Natural Killer Cells and Cytotoxic T Cells: Complementary Partners against Microorganisms and Cancer.

Natural killer (NK) cells and cytotoxic T (CD8+) cells are two of the most important types of immune cells in our body, protecting it from deadly invaders. While the NK cell is part of the innate immune system, the CD8+ cell is one of the major components of adaptive immunity. Still, these two very different types of cells share the most important function of destroying pathogen-infected and tumorous cells by releasing cytotoxic granules that promote proteolytic cleavage of harmful cells, leading to apoptosis. In this review, we look not only at NK and CD8+ T cells but also pay particular attention to their different subpopulations, the immune defenders that include the CD56+CD16dim, CD56dimCD16+, CD57+, and CD57+CD16+ NK cells, the NKT, CD57+CD8+, and KIR+CD8+ T cells, and ILCs. We examine all these cells in relation to their role in the protection of the body against different microorganisms and cancer, with an emphasis on their mechanisms and their clinical importance. Overall, close collaboration between NK cells and CD8+ T cells may play an important role in immune function and disease pathogenesis. The knowledge of how these immune cells interact in defending the body against pathogens and cancers may help us find ways to optimize their defensive and healing capabilities with methods that can be clinically applied.

Autophagy dysregulation plays a crucial role in regulatory T-cell loss and neuroinflammation in amyotrophic lateral sclerosis (ALS).

OBJECTIVE: Neuroinflammation is the hallmark of amyotrophic lateral sclerosis (ALS) disease. Regulatory T cells (Tregs) are essential in immune tolerance and neuroinflammation prevention. It has been shown that a significant decrease in Treg and FoxP3 protein expression is observed in ALS patients. The main reason for the FoxP3+ Treg loss in ALS is unknown. In this study, the role of autophagy dysregulation in FoxP3+ Tregs in ALS was investigated. METHODS: Twenty-three ALS patients and 24 healthy controls were recruited for the study. Mononuclear cells (MNCs) were obtained from peripheral blood, and then Tregs were isolated. Isolated Tregs were stained with FoxP3 and LC3 antibodies and analyzed in flow cytometry to determine autophagy levels in FoxP3+ Tregs in patients and controls. RESULTS: The mean of FoxP3+ LC3+ cells, were 0.47 and 0.45 in patients and controls, respectively. The mean of FoxP3+ LC3- cells was 0.15 in patients and 0.20 in controls, p = 0.030 (p < 0.05). There is no significant correlation between ALSFRS-R decay rate and autophagy level in patients. Also, there is no significant difference between autophagy levels in FoxP3+ Tregs in patients with rapidly progressing ALS and slow-progressing ALS. CONCLUSION: Excessive autophagy levels in FoxP3+ Tregs in ALS patients can potentially be an explanation for an increased cell death and result in worsened neuroinflammation and disease onset. However, the disease progress is not attributable to autophagy levels in FoxP3+ Tregs.

Regulatory B Cells Profile in Kidney Transplant Recipients With Chronic-Active Antibody-Mediated Rejection.

This study aims to reveal the relationship between regulatory B cell (Breg) subsets and chronic-active antibody-mediated rejection (c-aABMR) in renal transplant recipients. Our study involved 3 groups of participants: renal transplant recipients with biopsy-proven c-aABMR as the chronic rejection group (c-aABMR, n = 23), recipients with stable graft functions as the patient control group (PC; n = 11), and healthy volunteers (HV; n = 11). Breg subsets, immature/transitional B cells, plasmablastic cells, B10 cells, and BR1 cells were isolated from venous blood samples by flow cytometry. The median values of Breg frequencies in the total lymphocyte population were analyzed. There were no significant differences between the study groups for immature and/or transitional B cell frequencies. Plasmablastic cell frequencies of the c-aABMR group (7.80 [2.10-27.40]) and the PC group (6.00 [1.80-55.50]) were similar, but both of these values were significantly higher than the HVs' (3.40 [1.20-8.50]), (respectively, P = .005 and P = .039). B10 cell frequencies were also similar, comparing the c-aABMR (4.20 [0.10-7.40]) and the PC groups (4.10 [0.10-5.90]), whereas the HVs (5.90 [2.90-8.50]) had the highest B10 cell frequency with an only statistical significance against the PC group (respectively, P = .09 and P = .028). The c-aABMR and the PC groups were similar regarding BR1 cell frequencies. However, the HV group significantly had the highest frequency of BR1 cells (5.50 [2.80-10.80]) than the other groups (P < .001 for both). We demonstrated that frequencies of B10 and BR1 cells were higher in HVs than in transplant recipients, regardless of rejection state. However, there was no significant relation between Breg frequencies and the c-aABMR state.

Relationship of HLA-B alleles on susceptibility to and protection from HIV infection in Turkish population.

OBJECTIVE: Many human leukocyte antigen (HLA)-B alleles are associated with an increased risk of Acquired Immune Deficiency Syndrome (AIDS) and Human Immunodeficiency Virus (HIV) progression; however, their distribution varies among different racial/ethnic groups. Abacavir used in the treatment of AIDS significantly increases the risk of hypersensitivity reactions in patients with HLA-B*57:01. The aim of this study was to determine the distribution of HIV-associated HLA-B subgroups (high and low resolution) and HLA-B*57:01 associated with Abacavir sensitivity in Turkiye. METHODS: This retrospective case-control study consisted of 416 (F/M:111/305) HIV positive patients and 416 (F/M:111/305) healthy controls. HLA-B alleles were identified using Luminex based low-resolution method and further subgrouped by sequence-based high-resolution typing. RESULTS: Our data showed that in patients with HIV-1 infection, HLA-B*15, *35, and *51 allele frequencies were higher, while the HLA-B*07, *14 and *55 allele frequencies were lower as compared to the controls. It was determined that HLA-B*15:01, *35:01, *35:08, and *51:01 alleles frequencies were higher in the patients with HIV-1 infection compared to the controls as HLA-B*07:02, *14:01, *44:01, and *55:01 allele frequencies were detected low. HLA-B*57:01 allele positivity, which is important in Abacavir hypersensitivity, was lower than controls, and this difference was not statistically significant. CONCLUSION: Our results suggest that, HLA-B*07, *14, and *55 alleles and HLA-B*07:02, *14:01, *44:01, and *55:01 subgroups might have a protective effect, while HLA-B*15, *35, and *51 alleles and HLA-B*15:01, *35:01, *35:08, and *51:01 subgroups might play a role in susceptibility to HIV-1 infection.

Evaluation of the effect and predictive role of vitamin D and vitamin D receptor expression in CD4 + , CD8 + , CD14 + , CD56 + cells on the development of chronic rejection and graft functions in kidney transplant patients.

PURPOSE: It is known that vitamin D has positive effects on graft functions (reduce fibrosis, suppress excessive inflammatory response, improve graft functions). In our study, it was aimed to evaluate the effects and predictive roles of vitamin D, the expression of vitamin D receptor (VDR) in lymphocytes, monocytes, natural killer cells on chronic rejection and graft functions in kidney transplant patients. METHODS: Seventy one people were included in the study and analyses were made by dividing them into 3 groups. Group 1: Healthy control (n = 29), Group 2: Kidney transplant patients with stable kidney function (n = 17), and Group 3: Kidney transplant patients with chronic rejection diagnosis (n = 25). Serum 25-hydroxycholecalciferol, 1.25 dihydroxycholecalciferol levels and VDR percentages in CD4 + , CD8 + , CD14 + , CD56 + cells were measured in 3 groups. ROC analyses and logistic regression models were performed to predict rejection and long-term graft functions. RESULTS: The percentage of VDR expression in CD4 + lymphocytes (p < 0.001) and CD14( +) monocytes (p < 0.001), 25-hydroxycholecalciferol and 1.25 dihydroxycholecalciferol levels were lower in group 3 was detected. In ROC analyses and logistic regression models, VDR expression in CD4( +)T lymphocytes was shown to have a statistically significant value in the development of chronic rejection (Odds ratio 0.86: 0.76-0.92; p = 0.001/AUC = 0.941, p < 0.001) and prediction of 5th-year graft functions (Odds ratio 0.93: 0.88-0.98; p = 0.017/AUC = 0.745, p = 0.007). CONCLUSION: In our study, it was shown that low vitamin D and VDR expression is associated with poor outcome and VDR expression in CD4( +)T lymphocytes is predictive in terms of graft function and rejection.

C allele in transforming growth factor-ß1 rs1800471 gene polymorphisms might indicate a protective feature in encapsulating peritoneal sclerosis development.

INTRODUCTION: Peritoneal fibrosis may progress in peritoneal dialysis (PD) patients to a fatal clinical condition called encapsulating peritoneal sclerosis (EPS). Transforming growth factor (TGF)-ß plays a pivotal role in the pathogenesis of peritoneal fibrosis. We aimed to investigate the association among polymorphisms in the gene encoding TGF-ß1, which were -509C/T (rs1800469), +869T/C (rs1982073), and +915G/C (rs1800471) in EPS patients. METHODS: A total of 16 PD patients who were clinically and radiologically diagnosed with EPS were enrolled and 22 age- and gender-matched PD patients were selected as the non-EPS group. RESULTS: G allele frequency at the rs1800471 gene polymorphism was significantly higher in the EPS group than non-EPS group (p = 0.005). Interestingly, the non-EPS group patients had CC or CG polymorphisms. CONCLUSION: C allele in TGF-ß1 rs1800471 gene polymorphisms might indicate a protective feature in EPS development. Knowing the presence of polymorphism may be effective in selecting renal replacement therapy in patients.

DETERMINATION OF THE PRA POSITIVITY PERCENTAGE IN MALE PATIENTS WITH CHRONIC KIDNEY DISEASE BY USING FLOW CYTOMETRY TECHNIQUE.

The antibodies directed against human leukocyte antigen (HLA) molecules, which play a crucial role in allograft histocompatibility, are called anti-HLA antibodies. Anti-HLA antibodies against foreign HLA molecules may be present in patients with chronic kidney disease even before transplantation. The panel reactive antibody (PRA) test is used to measure the renal transplant candidate's immune sensitivity to HLA molecules other than their own HLA molecules by assessing the diversity of anti-HLA antibodies in the blood of these patients. This study aimed to determine the PRA values and the percentage of PRA positivity of Turkish male patients with chronic kidney disease (CKD), who had not been sensitized by the major known causes (those with no history of organ or tissue transplantation, those with no history of blood transfusion), who had not been diagnosed with any autoimmune diseases, and who had not been under immunosuppressive treatment. The study included 60 male patients aged over 18 years. All of the patients were followed up with a diagnosis of CKD at the Nephrology Clinic, Internal Medicine Department, Akdeniz University Medical Faculty Hospital. None of the patients included in the study was sensitized by a known mechanism previously (they did not have blood transfusion or organ transplantation). Glomerular filtration rate (GFR) levels of all patients were below the level of 60 mL/min/1.73 m2. Patient data including their age information, etiology of CKD, accompanying diseases, and information about dialysis modalities were recorded. HLA antibody percentage was determined by the flow cytometry technique. Statistical data analysis was performed by using SPSS 22.0 (Statistical Package for Social Sciences, Version 22.0). The values of p less than 0.05 were considered statistically significant. Twenty patients were receiving dialysis treatment due to end-stage renal disease. Of the 60 patients included in the study, 25% showed PRA positivity; 28.3% of all study patients were found to be positive for anti-HLA class I antibodies and 26.7% of all study patients were found to be positive for anti-HLA class II antibodies on separate analysis for anti-HLA class I and anti-HLA class II antibody positivity. When the patients were categorized as PRA negative and PRA positive in two groups, there were no differences between the groups according to mean age, percentage of hemodialysis patients, percentage of peritoneal dialysis patients and presence of accompanying chronic diseases (such as hypertension, type 2 diabetes mellitus, hyperlipidemia, nephrolithiasis, coronary artery disease). In addition to this, evaluation of the GFR levels showed that the PRA positive group contained a significantly higher percentage of end-stage renal disease patients (GFR <15 mL/min/1.73 m2) as compared with the PRA negative group. Detailed analysis of the percentages of PRA levels in the PRA positive patients, which was carried out to determine the degree of sensitization, showed that the PRA values were over 80% in 11.77% of the patients positive for anti-HLA class I antibodies. On the other hand, PRA values were within the range of 15%-80% in 88.23% of the patients who had anti-HLA class II antibodies. The PRA values were below 80% in all of the patients positive for anti-HLA class II antibodies and those positive for both anti-HLA class I and class II antibodies. In conclusion, PRA levels of the candidates for kidney transplantation should always be measured to assess their state of sensitization before transplantation, even though they have no risk factors known to cause anti-HLA antibody development.

Prognosis and Treatment for Active and Chronic Antibody-Mediated Rejection in Renal Transplant Recipients; Single Center Experience.

BACKGROUND: The aim of the study was to evaluate the prognostic factors and treatment alternatives of antibody-mediated rejection (ABMR) in renal transplant patients. METHODS: Three thousand renal transplant patients were included in the study. The patients were first divided into 2 groups. Group 1: ABMR [-] recipients (n = 2871), Group 2: ABMR (+) recipients (n = 129). ABMR patients were compared among themselves by dividing them into 3 subgroups (early-active, late-active, chronic-active). The study was performed retrospectively. Different combinations of methylprednisolone, intravenous immunoglobulin (IVIG), rituximab, plasmapheresis (PP), anti-thymocyte globulin (ATG) were used in the treatment and the results were compared. RESULTS: Graft survival and functions were worse and the rates of CAD, delayed graft function, BK virus, and cytomegalovirus higher in patients with ABMR. Also, graft survival was lower in patients with serum creatinine ≥3 (P = 0.001), GFR <30 (P <0.001), and spot urine protein to creatinine ratio ≥1 (P = 0.042) at the time of diagnosis. High interstitial fibrosis and tubular atrophy scores in chronic ABMR cases and high intimal arteritis scores in active ABMR cases were poor prognostic factors. CONCLUSIONS: The study showed that ABMR has a poor prognosis in terms of clinical parameters, and treatment should be individualized according to pathologic findings and graft functions at the time of diagnosis. Pulse methylprednisolone and IVIG should be used in the treatment of all ABMR patients, but PP, rituximab, and ATG should be used in selected cases. ABMR has a poor prognosis and treatment should be individualized.

Diclofenac down-regulates COX-2 induced expression of CD44 and ICAM-1 in human HT29 colorectal cancer cells.

Cyclooxygenase-2 (COX-2) is expressed in a variety of human colorectal cancer cells and can contribute to carcinogenesis. This study aimed to investigate the effect of diclofenac (DCF), a selective COX-2 inhibitor, on cell adhesion molecules and apoptosis in human colon adenocarcinoma cells. Levels of homing cell adhesion molecule (H-CAM, CD44), intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule-1 (VCAM-1, CD106), and epithelial cell adhesion molecule (EpCAM, CD326) were evaluated in cancer cells overexpressing (HT29) or not expressing (HCT116) COX-2. Cell viability was determined by MTT assay, COX-2 protein levels and activity were assessed by immunofluorescence and fluorometric analysis, respectively. Endogenous levels of polyunsaturated fatty acids (PUFAs) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) while expression of cell adhesion molecules was analyzed by flow cytometry. Annexin V-FITC/propidium iodide-labelling and fluorometric caspase-3 activity measurements were carried out to determine apoptosis. Flow cytometry analysis revealed that the percentage of CD44 and ICAM-1 staining in HCT116 cells was significantly lower compared to HT29 cells. In HT29 cells, phorbol 12-myristate 13-acetate (PMA) induced COX-2 expression and increased CD44 and ICAM-1 levels were down-regulated by diclofenac. Stimulation of COX-2 activity in HT29 cells via PMA significantly decreased diclofenac associated increase in PUFA levels. Treatment with both diclofenac and PMA significantly increased the number of apoptotic cells and caspase-3 activity in colon adenocarcinoma cells compared to control groups. In conclusion, diclofenac's effect to retard colorectal tumor growth and metastasis occurs in COX-2 overexpressing colon cancer cells by increased apoptosis and decreased expression of CD44 and ICAM-1.

Levels of lymphocyte-associated regulators of complement system CD55 and CD59 are changed in schizophrenia patients.

OBJECTIVE: Although pathological mechanisms of schizophrenia are unknown, evidence in the literature suggests that the immune system might be involved in the pathogenesis. Complement is an important part of the immune system and it has been suggested to play role in the pathogenesis of schizophrenia. We aimed to investigate the potential involvement of the complement system in schizophrenia by the determination of peripheral concentrations of certain complement proteins and their regulators in patients. METHODS: Plasma concentrations of complement C3, C4, and C1 inhibitory protein were measured by chemiluminescence in 41 schizophrenia patients and 39 healthy controls. Expression of CD55, CD59, and CD46 proteins on peripheral blood mononuclear cells were determined by flow cytometry in the same groups. RESULTS: Frequencies of peripheral immune cells expressing CD55 were determined to be significantly higher in schizophrenia patients than in healthy people (p = 0.020). Frequencies of peripheral immune cells expressing CD59 was determined to be significantly higher in healthy people than in schizophrenia patients (p = 0.012). The expression level of CD55 per cell was measured to be significantly elevated in patients compared to healthy controls (p = 0.026). CONCLUSIONS: Our data clearly demonstrate an elevated complement activity in schizophrenia and points to a possible complement association in the pathogenesis.Key pointsIncreased the expression level, and frequency of CD55 in schizophrenia patients.Decreased frequency of CD59 in schizophrenia patients.No difference in the expression level of CD59; the expression level, and frequency of CD46; frequency of complement C3, C4, and C1 inhibitory protein.

Effects of amnion derived mesenchymal stem cells on fibrosis in a 5/6 nephrectomy model in rats.

Chronic kidney disease (CKD) is characterized by disruption of the glomerulus, tubule and vascular structures by renal fibrosis. Mesenchymal stem cells (MSC) ameliorate CKD. We investigated the effects of human amnion derived MSC (hAMSC) on fibrosis using expression of transforming growth factor beta (TGF-ß), collagen type I (COL-1) and bone morphogenetic protein (BMP-7). We also investigated levels of urinary creatinine and nitrogen in CKD. We used a 5/6 nephrectomy (5/6 Nx) induced CKD model. We used 36 rats in six groups of six animals: sham group, 5/6 Nx group, 15 days after 5/6 Nx (5/6 Nx + 15) group, 30 days after 5/6 Nx (5/6 Nx + 30) group, transfer of hAMSC 15 days after 5/6 Nx (5/6 Nx + hAMSC + 15) group and transfer of hAMSC 30 days after 5/6 Nx (5/6 Nx + hAMSC + 30) group. We isolated 106 hAMSC from the amnion and transplanted them via the rat tail vein into the 5/6 Nx + hAMSC + 15 and 5/6 Nx + hAMSC + 30 groups. We measured the expression of BMP-7, COL-1 and TGF-ß using western blot and immunohistochemistry, and their gene expressions were analyzed by quantitative real time PCR. TGF-ß and COL-1 protein, and gene expressions were increased in the 5/6 Nx +30 group compared to the 5/6 Nx + hAMSC + 30 group. Conversely, both protein and gene expression of BMP-7 was increased in 5/6 Nx + hAMSC + 30 group compared to the 5/6 Nx groups. Increased TGF-ß together with decreased BMP-7 expression may cause fibrosis by epithelial-mesenchymal transition due to chronic renal injury. Increased COL-1 levels cause accumulation of extracellular matrix in CKD. Levels of urea, creatinine and nitrogen were increased significantly in 5/6 Nx + 15 and 5/6 Nx + 30 groups compared to the hAMSC groups. We found that hAMSC ameliorate CKD.

Regulation of E2F1 activity via PKA-mediated phosphorylations.

E2F1 becomes activated during the G1 phase of the cell cycle, and posttranslational modifications modulate its activity. Activation of G-protein coupled receptors (GPCR) by many ligands induces the activation of adenylate cyclases and the production of cAMP, which activates the PKA enzyme. Activated PKA elicits its biological effect by phosphorylating the target proteins containing serine or threonine amino acids in the RxxS/T motif. Since PKA activation negatively regulates cell proliferation, we thought that activated PKA would negatively affect the activity of E2F1. In line with this, when we analyzed the amino acid sequence of E2F1, we found 3 hypothetical consensus PKA phosphorylation sites located at 127-130, 232-235, and 361-364 positions and RYET, RLLS, and RMGS sequences. After showing the binding and phosphorylation of E2F1 by PKA, we converted the codons of Threonine-130, Serine-235, and Serine-364 to Alanine and Glutamic acid codons on the eukaryotic E2F1 expression vector we had previously created. We confirmed the phosphorylation of T130, S235, and S364 by developing monoclonal antibodies against phospho-specific forms of these sites and showed that their phosphorylation is cell cycle-dependent. According to our results, PKA-mediated phosphorylation of E2F1 by PKA inhibits proliferation and glucose uptake and induces caspase-3 activation and senescence.

Concealed role of red blood cells in pathogenesis of pulmonary arterial hypertension: Decreased red blood cell nitric oxide generation and effect of Rho-Kinase inhibitor fasudil.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a devastating disease characterized with alterations in pulmonary vasculature yielding increased pulmonary arterial resistance. Emerging evidences suggest important regulatory roles of red blood cells (RBCs) on nitric oxide (NO) bioavailability, mainly by modulating their endothelial nitric oxide synthase (eNOS) enzyme activity. OBJECTIVE: The aim of this pilot study was to evaluate the alterations in RBC eNOS activity and intracellular NO generation in PAH patients and the modulatory effects of Rho-Kinase (ROCK) inhibitors. METHODS: RBCs were isolated from patients with PAH and age-matched healthy subjects and were analyzed for their eNOS activity and NO generation capacity under the conditions of the presence or absence of ROCK inhibitor, fasudil. Phosphotidylserine (PS) exposure was also defined. RESULTS: eNOS activity and intracellular NO generation were lower in RBC from PAH patients. ROCK inhibitor increased basal eNOS activity and improved NO generation capacity of RBC of PAH patients to healthy control levels. PS exposure levels were also higher in RBC of PAH patients. CONCLUSIONS: This study provides first evidences for decreased RBC eNOS activity due to its ROCK mediated negative regulation in PAH patients. Considering increased ROCK activity contribution to progression of PAH, ROCK inhibition influences NO bioavailability through RBC eNOS, in addition to endothelial eNOS.

Lymphocyte and monocyte vitamin D receptor expression during paricalcitol or calcitriol treatments in patients with stage 5 chronic kidney disease.

PURPOSE: In this study, we aimed to investigate the effect of paricalcitol and calcitriol usage on vitamin D receptor (VDR) contents of CD8+ , CD4+ lymphocytes and monocytes in stage 5d chronic kidney disease (CKD) patients. METHODS: Thirty-six hemodialysis patients older than 18 years of age and 19 healthy controls (group HC) without any known acute or chronic diseases were included in the study. The group of patients undergoing scheduled hemodialysis comprised three subgroups: group CL: patients on calcitriol (n: 10), group PC: patients on paricalcitol (n: 13), and group NT: patients not taking any vitamin D or VDR activating medications (n: 13). CD8+/VDR, CD4+/VDR and MONO/VDR values were representing the ratio of VDR representing cells among related cell group. On the other hand, values of CD8+/MFI, CD4+/MFI and MONO/MFI have shown the total amount of cellular VDR content per cell which has been given as of mean fluorescence intensity in the flow cytometric process. Main CKD mineral bone disorder parameters such as a hemogram, serum BUN, creatinine, albumin, Ca, iP, iPTH, 25(OH)D3 levels were also measured. RESULTS: Average VDR contents in CD8+, CD4+ and monocytes were not different among three patient groups on hemodialysis. But in all hemodialysis subgroups, CD8+/VDR, CD4+/VDR, MONO/VDR, CD8+/MFI, CD4+/MFI and MONO/MFI levels were found to be higher compared with the healthy control subjects (p < 0.001). Among hemodialysis groups, no significant CD8+/VDR, CD4+/VDR, and MONO/VDR content differences were found with regard to the type of VDR activator agent used. There was no difference in serum levels of 25(OH)D3 and CRP among groups participating in the study. CONCLUSION: There was no difference between CD8+/VDR, CD4+/VDR, and MONO/VDR levels in hemodialysis patients using calcitriol or paricalcitol, suggesting that both treatment agents may have a similar effect on VDR contents in lymphocytes and monocytes in that patient population. But in all hemodialysis subgroups, CD8+/VDR, CD4+/VDR, and MONO/VDR levels were found to be higher compared with the healthy control subjects, suggesting an overexpression of VDR through a non CRP and/or 25(OH)D3 dependent mechanism.

NK1R antagonist decreases inflammation and metastasis of breast carcinoma cells metastasized to liver but not to brain; phenotype-dependent therapeutic and toxic consequences.

Substance P a neuro-immune mediator acts on Neurokinin-1 and -2 receptors (NK1R and NK2R). Inhibitors of NK1R are considered to be safe and effective approaches for cancer treatment since Aprepitant, a non-peptide antagonist of NK1R is widely used for chemotherapy-induced emesis and has cytotoxic and antitumor effects in various models for cancer. On the other hand, our previous findings demonstrated that systemic inhibition of NK1R may decrease cytotoxic anti-tumoral immune response. Hence, actual consequences of inhibition of neurokinin receptors under in vivo conditions in a syngeneic model of carcinoma should be determined. The effects of highly potent and selective non-peptide mouse NK1R and NK2R antagonists RP 67580 and GR 159897, respectively, on metastatic breast carcinoma were evaluated. Specifically, 4T1 breast cancer cells metastasized to brain (denoted as 4TBM) and liver (denoted as 4TLM) were used to induce tumors in Balb-c mice. Changes in tumor growth, metastasis and immune response to cancer cells were determined. We here observed differential effects of NK1R antagonist depended on the subset of metastatic cells. Specifically, inhibition of NK1R markedly increased liver metastasis of tumors formed by 4TBM but not 4TLM cells. On the contrary, NK1R antagonist decreased inflammatory response and liver metastasis in 4TLM-injected mice. 4TLM tumors act more aggressively inducing more inflammatory response compared to 4TBM tumors. Hence, differential effects of NK1R antagonist are at least partly due to extend and type of the inflammatory response evoked by specific subset metastatic cells. These findings demonstrate the necessity for understanding the immunological consequences of tumor-microenvironment interactions.

Role of the Th1 and Th17 Pathway in Subacute Sclerosing Panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a progressive and fatal disease caused by reactivation of a mutated measles virus in brain tissue. The process of reactivation is yet to be elucidated. In this study, the possible roles of the Th1 (interleukin [IL]-12, interferon [IFN]-γ) and the Th17 axis (IL-23, IL-17, IL-22), particularly of IL-17, in the pathogenesis of SSPE were investigated. Briefly, mononuclear cells from SSPE patients were stimulated using measles virus peptide, and the release of IL-12, IL-23, IL-22, IFN-γ, and IL-17 cytokines was measured using enzyme-linked immunosorbent assay and/or enzyme-linked immunosorbent spot assay (ELISpot). We found that in comparison to the mononuclear cells obtained from healthy donors, cells from SSPE patients exhibited increased levels of IL-12, IL-23, IL-17, IL-22, and IFN-γ cytokines in response to measles virus stimulation. However, the same result was not obtained with cytomegalovirus and phytohemagglutinin. Using flow cytometry, mononuclear cells obtained from SSPE patients and healthy controls were also analyzed for the presence of intracellular IL-17 in response to measles virus stimulation. On stimulation, the number of IL-17-positive cells were found to be higher among mononuclear cells obtained from the patients. In addition, the numbers of IL-17- and IFN-γ-positive cells were significantly increased in SSPE patients. In conclusion, this study demonstrates that both the IL-12/IFN-γ and the IL-23/IL-17/IL-22 pathways are functionally abnormal in SSPE pathogenesis. Targeting these pathways and their specific pro-inflammatory mediator production may provide a new strategy to suppress SSPE development.

Rho-kinase is a negative regulator of red blood cell eNOS under basal conditions.

BACKGROUND: Rho-kinase, an effector of the small GTPase RhoA, is known to be a novel inhibitory regulator of eNOS in endothelial cells under basal conditions and disease states. However, although RBC possesses active RhoA/Rho-kinase pathway, Rho-kinase mediated eNOS regulation has not been investigated in RBC, so far. OBJECTIVE: The aim of the present study is to investigate whether eNOS activity is regulated by Rho-kinase under basal conditions and to evaluate whether inhibition of this enzyme causes eNOS activation and intracellular NO production in RBC. METHODS: RBC packeds were isolated from healthy volunteers and resuspended in Hepes solution at a hematocrit of 0.01 l/l. Intracellular NO and Ca+2 levels and eNOS activation measured by flow cytometry in response to Rho-kinase inhibitors, fasudil and Y-27632, in the absence and presence of NOS, and PI3K inhibitors. RESULTS: Rho-kinase inhibitors fasudil and Y-27632 found to increase intracellular NO concentrations. These inhibitors also cause enhancement of intracellular Ca+2 and serine 1177 phosphorylated eNOS levels. Besides, although these responses have shown to be suppressed by NOS enzyme, PI3K inhibition had no effect on this mechanism. CONCLUSIONS: The results of the present study demonstrated that RBC eNOS enzyme activity is regulated by inhibitory Rho-kinase pathway under basal conditions and inhibition of this pathway enhances the activity of eNOS in RBC. This activation is mediated by both intracellular Ca+2 and Serine 1177 phosphorylated eNOS increment, with no contribution of AKT activation, in RBC. The mechanism we described here gives first evidences about Rho-kinase mediated eNOS regulation in RBC under basal conditions. This pathway could also be more important under disease states.

Effects of Human Placental Amnion Derived Mesenchymal Stem Cells on Proliferation and Apoptosis Mechanisms in Chronic Kidney Disease in the Rat.

BACKGROUND AND OBJECTIVES: The feature of chronic kidney failure (CKF) is loss of kidney functions due to erosion of healthy tissue and fibrosis. Recent studies showed that Mesenchymal stem cells (MSCs) differentiated into tubular epithelial cells thus renal function and structures renewed.Furthermore, MSCs protect renal function in CKF. Therefore, we aimed to investigate whether human amnion-derived mesenchymal stem cells (hAMSCs) can repair fibrosis and determine the effects on proliferation and apoptosis mechanisms in chronic kidney failure. METHODS AND RESULTS: In this study, rat model of CKF was constituted by applying Aristolochic acid (AA). hAMSCs were isolated from term placenta amnion membrane and transplanted into tail vein of rats. At the end of 30 days and 60 days of recovery period, we examined expressions of PCNA, p57 and Parp-1 by western blotting. Immunoreactivity of PCNA, Ki67, IL-6 and Collagen type I were detected by immunohistochemistry. Besides, apoptosis was detected by TUNEL. Serum creatinine and urea were measured. Expressions of PCNA and Ki67 increased in hAMSC groups compared with AA group. Furthermore, expressions of PARP-1 apoptosis marker and p57 cell cycle inhibitory protein increased in AA group significantly according to control, hAMSC groups and sham groups. IL-6 proinflammatory cytokine increased in AA group significantly according to control, hAMSCs groups and sham groups. Expressions of Collagen type I protein reduced in hAMSCs groups compared to AA group. After hAMSC treatment, serum creatinine and urea levels significantly decreased compared to AA group. After injection of hAMSC to rats, Masson’s Trichrome and Sirius Red staining showed fibrosis reduction in kidney. CONCLUSIONS: According to our results hAMSCs can be ameliorate renal failure.

Extracellular ATP activates eNOS and increases intracellular NO generation in Red Blood Cells.

BACKGROUND: It has been well documented that ATP activates NOS enzymes and causes increased NO production in several cell types. Although RBC known to possesses eNOS enzyme activity, it has not been investigated whether RBC eNOS could be induced by extracellular ATP. OBJECTIVE: The aim of the present study is to evaluate extracellular ATP mediated eNOS activation and NO production in RBC. METHODS: RBC packed were isolated from healthy volunteers and re-suspended in Hepes solution at a hematocrit of 0.01 l/l. Intracellular NO and Ca+2 levels and eNOS activation measured by flow cytometry in response to P2X receptor agonist, Bz-ATP, in the absence and presence of NOS, P2 receptors and PI3K inhibitors. RESULTS: P2X receptor agonist Bz-ATP found to increase intracellular NO, Ca+2 and serine 1177 phosphorylated eNOS levels and these responses have shown to be suppressed by NOS enzyme, P2 receptors and PI3K inhibitors. CONCLUSIONS: The results of the study clearly demonstrated extracellular ATP induced NO generation in RBC through intracellular Ca+2 and PI3K/Akt pathways. The mechanism we described here might be important at basal conditions and also in conditions with increased ATP release.

[Monitoring of cytomegalovirus-specific CD4+ and CD8+ T cell responses by cytokine flow cytometry in renal transplant recipients]. / Böbrek nakil hastalarinda sitomegalovirusa özgül CD4+ ve CD8+ T hücre yanitinin sitokin akim sitometri yöntemiyle izlemi.

In spite of the improvements in the clinical management of solid organ transplant (SOT) recipients provided by immunosuppresion and universal prophylaxis, human cytomegalovirus (CMV) infections continue to be one of the most leading causes of morbidity and mortality. Cell-mediated immunity specific to CMV (CMV-CMI) plays an important role in the control of CMV replication. Therefore, monitoring of CMV-specific T-cell response can be used to predict individuals at increased risk of CMV disease. The aim of this study was to investigate the levels of CMV-specific interferon (IFN)-γ producing CD4(+) and CD8(+) T cells in kidney transplant recipients before and after the transplantation, by cytokine flow cytometry. A total of 21 kidney transplant recipients (14 male, 7 female; age range: 18-66 years, mean age: 34.5 ± 9.9) who were all CMV seropositive have been evaluated in the study. Blood samples from the patients were obtained before and at the 1(st), 3(rd) and 6(th) months after transplantation. CMV seropositive healthy kidney donors (n= 20) constituted the control group. The main stages of our procedure were as follows; isolation of peripheral blood mononuclear cells from whole blood, freezing and storing of the samples, later on thawing the samples, ex vivo stimulation of lymphocytes with pooled CMV peptides and counting CMV-specific IFN- producing CD4(+) and CD8(+) T cells by flow cytometry following surface and intracellular cytokine staining. Monitoring of the viral load (CMV-DNA) was performed in 10 days intervals in the first 3 months followed by 3 week intervals until 6 months using COBAS AmpliPrep/COBAS TaqMan CMV test system (Roche Diagnostics, USA). The frequencies of pretransplant CMV-specific IFN-γ producing CD8(+) T cells in patient (3.53 ± 4.35/µl) and control (4.52 ± 5.17/µl) groups were not statistically different (p= 0.266). The difference between the number of virus-specific CD4(+) T cells in patients (8.84 ± 9.56/µl) and those in the control group (8.23 ± 11.98/µl) was at the borderline of significance (p= 0.057). The age and gender of the patients and type of antiviral prophylaxis protocols [valgancyclovir (n= 4); valacyclovir (n= 17)] did not have any significant effect on CMV-CMI (p> 0.05). Similarly, induction therapy administered to four patients did not show any effect on CMV-CMI (p> 0.05). CMV-specific immune responses of patients who received different immunosuppression protocols [tacrolimus + mycophenolate mofetil (MMF) + steroid (n= 17); cyclosporine + MMF + steroid (n= 2); mTOR inhibitor + MMF + steroid (n= 2)] were not different (p> 0.05). The number of CMV-specific CD4(+) T cells in all patients were significantly decreased in the 3rd month compared to the 1st month after the transplantation (p=0.003), indicating a relationship with the period of immunosuppressive therapy. In one of the patients who did not have CMV-specific CD4+ T-cell response but had cytotoxic T-cells (CD8(+) T= 0.6%) before transplantation, CD4(+) T-cell response have developed during monitorization (1.4%, 1.5% and 0.5% in 1st, 3rd and 6th months, respectively), and no viral reactivation was detected. Out of the two patients who had no CD4(+) and CD8(+) T cell response in the 3rd month, one of them developed low level viremia (150 copies/ml) in the 6th month. In this patient the level of CMV-CMI in the 6th month (CD4(+)T + CD8(+)T= 0.9%), have reached higher values than the values obtained before the transplantation (CD4(+) T + CD8(+) T= 0.5%). The viremia was cleared spontaneously in this patient and no antiviral therapy was required. In conclusion, our results suggested that pretransplant and posttransplant monitoring of CMV-specific T-cell responses might be helpful as well as viral load in the clinical management of CMV infection in SOT patients.