IDENTIFICATION AND ASSESSMENT OF PREDIABETES-A RURAL INDIAN STUDY (A CORRELATIVE STUDY BETWEEN QUESTIONNAIRE AND BIOCHEMICAL ANALYSIS)

Objective: To identify and prevent the vulnerable prediabetic population becoming diabetic patients in the future using the Indian Diabetic Risk Score (IDRS) and to evaluate the performance of the IDRS questionnaire for detecting prediabetes and predicting the risk of Type 2 Diabetes Mellitus in Chidambaram rural Indian population. Methods: A cross-sectional descriptive study was carried out among patients attending a master health check-up of RMMCH hospital located at Chidambaram. The IDRS was calculated by using four simple measures of age, family history of diabetes, physical activity, and waist measurement. The relevant blood test, like Fasting plasma glucose (FBS), Glycated hemoglobin (HbA1C) test, were observed for identifying prediabetes. Subjects were classified as Normoglycemic, prediabetics, and diabetics based on the questionnaire and diagnostic criteria of the Indian Council of Medical Research (ICMR) guidelines. Results: In the study, sensitivity and specificity of IDRS score were found to be 84.21% and 63.4% respectively for detecting prediabetes in community with the positive predictive value of 51.6% and negative predictive value of 89.6% and prevalence of prediabetes in the Chidambaram rural population is 31.6% among the 60 participants. Conclusion: The Indian diabetic risk score questionnaire designed by Ma­dras diabetic research federation is a useful screening tool to identify unknown type 2 diabetes mellitus. The question­naire is a reliable, valuable, and easy to use screening tool which can be used in a primary care setup.&nbsp

Urinary cell mRNA profiles predictive of human kidney allograft status

Kidney allograft status is currently characterized using the invasive percutaneous needle core biopsy procedure. The procedure has become safer over the years, but challenges and complications still exist including sampling error, interobserver variability, bleeding, arteriovenous fistula, graft loss, and even death. Because the most common type of acute rejection is distinguished by inflammatory cells exiting the intravascular compartment and gaining access to the renal tubular space, we reasoned that a kidney allograft may function as an in vivo flow cytometer and sort cells involved in rejection into urine. To test this idea, we developed quantitative polymerase chain reaction (PCR) assays for absolute quantification of mRNA and pre-amplification protocols to overcome the low RNA yield from urine. Here, we review our single center urinary cell mRNA profiling studies that led to the multicenter Clinical Trials in Organ Transplantation (CTOT-04) study and the discovery and validation of a 3-gene signature of 18S rRNA-normalized measures of CD3ε mRNA and IP-10 mRNA and 18S rRNA that is diagnostic and predictive of acute cellular rejection in the kidney allograft. We also review our development of a 4-gene signature of mRNAs for vimentin, NKCC2, E-cadherin, and 18S rRNA diagnostic of interstitial fibrosis/tubular atrophy (IF/TA)

Allograft rejection and tubulointerstitial fibrosis in human kidney allografts: Interrogation by urinary cell mRNA profiling

Because the kidney allograft has the potential to function as an in-vivo flow cytometer and facilitate the access of immune cells and kidney parenchymal cells in to the urinary space, we hypothesized that mRNA profiling of urinary cells offers a noninvasive means of assessing the kidney allograft status. We overcame the inherent challenges of urinary cell mRNA profiling by developing pre-amplification protocols to compensate for low RNA yield from urinary cells and by developing robust protocols for absolute quantification mRNAs using RT-PCR assays. Armed with these tools, we undertook first single-center studies urinary cell mRNA profiling and then embarked on the multicenter Clinical Trials in Organ Transplantation-04 study of kidney transplant recipients. We report here our discovery and validation of diagnostic and prognostic biomarkers of acute cellular rejection and of interstitial fibrosis and tubular atrophy (IF/TA). Our urinary cell mRNA profiling studies, in addition to demonstrating the feasibility of accurate diagnosis of acute cellular rejection and IF/TA in the kidney allograft, advance mechanistic and potentially targetable biomarkers. Interventional trials, guided by urinary cell mRNA profiles, may lead to personalized immunosuppression in recipients of kidney allografts

Hypertension, kidney disease, HIV and antiretroviral therapy among Tanzanian adults: a cross-sectional study.

BACKGROUND: The epidemics of HIV and hypertension are converging in sub-Saharan Africa. Due to antiretroviral therapy (ART), more HIV-infected adults are living longer and gaining weight, putting them at greater risk for hypertension and kidney disease. The relationship between hypertension, kidney disease and long-term ART among African adults, though, remains poorly defined. Therefore, we determined the prevalences of hypertension and kidney disease in HIV-infected adults (ART-naive and on ART >2 years) compared to HIV-negative adults. We hypothesized that there would be a higher hypertension prevalence among HIV-infected adults on ART, even after adjusting for age and adiposity. METHODS: In this cross-sectional study conducted between October 2012 and April 2013, consecutive adults (>18 years old) attending an HIV clinic in Tanzania were enrolled in three groups: 1) HIV-negative controls, 2) HIV-infected, ART-naive, and 3) HIV-infected on ART for >2 years. The main study outcomes were hypertension and kidney disease (both defined by international guidelines). We compared hypertension prevalence between each HIV group versus the control group by Fisher's exact test. Logistic regression was used to determine if differences in hypertension prevalence were fully explained by confounding. RESULTS: Among HIV-negative adults, 25/153 (16.3%) had hypertension (similar to recent community survey data). HIV-infected adults on ART had a higher prevalence of hypertension (43/150 (28.7%), P = 0.01) and a higher odds of hypertension even after adjustment (odds ratio (OR) = 2.19 (1.18 to 4.05), P = 0.01 in the best model). HIV-infected, ART-naive adults had a lower prevalence of hypertension (8/151 (5.3%), P = 0.003) and a lower odds of hypertension after adjustment (OR= 0.35 (0.15 to 0.84), P = 0.02 in the best model). Awareness of hypertension was ≤ 25% among hypertensive adults in all three groups. Kidney disease was common in all three groups (25.6% to 41.3%) and strongly associated with hypertension (P 2 years had two-fold greater odds of hypertension than HIV-negative controls. HIV-infected adults with hypertension were rarely aware of their diagnosis but often have evidence of kidney disease. Intensive hypertension screening and education are needed in HIV-clinics in sub-Saharan Africa. Further studies should determine if chronic, dysregulated inflammation may accelerate hypertension in this population

Development of a multivariable gene-expression signature targeting T-cell-mediated rejection in peripheral blood of kidney transplant recipinets validated in cross sectional and longitudinal samples

Background: Acute T-cell mediated rejection (TCMR) is usually indicated by alteration in serum-creatinine measurements when considerable transplant damage has already occurred. There is, therefore, a need for non-invasive early detection of immune signals that would precede the onset of rejection, prior to transplant damage. Methods: We examined the RT-qPCR expression of 22 literature-based genes in peripheral blood samples from 248 patients in the Kidney Allograft Immune Biomarkers of Rejection Episodes (KALIBRE) study. To account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene-expression residuals from linear mixed-effects models in stable patients. To select genes, we used penalised logistic regression based on 27 stable patients and 27 rejectors with biopsy-proven T-cell-mediated rejection, fulfilling strict inclusion/exclusion criteria. We validated this signature in i) an independent group of stable patients and patients with concomitant T-cell and antibody-mediated-rejection, ii) patients from an independent study, iii) cross-sectional pre-biopsy samples from non-rejectors and iv) longitudinal follow-up samples covering the first posttransplant year from rejectors, non-rejectors and stable patients. Findings: A parsimonious TCMR-signature (IFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A) showed cross-validated area-under-ROC curve 0·84 (0·77-0·88) (median, 2·5th-97·5th centile of fifty cross-validation cycles), sensitivity 0·67 (0·59-0·74) and specificity 0·85 (0·75-0·89). The estimated probability of TCMR increased seven weeks prior to the diagnostic biopsy and decreased after treatment. Gene expression in all patients showed pronounced variability, with up to 24% of the longitudinal samples in stable patients being TCMR-signature positive. In patients with borderline changes, up to 40% of pre-biopsy samples were TCMR-signature positive. Interpretation Molecular marker alterations in blood emerge well ahead of the time of clinically overt TCMR. Monitoring a TCMR-signature in peripheral blood could unravel Tcell-related pro-inflammatory activity and hidden immunological processes. This additional information could support clinical management decisions in cases of patients with stable but poor kidney function or with inconclusive biopsy results

Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation

T lymphocytes are defective in cystine uptake and thus require exogenous thiols for activation and function. Here we show that monocyte-derived human dendritic cells (DCs) release cysteine in the extracellular space. Cysteine generation is increased by lipopolysaccharide and tumor necrosis factor alpha, and by contact with T cells specifically recognizing soluble or alloantigens. These stimuli also induce thioredoxin (TRX) accumulation in DCs. However, only the contact with antigen-specific T cells triggers TRX secretion by the antigen-presenting cells. Fewer extracellular thiols are recovered after DC-T cell interactions when cystine uptake or TRX activity are inhibited. In addition, glutamate (Glu) and anti-TRX-inactivating antibodies inhibit antigen-dependent T lymphocyte proliferation. These findings indicate that, during antigen presentation, DCs uptake cystine and release cysteine and TRX, thus providing a reducing microenvironment that facilitates immune response

Urinary-Cell mRNA Profile and Acute Cellular Rejection in Kidney Allografts

Background—The standard test for the diagnosis of acute rejection in kidney transplants is the renal biopsy. Noninvasive tests would be preferable. Methods—We prospectively collected 4300 urine specimens from 485 kidney-graft recipients from day 3 through month 12 after transplantation. Messenger RNA (mRNA) levels were measured in urinary cells and correlated with allograft-rejection status with the use of logistic regression. Results—A three-gene signature of 18S ribosomal (rRNA)–normalized measures of CD3ε mRNA and interferon-inducible protein 10 (IP-10) mRNA, and 18S rRNA discriminated between biopsy specimens showing acute cellular rejection and those not showing rejection (area under the curve [AUC], 0.85; 95% confidence interval [CI], 0.78 to 0.91; P<0.001 by receiver-operatingcharacteristic curve analysis). The cross-validation estimate of the AUC was 0.83 by bootstrap resampling, and the Hosmer–Lemeshow test indicated good fit (P = 0.77). In an externalvalidation data set, the AUC was 0.74 (95% CI, 0.61 to 0.86; P<0.001) and did not differ significantly from the AUC in our primary data set (P = 0.13). The signature distinguished acute cellular rejection from acute antibody-mediated rejection and borderline rejection (AUC, 0.78; 95% CI, 0.68 to 0.89; P<0.001). It also distinguished patients who received anti–interleukin-2 receptor antibodies from those who received T-cell–depleting antibodies (P<0.001) and was diagnostic of acute cellular rejection in both groups. Urinary tract infection did not affect the signature (P = 0.69). The average trajectory of the signature in repeated urine samples remained below the diagnostic threshold for acute cellular rejection in the group of patients with no rejection, but in the group with rejection, there was a sharp rise during the weeks before the biopsy showing rejection (P<0.001). Conclusions—A molecular signature of CD3ε mRNA, IP-10 mRNA, and 18S rRNA levels in urinary cells appears to be diagnostic and prognostic of acute cellular rejection in kidney allografts

Stratification of pancreatic tissue samples for molecular studies: RNA-based cellular annotation procedure

Abstract Background/objectives Meaningful profiling of pancreatic cancer samples is particularly challenging due to their complex cellular composition. Beyond tumor cells, surgical biopsies contain desmoplastic stroma with infiltrating inflammatory cells, adjacent normal parenchyma, and "non-pancreatic tissues". The risk of misinterpretation rises when the heterogeneous cancer tissues are sub-divided into smaller fragments for multiple analytic procedures. Pre-analytic histological evaluation is the best option to characterize pancreatic tissue samples. Our aim was to develop a complement or alternative procedure to determine the cellular composition of pancreatic cancerous biopsies, basing on intra-analytic molecular annotation. A standard process for sample stratification at a molecular level does not yet exist. Particularly in the case of retrospective or data depository-based studies, when hematoxylin-eosin stained sections are not available, it supports the correct interpretation of expression profiles. Methods A five-gene transcriptional signature ( RNA CellStrat) was defined that allows cell type-specific stratification of pancreatic tissues. Testing biopsy material from biobanks with this procedure demonstrated high correspondence of molecular (qRT-PCR and microarray) and histologic (hematoxylin-eosin stain) evaluations. Results Notably, about a quarter of randomly selected samples (tissue fragments) were exposed as inappropriate for subsequent clinico-pathological interpretation. Conclusions Via immediate intra-analytical procedure, our RNA-based stratification RNA CellStrat increases the accuracy and reliability of the conclusions drawn from diagnostic and prognostic molecular information

Glutathione and Adaptive Immune Responses against Mycobacterium tuberculosis Infection in Healthy and HIV Infected Individuals

Glutathione (GSH), a tripeptide antioxidant, is essential for cellular homeostasis and plays a vital role in diverse cellular functions. Individuals who are infected with Human immuno deficiency virus (HIV) are known to be susceptible to Mycobacterium tuberculosis (M. tb) infection. We report that by enhancing GSH levels, T-cells are able to inhibit the growth of M. tb inside macrophages. In addition, those GSH-replenished T cell cultures produced increased levels of Interleukin-2 (IL-2), Interleukin-12 (IL-12), and Interferon-gamma (IFN-γ), cytokines, which are known to be crucial for the control of intracellular pathogens. Our study reveals that T lymphocytes that are derived from HIV infected individuals are deficient in GSH, and that this deficiency correlates with decreased levels of Th1 cytokines and enhanced growth of M. tb inside human macrophages