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CD8+CD28- T hücreleri, baskılayıcı özelliği en iyi tanımlanmış CD8+ supresör T hücre (Ts) alt grubudur. Doğal immün sistemin reseptörleri toll benzeri reseptörler (TLR), T hücreleri tarafından da eksprese edilmektedir. Çalışmamızda, CD8+CD28- Ts'nin fenotipik özellikleri ve baskılayıcı mekanizmalarını incelemenin yanı sıra TLR'lerin bu hücrelerin baskılayıcı işlevlerini düzenlemedeki rollerini incelemeyi amaçladık. Bu amaçla, CD8+CD28- Ts ve CD8+CD28+ efektör T hücrelerinde (Tef) HLA-DR, CD56 ve perforin ekspresyonları akım sitometri ile analiz edildi. Fitohemaglutinin (PHA) ile kültür edilen CD8+ T hücrelerinde uyarım öncesinde, 24 ve 72 saat uyarım sonrasında TLR ekspresyon kinetikleri incelendi. Manyetik boncuklar ile izole edilen Ts, TLR agonistleri ile uyarılarak CD4+ T hücrelerinin proliferasyonlarına etkileri 5-etinil-2´-deoksiüridin (EdU) ile incelendi. Bu hücrelerin salgıladıkları baskılayıcı sitokinler IL-10 ve TGF-ß, ELIZA yöntemi ile tayin edildi. CD8+CD28- Ts'de HLA-DR, CD56 ve perforin ekspresyonları Tef'e oranla yüksek bulundu. CD8+CD28-CD56+ ve CD8+CD28-HLA-DR+ olmak üzere iki farklı populasyonun bulunduğu gözlemlendi. Ts'lerin PHA uyarımı sonucunda TLR ekspresyonlarının artışının, Tef'lere oranla daha az olduğunu bulundu. Ts'lerin CD4+ T hücrelerinin proliferasyonlarını baskıladıklarını ve TGF-ß salınımlarının yüksek olduğu görüldü. TLR1/TLR2 ve TLR3 agonistlerinin, bu hücrelerin baskılayıcı kapasitelerini bozduğu gözlemlendi. TLR4 uyarımının Ts'nin baskılayıcı işlevini etkilemediği bulundu. TLR1/TLR2 uyarımı sonucunda, Ts'lerin ürettiği TGF-ß seviyelerinde bir değişim gözlenmedi. Ancak, TLR3 ve TLR4 agonistleri ile uyarım sonucunda, TGF-ß üretiminin azaldığı gözlemlendi. Çalışmamızda, CD8+CD28- Ts'nin, CD4+ T hücrelerini TGF-ß aracılığı ile baskıladıkları ancak bu baskılamanın TLR agonistleri ile uyarı sonrasında bozulduğu gösterilmiştir. Özellikle, TLR3 uyarımı gibi güçlü bir uyarımla karşılaştıklarında baskılama özelliklerinin kaybolması, efektör hücre olma yolunda plastisite göstererek immün savunmaya katkıda bulunduklarının bir kanıtıdır. The suppressive effects of CD8+CD28- T cells on the proliferation of T helper cells have been demonstrated. Although toll like receptors (TLRs) are potent activators of innate immune response, it has recently been shown that they are also expressed on T cells. We aimed to identify the phenotypic and functional characteristics of CD8+CD28- suppressive T cells (Ts) and how their functions are regulated by TLRs. Expressions of CD56, HLA-DR and perforin levels were analyzed. TLR expression kinetics of CD8+ cells were examined before and after stimulation with phytohemagglutinin (PHA). Magnetically isolated Ts were stimulated with TLR agonists and their suppressive capacity was investigated by using 5-ethynyl-2'-deoxyuridine (EDU). Suppressive cytokines IL-10 and TGF-ß were analyzed with ELISA. HLA-DR, CD56 and perforin expressions were found higher on Ts compared to Tef. Among the Ts, two distinct populations, CD8+CD28-CD56+ and CD8+CD28-HLA-DR+, were identified. TLR expressions of Ts after PHA stimulation were found lower compared to the TLR expressions on Tef. We also demonstrated that these cells indeed suppress the proliferation of CD4 T cells and secrete high levels of TGF-ß. TLR1/TLR2 and TLR3 agonists inhibited the suppressive function of Ts but TLR4 stimulation did not affect their function. Although we did not find any difference in the levels of TGF-ß secretion after TLR1/TLR2 stimulation, TGF-ß levels were reduced after TLR3 and TLR4 stimulations. We demonstrated that CD8+CD28- Ts suppress the CD4+ T cells via TGF-ß. However, this suppression can be inverted by TLR stimulation. When CD8+CD28- Ts encounter a strong stimulation like TLR3 stimulation, they lose their suppressive capacity and their plasticity enables them to differentiate into effector cells in order to sustain immune defense. 75

Objective: The survival of a semi-allogeneic fetus depends on several immunological mechanisms, and it has been suggested that recurrent pregnancy loss (RPL) could develop as a result of one or more immunological abnormalities.Methods: Compatibility between partners for human leukocyte antigen (HLA) genotypes and the relationships between maternal killer-cell immunoglobulin-like receptor (KIR) and paternal HLA-Bw4/Bw6 and HLA-C1/C2 supra-groups were investigated in 25 couples with RPL in comparison to healthy couples with children. HLA and KIR genotyping was performed using polymerase chain reaction with sequence-specific primers and/or sequence-specific oligonucleotides.Results: HLA class I incompatibility between partners, especially in HLA-B alleles, was more common in the RPL group (p= 0.01). HLA-C2 homozygosity was more frequent in the male partners of RPL couples than in other groups (p= 0.03). The KIR2DL5 gene frequency was significantly higher in both the female and male partners of RPL couples, whereas the KIR2DS3 gene frequency in male partners of RPL couples was significantly reduced (p= 0.03). The presence of KIR2DL3 in women with RPL was correlated with the presence of HLA-C2 alleles in their spouses (p= 0.03).Conclusion: Our data from a Turkish population suggest that male HLA-C2 homozygosity may play an important role in RPL. Additionally, an incidental match between male HLA-C2 and female HLA-C1 ligand KIR receptors might perturb the balance between activatory and inhibitory KIR-ligand interactions during pregnancy in couples affected by RPL. The roles of orphan KIR2DL5 and orphan KIR2DS3 in RPL remain obscure.

Amaç: Muskarinik reseptörler merkezi sinir sistemindeasetilkolinin çeşitli etkilerine aracılık ettiği gibi, parasempatik sinirsistemi ile etkileşen sinirsel olmayan dokulara da aracılık ederler.Çalışmamızda, M3 muskarinik reseptör alttipinin K562 kanserhücre çoğalması ve ölümündeki rolünü belirlemeye çalıştık.Gereçler ve Yöntemler: Hücre çoğalması bromodeoksiüridin(BrDU) yöntemi ile belirlenmiştir. Hücre ölümü, erken ve geçapoptoz Anneksin V ve propidyum iodid (PI) varlığında akışsitometrisi yöntemi ile gösterilmiştir. Nuklear dış sinyal düzenleyicikinaz (ERK/fosfo-ERK) ekspresyonu western emdirimi yöntemiile belirlenmiştir.Bulgular: Çalışmamızda 48 saat karbakol(CCh) ile muameleedilen K562 hücre sayısında azalma belirlenmiştir. Hücreler24 saat CCh ile muamele edildiklerinde erken apoptotik hücresayısında azalma gözlemlenirken geç apoptoz ve nekrotik hücresayısında değişim olmamıştır. Bununla birlikte, 48 saat boyuncaCCh ile muamele olan hücrelerde, erken ve geç apoptotik hücresayısı azalırken, nekrotik hücrelerin sayısı artmıştır. CCh ilemuamele edilen hücrelerde nüklear ERK ekspresyonu artarkenbu etki 1,1-dimetil-4-difenilasetoksipiperidinium iodid (4DAMP)ile geri çevrilmiştir. Aynı koşullarda CCh ile muamele edilenhücrelerde nuklear pERK ekspresyonu azalmış, bu etki 4DAMPile geri çevrilmemiştir.Sonuç: Bulgularımız, K562 hücre proliferasyonundakikolinerjik etkinin yalnızca muskarinik mekanizma ile değildiğer kolinerjik reseptörlerin de katkısıyla gerçekleştiğinidüşündürmektedir., Objectives: Muscarinic receptors mediate diverse actions ofacetylcholine in the central nervous system and in non-nervoustissues innervated by the parasympathetic nervous system.Our study aims to evaluate the potential association of theM3 muscarinic receptor with K562 cell proliferation and death.Materials and Methods: Cell proliferation was evaluatedby bromodeoxyuridine (BrDU) incorporation. To show early,late apoptosis and cell death, cells were labelled with AnnexinV, propidium iodide (PI) and analyzed by flow cytometry. Nuclearextracellular signal-regulated kinase (ERK/pERK) expressionwas measured by western blot analysis.Results: Treatment with carbachol (CCh) for 48h decreased cellnumber. Exposing K562 cells to CCh for 24h decreased the number ofearly apoptotic cells but did not change the number of late apoptotic andnecrotic cells. CCh treatment for 48h increased the number of necroticcells, but decreased the number of early and late apoptotic cells. Inresponse to CCh, nuclear ERK expression was increased and this effectwas reversed by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide(4DAMP). Nuclear pERK expression was decreased in CCh treatedcells, 4DAMP did not reverse the effect.Conclusion: Our data suggest that cholinergic agonist CChaffects cell proliferation in K562 cells not only through muscarinicreceptors but also through other cholinergic receptors.

Objectives: Activated innate immunity is implicated in the pathogenesis of Behcet’s Disease (BD). To clarify the mechanisms of innate immune responses, we investigated inflammasome activation in dendritic cells (DC) and neutrophils, following stimulation with two different pattern recognition receptors (PRR) “RIG-1 like” (RLR) and “NOD-like” (NLR) in patients with BD. Methods: Sixteen active BD patients with mucocutaneous lesions and 17 healthy controls (HC) were included in this study. Dendritic cells were generated from monocytes. DCs and isolated neutrophils were activated by RLR and NLR ligands. Caspase-1 activation and expressions of p38 and RIP2 were determined by flow cytometry. Levels of IL-1ß, IL-6, TNF-?, IFN-? and IL-18 in culture supernatants were measured by ELISA. Results: Activation of caspase-1 following intracellular PRR stimulation was found to be in similar levels in DCs and neutrophils of BD patients compared to HC. However, activation of DCs from BD patients to NOD2 stimulus measured with the expressions of RIP2, p38 as well as IL-18 levels was found to be slightly defective (p

Background: In this study, we compared the clinical and immunological efficacy of nebulized corticosteroid (CS) to systemic route during treatment of moderate asthma attack in children. Methods: In this randomized, placebo-controlled, double-blind, double-dummy, prospective study, 81 children aged 12 months to 16 years experiencing asthma attack randomized into two treatment groups to receive, either; nebulized fluticasone propionate (n = 39, 2000 mcg/day) or oral methylprednisolone (n = 41, 1 mg/kg/day). Pulmonary index scores (PIS) were assessed at admission and at 1st, 4th, 8th, 12th, 24th, 48th hours, as well as, on day 7 and peak expiratory flow (PEF) at baseline and at the 7th day. Daily symptom and medication scores were recorded for all subjects. Immunological studies included phytohemagglutinin induced peripheral blood mononuclear cells culture supernatant for cytokine responses and CD4(+) CD25(+) FOXP3(+) T regulatory cell (T reg) percentage at baseline and day 7. Results: The changes in PIS and PEF were similar in both treatment groups, with a significant improvement in both values at the 7th day, when compared to baseline. In both groups, significant reductions in symptom and medication scores were observed during the treatment period with no significant difference between the groups. At day 7 of intervention, phytohemagglutinin induced IL-4 level was significantly decreased only in the nebulized group compared to baseline (p = 0.01). Evaluation of cytokine responses by means of fold increase (stimulated (S)/unstimulated (US) ratio) revealed a significant reduction in IL-4, IL-5 and IL-17 only in nebulized group (p = 0.01, 0.01, 0.02; respectively). The fold increase value of IL-5 was significantly lower at 7th day in nebulized group when compared to systemic one (p = 0.02). At 7th day, although in both treatment groups the percentage of T reg cells was suppressed, it remained significantly higher in the nebule one when compared to systemic route (p = 0.04). Conclusion: In the management of moderate acute asthma attack, nebulized CS (2000 mcg daily) was found to be as effective as systemic route with regard to clinical improvement. In addition, immunological parameters were more in favor of nebulized route which may imply a salutary effect of local CS usage. (C) 2015 Elsevier Ltd. All rights reserved.

Background. Inflammation is one of the main contributors to atherosclerosis in haemodialysis (HD) patients. Activation of Toll-like receptors (TLRs) leads to inflammatory response. In this study, we aimed to evaluate the expression of TLRs on monocytes and relate their expression with inflammation in chronic kidney disease (CKD) and HD patients. Methods. Thirty-four age- and gender-matched controls and stage 3-4 CKD patients and thirty-two HD patients were included in each study group. The effect of HD on the expression of Toll-like receptor-2 (TLR-2) and Toll-like receptor-4 (TLR-4) on CD14(+) monocytes was determined at the beginning (baseline), during (120 min) and following (300 min and 24 h) HD and compared with control and stage 3-4 CKD groups. The HD procedure was performed by using low-flux polysulphone dialysers. In addition, serum IL-6 levels were evaluated in both groups at baseline and after a HD session. Results. The percentage of CD14(+) monocytes expressing TLR-2 were similar in all of the study groups, whereas the percentage of CD14(+) monocytes expressing TLR-4 were significantly lower in both stage 3-4 CKD and HD patients at baseline than in controls. The mean fluorescence intensities (MFI) of TLR-2 were significantly lower in controls than in stage 3-4 CKD and HD patients at baseline. The MFI of TLR-4 was similar in all of the groups. The percentage of CD14(+) monocytes expressing TLR-2 did not change during and after HD. The MFI of TLR-2 decreased at 120 min of HD compared with baseline (1837 +/- 672 vs 1650 +/- 578, P < 0.05), and recovered back to baseline values at 300 min and at 24 h post-HD. MFI of TLR-4 increased at 24 h compared with baseline (941 +/- 294 vs 1087 +/- 441, P < 0.05). Serum IL-6 levels correlated with MFI of TLR-2 and TLR-4 in stage 3-4 CKD patients and in HD patients at baseline and after HD in univariate analysis. Stepwise multiple regression analysis revealed that MFI of TLR-2 was an independent determinant of serum IL-6 concentrations in stage 3-4 CKD and in HD patients at baseline, at 300 min and at 24 h post-HD. Conclusions. Our study demonstrates that TLR-2 is associated with the inflammatory response of non-dialysed and dialysed CKD patients.