Tuberculosis verrucosa cutis lesions exhibit a greater microvessel count than lesions Sulochana Bhandarkar, Emory University Padmavathy Lanka, Veterans Administration Medical Center Lakshmana Rao Lanka, Vinayaka Mission’s Medical College Emir Veledar, Emory University Michael Y. Bonner, Emory University Jamie MacKelfresh, Emory University Jack Arbiser, Emory University

Journal Title: Experimental Dermatology Volume: Volume 25, Number 6 Publisher: Wiley: 12 months | 2016-06-01, Pages 479-480 Type of Work: Article | Post-print: After Peer Review Publisher DOI: 10.1111/exd.12980 Permanent URL: https://pid.emory.edu/ark:/25593/s6nz1

Final published version: http://dx.doi.org/10.1111/exd.12980

Accessed September 27, 2021 11:31 PM EDT HHS Public Access Author manuscript

Author ManuscriptAuthor Manuscript Author Exp Dermatol Manuscript Author . Author manuscript; Manuscript Author available in PMC 2017 June 01. Published in final edited form as: Exp Dermatol. 2016 June ; 25(6): 479–480. doi:10.1111/exd.12980.

Tuberculosis Verrucosa Cutis Lesions Exhibit a Greater Microvessel Count than Lupus Vulgaris Lesions

Sulochana S. Bhandarkar, MDa, Padmavathy Lanka, MDb, Lakshmana Rao Lanka, MDc, Emir Veledar, PhD, FAHAe, Michael Y. Bonner, BAa, Jamie MacKelfresh, MDa,d, and Jack L. Arbiser, MD,PhDa,c aDepartment of Dermatology, Emory University School of Medicine, Atlanta, GA bDepartment of Dermatology Veterans Administration Medical Center, Atlanta, GA cDepartment of Pathology Vinayaka Mission’s Medical College, Karaikal, Pondicherry UT, INDIA dDepartment of Pathology, Emory University School of Medicine, Atlanta, GA eBaptist Health South Flordia, Jacksonville, FL

BACKGROUND Tuberculosis is reported to be the second most common infectious cause of death after HIV. The causative organism of tuberculosis, tuberculosis (MTB) is capable of infecting virtually every organ of the body, including the skin (1). The top three most common cutaneous forms of tuberculosis are Tuberculosis Verrucosa Cutis (TBVC), Lupus Vulgaris (LV) and . TBVC is often self limited, and may clear without antibiotic treatment, while LV is progressive without treatment. TBVC may thus represent a Th1 immune response to MTB, while LV may represent a Th2 response. This is analogous to the differentiation between tuberculoid (Th1) and (Th2) (2).

Previously, we examined angiogenesis in differing stages of leprosy, caused by a Mycobacterium (3). We demonstrated that lepromatous leprosy had a higher microvessel count than . In the current study of cutaneous Mycobacterium tuberculosis infection, we found that TBVC had an elevated microvessel count compared to LV, despite LV having a clinically more aggressive course (3). This difference suggests that

Corresponding author: Jack L Arbiser, Department of Dermatology, Emory University School of Medicine, WMB 5309, 101 Woodruff Circle, Atlanta, GA 30322, Tel (404) 727-5063, Fax (404) 727-0923, ; Email: [email protected] Financial Disclosures: None Author Contributions: Dr. Arbiser had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept & design: JLA SSB Acquisition of data: JLA, SSB Analysis & interpretation of data: SSB, EV, JLA Drafting of the manuscript: JLA, MYB and SSB Critical revision of the manuscript for important intellectual content: JLA, JM, SSB Obtained funding: JLA Administrative, technical, or material support: PL, LRL, SSB, MYB and JLA Study supervision: JLA Conflict of Interest: There are no conflicts of interests for all authors: SSB, PL, LRL, EV, MYB, JM, and JLA. Bhandarkar et al. Page 2

Mycobacterium tuberculosis is controlled in the skin through a different mechanism than Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author . The increased microvessel count in TBVC compared to LV suggests a possible requirement for vessel associated cytokines in the control of MTB, and conversely, that tissue hypoxia may prevent an effective immune response to MTB.

QUESTIONS ADDRESSED We wanted to determine whether the two most common forms of cutaneous tuberculosis differ in terms of angiogenesis. This knowledge may be beneficial in modifying host responses to tuberculosis.

EXPERIMENTAL DESIGN We studied 14 cutaneous lesions, one from each of the 14 patients with cutaneous tuberculosis. The paraffin blocks were received from Dr. Padmavathy from Annamalai University, India. The diagnosis of cutaneous tuberculosis in the patients was supported by hematoxylin-eosin-stained histology of the biopsied lesions and the clinical history. Seven patients had lupus vulgaris and seven had tuberculosis verrucosa cutis.

A total of 14 sections (5 mm) of formalin-fixed, paraffin-embedded tissue were immunostained with monoclonal antibodies against CD31 (clone JC170A,1/80, Dako Corp, Carpinteria, CA) using a polymer with the pressure cooker heat–induced antigen retrieval and an autostainer (Dako Corp). Negative controls had primary antibody replaced by buffer. Sections of myometrium (blood vessels) were used as positive control for CD31.The CD31 mean and microvessel density were quantitated microscopically by 1 observer (J.M). The number of CD31 positive blood vessels in the sections and in two hotspots at a power of 20X according to the method of Weidner et al (4). Hot spots were areas determined by the pathologist to represent the fields of greatest vascular density within a given section.

We used the nonparametric Wilcoxon matched pairs test to compare the durations of efficacy between the first and last injections, with a significance threshold of 5%. Statistical analyses were carried out with SAS 9.3 software.

RESULTS The results of lesion vascularity evaluated with CD31 range from a mean of 4.8 in the Lupus Vulgaris spectrum and a mean of 10.1 in the Tuberculosis Verrucosa Cutis spectrum. The mean CD31 microvessel density in both types of lesions is shown in Figure 1. Both LV and TBVC demonstrated angiogenesis, but TBVC was significantly more angiogenic. CD31 values for TBVC were significantly higher than values for LV. There almost was no overlap in their distribution and test reported them as different with possible error of no more than 0.0106. Lupus vulgaris showed a significantly lower CD31 count when compared to Tuberculosis Verrucosa Cutis.

Exp Dermatol. Author manuscript; available in PMC 2017 June 01. Bhandarkar et al. Page 3

Author ManuscriptAuthor CONCLUSION Manuscript Author Manuscript Author Manuscript Author Cutaneous Tuberculosis is not as common as pulmonary tuberculosis, but it is still a significant infectious problem in developing countries (5). Lupus vulgaris is reported to be the most common in adults (6) in many countries and currently considered the most prevalent type (7). We decided to study angiogenesis patterns in these two forms to determine the role of angiogenesis, if any, in these two common types of cutaneous tuberculosis.

TNF increases the antimicrobial capacity of macrophages through the induction of reactive oxygen, but excess reactive oxygen can cause necroptosis of bacteria laden macrophages, leading to release and dissemination of MTB, while low concentrations of reactive oxygen favor enhanced intracellular growth (8, 9). Therefore a state of moderate elevation of reactive oxygen may be required for control of MTB. TNF is also a well-known inducer of angiogenesis and inducer of nitric oxide synthesis, which can be clinically observed in psoriasis. One possibility is that TBVC has increased blood vessels due to increased cytokines and reactive oxygen. The cytokines that have been implicated in control of MTB include TNF, superoxide and nitric oxide, all well-established mediators of angiogenesis (8,9).

We found that lupus vulgaris has significantly lower angiogenic vessel counts than tuberculosis verrucosa cutis. Clinically, LV is more aggressive than TBVC, a possible manifestation of invasive behavior. These differences may play a role in host control of tuberculosis infection. LV has relatively fewer vessels, suggesting that LV lesions may be relatively hypoxic compared with TBVC.

Acknowledgments

Funding/Support: JLA was supported by the grant RO1 AR47901and P30 AR42687 Emory Skin Disease Research Core Center Grant from the National Institutes of Health, a Veterans Administration Hospital Merit Award, as well as funds from the Margolis Foundation, Rabinowitch-Davis Foundation for Melanoma Research and the Betty Minsk Foundation for Melanoma Research.

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Figure 1. Distribution of Wilcoxon Scores for vessel count (CD31) in Lupus Vulgaris (LV) and Tuberculosis Verrucosa Cutis (TBVC) specimens TBVC was significantly more angiogenic than LV.

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Figure 2. Representative CD31 staining in TBVC (A.) and LV (B.) The mean staining of CD31 in TBVC was 10.1 compared to LV, which had a mean CD31 staining of 4.8.

Exp Dermatol. Author manuscript; available in PMC 2017 June 01.