High Frequency of Recessive WFS1 Mutations Among Indian Children With Islet Antibody-negative Type 1 Diabetes.

BACKGROUND: While the frequency of islet antibody-negative (idiopathic) type 1 diabetes mellitus (T1DM) is reported to be increased in Indian children, its aetiology has not been studied. We investigated the role of monogenic diabetes in the causation of islet antibody-negative T1DM. METHODS: We conducted a multicenter, prospective, observational study of 169 Indian children (age 1-18 years) with recent-onset T1DM. All were tested for antibodies against GAD65, islet antigen-2, and zinc transporter 8 using validated ELISA. Thirty-four islet antibody-negative children underwent targeted next-generation sequencing for 31 genes implicated in monogenic diabetes using the Illumina platform. All mutations were confirmed by Sanger sequencing. RESULTS: Thirty-five (21%) children were negative for all islet antibodies. Twelve patients (7% of entire cohort, 34% of patients with islet antibody-negative T1DM) were detected to have pathogenic or likely pathogenic genetic variants. The most frequently affected locus was WFS1, with 9 patients (5% of entire cohort, 26% of islet antibody-negative). These included 7 children with homozygous and 1 patient each with a compound heterozygous and heterozygous mutation. Children with Wolfram syndrome 1 (WS) presented with severe insulin-requiring diabetes (including 3 patients with ketoacidosis), but other syndromic manifestations were not detected. In 3 patients, heterozygous mutations in HNF4A, ABCC8, and PTF1A loci were detected. CONCLUSION: Nearly one-quarter of Indian children with islet antibody-negative T1DM had recessive mutations in the WFS1 gene. These patients did not exhibit other features of WS at the time of diagnosis. Testing for monogenic diabetes, especially WS, should be considered in Indian children with antibody-negative T1DM.

Carotid intima media as predictor of liver fibrosis in type 2 diabetes mellitus with NAFLD.

BACKGROUND AND AIMS: Non Alcoholic Fatty Liver Disease (NAFLD) is common in type 2 Diabetes Mellitus (DM) that might progress to advance liver fibrosis. Early recognition of liver fibrosis may have clinical implication. Non invasive assessment tool for severity of liver fibrosis in NAFLD is expensive fibroscan. An alternate method of diagnosis will be very useful innovation. We aimed to evaluate Carotid Intima Media Thickness (CIMT) and its association with severity of liver fibrosis in patients with type 2 DM and NAFLD. METHODS: Treatment naïve patients with type 2 DM were enrolled. Measurement of CIMT, hepatic ultrasound and fibroscan were done. Liver function tests included hepatic transaminases. The data obtained was subjected to statistical analysis using IBM SPSS version 20.0 software. RESULT: Prevalence of NAFLD was 76% including 12% with moderate to advance liver fibrosis in patients with type 2 DM. CIMT was significantly higher in patients with NAFLD than with normal liver. CIMT positively correlated with severity of liver fibrosis measured by fibroscan. ROC curve analysis showed right CIMT value of 0.575 mm predicting liver fibrosis with sensitivity of 91.7% and specificity of 78.9%. CONCLUSION: Three fourth of patients with type 2 DM had NAFLD but small proportion had moderate to advance liver fibrosis. CIMT increased more in patients with NAFLD than with normal liver in T2DM. CIMT value of 0.575 mm has a good sensitivity to predict liver fibrosis and therefore, it can be a reliable marker of severity of Non Alcoholic Steato Hepatitis (NASH) in diabetes with NAFLD.

Alterations in Bone Turnover during Chemotherapy in Children with Acute Lymphoblastic Leukemia.

Background Disturbances of bone metabolism frequently occur in children with acute lymphoblastic leukemia (ALL), leading to increased risk of osteopenia and osteoporosis at diagnosis, during and after completion of chemotherapy. The present study was performed to evaluate alteration in bone mineral metabolism in children with ALL during chemotherapy. Method Fifty newly diagnosed patients with ALL in the age group of 2 to 14 years were included. Relapsed and refractory cases were excluded. Enrolled children were stratified into standard and high risk according to National Cancer Institute criteria. Quantitative analysis of bone resorptive marker carboxyl-terminal telopeptide of human type 1 collagen (ICTP) was assessed at baseline and 3 months after chemotherapy by the sandwich enzyme-linked immunosorbent assay technique. Results Of 50 patients enrolled, 21 were standard and 29 were high risk. The mean age was 7.75 ± 4.0 years and the male-to-female ratio was 3.5:1. ICTP levels were analyzed in 44 patients, of which 37 (84%) showed significantly increased levels. The mean ICTP level in patients at diagnosis and controls was 1.78 ± 1.39 and 0.96 ± 0.32 µg/L, respectively ( p = 0.001). The mean ICTP level at 3 months after chemotherapy increased to 3.55 ± 1.40 µg/L ( p = 0.000). It was significantly increased in males ( p = 0.000) and in B cell ALL group ( p = 0.000) in comparison to females and T cell group. Both standard and high risk groups were equally affected ( p = 0.000). On multivariate analysis, no single risk factor could be identified. Conclusion The marker of bone resorption (ICTP) in children with ALL was increased at diagnosis, which further increased during chemotherapy. The disease itself and the intensive chemotherapy both contributed to the increased levels.

Anti-oxidant, anti-apoptotic, anti-hypoxic and anti-inflammatory conditions induced by PTY-2 against STZ-induced stress in islets.

The earlier assessment of Pueraria tuberosa (PT) has shown anti-diabetic effects through enhancing incretin action and DPP-IV (Dipeptidyl peptidase-IV) inhibition. The aim of this work was to further explore the protective role of aqueous extract of Pueraria tuberosa tuber (PTY-2) against streptozotocin (STZ) induced islet stress in rats. Diabetes was induced by STZ (65 mg/kg body weight) in charles foster male rats. After 60 days of STZ administration, animals with blood glucose levels > 200 g/dL were considered as diabetic. All the rats were later divided into three groups: Group-1 (STZ untreated normal rats), Group-2 (Diabetic control), and Group-3 (PTY-2 [50 mg/100 g bw treatment for next 10 days to diabetic rats). The rats were then sacrificed after the 10th day of treatment accordingly. STZ treatment led to an increase in expression of Matrix metalloproteinases-9 (MMP-9), Tumour necrosis factor-α (Tnf-α), Hypoxia inducible factor-α (HIF-1α), Vascular endothelial growth factor (VEGF), Interleukin-6 (IL-6), Protein kinase C-ε (PKC-ε), Nuclear factor kappa-light-chain-enhancer of activated B-cells (NFkB), and Caspase-3. Reverse Transcriptase-PCR (RT-PCR), Immunohistochemistry and Western-Blot analysis showed an increase in the expressions of Superoxide Dismutase (SOD) and Nephrin, and a decrease in the expressions of NFkB, PKC-ε, TNF-α, MMP-9, HIF-1α, VEGF, Caspase-3 and IL-6 after 10 days of PTY-2 treatment. The results showed that PTY-2 favorably changed all the expressions via anti-oxidant, anti-apoptotic, anti-hypoxic and anti-inflammatory pathways, making itself as a protective agent against STZ induced islet stress. Further evaluation of PTY-2 might be helpful in establishing its role in the management of diabetes mellitus.

GLP 1 Regulated Intestinal Cell's Insulin Expression and Selfadaptation before the Onset of Type 2 Diabetes.

Purpose: Basically insulin is known to be secreted by ß cells of the pancreas. Recently, it has also been found to be produced and expressed by intestinal epithelial cells with the help of L cells secreting glucagon like peptide 1 (GLP 1). Here, we have studied the same intestinal insulin expression property in T2D rats. Methods: Following 2 weeks of high fat diet (HFD) consumption, we have been given a single dose of streptozotocin (STZ) (35 mg/kg bw). Rats were then sacrificed after 1, 7 and 21 days. The GLP 1 analogue, liraglutide was also given to one group of diabetic rats, upto their respective durations. Intestinal cells apoptosis were checked by tunnel assay, Incretin hormones secretion and dipeptidyl peptidase 4 (DPP-IV) activity were analyzed through ELISA and immunohistochemistry was used to determine the insulin expression of intestine at different time interval during diabetes progression. Results: As compared to 1 and 21 days, we have found minor cells apoptosis in 7 days group along with high level of GLP 1 in diabetic model. Further, these effects were enhanced by liraglutide. In response to these we have found, decreased insulin expression after 21 days and with no significant effect upto 7 days in diabetic control groups. In contrast to this, GLP-1 level and insulin expression enhances prominently after 7 days of liraglutide treatment. Conclusion: These results explain the self-adapting approach of intestinal cells against diabetes onset and insulin expression enhancing property of liraglutide under stressful conditions. This study should be continued in future for the development of intestinal insulin producing drugs, to control diabetes under irreversible ß cells damage.

Evidence-based recommendations for insulin intensification strategies after basal insulin in type 2 diabetes.

Over the time due to progressive nature of diabetes, proactive intensification of the existing insulin therapy becomes imminent as it minimizes patients' exposure to chronic hypo/hyperglycaemia and reduces weight gain while achieving individualized glycaemic targets. This review focuses on the strength of evidence behind various options for intensification, primarily the insulins as also the GLP-1 analogues. The recommendations presented here are meant to serve as a guide for the physician managing type 2 diabetes patients requiring insulin intensification upon failing of basal insulin therapy.

Parenteral vitamin D supplementation is superior to oral in vitamin D insufficient patients with type 2 diabetes mellitus.

BACKGROUND/OBJECTIVES: Oral vitamin D supplementation is better than parenteral in improving vitamin D deficiency in individuals with no systemic illness. Our aim was to compare the efficacy of oral and parenteral routes of vitamin D supplementation on circulating serum 25(OH) vitamin D level in patients with type 2 diabetes mellitus. METHODS: Total 85 cases of with type 2 diabetes mellitus were screened for vitamin D status of which 71 patients were vitamin D insufficient/deficient. They were randomized into two intent to treat groups with different vitamin D supplementation protocols (a) Oral-60000 IU per day for 5days (group I; n=40) and (b) injectable-300000 IU intramuscularly once (group II; n=31). Baseline and one month post supplementation 25(OH) vitamin D levels were measured in both the groups. RESULTS: Baseline clinical characteristics and 25(OH) vitamin D levels were comparable in both the groups. Post treatment 25(OH) vitamin D level in group I was 26.06±9.06ng/ml and in group II was 49.69±18.92ng/ml. After one month of vitamin D supplementation, increment in 25(OH) vitamin D level from baseline was significantly higher in group II than group I (p<0.001). INTERPRETATION & CONCLUSIONS: Injectable method of supplementation was better than oral route in improving serum 25 (OH) vitamin D status in patients with type 2 diabetes. The study suggested impaired absorption of vitamin D from the gastrointestinal tract in patients with type 2 diabetes mellitus and a need for parenteral route of vitamin D supplementation in deficient patients with type 2 diabetes mellitus.

Effects of Azardirachta indica on Vascular Endothelial Growth Factor and Cytokines in Diabetic Deep Wound.

A chronic, unhealed diabetic wound is one of the severe complications of diabetes mellitus. Azadirachta indica has been reported to have antidiabetic and antiapoptotic properties. The present work incorporates the healing potential of 50 % ethanol A. indica leaves extract against deep surgical wounds in streptozotocin-induced mild diabetic rats. A. indica leaves extract (500 mg/kg) was administered orally, once daily for ten days. Serum glucose, cholesterol, and triglycerides as well as body weight, food, and water intake, and tissue antioxidants (catalase, superoxide dismutase and reduced glutathione), free radicals (lipid peroxidation and nitric oxide), myeloperoxidase, total collagens (hydroxyproline, hexuronic acid and hexosamine), protein, vascular endothelial growth factor, and cytokines (tumor necrotic factor-α and interleukin-1ß) were estimated. Histology was done for connective tissue formation and inflammatory and healing in deep granulation tissue after A. indica leaves extract treatment. Diabetic rats showed an increase in serum glucose, cholesterol, and triglycerides levels, food and water intake, and granular tissue free radicals, myeloperoxidase, and cytokines, but a decrease in body weight, total collagen, and vascular endothelial growth factor levels. A. indica leaves extract reversed the increased serum glucose, cholesterol, and triglycerides, food and water intake, and tissue free radicals, myeloperoxidase and, cytokines, but increased body weight, tissue antioxidants, total collagen, and vascular endothelial growth factor contents. The results thus indicated an improvement in wound healing by A. indica leaves extract in diabetic rats through enhanced angiogenesis mediated through the inhibition of hyperglycemia, oxidative stress, and down- and upregulation of inflammatory mediators and growth factor expression.

Vitamin D deficiency is associated with inflammatory cytokine concentrations in patients with diabetic foot infection.

Vitamin D has been recognised as a potent immunomodulator and its deficiency is common in different population groups including patients with diabetic foot infection. Diabetic foot infection reflects the altered immune status of the host. As cytokine regulation plays a significant role in infection and wound-healing processes, the present study aimed to evaluate the association between vitamin D status and inflammatory cytokine profiles in patients with diabetic foot infection. The serum concentrations of vitamin D (25-hydroxyvitamin D), IL-1ß, IL-6, TNF-α and interferon-γ (IFN-γ) were measured in 112 diabetic foot infection cases and 109 diabetic controls. Severe vitamin D deficiency (25-hydroxyvitamin D concentration < 25 nmol/l) was more common in cases than in controls (48.2 v. 20.5%). Although age, duration of diabetes, HbA1C (glycosylated Hb) concentration and BMI were similar, cases had significantly higher concentrations of IL-6 (P≤ 0.001), IL-1ß (P≤ 0.02) and TNF-α (P≤ 0.006) than controls. A significant negative correlation was also observed between 25-hydroxyvitamin D concentration and circulating concentrations of IL-1ß (r -0.323; P≤ 0.001) as well as IL-6 (r -0.154; P≤ 0.04), but not between 25-hydroxyvitamin D and TNF-α and IFN-γ concentrations. Furthermore, a significant difference in IL-1ß (P≤ 0.007) and IL-6 (P≤ 0.02) concentrations was observed in patients with severe 25-hydroxyvitamin D deficiency compared with patients with 25-hydroxyvitamin D concentration ≥ 25 nmol/l, and this difference was remarkable for TNF-α. In conclusion, severe vitamin D deficiency is associated with elevated inflammatory cytokine concentrations in diabetic patients, particularly in those with foot infection. A 25-hydroxyvitamin D concentration value < 25 nmol/l is suggested as the 'cut-off' for such immunological alterations in patients with diabetes mellitus.

Vitamin D status of patients with type 2 diabetes and sputum positive pulmonary tuberculosis.

INTRODUCTION: Vitamin D deficiency is expected to be higher in patients with diabetes and pulmonary tuberculosis (TB). Studies estimating prevalence in the subset of patients with both diabetes and pulmonary TB are scarce. MATERIALS AND METHODS: A total of 155 subjects were recruited; 46 patients with type 2 diabetes, 39 non-diabetic healthy controls, 30 patients of pulmonary TB and 40 patients with both pulmonary TB and type 2 diabetes. Vitamin D level (25 OH vitamin D) levels were done for all the 4 groups. RESULTS: Mean vitamin D levels were not different between groups with TB, diabetes mellitus or combination of both, but the prevalence of severe vitamin D deficiency was higher in the group with both diabetes and TB (45%) as compared with the group with only TB (26.66%) and diabetes (17.39%) and healthy controls (7.69%). CONCLUSION: The prevalence of patients with severe vitamin D deficiency is higher in patients with dual affection of TB and diabetes mellitus as compared with either disorder alone implying that patients with type 2 diabetes with the most severe vitamin D deficiency are the one of the most predisposed to pulmonary TB.

Prevalence and severity of vitamin D deficiency in patients with diabetic foot infection.

The aim of the present research was to study the prevalence and severity of vitamin D deficiency in patients with diabetic foot infection. Patients were enrolled in two groups: diabetic patients with foot infection (n 125) as cases and diabetic patients without the infection as controls (n 164). Serum 25-hydroxyvitamin D (25(OH)D) was measured by RIA. Data were presented as means and standard deviations unless otherwise indicated and were analysed by SPSS. Results revealed that 25(OH)D (nmol/l) was significantly lower (40·25 (sd 38·35) v. 50·75 (sd 33·00); P < 0·001) in cases than in controls. Vitamin D inadequacy (25(OH)D < 75 nmol/l) was equally common in cases and controls (OR 1·45, 95 % CI 0·8, 3·0; P = 0·32), but cases had a greater risk of vitamin D deficiency (25(OH)D < 50 nmol/l) than controls (OR 1·8, 95 % CI 1·1, 3·0; P = 0·02). Risk of severe vitamin D deficiency (25(OH)D < 25 nmol/l) was significantly higher in cases than in controls (OR 4·0, 95 % CI 2·4, 6·9; P < 0·0001). Age, duration of diabetes and HbA1c were significantly higher in cases than in controls and therefore adjusted to nullify the effect of these variables, if any, on study outcome. The study concluded that vitamin D deficiency was more prevalent and severe in patients with diabetic foot infection. This study opens up the issue of recognising severe vitamin D deficiency (< 25 nmol/l) as a possible risk factor for diabetic foot infections and the need for vitamin D supplementation in such patients for a better clinical outcome. This could be substantiated by similar data from future studies.

Sitagliptin: anti-platelet effect in diabetes and healthy volunteers.

Sitagliptin, a selective dipeptidyl peptidase-4 inhibitor drug is used to treat type-2 diabetes (T2DM). We investigated the anti-platelet activity of sitagliptin in patients with T2DM and in in vitro samples obtained from healthy humans. Patients with T2DM (27 male + 23 female) were selected and followed up before (control) and after treatment with sitagliptin for up to 3 months. Platelets were isolated from the blood of sitagliptin treated patients and controls. Patients with T2DM treated with sitagliptin for 1and 3 months, showed 10 ± 2% and 30 ± 5% inhibition of platelet aggregation, respectively. For the in vitro study, platelets from 10 normal humans (n = 10) were isolated. Platelet aggregation, intracellular free calcium and tyrosine phosphorylation of multiple proteins were measured by aggregometer, spectrofluorometer and western blotting, respectively. Platelets pre-treated with 5 and 10 µg/ml of sitagliptin, showed 25 ± 4% and 40 ± 6% inhibition of thrombin-induced platelet aggregation, respectively. Sitagliptin decreased intracellular free calcium (2.5-fold) and tyrosine phosphorylation of multiple proteins in thrombin-induced platelet activation. Sitagliptin inhibited platelet aggregation in T2DM as well as in healthy humans. Sitagliptin has significant concentration-dependent anti-platelet activity. This activity was due to its inhibitory effect on intracellular free calcium and tyrosine phosphorylation.

Waist circumference cutoff and its importance for diagnosis of metabolic syndrome in Asian Indians: A preliminary study.

BACKGROUND: There is inconsistency in accepting waist circumference (WC) as mandatory and also regarding its significance for diagnosis of metabolic syndrome (MetS) for different populations. AIM: To study the association of individual parameters of MetS with WC cutoffs suitable for South Asian Indians. MATERIALS AND METHODS: From an ongoing hospital-based study on MetS as per the criteria of diagnosis of modified NCEP ATP III, 713 subjects having a minimum three of the four parameters, i.e., dyslipidemia [low high density lipoprotein (HDL), high triglycerides], dysglycemia and hypertension, without regard to cutoffs of WC, were included in the present study. RESULTS: Receiver operator characteristic curve analysis of WC cut-off points for males was 90 cm with a sensitivity and specificity of 71% and 96%, respectively, and for females was 85 cm with a sensitivity and specificity of 86% and 93%, respectively, associated with the risk factors of MetS. Multiple logistic regression analysis for low density lipoprotein (LDL) cholesterol concentration of ≥3.38 mmol/l showed an odds ratio of 5.03 (95% CI = 1.29-19.5) in males and 3.17 (95% CI = 1.14-8.76) in females which was statistically significant (P < 0.02); in addition to higher WC, higher level of triglyceride (P ≤ 0.0001) and lower level of high density lipoprotein cholesterol (P ≤ 0.02) were observed. CONCLUSION: This study suggests that WC of 90 cm in males and 85 cm in females should be a mandatory criterion of MetS in our subset of population. LDL may be considered one of the components of MetS along with the currently defined WC cutoffs.

Detection of anaerobic infection in diabetic foot ulcer using PCR technique and the status of metronidazole therapy on treatment outcome.

UNLABELLED: Metronidazole is the drug of choice for anaerobic infection in diabetic foot ulcers (DFU) for a majority of clinicians. The present study was conducted to determine if Metronidazole is really making a difference in the healing of DFU. METHODS: Deep tissue samples from the wound area of 61 diabetic foot patients were tested for anaerobic bacterial infection (Peptostreptococcus productus, Bacteroides, and Clostridium) by polymerase chain reaction (PCR). PCR-positive patients were randomized into 2 groups: Metronidazole and non-Metronidazole. Antibiotics for the control of infection were given in both groups as per clinical condition of patients. Treatment outcome was assessed by complete healing of the wound. RESULTS: Out of 61 patients, PCR detected evidence of anaerobic infection in 32 (52%), while culture methods detected only 5 (8%) (Clostridium spp.), hence emphasizing the significance of the PCR technique over culture methods in detection of microbes. In this study, Clostridium was found with maximum prevalence of n (75%), followed by Bacteroides with n (53.1%), and Peptostreptococcus productus with n (40.6 %). Across all Wagner Ulcer Classification grades, Clostridium was the most prevalent anaerobe, and significantly associated with wound age and total leukocyte count. No difference was noted in wound healing in both groups at the end of 16 weeks. CONCLUSIONS: The authors propose that it is not mandatory to supplement Metronidazole in antibiotic regime for treatment of DFU.

Antitubercular drug resistance in four healthcare facilities in North India.

Tuberculosis (TB) is a major public-health problem in India, having the highest number of incident and multidrug-resistant (MDR) TB cases. The study was carried out to appraise the prevalence of first-line anti-TB drug resistance in Mycobacterium tuberculosis (MTB) and its patterns among different types of TB patients from different settings in a province of North India. Of 3,704 clinical specimens, 345 (9.3%) were culture-positive, and drug-susceptibility testing was carried out for 301 MTB strains. A high level of primary and acquired drug resistance of MTB was observed in the region studied, with weighted mean of 10.5% and 28.08%, 12.81% and 29.72%, 17.12% and 29.94%, 11.97% and 27.84%, and 10.74% and 23.54% for rifampicin, isoniazid, streptomycin, ethambutol-resistant and MDR cases respectively. Drug resistance was significantly higher in pulmonary (p = 0.014) and acquired drug-resistant TB cases (p < 0.001). Any drug resistance (p = 0.002) and MDR TB were significantly (p = 0.009) associated with HIV-seropositive cases. An urgent plan is needed to continuously monitor the transmission trends of drug-resistant strains, especially MDR-TB strains, in the region.