Lata Rani

Secretion of testicular steroids and gonadotrophins in hypothyroidism

Inconsistent alterations in gonadal steroidogenesis and pituitary functions have been reported in hypothyroid males. We have compared the lipid and endocrine profiles of the euthyroid and hypothyroid [thyroid‐stimulating hormone (TSH) >100 mIU l−1] males. Hypothyroidism was found to be associated with an increase in the circulating level of total cholesterol and LDL‐cholesterol (LDL‐C) and a reduction in the levels of progesterone and testosterone, without any change in the serum levels of oestradiol and gonadotrophins. The failure of gonadotrophins to rise could be accounted by a normal level of serum oestradiol in the hypothyroid male. A mild hyperprolactinaemia was also noted in the hypothyroid patients. The reduction in serum testosterone level could be explained by (i) a reduced uptake of LDL‐C by the Leydig cells and thereby a reduction in the synthesis of progesterone and consequentially testosterone, (ii) a further reduction in the rate of conversion of progesterone to testosterone, (iii) a higher rate of conversion of testosterone to oestradiol, (iv) a decrease in serum triiodothyronine and (v) hyperprolactinaemia. Rise in TSH needs to be investigated as a cause of the suppression of gonadal steroidogenesis.

Role of oxygen in the regulation of Leydig tumor derived MA-10 cell steroid production: the effect of cobalt chloride

Abstract We have earlier shown that cobalt chloride (CoCl2)-induced hypoxia and second messenger 8-bromoadenosine 3′, 5′-cyclic adenosine monophosphate (8-Br-cAMP) stimulates vascular endothelial growth factor (VEGF) production in Leydig tumor cell derived MA-10 cells. Both stimuli follow common signal transduction pathways including protein kinase A (PK-A), extracellular regulated kinase 1/2 (ERK1/2), and phosphatidyl inositol-3 kinase/akt (PI3-K/Akt) pathways in the stimulation of VEGF by MA-10 cells. In the present study we investigated the role of CoCl2 and 8-Br-cAMP on steroid production in MA-10 cells. The MA-10 cells were cultured in Waymouth MB 752/1 medium, supplemented with 15% heat inactivated horse serum. Progesterone was estimated by radioimmunoassay (RIA).We report that 8-Br-cAMP stimulated progesterone production by the MA-10 cells whereas CoCl2 inhibited the same. Also, 8-Br-cAMP stimulated steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) mRNAs expression. However, CoCl2 had no effect on StAR mRNA. Cobalt chloride directly inhibited the expression of P450scc mRNA. The decrease in progesterone production could be attributed to three different mechanisms, (1) an increase in production of reactive oxygen species (ROS), (2) an increase in HIF-1α activity, and (3) ultimately a decrease in the level of cytochrome P450 side chain cleavage (CYT P450scc). Hypoxia has an action and mechanism of action similar to that of gonadotropins on VEGF production, whereas they have a contrasting effect on steroidogenesis. This study suggests that hypoxia could be as important as gonadotropins in regulating Leydig cell steroidogenesis.

Synergistic effect of vascular endothelial growth factor and angiopoietin-2 on progression free survival in multiple myeloma

Bone marrow neoangiogenesis plays an important role in multiple myeloma (MM) and depends on the interplay of angiogenic cytokines. We investigated the levels of angiogenic cytokines such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin (Ang)-1, Ang-2 and hypoxia inducible factor-1 alpha (HiF-1α) in MM patients and their association with treatment outcome. Serum levels and mRNA expression of VEGF, Ang-2, Ang-1, bFGF and HiF-1α were evaluated in 71 MM patients using enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction. In multivariate Cox regression analysis, serum levels of VEGF≥756 pg/ml (HR 2.2, 95% CI 1.02-4.91; p=0.045) and relative mRNA expression levels of Ang-2≥0.93 (HR 21.0, 95% CI 6.27-70.45; p<0.001) were predictive of inferior progression free survival (PFS) and patients with concomitant increase in VEGF and Ang-2 had poor outcome compared to the rest of the patients (HR 32.6, 95% CI 7.20-148.36; p<0.001). These results suggest that VEGF and Ang-2 act in synergy and their expression levels at presentation are predictive of PFS in MM.

Oxygen as a regulator of MA-10 cell functions: effect of cobalt chloride on vascular endothelial growth factor production.

Mammalian testis functions at a temperature and oxygen tension (pO2) lower than the core body. Hypoxia‐inducible factor‐1α (HIF‐1α) mediates the adaptive responses to hypoxia such as production of angiogenic vascular endothelial growth factor (VEGF) in a variety of cells and tissues. VEGF production in Leydig cells is stimulated by luteinising hormone (LH)/cAMP. We have conducted experiments to find out whether HIF‐1α is involved in LH/cAMP‐induced secretion of VEGF by Leydig cell‐derived MA‐10 cells. Both cobalt chloride (CoCl2), an inducer of hypoxia, and 8‐Br‐cAMP enhanced HIF‐1α activity followed by an increase in VEGF secretion. However, there was no change in mRNA levels of HIF‐1α. Inhibition of HIF‐1α activity by cyclosporine A (CsA) inhibited a rise in VEGF production in response to CoCl2 as well as 8‐Br‐cAMP. Inhibitors of protein kinase A (PKA), extracellular regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol‐3 kinase/Akt (PI3‐K/Akt) inhibited the increase in VEGF levels in response to both CoCl2 and 8‐Br‐cAMP. The data suggest that HIF‐1α is a mediator of hypoxia‐ as well as 8‐Br‐cAMP‐stimulated production of VEGF in MA‐10 cells; both the stimuli act through a common signalling cascade.

Genome-wide DNA methylation profiling integrated with gene expression profiling identifies PAX9 as a novel prognostic marker in chronic lymphocytic leukemia

BackgroundIn chronic lymphocytic leukemia (CLL), epigenomic and genomic studies have expanded the existing knowledge about the disease biology and led to the identification of potential biomarkers relevant for implementation of personalized medicine. In this study, an attempt has been made to examine and integrate the global DNA methylation changes with gene expression profile and their impact on clinical outcome in early stage CLL patients.ResultsThe integration of DNA methylation profile (n = 14) with the gene expression profile (n = 21) revealed 142 genes as hypermethylated-downregulated and; 62 genes as hypomethylated-upregulated in early stage CLL patients compared to CD19+ B-cells from healthy individuals. The mRNA expression levels of 17 genes identified to be differentially methylated and/or differentially expressed was further examined in early stage CLL patients (n = 93) by quantitative real time PCR (RQ-PCR). Significant differences were observed in the mRNA expression of MEIS1, PMEPA1, SOX7, SPRY1, CDK6, TBX2, and SPRY2 genes in CLL cells as compared to B-cells from healthy individuals. The analysis in the IGHV mutation based categories (Unmutated = 39, Mutated = 54) revealed significantly higher mRNA expression of CRY1 and PAX9 genes in the IGHV unmutated subgroup (p < 0.001). The relative risk of treatment initiation was significantly higher among patients with high expression of CRY1 (RR = 1.91, p = 0.005) or PAX9 (RR = 1.87, p = 0.001). High expression of CRY1 (HR: 3.53, p < 0.001) or PAX9 (HR: 3.14, p < 0.001) gene was significantly associated with shorter time to first treatment. The high expression of PAX9 gene (HR: 3.29, 95% CI 1.172–9.272, p = 0.016) was also predictive of shorter overall survival in CLL.ConclusionsThe DNA methylation changes associated with mRNA expression of CRY1 and PAX9 genes allow risk stratification of early stage CLL patients. This comprehensive analysis supports the concept that the epigenetic changes along with the altered expression of genes have the potential to predict clinical outcome in early stage CLL patients.

Immunoglobulin heavy chain variable region gene repertoire and B-cell receptor stereotypes in Indian patients with chronic lymphocytic leukemia

Abstract In chronic lymphocytic leukemia (CLL), the geographical bias in immunoglobulin heavy-chain variable (IGHV) gene usage lead us to analyze IGHV gene usage and B-cell receptor stereotypy in 195 patients from India. IGHV3, IGHV4, and IGHV1 families were the most frequently used. 20.5% sequences had stereotyped BCR and were clustered in 12 pre-defined and 6 novel subsets. Unmutated IGHV was significantly associated with reduced time to first treatment (p < 0.033) and poor overall survival (OS; p = 0.01). We observed a significant difference in OS between IGHV1, IGHV3, and IGHV4 family cases (p = 0.045) in early stage patients. Regarding subfamily usage, only IGHV1-69 expression was found to have statistically significant poor outcome (p = 0.017). Our results from the analysis of various molecular and clinical features suggest that the expression of specific IGHV gene influences the outcome in early stage CLL, and hence its assessment may be added to the clinical leukemia laboratory armamentarium.

Comparative assessment of prognostic models in chronic lymphocytic leukemia: evaluation in Indian cohort

Prognostic indices combining several clinical and laboratory parameters have been proposed for prognostication in chronic lymphocytic leukemia (CLL). Recently, international consortium on CLL proposed an international prognostic index (CLL-IPI) integrating clinical, molecular, and genetic parameters. The present study was designed to evaluate the reproducibility of CLL-IPI in Indian CLL cohort. The prognostic ability of CLL-IPI in terms of overall survival (OS) and time to first treatment (TTFT) was investigated in treatment-naive CLL patients and also compared with other existing prognostic scores. For assigning scores, clinical and laboratory details were obtained from medical records, and IGHV gene mutation status, β2-microglobulin levels, and copy number variations were determined using c-DNA, ELISA, and multiplex ligation-dependent probe amplification (MLPA), respectively. The scores were generated as per the weighted grades assigned to each variable involved in score categorization. The predictive value of prognostic models was assessed and compared using Harrell’s C-index and Akaike’s information criterion (AIC). Stratification of patients according to CLL-IPI yielded significant differences in terms of OS and TTFT (p < 0.001). Comparative assessment of scores for OS suggested better performance of CLL-IPI (C = 0.64, AIC = 740) followed by Barcelona–Brno (C = 0.61, AIC = 754) and MDACC score (C = 0.59, AIC = 759). Comparison of predictive value of prognostic scores for TTFT illustrated better performance of CLL-IPI (C = 0.72, AIC = 726) followed by Barcelona–Brno (C = 0.68, AIC = 743), modified GCLLSG (C = 0.66, AIC = 744), and O-CLL1 index (C = 0.55, AIC = 773). The results suggest better performance of CLL-IPI in terms of both OS and TTFT as compared to other available scores in our cohort.

Vascular endothelial growth factor expression in pediatric non-Hodgkin lymphoma: A prospective study

Serum vascular endothelial growth factor (VEGF) level is higher in children with nonHodgkin lymphoma (NHL) than healthy controls, but its prognostic significance is unknown [1]. VEGF expression has been evaluated in serum [2] and in tissue using immunohistochemistry [3] and polymerase chain reaction (PCR) [4]. After institute ethical approval, treatment-naïve patients of pediatric NHL 18 years were included from July 2012 to March 2014. Patients with early-stage nonlymphoblastic lymphomas were treated with short-course chemotherapy [5]; advanced-stage nonlymphoblastic lymphoma received NHL-BFM-90 protocol [6] and lymphoblastic lymphoma BFM-90 ALLlike protocol [7]. Survival was censored on 15 September 2015. Baseline serum VEGF-A immune reactivity was assessed using quantitative sandwich enzyme immunoassay (Quantikine Human VEGF-A Immunoassay; R&D Systems, Minneapolis, MN, USA). Cytoplasmic VEGF expression in tumor cells was assessed using immunohistochemistry with mouse monoclonal VEGF antibody (VEGF:Ab-3, Clone:JH121; Neomarkers, Fremont, CA, USA). Intensity was graded negative (0), weak, moderate, or intense. VEGF staining intensity 1+ in 10% tumor cells was considered positive. Polymerase chain reaction (PCR) was performed using gene-specific primer pair for VEGF-A and glyceraldehyde-3-phosphate dehydrogenase. There were 50 patient samples and 10 healthy ageand sex-matched controls. Median age of patients was 11 years (2–18 years). Stage III/IV disease was present in 74% patients; bone marrow was involved in 9 patients. Median VEGF level in patients was 648.2 pg/mL (16.98–3335.5 pg/mL). Median serum VEGF in patients was higher than controls (P = .03). Bone marrow involvement in lymphoma was associated with increased serum VEGF (P = .03) (Table 1). Immunohistochemistry for VEGF was performed in 27/50 tissue blocks. Positivity was observed in 17/27 (63%) patients (Supplemental Figure 1). No significant correlation was observed with VEGF positivity and baseline characteristics (Table 1). The number of patients with moderate or intense VEGF expression was 3/7 in stages I and II and 5/20 in stages III and IV (P = .37). Serum VEGF versus VEGF positivity (plotted as positive/negative) using Spearman’s correlation showed positive correlation (r = .44; P = .02).

Serial assessment of circulating T regulatory cells and T helper 17 cells in pediatric non-Hodgkin lymphoma: a prospective study

T regulatory cells (Tregs) (CD4 + CD25 + cells) have immunosuppressive function and constitute 5–10% of all peripheral CD4 + lymphocytes. T-helper 17 (TH17) cells are new effector T-Helper cells that play an important role in autoimmunity and protection from bacteria. TH17 cells and Tregs constitute two opposing immune responses that are critically involved in modulation of inflammation induced by either autoimmunity or bacterial infection [1]. Studies of Tregs in adult patients with non-Hodgkin lymphoma (NHL) have reported variable results with regard to prognosis [2]. There is no existing data to describe the relevance of Tregs and TH17 cells in pediatric NHL. In view of this gap in our knowledge, this study was planned with the aim to determine Tregs and TH17 cells at baseline and on follow-up in children with NHL and compare the same with healthy controls and baseline demographics. The secondary objective was to evaluate interrelationship of Tregs and TH17 cells, and also to evaluate their impact on outcome. Treatment naive pediatric patients ( 18 years) of NHL were included in this study from October 2012–March 2014. The study was approved by Institute Ethics Committee. Informed consent was obtained from guardians of patients and control subjects. Patients with early stage non-lymphoblastic lymphomas were treated with short course chemotherapy [3], advanced stage non-lymphoblastic lymphomas were treated with NHL BFM-90 protocol [4] and patients with lymphoblastic lymphoma patients were treated with BFM-90 ALL like protocol [5]. Five millilitres of peripheral blood was collected in anticoagulant coated vacutainers from patients at baseline (Sample-0) prior to initiation of treatment, post treatment (Sample-1) and at relapse/progression (Sample-R). Sample-1 was obtained at the following time-points: in lymphoblastic lymphoma at end of induction; early stage non-lymphoblastic lymphomas at end of treatment; and in non-lymphoblastic stage 3 and 4 lymphoma after two cycles of chemotherapy. For Sample-1, the samples were specifically collected after recovery of blood counts prior to administration of next cycle of chemotherapy. Sample-R was obtained at relapse or progression prior to initiation of salvage chemotherapy. Samples were also collected from ten healthy controls. All samples were processed within 6 h. Mononuclear cells (MNCs) were separated from peripheral blood using density gradient media (Ficoll Histopaque-1077, Sigma Aldrich) and stained with CD4 Phycoerythrin carboxy flourescein 5 (PECF594), CD25 phycoerythrin cyanin 7 (PE-Cy7), CD127 PE and FoxP3 Fluorescein isothiocyante (FITC) (BD Biosciences, San Jose, CA). Briefly, 2 10 peripheral blood MNCs were incubated with antibodies for surface staining followed by fixation and permeabilization for FoxP3 staining as per manufacturer’s recommendations (BD Biosciences). Minimum of 2 10 events were acquired on flowcytometer (FC500, Beckman Coulter) and analyzed using FCS expression ver 3.0 (denovo software). Using sequential gating strategy, Tregs were identified as CD4 + CD127 CD25 high cells expressing FoxP3. Treg events were the positive cells from total CD4 + cells of gated lymphocytes, while absolute numbers of Tregs per cubic millimeter were calculated from absolute lymphocyte counts. For TH17 cell, MNCs were incubated in Rosewell Park Memorial Institute medium and 5% CO2 at 37 C with leucocyte activation cocktail and golgi plug (BD Biosciences), followed by fluorochrome staining with CD3 (PC5), CD8 (FITC), IL-17 (PE) and anti-human IFN-g monoclonal antibody (PECy 7) after fixation and