz, 2009). This situation is rightly criticised by them, calling for the introduction of a wording: "Collection of cells, tissues or organs from human corpses for their transplantation or for diagnostic or scientific purposes can be done if the deceased person did not express opposition during his or her lifetime" (Nesterowicz, 2009; Krekora-Zając, 2017) into Art. 5 sec. 1 of the Transplantation Act. This would allow the scope of the implied consent of the potential donor to be extended beyond medicinal purposes while maintaining the consent granted "explicitly" for didactic purposes. The question should definitely be asked whether using bodies of the deceased is the only possible way to acquire new knowledge. Some people, especially bioethics, but not researchers of medical sciences, believe that with the technological advances of laboratory methods, the use of human corpses may be limited (Krekora-Zając, 2017; Morciniec, 2009; Birnbacher, 1998; Meller, 2007). However, it may not. The possibility of extensive interference in the body of the deceased and collecting material impossible to obtain from living people opens new clinically unavailable research perspectives. Much more tissues can be collected form a deceased person, tissues that are difficult or even impossible to collect from living persons without the risk of causing death. The progress of research methodology and technological revolution in medicine will not replace the access to biological samples – on the contrary, it "demands" material from a human being that could be examined. While the introduction of legal provisions prohibiting the use of human corpses in cases where the results of potential research may be obtained otherwise seems reasonable, complete exclusion of the possibility of scientific examination of corpses is like "shackles" for science, which is, after all, developed for the good of the entire population (Pounder, 2005; Besley 2016, Kaliszan 2019; Nesterowicz 2009). BODY DONATION – UNUSUAL PRACTICES When considering the issue of donation in the context of legal, ethical and moral norms, activities that are on the edge of all the rules discussed cannot be omitted. It is worth noting that despite the enormous contribution that medical science made or is still making, it still causes huge controversies and discussions (Mitscherlich, 1963; Meler, 2014). The first and the most shocking example is all research carried out during the Second World War. Until 1933, there was a huge problem with access to corpses for scientific purposes on the territory of the Third Reich. Only later the rule of NSDAP, that restored the death penalty, changed the situation. Nevertheless, the number of donations was still low (as a result of legal regulations), which affected the troublesome situation of medical universities. It is worth noting, that bodies of prisoners after decapitation were mostly preferred (Meler, 2014; Mitscherlich, 1963). The situation changed only during World War II, when the extermination policy of the Third Reich resulted in sending masses of people to concentration camps, extermination camps, prisons and shelters. According to the Regulation of the Minister of Justice of the Third Reich dated 18 February 1939 (Meler, 2014), corpses were transported (mainly from penal prisons) to specific scientific institutions in Germany,

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Body donation as the basis for advance in medicine in past and today – meaning, principles, controversies and after the occupation of Poland, then the authorities created subsequent places of execution also on the incorporated lands. Anatomical institutes extremely objectified the received bodies and treated them inhumanly, carrying out the inhuman procedure before the eyes of the whole world. Nevertheless, in the discussed period there was a significant development of medical sciences, also in Poland (Meler, 2014). In addition to typical autopsy practices, organs or tissues were also dissected, which were later preserved and closed in sealed jars. Many collections of anatomical preparations were created this way and they are exhibited in university museums to this day, among others in Poland in Wrocław Medical University or Medical University in Kraków. These exhibitions are particularly valuable due to their unique character, because for bioethical reasons, no more such exhibits will be made (Pluta, 2017). It is also worth to mention the cruel medical practices that were used in concentration camps, both on living people and corpses, breaking all the principles of humanity and bioethics. The described activities from 1945-46 were referred to the International Military Tribunal at Nuremberg by conducting 12 trials, including the trials of doctors participating in this inhuman procedure (Spitz, 2014). In the context of donation, one should also discuss the recently popular exhibition of human bodies subjected to the plastination process. The author of the technique is a German physician, pathologist and anatomist – Gunther von Hagens, who has been constantly improving his procedure since obtaining the patent (1977-1982). The donation programme run by his Plastinarium was launched in the 80s of the twentieth century, to date it has gathered over 14 thousand donors. Persons interested in donating their body for plastination are required to fill in a document, which is called not a "contract", but "a declaration of will", subject to immediate cancellation at the time of the donor's resignation (Plastinarium, 2020). The German scientist is also the originator and initiator of the series of exhibitions entitled "Body worlds" which are being presented all over the world since 1995 (also in Poland), both causing countless protests and arousing huge interest. The preparations collected in this way are not used for the training of medical staff and were made public on a large scale for everyone interested. Therefore, it is a typically commercial activity, although according to the assurances of the organisers, the above activities are aimed primarily at raising public awareness and broadening the knowledge of the human organism (Czakon, 2012). An unusual example of donations for typically scientific purposes are the so-called "Body Farms". Currently there are several of them in different locations, although the first and also the most famous of them was established in Knoxville (USA) on the initiative of an outstanding anthropologist Dr. William M. Bass. In this unique place founded in the autumn of 1980 and covering around 2 ha, research is carried out on the decomposition of corpses in the natural environment. These observations are extremely useful for determining the time elapsed from the death to the time of finding corpses, which is used in many investigative cases. The first body was donated to scientists in 1981 and it is estimated that from this point up to 1999, research was carried out on more than 300 corpses. It was also calculated that more than 100 people sign the act of donation for the Body Farm every year (Gawliński, 2016; Bass, 2012). There are also projects to create a Body Farm in Poland (what is also a will of authors of this work), but for now this is not allowed by law (Pruszkowska, 2012).

195 Jędrzej Siuta, Zygmunt Domagała, Dominika Pluta, Miron Tokarski, Dariusz Iwan, Tomasz Jurek, Tadeusz Dobosz

CONCLUSION The importance of research carried out on human corpses is invaluable for the development of medical science. This applies not only to forensic medicine and pathology, which are typically associated with autopsy and examination of tissues collected from the human body, but also to almost all other branches of medicine. As the medical practice shows, replacing the human body with other models, including animals, computer models or various manikins, which is widespread in science and didactics, ultimately always requires verification by carrying out activities on the human body. Anatomy – the foundation of medical science – cannot be learned well solely only from textbooks; this also applies to other branches of medicine, such as pathology and forensic medicine. It should be noted that both medical-legal and pathological autopsies performed as part of didactic classes for medical students often do not allow any extension of autopsy techniques or the use of bodies for experimental purposes and thus are not able to replace bodies donated to the universities. The need to carry out extensive didactic and scientific activities on human corpses generates the eternal problem of acquiring bodies that could be used for this purpose. The moral and religious canons, and often the legal aspects resulting from them, have always been an obstacle to the use of corpses. Over the last century, this state of affairs has improved significantly, which – it is worth noting – coincided with the significant development of medical sciences. Currently, the main source of acquiring corpses by scientific medical institutions in Poland is body donation, both by authorised entities and the deceased persons themselves, by expressing voluntary will to donate the body before dying. In Poland, the development of programmes of conscious body donation run by various Universities was the factor that most effectively contributed to the increase in the number of people donating their bodies for medical scientific research. The number of bodies donated to universities is still too low to fully cover the existing needs, therefore it is important that doctors know the principles and possibilities of body donation in Poland and promote them not only among their patients, but also wherever possible. REFERENCES Estai M., Bunt S., Best Teaching Practices in Anatomy Education: A Critical Review, In: Ann Anat. 2016; 208: 151-157. Pilonis H., Za ścianami prosektorium, In: Medonet.pl 2012; https://www.medonet.pl/zdrowie/zdrowie- dla-kazdego,za-scianami-prosektorium,artykul,1663565.html [accessed on 07.07.2020]. https://www.rynekzdrowia.pl/Nauka/Specjalista-lekarz-musi-oswoic-sie-z-cialem-zmarlego,199983,9.html [accessed on 07.07.2020]. Gluczyński J, Iżycka-Świeszewska E., Wspólne początki anatomii weterynaryjnej i anatomii człowieka, In: Felsmann Z.M., Szarka J, Felsmann M. (eitors), Dawna medycyna i weterynaria, 2011 Muzeum Ziemi Chełmińskiej w Chełmnie (Chełmno, Poland) 2011; pp. 469-483, ISBN: 83-919037-2-9. Szumowski W., Historia medycyny, In: PZWL 1961 (Warszawa). Bilikiewicz T., Rys historii anatomii, In: Bochenek A. Reicher M. Anatomia człowieka T.1, 1978; PZWL (Warszawa, Poland). https://www.bbc.com/news/health-49294861 [accessed on 07.07.2020]. Giżewski D., Pozycja Jana Tomickiego w anatomii polskiej XVII wieku, In: Kwartalnik Historii Nauki i Techniki 1978; 23/1: 101-118.

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Birnbacher D., Philosophisch-ethische Überlegungen zum Status des men – schlichen Leichnams, In: N. Stefenelli (editor), Körper ohne Leben: Begegnung und Um- gang mit Toten. 1998; Böhlau Verlag (Wien, Austria). ISBN: 978-3205988830. Ingelheim F.A., Zur geschichtlichen Entwicklung des ärztlichen Leichenschaubestimmungen in Deutschland, Med. Dissertation. Uni.Marburg. 1981. Katechizm Kościoła katolickiego, 1994; Pallotinum (Poznań, Poland), pp. 2300-2301. ISBN: 978-83- 7014-430-2. Conte a Coronata M., Institutiones iuris canonici ad usum utriusque cleri et scholarum, vol. 2: De rebus. 1951; (Taurini-Romae, Italy). Majer P., Pogrzeb symboliczny po donacji zwłok na cele naukowe – aspekty prawno kanoniczne, Annales Canonici 2016; 12: 91-114. Nesterowicz M., Śliwka K., Pobieranie ze zwłok komórek, tkanek i narządów do celów naukowych – problem prawny i medyczny [online], Prawo i Medycyna, 2009; 4: 5-12, [accessed on: 2018-02-01 21:32], Accessible on the Internet:mhttps://sip.lex.pl/#/publication/151103899. Żelichowski M., Pobranie komórek, tkanek i narządów: zgoda jest regułą, sprzeciw wyjątkiem [online], Rzeczpospolita – Prawo co dnia, 2007;. 9: 25, [accessed on: 2018-02-01 21:45], Accessible on the Internet: https://sip.lex.pl/#/publication/151058147. Krekora-Zając D., Akt donacji zwłok ludzkich (uwagi de lege lata i de lege ferenda) [online], Państwo i Prawo, 2017; 11;85-98, [accessed on: 2018-02-01 22:04], Accessible on the Internet: https://sip.lex.pl/#/publication/151323917 Meller J., Moralny aspekt wykorzystania zwłok ludzkich [online], Studia Gdańskie, 2007; XXI [accessed on 07.07.2020]. Pounder D.J., Postmortem interval, In: Payne-James J. (editor) Encyclopedia of Forensic and Legal Medicine, 2005; Elsevier (Oxford, UK); ISBN 9780128000342. Besley S.L., Flanagan R.J., Postmortem Biochemistry: Current Applications, J Forensic Leg Med. 2016; 41: 49-57. Kaliszan M., Teresiński G., Metody ustalania czasu zgonu, In: Teresiński G. (Editor) Medycyna sądowa T.1, 2019; PZWL (Warszawa, Poland); ISBN: 978-83-200-5856-7. Mitscherlich A., Mielke F., Nieludzka medycyna: dokumenty procesu norymberskiego przeciwko lekarzom, 1963; PZWL (Warszawa, Poland). Meler J., Wykorzystywanie zwłok więźniów do badań w instytutach anatomicznych III Rzeszy, Studia Iurdica Toruniensia 2014; 14: 197-224. Spitz V., Doktorzy z piekła rodem. Przerażające świadectwo nazistowskich eksperymentów na ludziach, 2013; Replika (Zakrzewo, Poland). ISBN: 978-83-767-4547-3. Plastinarium, Dawcy ciał, 2020, http://www.plastinarium.de/pol/plastinariump/dawcy_cial.html [accessed on: 14.06.2020 ]. Czakon D., Body Worlds Guinthera von Hagensa, Estetyka i Krytyka 2012; 1(24):223-230. Gawliński A., Nekrofilia jako problem interdyscyplinarny, Studia Prawnoustrojowe 2016; 34: 35-48. Bass B., Jefferson J., Trupia Farma. Sekrety legendarnego laboratorium sądowego, gdzie zmarli opowiadają swoje historie, 2012; Wydawnictwo Znak, (Kraków, Poland); pp. 49-51, 93, 110, ISBN: 978- 83-240-4662-1. Pruszkowska K., Czubak A., W Polsce "Trupich Farm" nie będzie, 2012; https://menway.interia.pl/ obyczaje/nauka/news-w-polsce-trupich-farm-nie-bedzie,nId,596130 [Accessed on 07.07.2020].

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An image of a first-year student at the Faculty of Medicine. Qualitative research based on written work

Kamila Łampika* Wroclaw Medical University, Wybrzeże L. Pasteura 1, 50-367 Wrocław, Poland Corresponding author* Wydział Lekarski, Zakład Humanistycznych Nauk Lekarskich, ul. J. Mikulicza-Radeckiego 7, 50-368 Wrocław, tel.: 71 784 01 02, e-mail: [email protected], Kamila Łampika e-mail: [email protected] ABSTRACT: The development of biomedicine depends on the people who work and will work in this area. People in occupational groups are expected to have specific personality traits. The article presents a qualitative research based on an analysis of 380 written essays of first-year students of the Faculty of Medicine at the Medical University in Wroclaw. Written papers were prepared as a part of the seminar on the subject of medical ethics. First-year students of the Faculty of Medicine took part in the seminars in the winter semester 2019/2020 divided into 16 seminar groups. Each student had several weeks to complete the assignment and provide a typescript. The works had on average from 4 to 8 pages. The main goal for the student was to analyse a fragment of the court decision provided by the teacher in terms of compliance of medics' behaviour with the Code of Medical Ethics in force in Poland. In cases, patients demanded compensation for the damage they had suffered as a result of medical interventions. The student was asked to refer to a specific article of the Polish Code of Medical Ethics and motivate his/her position. The article gives an insight into the attitudes presented. The form of completed work proves compliance with the rules, shows diligence, accuracy and meticulousness when performing a task. Analysing the content, we see a similar level of clarity in expressing thoughts in writing. Different levels of argumentation skills and logical conclusions. There are assessments and opinions in the works. Students share their own thoughts and even experiences. They express empathy, and sometimes identify with patients or the professional group of medics. Based on the collected research material features common to the group of students – future medics – were distinguished. INTRODUCTION Biomedicine is a rapidly growing field of science thanks to the people who create it. Young people starting their studies in medical fields may have some predispositions to pursue the professions in which they started their education. The subjective factors significantly affect the level of the professional satisfaction achieved. Satisfaction is an important subjective feeling of an employee, leading to many positive effects in human functioning at work. These positive effects can directly translate into the benefits of the organization, for example, they increase commitment to the duties performed (Letwak, 2012). Patients and other people making up this professional group will expect commitment from current students who will fulfil themselves professionally as physicians (Bajcar, 2011). Of course, expectations towards doctors are much broader. In the future, society and, above all, other people who make up this professional group will have expectations of the behaviour of these people practicing the medical profession. Persons who form the professional group of doctors should carry out their activities in accordance with the medical code of ethics. We understand the Code of Ethics as a specific set of professional ethics standards that has been officially announced by an authorized body and applies – under specific sanctions – in a given professional

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Kamila Łampika environment. The Code is a narrower concept than professional ethics in the sense that it contains very specific norms, written in the form of an order or prohibition. Therefore, the Code does not contain a broad analysis of moral motivation and axiological background. Instead, it covers all areas and areas of interest to professional ethics (Sikora, 2002). The principles of medical ethics currently in force in Europe and in many other countries in the world are derived from the laws written by the Greek physician, Hippocrates, in the 5th century BC. As R. Gillon has written: "a particularly valuable role in medical and other health care ethics for it systematises the traditional core components of medical ethics (what I am inclined to call the Hippocratic moral commitment of providing health benefits with minimal harm) and adds to them the key concerns of respect for autonomy and justice, neither of which have, until fairly recently, been notable commitments in medical ethics" (Gillon, 2003). Today, all graduates of medical faculties in Poland make a medical promise, which is included in the introduction to the Polish Code of Medical Ethics (further in the text as KEL). The Medical University of Silesia in Wroclaw in the academic year 2018/2019 as a part of full-time uniform studies at the Faculty of Medicine 1749 people were educated, including 1073 women. (Central Statistical Office of Poland, 2019). Students prepare for the profession before starting their career they will make a promise: "I accept with respect and gratitude for my Masters the title of doctor granted to me and fully aware of my duties, I promise: • these duties are conscientiously fulfilled; • serve human life and health; • to the best of my knowledge, prevent suffering and prevent disease, and help the sick without any differences, such as race, religion, nationality, political views, property status and other, aiming solely for their well-being and showing them due respect; • do not abuse their trust and keep medical confidentiality even after the death of the patient; * protect the dignity of the medical condition and do not stain it with anything, and treat their fellow doctors with their due kindness, without undermining their trust in them, but acting impartially and with a view to the good of the sick ; • constantly broaden my medical knowledge and make available to the medical world everything that I can invent and improve". Are first-year medical students ready to learn about the principles of medical ethics written at KEL? It can be argued that the sooner a student becomes familiar with and understands the essence of the principles contained in KEL, the faster the chance to become aware of which personality traits should be developed. (Barański, 1998) The study is based on the written work of first-year students of the medical faculty based on knowledge of KEL. Based on the texts, the author of the study attempts to interpret the statement and assign three features to a group of students. These are the ability to 1. meet formal conditions, 2. reflectiveness and 3. emotionality.

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An image of a first-year student at the Faculty of Medicine. Qualitative research based on written work.

MATERIALS AND METHODS The materials analysed are written essays of the first-year students of the Faculty of Medicine at the Medical University of Silesian Piasts in Wroclaw. 380 students were divided into 16 seminar groups. On average, the group consisted of 24 people. As part of the subject of medical ethics, students were obliged by the teacher to write an essay. The teacher provided materials in the form of cases, which are fragments of court decisions, taken from the database available on-line www.saos.org.pl. Each of the 16 groups received a different case. Sample judgments: Ref. act II K 496/17, reference number act VII Ka 68/19, reference number act XII C 1793/17, reference number act I C 947/17. After learning about the case, the student was to analyse the correctness of the behaviour of medical workers in the context of the requirements of the Polish Code of Medical Ethics. The teacher formulated the editorial requirements thoroughly. Students had three to six weeks to complete the task. The essay was to consist of two parts. The first part is a description of the facts, i.e. the isolation of the most important events described in the case study. Below is an example of the correctly prepared first part: "The facts presented in the case report pointed to the following key issues: • diagnosis of bladder malignancy, hypertension and diabetes; • october 1, 2013 – surgery to remove the bladder and prostate, losing large amounts of blood to the patient; • decision to transfuse two blood units; • failure to perform patient identification, transfusion of a second unit of blood of incompatible group; • shivering, chest pain and lumbar pain; • stopping the transfusion by medical staff and notifying the patient about the rounds of the rallying patient; • no response from doctors; • re-notify your physician about your symptoms; • conducting patient examination, decision on further blood transfusion; • noticing incorrect transfusions by doctors during the walk; • treatment of a patient after transfusion of group mistaken blond; • deterioration of patient's health; • patient's death". The second part is the statement referring to the content of the case study. The main core of the work was to refer to the Polish code of medical ethics. First of all, choosing articles from the Code of Medical Ethics that are adequate to the discussed situation. Each article was to be explained and analyzed in the context of the discussed case

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Kamila Łampika study. The student was to discuss at least 6 articles. The student had the opportunity to refer to his general knowledge, experience and insights. Students received the instructions below: "Introduction – in this part the student introduces the issues raised in the work. The introduction is to encourage the reader to get acquainted with the whole work, I show key issues, it can contain questions that will be answered in the next parts of the text. We edit or even write the entry in the last stage of work. Development – in this part the student refers to the Code of Medical Ethics (hereinafter KEL). The following follows: analysis of the actual state and its distribution into prime factors. The student's task is to list a minimum of 6-10 articles from the KEL, each article referred to the actual state discussed. The student explains how he understands a given article from KEL, explains whether a given rule has been broken, and then you can learn what the consequences are (e.g. for a doctor, other medical staff, patient, family patient). The student may also refer to KEL articles that were implemented in the described situation. Summary – in this part the student shares his thoughts and conclusions". The teacher pointed out that it is worth dividing the work into several stages in order to be able to improve your essay from week to week. Essays were submitted within the prescribed period. 15 out of 16 groups submitted their work within the originally set deadline. One group two weeks before the agreed date asked to extend the deadline for another week. All essays from the group were to be in one folder or file if the work was sent electronically. It therefore required cooperation from the whole group. The collected material has been read, analyzed and subjected to qualitative interpretation. In qualitative research, interpretations should be understood as the discovery of meaning and meaning (Żurko, 2016). The following categories were interpreted: 1. Compliance with formal requirements (hereinafter form) 2. Reflexivity 3. Emo- tionality. A rating scale of 200 essays was used. The use of scales is a significant reduction of data, but it allows you to perform statistical analyzes necessary to compare data from different authors. [1] A scale of 0-2 was used for formal requirements, where 0 – more than one error, 1 – one error, 2 – full implementation of formal requirements. To reflectivity 0-2, where 0 – no reflection, restoration work, 1 – simple statements (e.g. "This situation shows the need for attention to detail and constant caution in the medical profession"), 2 – complex, in-depth reflection. To emotionality 0-2, where 0 no emotional relation, event reporting, 1 – shallow emotional relationship 2 – deepened, developed emotional relationship. CONCLUSIONS FROM THE ANALYSIS AND INTERPRETATION OF 200 ESSAYS In this chapter, the results of the interpretation will be presented, starting from the form category. Completion of formal conditions meant that the author referred to at least 6 articles of his choice from the KEL and discussed them. The form is also the use of the required font, line spacing, edge alignment on both sides. The specified requirements were met by 166 people out of 200 receiving 2 points according to the adopted scale.

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In a dozen or so works from 166 punctuation errors appeared. 34 students, i.e. up to 17%, did not meet all formal requirements. The most common deviation from the requirements was the lack of alignment to both edges in the second place, the lack of required spacing. It is difficult to unequivocally find the reason for not meeting these requirements. It should be noted that in works sent electronically failure to meet formal conditions was marginal. Which leads to the conclusion that the students gave each other tips on what to improve or the person responsible for formatting one pdf file made editorial corrections. The percentage distribution of points awarded is presented in Chart 1. Fulfilling the abovementioned requirements required reading the instructions with understanding and attentiveness in carrying out the task.

Chart 1. Percentage distribution of points awarded in the "form" category

Chart 2. Percentage distribution of points awarded in the reflexivity category

The concept of reflectivity is worth a closer look. Reflectivity is discussed in various ways in the philosophical, sociological, theological, psychological and pedagogical aspects. In a general sense, the term reflectivity is defined as a tendency to reflect, ponder and analyze. Marzena Chrost organizes numerous interpretations and reduces them to three basic shots. A view of the cognitive charter – highlighting the relationship of reflectivity with forms of thinking, learning processes and professionalization.

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Another saying that this is a general human quality that leads to self-awareness and subjectivity. The third recognizes reflectivity as a requirement of modern times. (Chrost, 2018) In this article, where we analyze students' essays, the cognitive approach is the closest. Lucyna Myszka-Strychalska notices that adolescence is the moment when a person acquires the ability to reflectively think. She writes: "The interest in the issue of adolescent reflectivity is justified by the fact that an individual achieves the ability to reflective awareness during adolescence" (Myszka-Strychalska, 2017) feature develops. What characterizes reflective people? "The tendency to carefully, accurately, and thoroughly analyze the task and potential solutions in order to avoid error and find the most correct solution. In task-oriented situations, these people delay answering in order to consider the various solutions that arise, compare them in terms of accuracy, and select the best one" (Jankowska, 2017). This could be seen in the essays in which the authors considered various scenarios and described their observations in detail. Other features related to reflectivity are "regularity, rigor and discipline" (Gara, 2017). It is important to note after Jarosław Gara that reflective thinking" also has a social dimension, because it takes place in interaction with others" (Gara, 2017). Let me remind you that the task that the students were carrying out was extended over a period of several weeks, and the students were encouraged to discuss the given topic with each other. Chart 2 shows the results for the reflectivity category. Three people received 0 in the category of reflectiveness. These works stood out from the rest, the authors quoted the content of selected KEL articles in a reproductive way. The task required reflectiveness so it is not surprising that the marginal part of the group did not show this feature. Analyzing essays in terms of reflectivity could be used a much wider scale (Perkowska-Klejman, 2013). However, the division into a two-stage scale showed a very interesting result. The use of simple constellations (1 point in the adopted scale) was assigned to 84 student works. In these works questions appeared, for example: "But is it really their fault?", "Was this behavior ethical?", "Was the trust between the doctor and the patient not significantly violated?", "Why did the doctor not reach for this method?", "What if someone doesn't want to be healthy at all?" However, the authors did not develop thoughts and did not look for answers. At other times, the authors marked areas on the basis of which they could build a rich narrative, but they did not. They wrote: "It is worth considering ...", "Doubts raise a lot of doubts ...", "An aspect worth considering is also ..." Another common feature for this work were unambiguous opinions and assessments of both the behavior of doctors and patients. There were such statements: "The conduct of doctors in the abovementioned the situation is flawless in my opinion", "Most importantly, the doctors admitted their mistake (...)", "In this situation, the doctor showed a lack of professionalism and proper medical knowledge", The reaction of the hospital and its employees to medical malpractice was correct and enabled the patient to recover". In essays rated at 1 point, such truisms were as: "Doctor's work is one of the most responsible jobs", "Doctors are obliged to follow the rules of professional ethics", "Helping a patient is the doctor's primary task".

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113 of 200 works were characterized by in-depth analysis. Argumentation was extensive, logical conclusions were drawn, consistent, extensive thought strings were used. Students eagerly invoked the authorities in the Medicine child of both Hippo- crates and modern doctors promoting professional ethics. They quoted the maxims, for example "Primum non nocere", "Bene diagnoscitur, bene curatur", "Bona valetudo melior est wuam maximae divitiae". Most importantly, the quote was the starting point for deeper analysis (Johns, 2009). One of the students wrote in the introduction of his work: "It is impossible to miss the first KEL article, point 1 and point 3. They talk about the dignity of the medical profession. Every carelessness of a doctor is a fault that violates the dignity of this profession. Remember that carelessness weakens the patient's confidence in the doctor. (...) In private life, we surround ourselves with people we trust. We call them friends. The same should be in patient-doctor contacts. We entrust to doctors what we have the most valuable health and life. Confidence in KEL comes first. When we trust, we are honest. As patients, we cannot be ashamed because the omission of not always pleasant details can lead to an incorrect diagnosis. Just how to trust the doctor who caused my neighbour to be disabled?" The essay was evaluated at 2 points, when, among others, the author specified the keynote and developed it. At this point, it is worth referring to the publication in which Anna Perkowska- Klejman presents a study carried out on a group of 300 students in three stages of their studies. It was about measuring reflective thinking. The research was carried out using the "Questionnaire to Measure the Level of Reflective Thinking" by D. Kember, adapted to Polish conditions. "The analysis of the research results leads to the conclusion that reflectivity as the basis of the cognitive development of students develops along with reaching the next levels of education. Graduates showed higher results in terms of critical reflection compared to younger colleagues". The author draws attention to similar results of students from Great Britain and Australia to the results of Polish students participating in the study. One can risk a thesis that tasks such as writing an essay for a first-year medical student is a great opportunity to stimulate the process of reflexivity development in terms of the chosen career path. (Perkowska-Klejman, 2016) Lucyna Myszka-Strychalska notes that adolescence is the moment when a person acquires the ability to reflectively think. "The interest in the issue of adolescent reflectivity is justified by the fact that an individual achieves the ability to reflective awareness during adolescence" (Myszka-Strychalska, 2017). Summarizing, I would like to mention the words of Marzena Chrost: "It should be emphasized that reflective competences largely determine the overall personality development, including ethical and moral development, because the tendency to deeply reflect on one's own actions and the essence of individual elements of the experienced reality facilitates changing existing habits. , it allows freeing oneself from various types of cognitive deformations, increases openness to different perspectives and views, teaches responsibility, directs attention to the world of values" (Chrost, 2018).

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Another category interpreted is emotionality. Emotionality, understood as ways of manifesting emotions in language (linguistic methods of describing e.g. anger, contempt, satisfaction, etc.). You can also define emotionality as the emotional attitude of the sender to reality, expressed through linguistic means. (Kosek, 2020) Andrzej Dąbrowski in his article draws attention to the phenomenon of the influence of emotions on cognition. It turns out that emotions fundamentally affect cognitive processes, including: perception, attention, memory and thinking. The author states that: "Emotions direct attention. By attention we mean here the standard state of increased alertness (concentration) of consciousness with the possibility of selection and searching, and concentration on something specific that appears in the perceptual field, on some element in a wider background. Attention helps organize perception, cognition, and action. Emotions draw our attention to the issues that are currently important". Such an approach to the conducted analysis is justified, it can be said that if the student felt intense emotions towards a specific problem mentioned in the case, he focused on this problem and it intensified his emotionality (Dąbrowski, 2012). Emotionality can be distinguished on the basis of expressiveness of utterance, characterized by: exclamation point intonation; accumulation of names of various emotional states (both positive and negative); the use of other emotive markers in the immediate or further vicinity (Czajkowski, 2019).

Chart 3. Percentage distribution of points awarded in the emotionality category

In the analyzed texts, emotionality was also noticed through expressing empathy. Scientific empathy is a broadly defined concept that combines emotions with cognition. It includes the following categories: empathetic concern, personal distress, taking perspective (Kazimierczak, 2007). During the interpretation 0 points were assigned for 16% of the papers, i.e. 33 people did not show an emotional relation to the topic when writing the essay. In such work the student wrote about facts, e.g. "His condition is getting worse. After a few months, the patient dies". There were no further thoughts about what the death of this man was for his family or what dilemmas the doctor whose patient died had to deal with. It did not assess events but only undertook analysis against the principles of the KEL. The works had substantive value. Showing an emotional relationship could occur

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An image of a first-year student at the Faculty of Medicine. Qualitative research based on written work. but there was no such expectation. It can therefore be argued that in this aspect, the student revealed the closest way of seeing the world around him. The data presented in Chart 3 show that the vast majority of 84% of first-year students of the Medical Faculty showed an emotional relationship to the events or people described in the case report. 51% interpreted emotionality as superficial. Which means the student used emotionally marked words and phrases. One of the most often used words in the essay several times was: "Unfortunately...". At other times, students drew conclusions revealing their perception of events from the case report: "The whole situation is very unfortunate and very unhappy for a minor", "The case we have seen today has particularly unpleasant consequences (...). Revealing the emotionality interpreted according to the adopted scale on 1 point was manifested in perceiving the perspective of other people, you can read such sentences: "The situation also negatively affected the mental state of this man, as well as the suffering of the family". Another student wrote: "This behavior had consequences for both doctor and patient and his family". In the next work, we have read: "M.M. met bad news caused by the diagnosis". In the next quote you can see that the author describes the situation from the perspective of the patient's forgiving parents. Parents could say that they are relieved and even happy about their child's situation. At the same time, they are sorry that their child was exposed to incidents that could have been avoided. "Fortunately, the consequences of the neglect of the doctor L.P. they were reversible, unfortunately the minor was exposed to prolonged pain and additional stress by forcing her to stay in the hospital several times and a fairly long-term treatment process. "Emotionality as interpreted also means paying attention to doctors' skills such as building trust, communication, and diligence. Below are examples of fragments of works in which attention was paid to it: "Empathy is also important, because the doctor is a person who has direct contact with the patient's family, is responsible for informing loved ones about the patient's condition. That is why qualities such as dignity, duty, kindness, communication skills, responsibility, diligence, accuracy and perceptiveness, as well as willingness to bring relief to those suffering without profit are as important as medical knowledge". In another essay: "A doctor's work with a child requires due diligence and great attention and empathy". In the next: "Closeness and friendship were established between him and the patient". This indicates the emotional involvement of the doctor in the relationship with the patient, which is a very important element during treatment". Reference to the subject "I" "(...), by which we unintentionally commit the error undoubtedly later on the conscience" One point was also given to works where the emotionality was inexperienced, which could be seen when the author used numerous exclamation marks, and sentences were formulated with full conviction what emotions the case-maker was experiencing. Emotionality assessed at 2 points according to the adopted scale occurred in 33% of cases. The authors of these works were able to look at events from different perspectives. They analyzed events in terms of the emotions that individuals could experience. They called the emotions, pointed to the long-term emotional consequences of the event for each party. Not only did they speak of empathy as a feature of lust in the medical profession but they were able to express it. The highly reflective

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Kamila Łampika character of the work was associated with the title given to it; further examples: "The smallest medical mistake matters", "In prison of your own dilemmas", "Carelessness and recklessness – the greatest enemies of the medical profession", "Can you forget that this is a mission?", "Carefulness – the doctor's middle name?", "The condition and progress of medicine should always be assessed from the position of a suffering patient, never from the position of a patient who has never suffered" – Jürgen Thorwald. It is noteworthy that there is a positive correlation between emotionality and reflectivity, as it is illustrated in Chart 4. A positive correlation in this case means that the increase in emotionality scores is associated with an increase in reflectivity. The exact data was as follows, 65 essays received 2 points for emotionality out of which 55 essays received 2 points for reflectivity, the average score was 1.8. Thus, more than 1/4 of students showed both traits that are desirable in the medical profession by deeper reflexivity and emotionality.

Chart 4. Dependence between the level of emotionality and other criteria

In the works of students who received 2 points from these categories, the following considerations could be read: "On the other hand, the physician must take into account that every detail is important in his work, and every mistake will have specific consequences. They can not only be legal, but in particular affect the patient's health and future life. He can also have emotional and psychological problems related to guilt and remorse through a moment of inattention or carelessness, carelessness, lack of compliance with accepted guidelines". Example two: "This man's story is extremely depressing. (hereinafter) The man experienced many negative emotions, he survived stress related to the endlessly prolonged treatment process has been painfully stripped of its previous fitness. Numerous complications being the guilt of doctors'

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An image of a first-year student at the Faculty of Medicine. Qualitative research based on written work. diligence have left not only postoperative scars on the body, but also irreversible traces on the psyche. The patient is still struggling with the burden of experiences. Doctors who, like Hippocrates, swore to counteract suffering, condemned the patient to unimaginable suffering that would remain a mark on his personality for the rest of his life. How will they deal with this awareness? People who became doctors to protect against suffering". Referring to the correlation between subsequent categories, the following conclusions can be made: (1) The increase of points in the reflexivity category has a positive effect on the scores in both the emotional category and the form category. (Chart 5). It should be added that 3 papers were rated 0 in the reflexivity category and at the same time all of them were rated 0 in the emotionality category. (2) The highly reflective nature of the work was also associated with the title given to it – examples below: "Accuracy as an ethical aspect of medical art", "Cooperation of specialists as a key element of treatment", "Accuracy and insight, i.e. the foundation of a doctor's work", "From detail to the general", "Correct diagnosis as the key to good treatment".

Chart 5. Dependence between the level of reflexivity and other criteria

(3) It was also noted that the higher grades in the form category are associated with the higher grades in the reflectivity category, but they do not affect the emotional grade (Chart 6).

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Chart 6. Dependence between the level of form and other criteria

SUMMARY Medical students were surprised to hear the need to write essays. By passing the information on to group 16, various reactions could be observed. Usually there was at least one person or a group of two-three people who openly protested. Very interesting and the most repetitive arguments was "we are not humanists". Other people in the group gladly confirmed this thesis. For the teacher, it was always a good starting point for conversation. The conversation in which the question "What features should a doctor have?" The confrontation of the doctor's silhouette created by students with a short definition of the slogan 'humanism' resulted in the desired result by the teacher, students were encouraged to self-development. The reactions of students after writing the papers were often a surprise to the students themselves, who did not expect that the assignment could benefit them. Most students admitted that it was a great opportunity for them to see the profession they are preparing for in a broader perspective. An opportunity to think about what traits they should cultivate in order to consciously create their career path from the first months. The French philosopher Emmanuel Levinas (1906-1995) points out that humanism consists in extreme openness to and serving the Other (Włodarczyk, 2014). For future medical students in the future, when they become doctors, there will be social expectation that the doctor will be "human". Capacity and wide significance can sharpen how much, apart from medical knowledge, a doctor should offer to his patient. To be human is to have "the ability to wish good, help and support, being tender, compassionate or compassionate; it is showing heart, kindness directed towards everyone, the ability to feel the needs of others and sensitivity to their harm; it is also being good, obliging, prone to love and – finally – useful, kind to others and sincerely dealing with the fate of our neighbor, simply – favorable to all people" (Barański J., 2019).

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REFERENCE Bajcar B., Borkowska A., Czerw A., Gąsiorowska A., Satysfakcja z pracy w zawodach z misją społeczną, GWP, Gdańsk 2011, 59-66, ISBN: 978-83-7489-351-0. Barański J., On Moral Virtue in Practice: Essay About Medical Ethic, in: Prace Naukowe Akademii Ekonomicznej we Wrocławiu. Nauki Humanistyczne, 1998; 4, 804 (4):18-27. Barański J., Życzliwość Jako Cnota Moralna W Polskiej Filozofii Moralnej, In: Etyka 58, 2019, (2), 50-70, ISSN online: 2392-1161. Chrost M., Refleksyjność wyzwaniem współczesnego świata, In: Studia Paedagogica Ignatiana, 21(4), 2019;55-66, doi:http://dx.doi.org/10.12775/SPI.2018.4.003, ISSN 2450-5358, e-ISSN 2450-5366. Czajkowski P., Leksykalne wykładniki bezpośredniej ekspresji emocjonalnej w komunikacji, in: Studia methodologica, 2019;49, ISSN 2304-1222, DOI 10.25128/2304-1222.19.49.10. Dąbrowski A., Wpływ emocji na poznawanie, in: Przegląd Filozoficzny – Nowa Seria, R. 21: 2012, Nr 3 (83), ISSN 1230-1493, DOI: 10.2478/v10271-012-0082-6. Gara J., Egzystencjalna problematyzacja poszukiwania; poznawania i tworzenia samego siebie; in: Przegląd Pedagogiczny, 2017;(2);9-33, Wydawnictwo Uniwersytetu Kazimierza Wielkiego w Bydgoszczy, Bydgoszcz, ISSN 1897-6557 Gillon R., Ethics needs principles – four can encompass the rest – and respect for autonomy should be "first among equals", In: Journal of Medical Ethics 2003; 29:307-312. Jankowska, A., (Nie) pożądana refleksyjność. In: Paszenda I. (editors), Codzienność jako wyzwanie edukacyjne, T. 2, 2017;218-232 Instytut Pedagogiki Uniwersytetu Wrocławskiego, Wrocław; oai:repozytorium.uni.wroc.pl:89349 ; ISBN 978-83-62618-35-4 Johns C., Becoming a Reflective Practitioner, Blackwell Science 2009;5-23, Oxford, ISBN 978-1-4051- 8567-7 Kaźmierczak M., Plopa M., Retowski S., Skala Wrażliwości Empatycznej, in: Sękowski A. (Editors), Przegląd Psychologiczny, 2007, T. 50, Nr 1, 9-24, PTP & Wyd.Nauk.KUL, ISBN:0048-5675 Kodeks Etyki Lekarskiej, 2003, Naczelna Izba Lekarska. Kosek I., A to prostak jeden! – o ekspresywnej jednostce jeden, in: Papers in Linguistics, 2020; 22(1):119-130. Lewtak K., Poznańska A., Wysocki M.J., Predyktory satysfakcji zawodowej lekarzy podstawowej opieki zdrowotnej, in: Problemy Higieny i Epidemiologii, 2012, 93(3), s. 558-567. Myszka-Strychalska L., Refleksyjność młodzieży wobec własnej przyszłości zawodowej, in: Paszenda I. (editors), Codzienność jako wyzwanie edukacyjne. Refleksyjność w codzienności edukacyjnej (T. 2), 2017;265-282, Instytut Pedagogiki Uniwersytetu Wrocławskiego, Wrocław; oai:bibliotekacyfrowa.pl:89354 ; ISBN 978-83-62618-31-6. Perkowska-Klejman A., Modele refleksyjnego uczenia się, In: Teraźniejszość – Człowiek – Edukacja tom 16 nr 1 (61), 2013, ISSN online 2450-3428. Sikora A., Pojęcie, rozwój i struktura polskich kodeksów etyki lekarskiej na tle etyki zawodowej, In: Poznańskie Studia Teologiczne, T. 13, 2002, s. 95-109. Księgarnia Św. Wojciecha (PL) ISSN: 0209-3472. Włodarczyk A., Etyka interpretacji tekstu literackiego. Postmodernizm. Humanizm. Dydaktyka, 2014;63-65, Wydawnictwo Uniwersytetu Jagiellońskiego, ISBN 978-83-233-3717-1. Żurko M., Wybrane psychologiczne metody jakościowe stosowane w badaniach nad tekstem pisanym, In: Tom 7 Dokumenty osobiste w doświadczeniach badawczych – od (auto)biografii do fikcji (nie)literackiej, 2016, 85-100, Forum Socjologicze, ISSN: 2083-7763.

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Index of authors

Bartkowiak K...... 134 Królewicz E...... 52, 173

Bartkowiak M...... 134 Kulbacka J...... 97, 161

Chojdak-Łukasiewicz J...... 43 Kustrzeba-Wójcicka I...... 52, 173

Cyran A...... 149 Lang M...... 161

Detyna J...... 97 Łabowska M.B...... 97

Dębska-Łasut K...... 113 Łampika K...... 199

Dobosz T...... 187 Michalak I...... 97

Domagała Z...... 187 Mysiak A...... 113

Franciszek Burzynski P...... 16 Pilaczyńska-Cemel M...... 7

Gebuza M...... 161 Pluta D...... 187

Golema W...... 35 Przybylski G...... 7

Iwan D...... 187 Radomska-Leśniewska D.M...... 149

Jagodziński A...... 142 Rembiałkowska N...... 161

Jankowska A.M...... 97 Siuta J...... 35, 187

Jankowska-Steifer E...... 134 Sobiepanek A...... 122

Jurek T...... 187 Soszyńska M...... 122

Kimsa-Dudek M...... 88 Synowiec-Wojtarowicz A...... 88

Kobusiak-Prokopowicz M...... 113 Szlasa W...... 161

Kowalik-Jagodzińska M...... 142 Ścieżyńska A...... 122

Kowalska P.D...... 122 Tokarski M...... 187

Kozanecka A...... 7 Turek K...... 161

Krawczyk A...... 88 Zarek A...... 60

Kroll A...... 60 Ziętek M...... 113

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