The Expression Levels of MicroRNAs Associated with T and B Cell Differentiation/stimulation in Ankylosing Spondylitis.

Spondyloarthropathies (SpAs), are a group of chronic inflammatory diseases with a number of genetic, physiopathological, clinical and radiological features. Ankylosing spondylitis (AS) is the most common type of spondylo-arthropathies, and >90.0% of patients with ankylosing spondylitis are human leukocyte antigen-B27 (HLA-B2 7)-positive. In recent years, non-HLA genetic factors have been reported to have an effect on ankylosing spondylitis. MicroRNAs (miRNAs), are endogenous non coding RNA molecules containing 18-23 nucleotides that play a role in the post-transcriptional regulation of gene expression. In this study, we aimed to determine the expression levels of miRNAs associated with T- and B-cell differentiation/stimulation in peripheral blood mononuclear cells and their relationship with the etiology of the AS in patients and healthy controls. In a molecular study, peripheral blood mononuclear cell isolation, and total RNA isolation were performed first. In the second step, cDNA synthesis and quantitative real-time PCR (qPCR) expression analysis were completed. Ultimately, in the patient and control group, the expression levels of miR-142-5p and miR-143 were found to be significantly different (p <0.05). According to current knowledge, miR-142-5p andmiR-143 expressions were found to be important for those diseases that share similar etiology with AS. We suggest that miR-142-5p and miR-143 may play a role in the pathogenesis, especially miR- 142-5p may be a potential biomarker and a target molecule for the treatment.

Cytotoxic Antibody Detection by Means of Flow-Cytometric Cross-Match.

BACKGROUND: Complement-dependent lymphocytotoxicity (CDC-XM) and flow-cytometric (FCXM) cross-match are analyzed individually for each donor and recipient pair, because these techniques have fundamental differences for the evaluation of histocompatibility. Lately, cytotoxic flow-cytometric cross-match (cFCXM) has been developed as an alternative to both CDC-XM and FCXM techniques. We evaluated the limits of cFCXM with the use of different positive serum dilutions. METHODS: CDC-XM, FCXM, and cFCXM tests were performed with the use of commercially available negative and positive serum samples and lymphocytes from healthy donors. RESULTS: Complement-dependent cell death was successfully detected with the use of cFCXM. Complement-dependent cell death ratios in cFCXM were similar those in CDC-XM. With cFCXM, not only complement-dependent cell death but also IgG binding could be detected within a single assay. At higher concentrations of the positive serum, IgG-fluorescein isothiocyanate (FITC) mean fluorescent intensity (MFI) values detected with the use of cFCXM were less than those of conventional FCXM. Correspondingly, for dead cells, MFI values of IgG-FITC were less than those of live cells in higher positive serum concentrations in the cFCXM assay. Moreover, our results demonstrated that in cFCXM analysis, the decreasing ratio of dead cells at increasing positive serum dilutions was not in parallel with the same decrease in IgG-FITC MFI values. CONCLUSIONS: The cFCXM technique detects complement-mediated cytotoxic cell death with the additional ability to show IgG binding in the same tube and therefore may reduce the necessary bench time and workload.

Factors Affecting the Selection of Patients on Waiting List: A Single Center Study.

INTRODUCTION: There is an increasing gap between organ supply and demand for cadaveric transplantation in our country. Our aim was to evaluate factors affecting selection of patients on waiting list at our hospital. METHOD: Patients who have been waiting on list and who were transplanted were compared in order to find factors, which affected the selection of patients. Non-parametric Mann-Whitney U test was used for comparison and cox regression analysis was used to find the risk factors that decrease the probability of transplantation in this retrospective case-control study. RESULTS: Patients in the transplanted group were significantly younger, had relatively lower body mass index than the awaiting group. Cardiovascular diseases were more in the awaiting group than the transplanted group. There was no patient with diabetes in transplanted group, despite fifteen diabetic patients were in the awaiting group. Selected patients had lower immunologic risk with regard to peak panel reactive antibody levels. No significant difference was found for gender, hypertension, hyperlipidemia, viral serology, time spent on dialysis and on waiting list between two groups. With cox regression analysis female gender, older age, diabetes mellitus, high body mass index, positive hepatitis B serology and high levels of peak class 1-2 peak panel reactive antibody positivity were found as risk factors that decrease the probability of transplantation. CONCLUSION: A tendency for selection of low risk patients was found with this study. Time and energy consuming complications and short allograft survival after transplantation in high risk patients and the scarcity of cadaveric pool in our country may contribute to this tendency.

Relationship of Urothelial Gene Expressions in Urine-Deprived Bladders of Renal Recipients With Posttransplant Urinary Infections.

OBJECTIVE: In this study, we analyzed gene expression levels of apoptotic (Fas, FasL, Bcl-2, Bax) and survival (CXCR1, CXCR2, IL-8) signal pathways of the urine-deprived bladder tissues and the relation of urinary tract infections with these pathways. MATERIAL AND METHODS: We included 37 patients admitted for renal transplantation between December 2009 and December 2012. Bladder mucosal samples were obtained at the time of transplantation and 6-8 weeks posttransplantation, at the time of ureteral catheter removal. RNA extraction and cDNA synthesis were done using guanidium-thiocyanate and colon filter methods. Expression analysis was studied with quantitative real-time polymerase chain reaction optimized with ROX dye and internal control ß-actin. RESULTS: According to our findings Fas, FasL, Bcl-2, and Bax expression was higher in urine-deprived bladder samples than those in the posttransplant samples (P < .05). Although Fas, FasL, Bcl-2, and Bax expression levels increased in pretransplant samples, there was an increase in posttransplant bladder samples; however, this increase was not as marked as those of pretransplant samples. IL-8, CXCR1, and CXCR2 expression was decreased at the pretransplant samples and increased in posttransplant bladder samples. CONCLUSIONS: We have found an upregulated apoptotic process and decreased survival signals at the urine-deprived bladder tissue. After transplantation, bladder tissue survival parameters were increased, demonstrating the importance of urinary flow for bladder tissue.

Cancer Screening of Renal Transplant Patients Undergoing Long-Term Immunosuppressive Therapy.

OBJECTIVE: With this study we aimed to research the effects of immunosuppressive drugs, their cumulative doses, and viral infections on development of malign tumors in patients who have undergone treatment for 5 years. METHODS: We examined 100 patients who underwent renal transplantation from 2004 to 2009. Patients had mycophenolate mofetil and steroid in addition to cyclosporine, sirolimus, or tacrolimus as immunosuppressive treatment. For malignancy screening, physical examination, radiologic and endoscopic screening were done, and immunosuppressive drugs and their cumulative doses, age, sex, body mass index (BMI), dialysis history, and viral infection history were investigated. RESULTS: The mean age of patients was 42.03 ± 11.30 years. There were 1 colon cancer patient, 1 retroperitoneal liposarcoma, 1 renal oncocytoma, 3 Kaposi sarcoma patients treated with cyclosporine; in those treated with Tac there were 1 basal cell carcinoma, 1 Kaposi sarcoma, 2 thyroid carcinoma, 1 breast carcinoma, 1 bladder carcinoma, 1 renal cell carcinoma, and 1 colon carcinoma patients. The mean age of patients having carcinoma was statistically significant compared with those without cancer (P < .01). The prednisolone cumulative dose was significantly higher in carcinoma patients than in patients without carcinoma (P < .01). RESULTS: The use of long-term chronic immunosuppressive therapy may increase the development of cancer. The risk of carcinoma increases with increasing drug dose and time period of the immunosuppressive drug. There was not a negative effect on cancer prevalence in patients with cyclosporine or tacrolimus. But the cumulative dose of steroids significantly increased malignancy occurence.

Monitoring of CD3(+) T-cell count in patients receiving antithymocyte globulin induction after cadaveric renal transplantation.

AIM: Although antithymocyte globulin (ATG) has been used for years, its ideal dose and administration period is obscure. Herein, we sought to use the CD3(+) cell count to detect the optimal ATG dosage. MATERIAL AND METHODS: Twenty-one patients who underwent cadaveric donor renal transplantation from January 2009 to January 2012 received a 1 mg/kg ATG initial dose at the time of the operation. Patients were randomized into 2 cohorts. Group 1 (n = 11) received ATG according to the clinical and total lymphocyte count and group 2 (n = 10), the dose was tailored according to the CD3(+) cell count. We compared the total and daily ATG dosages, ATG administration period, side effects of ATG, the number of days to a serum creatinine level <2 mg/dL, graft function at 3 months, acute rejection episodes, infection rates, costs of CD3(+) analysis, and ATG amounts. RESULTS: Both groups showed similar gender, age, and human leukocyte antigen matching data. There was no difference in presensitizing events or panel-reactive antibody class 1 and 2 levels. The number of days to a serum creatinine level of <2 mg/dL was 11 ± 1.5 for group 1 versus 10.4 ± 0.8 for group 2 (P = .45). Between groups 1 and 2, there was a significant difference between the mean total (P = .031) and mean daily ATG dosages (P = .006). We used a total dose of 3800 mg ATG for group 1 and 2200 mg for group 2 and for the group 2 who underwent 43 CD3(+) cell counts. The expenditure per patient was 20% higher among group 1 than group 2. CONCLUSION: Determination of appropriate ATG dosages by CD3(+) cell counts was useful, reliable, and cost effective.

Serum flow cytometric C1q binding antibody analysis of renal recipients with low levels of sensitization.

AIM: Patients displaying flow cytometric crossmatch results within the grey zone of positivity are hard to evaluate, especially if they are undergoing their first transplantation. For these patients assays of donor-specific anti-HLA (human leukocyte antigen) antibodies with complement-fixing properties to cause cell lysis are important for antibody-mediated rejection and graft failure. The aim of this study was to detect the relevance of serum C1q-binding antibodies detected in renal recipients with grey zone crossmatch reactivity who were considered to show low levels of sensitization against their potential donors. METHOD: This study includes 114 patients who were admitted for their first renal transplantation between September 2009 and August 2011, including 33 subjects considered by flow cytometric cross-match to be the sensitized group, whereas the remaining 81 recipients had negative results. We analyzed the accumulation of serum the immunoglobulin (Ig)G bound C1q on HLA-coated flow cytometric panel reactive antibody (FlowPRA) beads. The serum samples were retrospectively analyzed with [C1q]FlowPRA (HLA class I and II), which were collected during the pretransplantation period every 6 months and every week posttransplantation within the first month and every 3 months thereafter. All serum samples were analyzed for the presence of anti-FlowPRA IgG alloantibody. We compared the C1q FlowPRA-positive and-negative groups for the number of posttransplantation days that the serum creatinine level was below <2 mg/dL as a metric of graft function. RESULTS: With a mean follow-up of 492 ± 84 days, there was a significant difference between flow cytometric crossmatch results and creatinine decrease rate (P = .02). The serum creatinine decrease rates of the 9 C1q-positive versus the 15 C1q-negative subjects showed significant difference (P < .05). CONCLUSION: C1q-binding antibody analysis shows the presence of serum antibodies capable of complement binding and antibody-mediated rejection, which could be useful to assess rejection risk among the "grey zone" of renal recipients with low levels of sensitization against their donors.

The impact of C4d staining as a humoral injury marker.

PURPOSE: Acute and chronic humoral injuries in renal transplant recipients are the main reasons for graft rejection and failure. Histological and clinical characteristics of humoral rejection and symptoms are variable and not always helpful for differential diagnosis. Clinical monitoring of the allograft, an elevated serum panel-reactive antibody (PRA), and the presence of donor-specific antibody (DSA) during immune monitoring as well as C4d staining of biopsy material can establish the differential diagnosis. Even without a cellular component, humoral rejection reaction is serious because the target tissue is the graft endothelium. Because the kidney graft has a rich vascular structure this attack causes permanent injury to the kidney in the long term. Graft dysfunction in this setting is usually more severe, requiring dialysis therapy, compared with acute cellular reactions. Positive C4d staining of peritubular capillaries in biopsy material represent a hallmark of complement-dependent cytotoxicity, supporting the diagnosis of humoral rejection. We analyzed C4d staining as a hallmark of humoral rejection. METHODS: From 2009 to 2011, we analyzed the relationship between pathological findings of C4d immunohistochemistry staining and the clinical outcomes of 45 kidney transplant recipients who underwent a kidney biopsy because of graft dysfunction due to possible humoral rejection. RESULTS: Biopsy specimens of 15 patients stained C4d positive; the remaining 30 showed negative results. Intravenous steroids, PP + IVIG with or without antithymocyte globulin (ATG), was administered for treatment. Sixty six percent (n = 10) of patients were C4d positive with 16% (n = 5) of those showing C4d-negative biopsy results, losing their grafts, and returning to hemodialysis. CONCLUSIONS: C4d staining refractory humoral rejection injury was related to poor graft outcomes.

Flow cytometric detection of anti-AB antibody titers in blood group O recipients of blood group A2 donor kidneys.

AIM: ABO-incompatible kidney transplantation has been accepted for end-stage renal failure patients who have no ready opportunity for a deceased or living donor. Antibody titration for ABO-incompatible renal transplantation is not only difficult but also lacks conformity among laboratories. Herein we analyzed 20 living related renal transplant couples to detect recipient anti-A2 antibody using flow cytometric analysis. MATERIALS AND METHODS: Patients were admitted to our center for renal transplantation between January 1999 and December 2010. All but four of them had undergone a previous renal transplantation from an ABO-compatible donor but experienced graft failure. All donor blood groups were subtyped by our blood bank using a lectin-based dilution assay. To detect recipient anti-A2 antibody titers we used a tube hemagglutination method. A/B antibody titer analysis by flow cytometry incubated serially diluted serum samples with donor erythrocytes. Each analysis was repeated three times over a 2-week period using an older and the last sera simultaneously. RESULTS: The 13 male and 7 female patients showed our overall mean age of 32 ± 12 years. All patients had panel-reactive antibody levels below 15%. The level of flow cytometric antibody titers did not vary upon repeated analysis (P = .01). When compared with the tube method there was a discrepancy of the level at which the antibody titer became negative. DISCUSSION: Flow cytometric antibody titration is a practical and rapid technique to determine the amount of anti-A2 antibody in renal recipients.

Effects of intra- and extracellular factors on anti-aging klotho gene expression.

Inactivation of the klotho gene in mice causes serious systemic disorders, resembling human aging. However, at the molecular level, its action mechanisms are not well understood. The stimulatory or inhibitory effects of cis- and trans-regulatory factors on the klotho gene expression are also still unclear. We studied the effects of intra- and extracellular factors on human klotho gene expression. For this purpose, pHKP-Luc and pHKP-GFP reporter vectors were constructed with the 2.1-kbp upstream region of human klotho, covering its promoter region, using luciferase and GFP genes as the reporter. A series of vectors that have deletions in the upstream region of the klotho gene were constructed to assay cis-acting factors. Deletion of some parts of the klotho gene upstream region significantly affected reporter gene expression in HEK293 cells. p16 and p53 proteins inhibited reporter luciferase expression under the control of human klotho promoter in a dose-dependent manner. Calcium and phosphate ions stimulated klotho expression. p21, PTH, IGF-1, and angiotensin-II had no significant effect on klotho expression in HEK293 cells.

Risk factors for osteoporosis after renal transplantation and effect of vitamin D receptor Bsm I polymorphism.

OBJECTIVE: Rapid loss of vertebral or hip mineral density after renal transplantation is a major complication which occurs within 6-12 months. The aim of this study was to evaluate risk factors contributing to bone disease in the early stage after renal transplantation and the effect of vitamin D receptor (VDR) gene polymorphisms. METHODS: We prospectively followed for up to 12 months 44 patients (29 men and 15 women) with end-stage renal disease who underwent kidney transplantation. All patients received prednisone with either cyclosporine (CsA)/mycophenolate mofetil (MMF) or tacrolimus (Tac)/MMF therapy. Spine, hip, and whole body bone mineral density (BMD) was measured at 12 months after transplantation. According to World Health Organization recommendations, our patients were categorized as normal, osteopenic, or osteoporotic BMD levels. VDR alleles were genotyped as BB, Bb, or bb by polymerase chain reactions based on polymorphism at the Bsm I restriction site. RESULTS: Forty-six percent of patients were normal, 43% osteopenic, and 11% osteoporotic. Significant risk factors for osteoporosis among renal transplant recipients were younger age and pretransplant high intact parathyroid hormone (iPTH) levels. (P values .045 and .027, respectively). According to polymorphic group categorization, posttransplant serum Ca was significantly higher in patients with BB or Bb genotype than in those with bb genotype (P = .012). Although there was no statistical significance regarding iPTH levels, it was higher among Bb+BB than the bb genotype group. Also, first-year BMD analysis after transplantation according to Bsm I polymorphism showed significant differences in femur BMD levels according to the dual classification of polymorphism (P < .05). The BMD levels in the bb group was higher than in the Bb+BB group. CONCLUSIONS: Although high pretransplant iPTH levels and younger age enhanced posttransplant bone loss, functionally different alleles of the VDR gene may modulate bone turnover during the first year after renal transplantation.