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Immunoreactivity for Thymosin Beta 4 and Thymosin Beta

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Immunoreactivity for Thymosin Beta 4 and Thymosin Beta European Journal of Histochemistry 2013; volume 57:e17 Immunoreactivity for thymosin Introduction Correspondence: Sonia Nemolato, Istituto di beta 4 and thymosin beta 10 Anatomia Patologica, Dipartimento di Scienze in the adult rat oro-gastro- Beta thymosins are a versatile family of Chirurgiche, PO S. Giovanni di Dio, Università di intestinal tract small peptides expressed in multiple tissues in Cagliari, via Università 60, 09124 Cagliari, Italy. mammals, that show many intracellular and Tel. +39.070.6092370 - Fax: +39.070.657882. extracellular activities.1,2 These peptides are E-mail: [email protected] S. Nemolato,1 J. Ekstrom,2 T. Cabras,3 named thymosins after their first isolation in C. Gerosa,1 D. Fanni,1 E. Di Felice,1 Key words: rats, thymosin beta 4, thymosin beta the calf thymus.3 Fifteen highly homologous 1 3 4 10, immunohistochemistry, gastrointestinal tract. A. Locci, I. Messana, M. Castagnola, beta thymosin variants, containing 40 to 44 G. Faa1 4 amino acid residues, have been described. Contributions: SN, GF, research design and man- 1Istituto di Anatomia Patologica, Among beta thymosins, thymosin beta 4 uscript writing; JE, CM, IM, critical review; TC, 5,6 7 Dipartimento di Scienze Chirurgiche, PO (Tβ4) and thymosin beta 10 (Tβ10) are the CG, DF, EDF, AL, data analysis and research per- 8 forming. S. Giovanni di Dio, Università di Cagliari, most abundant in human cells and rat tissues. During the years, beta thymosins have been Italy; 2Division of Pharmacology, Institute detected inside of cells of different organs,9-14 Conflict of interests: the authors declare no con- of Neuroscience and Physiology, as well as in human blood,15 in human saliva,16 flict of interests. Sahlgrenska Academy, University in tears17 and in wound fluid after abdominal Acknowledgments: this work has been supported of Gothenburg, Sweden; 18 surgery. Many physiological properties and by “Fondazione Banco di Sardegna”. The authors 3Dipartimento di Scienze della Vita e cellular functions are connected to Tβ4: G- would like to thank Mr. Ignazio Ferru for the sec- dell’Ambiente, Università di Cagliari, actin-sequestering,1 promotion of cell migra- retarial assistance. The authors also gratefully Italy; 4Istituto di Biochimica e Biochimica tion,8 angiogenesis,9,19 stem cell differentia- acknowledge the Sardinia Regional Government Clinica, Facoltà di Medicina, Università tion,11 modulation of cytokines and for the financial support (P.O.R. Sardegna F.S.E. 20 Cattolica di Roma, Italy chemokines. This peptide is also involved in Operational Programme of the Autonomous lesion-induced neuroplasticity through Region of Sardinia, European Social Fund 2007- microglia upregulation and it participates in 2013 - Axis IV Human Resources, Objective l.3, the growth of neuronal processes.21 Therefore, Line of Activity l.3.1 Avviso di chiamata per il finanziamento di Assegni di Ricerca). Abstract the mRNA encoding for Tβ4 is expressed in mouse embryonic stem cells and in mesoder- Received for publication: 21 January 2013. 22 mal-like cells (cardiac and skeletal muscle). Accepted for publication: 5 April 2013. Thymosin beta 4 (Tβ4) and thymosin beta Moreover, recent studies demonstrated a role 10 (T 10) are two members of the -thymosin This work is licensed under a Creative Commons β β of Tβ4 in inducing the expression of the vascu- family, involved in multiple cellular activities lar endothelial growth factor (VEGF) in colon Attribution NonCommercial 3.0 License (CC BY- in different organs in multiple animal species. 19 NC 3.0). cancer cells in experimental models. Tβ4 also Here we report the expression pattern of T 4 β partecipates to the modulation of human ©Copyright S. Nemolato et al., 2013 23 and Tβ10 in rat tissues, in the gut and in colonic immune system probably through Licensee PAGEPress, Italy annexed glands. The two peptide were differ- degranulation of mucosal mast cells.24 European Journal of Histochemistry 2013; 57:e17 ently expressed: Tβ4 was absent in salivary Contrasting results have been published on doi:10.4081/ejh.2013.e17 glands whereas Tβ10 was expressed in parotid the role of Tβ10 in tumour progression. On and in submandibular glands. Tβ4 was mildly one hand, Tβ10 diminishes tumor growth, 25 expressed in the tongue and in the esophagus, angiogenesis and proliferation and Tβ10 over-expression has been related to the where Tβ10 was absent. A similar expression was found in the stomach, ileum and colon increase of apoptosis in human ovarian cancer Materials and Methods cells.25 On the other hand, T 10 has been asso- mucosa. In pancreas Tβ4 reactivity was β restricted to the Langerhans islet cells; T 4 ciated to the progression of papillary thyroid In order to test Tβ4 and Tβ10 immunoreac- β 26 was also detected in the exocrine cells. Both carcinoma and over-expression of the peptide tivity in animals, 20 male wistar rats were the object of our study, divided into 10 male and 10 peptide were not expressed in liver cells. When has been observed in non-small cell lung can- cer27 and in pancreatic cancer. 28 The recent female. Tissues were obtained from each ani- the rat expression pattern in rat organs was report by our group of a strong expression for mal including samples from tongue, esopha- compared to reactivity for T 4 and T 10 in β β T 10 in the human salivary glands during gus, stomach, ileum, colon, parotid gland, sub- humans, marked differences were found. Our β development,29 induced us to better analyse mandibular gland, sublingual gland, liver and data clearly indicate a species-specific expres- the protein expression pattern for Tβ10 in the pancreas. All samples were fixed in 10% forma- sion of Tβ4 and Tβ10, characterized by the oro-gastro-intestinal tract in adult rats, in lin, paraffin-embedded and routinely processed. actual unpredictability of the expression of order to show if T 10 is expressed in the gas- Paraffin sections were immunostained with these peptides in different cells and tissues. β trointestinal tract in adulthood, and to com- anti-Tβ4 and anti-Tβ10 antibodies, using the The common high expression of Tβ4 in mast pare the expression of this peptide with that labeled streptavidin-biotin complex system cells, both in humans and in rats, represents reported in the human digestive tract and in (LSAB2, Dako, Cytomation, Carpinteria, CA, one of the few similarities between these two annexed glands.24,30,31 Moreover, rat tissues USA) in a Dako Autostainer (Dako). Briefly, species. were immunostained for Tβ4, with the aim of samples were deparaffinized, rehydrated, and verifying the reciprocal interactions of Tβ4 endogenous peroxidase activity was quenced and Tβ10 in the oro-gastrointestinal tract. (30 min) by 0.3% hydrogen peroxide in [page 106] [European Journal of Histochemistry 2013; 57:e17] Original Paper methanol. Slides were then subjected to heat- Table 1. Immunoreactivity for thymosin beta 4 (Tβ4) and thymosin beta 10 (Tβ10) in induced antigen retrieval by steaming rat tissues and humans. unstained sections in a Target Retrieval Organ Tβ4 Rat Tβ10 Rat Tβ4 Human Tβ10 Human Solution (Dako TRS pH 6.1) for 30 min. Slides were then incubated with 10% normal goat Parothyd - +++ ++ serum in phosphate-buffered saline (PBS) for Submandibular gland - +++ ++ 60 min to block non-specific binding, followed Sublingual gland --++ by incubation (60 min at room temperature) Tongue +-+++ with a monoclonal anti-Thymosin Beta 4 anti- Oesofagus --+- body (Bachem-Peninsula Lab, San Carlos, CA, Stomach +++- USA) and with a monoclonal anti-Thymosin Ileum +++- Beta 10, respectively diluted 1:600 and 1:500 in Colon ++++ - the blocking solution. Slides were extensively washed with PBS containing 0.01% Triton X- Pancreas + ++ ++ + 100 and incubated with a secondary reagent Liver --+++ +++ Figure 1. Parotid. a) No immunoreactivity for T 4 is detected in Figure 2. Submandibular glands. a) T 4 is not expressed in the the parotid gland; scattered mast cells are stronglyβ immunoreac- structures of the submandibular glands;β only a fine positivity tive for the peptide; OM 250x. b) A granular and diffuse positiv- could be detectable in the surrounding stroma; mast cells show a ity for T 10 is observed in all the acinar structures; ductal cells strong granular cytoplasmic positivity (yellow arrows); OM 400x. show an βapical and homogeneous reactivity for the peptide; OM b) Coarse granules of T 10 are observed in the acini of the sub- 400x. mandibular gland (yellowβ arrows); ductal cells present a fine cyto- plasmic immunoreactivity for the peptide mainly localized in the lumen (red arrows); OM 400x. [European Journal of Histochemistry 2013; 57:e17] [page 107] Original Paper (En Vision kit) according with the manufactur- er instructions (Dako, Glostrup, Denmark). Diaminobenzidine (DAB) was used as chro- mogen. After additional washes, colour was developed using the AEC reagent (Dako), sec- tions were counterstained with Mayer’s hema- toxylin and mounted. Sections of reactive lymph nodes with Tβ4-immunoreactive histio- cytes were utilized as a positive control. As a negative control, the same procedure was applied omitting the primary antibody. Results Parotid Tβ4 immunoreactivity was completely Figure 3. Tongue. A weak immunoreactivity for T 4 is observed only in the superficial absent both in acini and in ducts (Figure 1a). layers of the tongue’s epithelium; a weak immunostainingβ for the peptide was observed in cell membranes of muscle cells (see inset, yellow arrows); OM 250x. Tβ10 was expressed both in acinar serous Figure 4. Stomach. a) Immunoreactivity for T 4 is expressed in Figure 5. Ileum. a,b) T 4 and T 10 are detected in the surface scattered foveolar cells (see inset, yellow arrows)β and in the lumi- epithelium of enterocytesβ coveringβ the villi of the ileum (see nal surface of the stomach; OM 400x. b) T 10 is mainly localized inset, black arrows); OM 400x.
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