Association between matrix metalloproteinase-9-1562C/T gene polymorphism and MMP-9 serum level in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease indicated by joint inflammation and cartilage destruction. Matrix metalloproteinase (MMP) enzymes play an influential role in inflammation by affecting the invasion and degradation of anatomical barriers. In this way, the current study investigated the relationship between the MMP-9-1562C/T gene polymorphism and this enzyme's serum level in RA. METHODS: The serum levels of MMP-9 in RA patients and healthy controls were measured using the enzyme-linked immunosorbent assay (ELISA). RA was confirmed using rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), and C-reactive protein (CRP). Then the MMP-9-1562C/T gene polymorphism was analyzed utilizing polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Also, multivariate analysis investigated the connection between this polymorphism and the risk of RA. RESULTS: In this study, the increase of MMP-9 in patients due to the development of single nucleotide polymorphism in the promoter region of this gene (-1562 C→T) was confirmed by increasing the frequency of heterozygous genotype (CT). Logistic regression analysis also demonstrated that the chance of development of RA is higher in people with CT/CC genotype than in other alleles. CONCLUSIONS: We demonstrated that MMP-9-1562C/T gene polymorphism can play a significant role in the occurrence of RA.

The Association of Methylation Status and Expression Level of MyoD1 with DNMT1 Expression Level in Breast Cancer Patients.

Background: Breast cancer (BC) is the most common malignancy in women worldwide. The methylation status of MyoD1, a tumor suppressor gene, is enrolled in various cancers, i.e., BC. Various studies showed the impact of MyoD1 epigenetic dysregulation in BC. This study aimed to investigate the methylation status and expression level of MyoD1 in BC patients and its association with the expression of DNMT1. Materials and Methods: This case-control study was conducted on 30 cases (pathology-confirmed ductal carcinoma) and 18 controls (fibroadenoma and fibrocystic masses), referred to Velayat Hospital, Qazvin, Iran. The expression of the MyoD1 and DNMT1 and the promoter methylation of the MyoD1 were evaluated in tissue blocks of BC patient masses using qRT-PCR and MS-PCR assays, respectively. SPSS 24.0 was used to analyze the data. Results: The MyoD1 promoter is hypermethylated in BC patients compared to controls (p =0.001). The expression level of MyoD1 in BC patients was significantly reduced compared to controls (fold change =0.13, p =0.042). In addition, in BC patients, the reduced expression level of MyoD1 was significantly associated with methylation of the MyoD1 promoter (p =0.001). There is no significant difference between the expression level of DNMT1 in BC patients and controls (p =0.197). A significant association is found between the expression of DNMT1 and the methylation status of the MyoD1 promoter (p =0.038). Discussion: The expression level of MyoD1 is affected by the methylation status of the promoter of this gene. Moreover, the expression level and methylation status of MyoD1 are correlated with clinical parameters.

Characterization of novel bacteriophage PSKP16 and its therapeutic potential against ß-lactamase and biofilm producer strain of K2-Hypervirulent Klebsiella pneumoniae pneumonia infection in mice model.

BACKGROUND: Severe infections caused by ß- lactamase producers, hypervirulent Klebsiella pneumoniae (BhvKp) with K2 serotype, highlight emergency need for new therapeutic strategies against this pathogen. We aimed to assess the efficacy of a novel phage, PSKP16, in the treating of pneumonia induced by BhvKp in mice models. METHOD: Genome sequences of PSKP16 were analyzed, and associated information can be found in NCBI. We applied treatment in two ways: by using mice for immediate and delayed treatments. Moreover, acute pneumonia obtained by BhvKp with intranasal method, was characterized in terms of histopathology of pulmonary lesions, biomarkers of inflammation level, leukocytes cells infiltration extent in mice, and was assessed treatment of them with PSKP16 multiplicity of infection (MOI: 10), either individually or in combination with gentamicin. Assessment of the ability of PSKP16 to inhibit BhvKp biofilm was studied. RESULTS: PSKP16 was associated with the Drexlerviridae family, and had a genome size of 46,712 bp, and 67 predicted ORFs. Herein, prompt phage administration's efficacy to decrease bacterial load and improve the survival rate in pneumonia models was faster than the synergism model with delay, but both almost displayed similar endpoints. The distribution of BhvKp strains in the lung was consistent with the histopathological findings, simultaneous inflammation, and level of serum tumor necrosis factor-α (TNF α). The phage treatment presented a lack of severe lesions and alveolar edema, reduction of inflammatory cell infiltration, which not only was it not associated with an over-inflammation but also provided a faster correction of blood cell count abnormalities compared to gentamicin. Phage with a high concentration in in vitro model effectively eliminated biofilms. CONCLUSION: It is essential to raise clinical awareness and management of BhvKp infections, signaled as the next superbug in waiting. The results of our study underscore the importance of PSKP16 as a phage with promising therapeutic potential in treating BhvKp-induced pneumonia.

Survey of the association between polymorphisms of CTLA-4 exon 1 49 A/G genes with rheumatoid arthritis in Iran.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which suppresses T cell proliferation, is a promising candidate for the susceptibility genes to rheumatic arthritis diseases (RA). This study aims to examine the association between the polymorphisms of the CTLA-4 exon 1(+ 49) genes with RA in the Qazvin city of Iran population. The polymerase chain reaction of genomic DNA-restriction fragment length polymorphism (PCR-RFLP) was applied to genotype the CTLA-4 exon 1(+ 49) polymorphisms in 105 RA patients and 90 control subjects. Laboratory diagnostic tests were also measured for RA and control groups. Our results did not demonstrate a significant difference in allele and genotype frequencies of the CTLA-4 exon 1(+ 49) between RA patients and the control group (p < .0001). There was no significant difference in age at onset, CRP, RF value in patients with RA according to the CTLA-4 polymorphisms; just anti-CCP showed a significant difference. Our data declared that polymorphisms of CTLA-4 exon 1(+ 49) genes are not correlated with RA susceptibility and its clinical and paraclinical manifestations.

Evaluation of expression level and methylation profile of CXX1 gene in breast cancer tissue blocks.

AIMS: The hypermethylation of CpG islands in the promoter of tumor-suppressor genes (TSGs) leads to silencing the transcription of tumor suppressors, which lead to the development of cancer. The hypermethylation of CXX1 and CDH1 genes, as TSGs, plays an essential role in the development of various types of cancer, i.e., colorectal and gastric cancer. This study aims at evaluating the expression level of CXX1 and CDH1 genes and the methylation status of CXX1 CpG island's promoter in breast cancer (BC). MATERIALS AND METHODS: In this study, the expression level of the CXX1 and CDH1 genes and the promoter methylation status of the CXX1 gene were evaluated in 30 paraffin-embedded tissue blocks of malignant BC and 18 benign breast lesions, using quantitative reverse transcription-PCR and methylation-specific (MS)-PCR assays, respectively. RESULTS: The CXX1 gene was downregulated in the malignant tissues due to the hypermethylation of the CpG islands in the promoter, compared to the control group (P = 0.031). The downregulation of CDH1 gene expression was observed in the patient group compared to control, but this reduction was not statistically significant. The results show that the risk of BC is increased with aging (P < 0.001). Furthermore, the benign breast lesions (controls) had more mobility in comparison with the malignant breast tumors (P < 0.001). In the malignant samples, the size of the mass was larger than control's mass samples (P = 0.006). CONCLUSIONS: In the pathophysiological state of BC, the aberrant DNA hypermethylation in CpG island of CXX1 promoter is responsible for the reduction of its expression level in BC patients.

Cloning and expressing of interleukine 2 in amniotic membrane-derived mesenchymal stem cells, as a potent feeder layer.

The application of mesenchymal stem cells (MSCs) is rapidly expanding due to their unique properties in cell therapy, especially as the feeder layer in the ex-vivo expansion of immune cells. Also, Interleukin 2 (IL-2) is an essential human cytokine in the expansion of hematopoietic precursors and progenitors, i.e., NK cells and T cells, while there is no endogenous expression of IL-2 in MSCs. This study aimed to examine the potency of amniotic membrane (AM)-MSCs as the IL-2 secretory cells. IL-2-containing pCMV3-C-GFPspark shuttle vector was transformed in E.coli DH5-alpha. After cloning, the plasmid DNA was extracted and transfected in isolated AM-MSCs, by lipofectamine-2000. Then, the RNA and protein expression levels of exogenous IL-2 were evaluated 3 to 15 days after transfection, using ELISA and qRT-PCR. Fluorescent microscopy and flowcytometry assays were used for evaluating the GFP-positivity of transfected AM-MSCs, as IL-2 expression control. There was a significant increase in RNA expression of exogenous IL-2 in transfected AM-MSCs in 3 to 15 days after transfection. (p<0.001) Also, IL-2 concentration released in the medium was increased in 3rd day after transfection (611 pg/ml). However, the RNA and protein expression of IL-2 was reduced through passing the time. The results show AM-MSC is a suitable host for the expression and secretion of IL-2 as a critical cytokine in the ex-vivo expansion of hematopoietic precursors and progenitors, i.e., NK cells and T cells. Also, the survival time of IL-2 expression in AM-MSCs was long enough for use as a feeder layer.

Decreased insulin resistance in diabetic patients by influencing Sirtuin1 and Fetuin-A following supplementation with ellagic acid: a randomized controlled trial.

BACKGROUND: The beneficial effects of polyphenols have been reported. This study aimed to investigate the effect of oral Ellagic acid (EA) supplement on insulin resistance (IR) and Fetuin-A and serum sirtuin1 (SIRT1) in type 2 diabetics. METHODS: In this double-blind, randomized clinical trial, 44 diabetic patients were selected. Patients were assigned to the intervention group (22 subjects) and placebo (22 subjects) and received a capsule containing 180 mg of EA per day or placebo for eight weeks, respectively. At the beginning and end of the study, anthropometric indices, fasting plasma glucose (FPG), plasma insulin level, IR, Fetuin-A, and SIRT1 were measured. Statistical analysis was performed using SPSS software. RESULTS: At the beginning and end of the study, there was no significant difference between the two groups regarding anthropometric indices (P > 0.05). At the end of the survey, EA supplementation significantly reduced FPG, insulin, IR, and Fetuin-A and increased SIRT1 levels compared with the placebo group (P < 0.05). However, these changes were not significant in the placebo group (P > 0.05). CONCLUSION: EA with antioxidant properties plays an essential role in reducing the macrovascular and microvascular complications of diabetes by reducing inflammation and insulin resistance. Trial registration The protocol of this clinical trial is registered with the Iranian Registry of Clinical Trials ( http://www.IRCT.IR , identifier: IRCT20141025019669N13).

Randomized double-blind clinical trial examining the Ellagic acid effects on glycemic status, insulin resistance, antioxidant, and inflammatory factors in patients with type 2 diabetes.

Oxidative stress can worsen glycemic status. Considering the antioxidant properties of Ellagic acid (EA), this study was designed to evaluate the effect of EA on glycemic indices, lipid profile, oxidative stress, and inflammation status in type 2 diabetic patients. Overall, 44 patients were recruited and were randomly allocated consumed 180 mg of EA per day (n = 22) or placebo (n = 22) for 8 weeks. The blood sugar (BS), insulin, insulin resistance (IR), hemoglobin A1c (HbA1 c), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), total antioxidant capacity (TAC), malondialdehyde (MDA), the activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD), C-reactive protein (CRP), TNF-α and interleukin 6 (IL-6) were measured at the beginning and end of the study. At the end of the study, the mean of BS, insulin, IR, HbA1 c, TC, TG, LDL, MDA, CRP, TNF-α, and IL-6 were significantly decreased in the intervention group (p < .05). Also, the mean of TAC (+0.8 ± 0.01) and activity of GPx (+10.26 ± 0.22) and SOD enzymes (+459.6 ± 9.76) significantly increased in the intervention group (p < .05). EA supplementation can be helpful as a diet supplement in patients with type 2 diabetes through improvement in chronic adverse effects.

Induced Pluripotent Stem Cell Meets Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) is classified as a primary immunodeficiency, which is characterized by impaired T-lymphocytes differentiation. IL2RG, IL7Ralpha, JAK3, ADA, RAG1/RAG2, and DCLE1C (Artemis) are the most defective genes in SCID. The most recent SCID therapies are based on gene therapy (GT) of hematopoietic stem cells (HSC), which are faced with many challenges. The new studies in the field of stem cells have made great progress in overcoming the challenges ahead. In 2006, Yamanaka et al. achieved "reprogramming" technology by introducing four transcription factors known as Yamanaka factors, which generate induced pluripotent stem cells (iPSC) from somatic cells. It is possible to apply iPSC-derived HSC for transplantation in patients with abnormality or loss of function in specific cells or damaged tissue, such as T-cells and NK-cells in the context of SCID. The iPSC-based HSC transplantation in SCID and other hereditary disorders needs gene correction before transplantation. Furthermore, iPSC technology has been introduced as a promising tool in cellular-molecular disease modeling and drug discovery. In this article, we review iPSC-based GT and modeling for SCID disease and novel approaches of iPSC application in SCID.

The Gene Expression Profile and DNA Methylation Pattern of CDH1 and DNMT1 Genes in Acute Promyelocytic Leukemia (APL).

BACKGROUND: DNA methylation is an epigenetic modification that has the ability to alter gene expression and function. These epigenetic changes have been associated with the development of cancer. Previous research has found that DNA methylation patterns can predict disease prognosis for patients with Acute Promyelocytic Leukemia (APL). The role of DNMT1 and CDH1 in regulating the extension of cells are studied in this study. METHODS: DNA was extracted from peripheral blood samples of APL patients and treated with bisulfite. DNMT1 and CDH1 gene promoter methylation was subsequently analyzed using methylation-specific PCR (MSP). Real-time PCR was used to measure the expression level of DNMT1 and CDH1 genes. RESULTS: Partial methylation of the CDH1 gene promoter was detected in 20% of APL patients and an unmethylated status was detected in 80% of patient samples. Additionally, an unmethylated status in the DNMT1 gene promoter was detected in 100% of APL patient samples. CONCLUSION: Our study found the CDH1 gene promoter to be unmethylated in almost all APL patients, while the DNMT1 promoter was unmethylated in all APL patients. Furthermore, we observed an increase in both CDH1 and DNMT1 gene expression in APL patients compared to healthy controls. These findings suggest that DNMT1 may not have a specific role in inhibiting CDH1 gene expression in APL. Applying higher resolution techniques would help to better uncover the DNA methylation patterns in patients with APL. Further research is required to determine the role of DNA methylation and CDH1 and DNMT1 gene expression in APL.

Application of PLGA nano/microparticle delivery systems for immunomodulation and prevention of allotransplant rejection.

INTRODUCTION: Allograft transplantation is an effective end-point therapy to replace the function of an impaired organ. The main problem associated with allotransplantation is the induction of immune responses that results in acute and chronic graft rejection. To modulate the response of the immune system, transplant recipients generally take high dose immunosuppressant drugs for life. These drugs are associated with serious side effects such as infection with opportunistic pathogens and the development of neoplasia. AREAS COVERED: We reviewed the obstacles to successful transplantation and PLGA-based strategies to reduce immune-mediated allograft rejection. EXPERT OPINION: Biomaterial-based approaches using micro- and nanoparticles such as poly (lactic-co-glycolic acid) (PLGA) can be used to achieve controlled release of drugs. This approach decreases the required effective dose of drugs and enables local delivery of these agents to specific tissues and cells, whilst decreasing systemic effects.

Observation of quantum many-body effects due to zero point fluctuations in superconducting circuits.

Electromagnetic fields possess zero point fluctuations which lead to observable effects such as the Lamb shift and the Casimir effect. In the traditional quantum optics domain, these corrections remain perturbative due to the smallness of the fine structure constant. To provide a direct observation of non-perturbative effects driven by zero point fluctuations in an open quantum system we wire a highly non-linear Josephson junction to a high impedance transmission line, allowing large phase fluctuations across the junction. Consequently, the resonance of the former acquires a relative frequency shift that is orders of magnitude larger than for natural atoms. Detailed modeling confirms that this renormalization is non-linear and quantum. Remarkably, the junction transfers its non-linearity to about thirty environmental modes, a striking back-action effect that transcends the standard Caldeira-Leggett paradigm. This work opens many exciting prospects for longstanding quests such as the tailoring of many-body Hamiltonians in the strongly non-linear regime, the observation of Bloch oscillations, or the development of high-impedance qubits.

Dynamic variation of kidney injury molecule-1 mRNA and protein expression in blood and urine of renal transplant recipients: a cohort study.

BACKGROUND: Acute renal dysfunction still constitutes a highly significant obstacle to renal transplantation outcome. Kidney injury molecule-1 is highly upregulated in proximal tubular cells and shed into the urine and blood circulation following kidney injury. The aim of current cohort study was to evaluate the urine KIM-1 (uKIM-1) mRNA expression level and its protein concentration in blood and urine samples to determine whether sequential monitoring of KIM-1 in renal allograft recipients is a reliable biomarker for predicting the clinical status and outcome. METHODS: Both uKIM-1 mRNA expression level and the level of serum and uKIM-1 protein concentration in the 52 renal transplant recipients were respectively quantified using real-time PCR and ELISA methods at 2, 90 and 180 days after transplantation. RESULT: KIM-1 mRNA and protein expression level in the blood and urine samples of patients with graft dysfunction was significantly higher than patients with well-functioning graft on days 2, 90 and 180 after transplantation. Receiver-operating characteristic curve analysis of mRNA and protein expression levels showed that urinary and blood KIM-1 at months 3 and 6 could predict acute renal dysfunction at 6 months and 1 year after transplantation. CONCLUSION: Sequential monitoring of uKIM-1 mRNA expression level and its protein concentration in the serum and urine samples of renal transplant patients suggests that KIM-1 could be a sensitive and specific biomarker for early diagnosis and prognosis of kidney allograft injury.

Effects of vitamin D supplementation on liver fibrogenic factors in non-alcoholic fatty liver patients with steatohepatitis: study protocol for a randomized clinical trial.

BACKGROUND: It has been suggested that vitamin D and its receptors involve in suppressing fibrogenic signaling in non-alcoholic fatty liver disease (NAFLD). However, the effect of vitamin D supplementation on fibrogenic factors has not been investigated in NAFLD individuals with steatohepatitis. This study was designed to examine the effects on vitamin D supplementation on serum levels of vitamin D receptor (VDR), fibrogenic factors, and fibrogenic microRNAs (MiR) in NAFLD patients. METHODS: Forty-six NAFLD patients will be recruited in this study. After block matching for sex and BMI, they will be randomly assigned to receive 4000 IU/day vitamin D or placebo for 12 weeks. Weight, height, and waist circumference will be measured. Determination of serum fibrogenic MiRs, laminin, collagen type IV, hyaluronic acid, vitamin D, VDR, calcium, blood glucose, serum insulin, lipid profile, liver markers (ALT, AST, total, direct, and indirect bilirubin) will be done at study baseline and at the end of the trial. Insulin resistance and insulin sensitivity will be determined using the HOMA-IR and QUICKI equation. DISCUSSION: This is the first randomized controlled trial that will determine the effect of vitamin D supplementation on serum levels of VDR, fibrogenic factors, and fibrogenic MiRs in NAFLD patients. The results of this trial will provide clinical evidence on the effectiveness of vitamin D supplementation in controlling liver fibrosis in NAFLD patients. TRIAL REGISTRATION: Iranian Registry of Clinical Trials, IRCT201405251485N13 . Registered on 14 March 2017.

Evaluating the Role of PTEN Promoter Methylation in Patients Predisposed to Hypercoagulable States via Methylation Specific PCR.

BACKGROUND: Hypercoagulable states (HS) can result from several different inherited and acquired disease conditions that cause abnormalities in the genes, proteins and cellular factors involved in the coagulation cascade. Novel insight into the molecular mechanisms involved in the coagulation pathways can provide a framework to develop improved therapeutics to treat patients with coagulation disorders. Therefore, investigating the genetic abnormalities present in patients with coagulation disorders can offer critical insight into disease pathogenesis. Our study aimed to assess the promoter methylation patterns of the phosphatase and tensin homologue (PTEN) gene as a potential underlying factor involved in HS. METHODS: To measure the differences between the mRNA expression of PTEN in HS patients and healthy individuals we used qRT-PCR. Following bisulfite conversion, the promoter methylation status was analyzed using methylation specific PCR. The two-tailed student t-test was used to analyze the quantitative data. The data was considered statistically significant with a p value <0.05. RESULTS: Our findings reveal PTEN to be down-regulated by 30% in the blood samples of HS patients when compared to healthy controls. The MSP data showed the PTEN promoter region to be un-methylated in both patients and healthy individuals. CONCLUSION: Since no differences in the methylation patterns of the PTEN gene was found between HS patients and controls, this suggests that DNA methylation of the PTEN promoter may not be a significant contributing epigenetic modification involved in the development HS. However, MSP may not be able to detect subtle changes in DNA methylation status. Thus, using an alternative high resolution technique may more accurately indicate differences in the PTEN promoter methylation status in HS patients.

Sequential monitoring of TIM-3 mRNA expression in blood and urine samples of renal transplant recipients.

BACKGROUND: T cell immunoglobulin and mucin domain 3 (TIM-3), as a co-inhibitory receptor expressed on Th1, Th17, CD8T, FoxP3 + Treg and innate immune cells, plays an important role in suppression of T cell-mediated immune responses, tolerance induction and T cell exhaustion. In this study, we evaluated sequential alterations of TIM-3 mRNA expression level in blood and urine samples of renal transplant recipients to predict approaching clinical episodes. METHODS: A total of 52 adult renal transplant recipients (31 male and 21 female) were enrolled in this study. All the patients received kidney transplant from living unrelated donors. TIM-3 mRNA expression in peripheral blood mononuclear cells (PBMCs) and urinary cells were quantified using Real Time TaqMan polymerase chain reaction (PCR) at 4 different time points (pre-transplantation, 2, 90 and 180 days post-transplantation). RESULT: TIM-3 mRNA expression level on days 2, 90 and 180 after transplantation was significantly higher in blood and urine samples of patients with graft dysfunction (GD) compared with patients with well-functioning graft (WFG). Our results also showed a high correlation between blood and urinary level of TIM-3 mRNA expression. The data from Receiver Operating Characteristic (ROC) Curve Analysis showed that blood and urinary TIM-3 mRNA expression level at month 3 and 6 could discriminate graft dysfunction (GD) from well-functioning graft (WFG) with high specificity and sensitivity. CONCLUSION: Our data suggested that serial monitoring of TIM-3 mRNA level in the blood and urine samples of renal transplant recipients could be a useful non-invasive biomarker for prediction and diagnosis of allograft dysfunction.

High Expression of FOXP3 mRNA in Blood and Urine as a Predictive Marker in Kidney Transplantation.

BACKGROUND: Diagnosis of allograft dysfunction by noninvasive biomarker tests is preferable to invasive allograft biopsies and has been extensively considered in recent years. This study aims to evaluate blood and urinary forkhead box P3 (FOXP3) messenger RNA (mRNA) expression in renal transplant recipients in an attempt to determine whether differential diagnosis of graft dysfunction is feasible using mRNA profiles. METHODS: We analyzed FOXP3 mRNA expression in paired urinary and peripheral blood mononuclear cell (PBMC) samples. A total of 91 kidney transplant recipients enrolled in this study that were classified into 3 groups: biopsy-proven acute rejection (AR; n = 27), chronic allograft nephropathy (n = 19), and well-functioning graft (n = 45). The FOXP3 mRNA expression was quantified by TaqMan probe real-time polymerase chain reaction. RESULTS: Acute rejection patients had a higher expression level of transcription factor FOXP3 compared to the chronic nephropathy and control groups. Analysis of receiver operating characteristic curves showed that rejection could be diagnosed with 100% sensitivity and 96% specificity in urine, and 92% sensitivity and 86% specificity in PBMC samples using the optimal FOXP3 mRNA cutoff value. We subdivided the AR group into progressive and nonprogressive patients, which showed a significant difference in FOXP3 mRNA expression. This result confirmed the role of FOXP3 as a diagnostic marker in predicting transplantation outcomes. CONCLUSION: Our results suggested that elevated expression of FOXP3 in blood and urine samples from kidney transplant recipients could be a useful noninvasive biomarker to diagnose graft dysfunction.

Expression patterns of Toll like receptor (TLR)-2, TLR-4 and myeloid differentiation primary response gene 88 (MYD88) in renal transplant patients developing allograft dysfunction; a cohort study.

This cohort intends to determine the sequential dynamic changes in Toll-like receptor (TLR)-4, TLR-2, and myeloid differentiation primary response gene 88 (MYD88) mRNA expressions in PBMCs and biopsy samples from kidney allograft recipients in relation to graft function. This study enrolled 52 renal transplant patients, 27 with well functioning graft (WFG) and 25 graft dysfunction (GD). Peripheral blood samples pre- and post-transplantation (days 2, 90 and 180) were collected to analyze mRNA expression levels of TLR-2, TLR-4, and MYD88 genes in relation to allograft function during one-year follow up. The mean dynamic changes of post-transplant TLR-2, TLR-4, and MYD88 mRNA expressions were significantly higher in GD compared to WFG patients (P = .001). ROC curve analysis based on glomerular filtration rate (GFR) index showed the area under curve (AUC) values for the genes: TLR-2(0.89;P < .001), TLR-4(0.86;P < .001), and MYD88(0.75;P = .003) in the third month post-transplantation for GD diagnosis. The calculated AUCs for the expressions of genes in allograft biopsies were 0.94(TLR-2), 0.95(TLR-4), and 0.98(MYD88) in the sixth month post-transplant based on pathology report (P < .001). Our results indicate that sequential monitoring of the expression patterns of TLR-2, TLR-4, and MYD88 in PBMCs and biopsy samples could be considered as predictive biomarkers for early and late kidney allograft function.

High expression of TIM-3 and KIM-1 in blood and urine of renal allograft rejection patients.

BACKGROUND: T cell immunoglobulin and mucin domain 3 (TIM-3) is involved in alloimmune and autoimmune responses, as well as tolerance induction in kidney transplantation. Kidney injury molecule-1 (KIM-1) is highly expressed in epithelial cells of the injured proximal tubule. In this study, we have investigated both urinary and blood TIM-3 mRNA expressions, urinary KIM-1 mRNA expression, and urinary and serum KIM-1 proteins in renal allograft recipients diagnosed with acute allograft rejection (AR) and chronic allograft dysfunction (CAD), as well as those with well-functioning transplants (WFG). METHODS: We divided 85 patients into the following groups: AR (n=24), CAD (n=19), and WFG (n=42). TIM-3 and KIM-1 mRNA expressions were quantified using real-time reverse-transcription TaqMan probe polymerase chain reaction (RT-PCR). An ELISA test was used to measure the amount of KIM-1 protein in serum and urine samples. RESULTS: AR and CAD patients had significantly greater urinary and blood TIM-3 mRNA expressions, urinary KIM-1 mRNA expression, and urinary and serum KIM-1 proteins compared to WFG patients. Receiver operating characteristic (ROC) analysis showed that these molecules discriminated Allograft rejections from WFG. CONCLUSION: Quantification of TIM-3 and KIM-1 mRNA expressions, along with KIM-1 protein measurements in urine and blood could be employed as promising tools for noninvasive diagnosis of allograft dysfunction.

Increased levels of CD4(+) and CD8(+) T cells expressing CCR1 in patients developing allograft dysfunction; a cohort study.

BACKGROUND: Leukocyte infiltration into the graft has pivotal effects on kidney transplantation outcome. The present study sought to determine whether the expression of sequential chemokine receptors on CD4(+) and CD8(+) T cells in human renal allograft can predict clinical episodes. METHODS: Blood samples from 52 consecutive renal transplant patients were evaluated at the time of transplantation and at three times (2, 90 and 180days) after transplantation to analyze the expression of CCR1 and CXCR3 on CD4(+) and CD8(+) T cells by flowcytometry. A total of 30 biopsies, including protocol biopsy (n=24) and cause biopsy (n=6), were investigated according to the Banff criteria. RESULTS: The mean percentage of CD4(+) and CD8(+) T cells expressing CCR1 was significantly increased in patients with allograft dysfunction (n=25) (p=0.006, p=0.004). The mean fluorescence intensity of CXCR3 on CD4(+) and CD8(+) T cells were found to be significantly higher in graft dysfunction than that in well-functioning grafts. (p<0.001, p=0.007). Receiver Operating Characteristic (ROC) Curve Analysis showed that the calculated AUC was 0.86 at the third month for CD4(+)CCR1(+) and CD8(+)CCR1(+) (p<0.001). Multiple logistic regression analysis showed that an increase in CD4(+) expressing CXCR3 leads to a lower risk of graft dysfunction (OR=0.37), while an increase in CD8(+) expressing CCR1 results in a higher risk of graft dysfunction (OR=3.66). CONCLUSION: During renal transplantation, CD4(+) and CD8(+) T cells expressing CCR1 were increased in patients who developed graft dysfunction. These findings may prospectively predict allograft dysfunction, and help elucidate the underlying pathogenic mechanisms.