Immunohistological characterisation of tumor infiltrating lymphocytes (TIL) in malignant melanoma and the evaluation of regulatory T cells in tumor tissue and circulation for the assessment of its relationship with prognosis

Malign melanomun diğer solid tümörlerden farklı olarak spesifik bir immün cevap sayesinde spontan regresyonun görülmesi immünoterapistlerin ilgisini bu hastalığa çekmiştir. Günümüzde de immünoterapinin başarıya ulaşan hedeflerinden biri haline gelmiştir. İmmün sistemdeki görevleri incelenen regülatör T hücrelerinin (Treg) melanomdaki rolünün anlaşılması için ayrıntılı çalışmalara gereksinim vardır. Bu araştırmanın temel amacı, malign melanom hastalarında tümör infiltre edici lenfositlerin immünohistolojik karakterizasyonu ve dolaşımdaki Treg hücrelerinin serum S100B ile karşılaştırarak prognozu saptamadaki gücünün araştırılmasıdır. Bunun için hem melanom hastalarında (n=30) hem de sağlıklı kontrollerde (n=24), günümüzde en uygun Treg markeri olan FOXP3 transkripsyon faktörünü kullanarak, Treg'lerin miktar ve fenotip analizi yapıldı. Ayrıca immünhistokimyasal TIL tayini yapıldı. Çalışmamızda serum S100B ve dolaşımdaki CD4+CD25+, CD4+CD25+FOXP3+ ve CD8+CD25+FOXP3+ hücrelerinin hasta grubunda kontrol grubuna göre yüksek olduğu bulundu. Serum S100B ile hastaların periferik kanlarında ölçülen CD4+ hücreleri ve de tümörü infiltre eden B lenfositleri arasında ters yönde korelasyon bulundu. Tümörü infiltre eden T lenfositlerinin tümörün Breslow kalınlığı arttıkça azaldığı gösterildi. Clark seviyesi yükseldikçe de tümörü infiltre eden CD4+ T lenfositlerin azaldığı görüldü. Sonuçlarımıza göre tümör progresyonu, hastalığın klinik seyri ile serum S100B ve dolaşımdaki Treg hücre ölçümü paralellik göstermektedir. Ayrıca, hastalık prognozu ile TIL varlığı arasında ters yönde bir ilişki olduğunu ve tümörü süprese edecek olan lenfositlerin azlığının bu tümörün gelişmesine neden olduğunu söylemek mümkündür. Bu çalışma sonunda elde ettiğimiz veriler, Treg'lerin anti tümör immünitesinde olası bir rolü olduğunu göstermektedir. Gelecekte Treg'ler için spesifik marker ekspresyonunu araştıran, inhibisyon mekanizmalarının aydınlatılmasına yönelik ve antijen özgünlüğünü dikkate alan çalışmalar dizayn edilmesinin yararlı olacağı öngörülmektedir. Bununla beraber, CD4+ T hücrelerini artırma ve Treg hücrelerini kısıtlamaya yönelik yeni kanser aşı programları hem CD8+ T hücreleri hem de antijen spesifik immün cevabı düşünerek değerlendirilmelidir. The immunotherapists have begun to focus on malignant melanoma, in which unlike most other solid tumors, spontaneous regression can be seen due to the specific immune response. Thus in nowadays, it has been listed among the successful goals of immunotherapeutic approaches. In order to clarify the role of regulatory T cells (Treg) in melanoma, further detailed studies should be performed. The main objective of this study is the immunohistological characterization of tumor infiltrating lymphocytes and to determine the prognostic efficacy of Treg cells in circulation by comparing serum S100B levels. Therefore, the analysis of phenotype and quantity of Treg's are performed in both melanoma patients (n=30) and healthy controls (n=24) by using the most suitable Treg marker FOXP3 transcription factor. Additionally, TILs are immunohistochemically evaluated. In this study, higher serum levels of S100B and CD4+CD25+, CD4+CD25+FOXP3+, and CD8+CD25+FOXP3+ cells are obtained in the patient group comparing to control group. Negative correlation is found between serum S100B and CD4+ cells and tumor infiltrating B lymphocytes in peripheral blood circulation. It was shown that tumor infiltrating T lymphocyte numbers were decreased when the Breslow thickness was increased. It was also seen that tumor infiltrating CD4+ lymphocyte numbers were decreased when the Clark level was increased. According to our results, there was a correlation between the tumor progression and the clinical status of the patients and the measurement of serum S100B and Treg's in blood circulation. Moreover, it is possible to say that, there is a negative correlation between the prognosis and presence of TIL's. The lower quantity of lymphocytes which should suppress the tumor is responsible for tumor development. The final data of this study suggest a possible role of Treg's in anti-tumor immunity. For future it can be said that it is useful to design studies to investigate Treg's in terms of specific marker expression, inhibition mechanism, and antigen specificity. Thus, novel cancer vaccine formulations, aiming to favor the expansion of CD4+ T cells, and to limit Treg cell expansion, should be evaluated in conjunction with CD8+ T cells and at the level of antigen specific immune responses

Anti-inflammatory and Anti-apoptotic Effect of Valproic Acid and Doxycycline Independent from MMP Inhibition in Early Radiation Damage

Background: Matrix metalloproteinase (MMP) inhibitors decrease inflammation in normal tissues and suppress cancer progress in normal tissues. Valproic acid (VA) and doxycycline (DX) are MMP inhibitors that have radio-protective effects. Their ability to inhibit MMPs in irradiated tissue is unknown and the role of MMPs in radio-protective effects has not been tested to date. Aims: The purpose of this study was to examine whether administration of VA and DX to rats before irradiation affects tissue inflammation and apoptosis in the early phase of radiation, and whether the effect of these drugs is mediated by MMP inhibition. Study Design: Animal experimentation. Methods: Twenty-six Wistar rats were randomized into four groups: control (CTRL), radiation (RT), VA plus radiation (VA+RT), and DX plus radiation (DX+RT).Three study groups were exposed to a single dose of abdominal 10 Gy gamma radiation; the CTRL group received no radiation. Single doses of VA 300 mg/kg and DX 100 mg/kg were administered to each rat before radiation and all rats were sacrificed 8 hours after irradiation, at which point small intestine tissue samples were taken for analyses. Levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and matrix metalloproteinases (MMP-2 and MMP 9) were measured by ELISA, MMP activities were measured by gelatin and casein zymography and apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results: VA decreased the levels of TNF-α and IL-1β proteins insignificantly and decreased apoptosis significantly in the irradiated tissue, but did not inhibit MMPs. In contrast, VA protected the basal MMP activities, which decreased in response to irradiation. No effect of DX was observed on the levels of inflammatory cytokines or activities of MMPs in the early phases of radiation apoptosis. Conclusion: Our findings indicated that VA protects against inflammation and apoptosis, and DX exhibits anti-apoptotic effects in early radiation and these effects are independent from MMP inhibition

Anti-inflammatory and Anti-apoptotic Effect of Valproic Acid and Doxycycline Independent from MMP Inhibition in Early Radiation Damage

Background: Matrix metalloproteinase (MMP) inhibitors decrease inflammation in normal tissues and suppress cancer progress in normal tissues. Valproic acid (VA) and doxycycline (DX) are MMP inhibitors that have radio-protective effects. Their ability to inhibit MMPs in irradiated tissue is unknown and the role of MMPs in radio-protective effects has not been tested to date. Aims: The purpose of this study was to examine whether administration of VA and DX to rats before irradiation affects tissue inflammation and apoptosis in the early phase of radiation, and whether the effect of these drugs is mediated by MMP inhibition. Study Design: Animal experimentation. Methods: Twenty-six Wistar rats were randomized into four groups: control (CTRL), radiation (RT), VA plus radiation (VA+RT), and DX plus radiation (DX+RT).Three study groups were exposed to a single dose of abdominal 10 Gy gamma radiation; the CTRL group received no radiation. Single doses of VA 300 mg/kg and DX 100 mg/kg were administered to each rat before radiation and all rats were sacrificed 8 hours after irradiation, at which point small intestine tissue samples were taken for analyses. Levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) and matrix metalloproteinases (MMP-2 and MMP 9) were measured by ELISA, MMP activities were measured by gelatin and casein zymography and apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results: VA decreased the levels of TNF-α and IL-1β proteins insignificantly and decreased apoptosis significantly in the irradiated tissue, but did not inhibit MMPs. In contrast, VA protected the basal MMP activities, which decreased in response to irradiation. No effect of DX was observed on the levels of inflammatory cytokines or activities of MMPs in the early phases of radiation apoptosis. Conclusion: Our findings indicated that VA protects against inflammation and apoptosis, and DX exhibits anti-apoptotic effects in early radiation and these effects are independent from MMP inhibition