ATP-sensitive K+ channels in the hypothalamus are essential for the maintenance of glucose homeostasis.

Glucose-responsive (GR) neurons in the hypothalamus are thought to be critical in glucose homeostasis, but it is not known how they function in this context. Kir6.2 is the pore-forming subunit of K(ATP) channels in many cell types, including pancreatic beta-cells and heart. Here we show the complete absence of both functional ATP-sensitive K+ (K(ATP)) channels and glucose responsiveness in the neurons of the ventromedial hypothalamus (VMH) in Kir6.2-/- mice. Although pancreatic alpha-cells were functional in Kir6.2-/-, the mice exhibited a severe defect in glucagon secretion in response to systemic hypoglycemia. In addition, they showed a complete loss of glucagon secretion, together with reduced food intake in response to neuroglycopenia. Thus, our results demonstrate that KATP channels are important in glucose sensing in VMH GR neurons, and are essential for the maintenance of glucose homeostasis.

Impact of combinatorial dysfunctions of Tet2 and Ezh2 on the epigenome in the pathogenesis of myelodysplastic syndrome.

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.

Frequent somatic mutations in epigenetic regulators in newly diagnosed chronic myeloid leukemia.

Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1-17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.

Inosithin neutralization test to measure lupus anticoagulants.

The inosithin neutralization test was performed in 14 patients in whom lupus anticoagulant was detected. To test its specificity, it was also performed in 10 patients with severe hemophilia and in three patients with Factor VIII inhibitors. Prolonged kaolin clotting time was corrected by adding varying amounts of inosithin (Asolectin, 0.19 to 100 micrograms), a soybean-derived phospholipid, in all patients with lupus anticoagulant but not in patients with hemophilia or in two patients with Factor VIII inhibitors. In one patient, both Factor VIII inhibitors and lupus anticoagulant were present. The concentration of lupus anticoagulant in a patient was expressed as the amount of inosithin (measured in micrograms) required to normalize the prolonged kaolin clotting time. This amount correlated significantly with the occurrence of thrombosis and/or recurrent abortion. The inosithin neutralization test is useful to measure lupus anticoagulant.

Evaluation of four coagulation tests to detect plasma lupus anticoagulants.

Lupus anticoagulant was detected in 205 newly diagnosed, untreated patients with systemic lupus erythematosus by the following tests: kaolin clotting time, activated partial thromboplastin time, plasma prothrombin time, and, in the last 99 patients, by dilute Russell's viper venom time. In 10 patients, lupus anticoagulant was detected by kaolin clotting time prolongation, corrected by inosithin but not by normal plasma; 12 and 6 of them had prolonged activated partial thromboplastin time and partial plasma prothrombin time, respectively. Only 10 patients had a history of recurrent abortions and/or thrombosis, nine of whom had lupus anticoagulant as shown by the kaolin clotting time test. Of the 99 patients studied by all four tests, 9 showed lupus anticoagulant by both kaolin clotting time and dilute Russell's viper venom time; 7 had a history of abortion and/or thrombosis. The dilute Russell's viper venom time test is easy to perform and not affected by inhibitors to factor VIII or IX. It is recommended as a primary screening test for lupus anticoagulant detection in a hospital clinical laboratory.

A study of serum ferritin in beta thalassemia. Iron deficiency and overload.

Serum ferritin, serum iron, and unsaturated iron binding capacity were studied in 64 patients with beta homozygous thalassemia (BHT), 120 patients with beta heterozygous thalassemia, and 46 normal subjects. Incidence of iron overload seen in 32 BHT cases was similar in untransfused and transfused cases. Among heterozygotes, iron stores were depleted in 24 (20%), mostly females (70.8%). Only male heterozygotes but not normals were iron deficient. In 18 (75%) heterozygotes with depleted iron stores, transferrin saturation (TS) was normal. It was also normal in 8 (25%) BHT patients and 5 (100%) heterozygotes with iron overload. In 13 (35.1%) BHT patients, it was raised in the absence of iron overload. It was concluded that iron deficiency in heterozygotes is of greater magnitude, especially in females, than hitherto known in India. Transferrin saturation is not a good indicator of either iron depletion or overload. Iron supplementation is recommended in heterozygous beta thalassemia in infants, children, and expectant mothers in geographic areas with high incidence of iron deficiency.

Vitamin B12, folate, and iron studies in homozygous beta thalassemia.

The efficacy of serum folate (SF), red blood cell folate (RCF), and serum B12 in diagnosing folate and B12 deficiency, and the effect of iron overload on hemoglobin, were studied in 157 cases of homozygous beta thalassemia (HBT). The patients had lower SF compared with normal subjects (NS) (P less than 0.001) and higher RCF than their parents (P less than 0.001) and NS (P less than 0.001). Forty percent of patients had both low SF (less than 3 ng/mL) and high RCF (greater than 600 ng/mL). Homozygous beta thalassemia patients are known to have folate deficiency. Yet, both folate and B12 deficiency status were similar in the authors' patients and NS. These deficiencies apparently were less in patients compared with their parents, who shared the same nutritional milieu (P less than 0.001). Mean hemoglobin in patients with iron overload (transferrin saturation, TS greater than 50%) was lower than in those without (P less than 0.005). The following is concluded: (1) diagnosis of folate and B12 deficiency based on SF, RCF and serum B12 is vitiated in HBT and needs a therapeutic trial; (2) iron overload of a magnitude indicated by TS greater than 50% can aggravate anemia in HBT.

Asymptomatic visceral leishmaniasis in a child with acute lymphoblastic leukaemia.

Visceral leishmaniasis was detected incidentally in a patient with acute lymphoblastic leukaemia in remission, during maintenance therapy. Absence of fever, a normal haemogram, normal serum globulins, a negative serology and testicular involvement were the hallmarks of the case. Treatment with sodium stibogluconate (20 mg/kg for 55 days) failed. Subsequent therapy with pentamidine resulted in complete parasite clearance. Prolonged therapy with pentavalent antimony compounds or a higher dose of second line drugs such as pentamidine are recommended for complete clearance.

A study of iron and protein deficiency in hookworm infestation.

PIP: 104 patients with positive stool for hookworm ova were studied in detail with regard to anemia, iron and protein deficiency, and their relation to hookworm ova load. A variable degree of anemia was seen in 80 of 104 patients. Hypochromia was noticed in 66 (64%). In 48 (46%), morphological changes due to vitamin B12 and/or folic acid deficiency were recorded. Iron deficiency was most common in anemic patients. Less than 15% saturation of transferrin was the most sensitive biochemical index of iron deficiency in these patients. Severity of anemia was significantly associated with iron deficiency. Hypoalbuminemia (serum albumin less than 3.25 g%) was found in 33 (32.6%) of the patients. Anemia and hypoalbuminemia were both significantly associated with the hookworm load. However, the association of hookworm load was seen with severe anemia (hemoglobin less than 5 g%) and hypoalbuminemia (serum albumin less than 2.75 g%). It has been suggested that besides parasitic factors, nutritional deficiencies of vitamin B12 and/or folic acid and protein are contributory factors in the pathogenesis of anemia and hypoalbuminemia respectively found in these patients.^ieng

Platelet function in type I (insulin dependent) diabetes without vascular disease.

In 10 patients of type I diabetes who were free from clinically apparent vascular disease, platelet functions were studied prior to control, and 10 days and 3 months after strict metabolic control. Platelet hyperactivity, seen as significant shortening of latent period and increase in rate and degree of aggregation, present in patients in the initial uncontrolled state, but absent at the subsequent two periods did not correlate with glycosylation of haemoglobin. Thus control of diabetes mellitus, capable of reversing platelet hyperactivity, is reflected by blood sugar levels, and not by the glycosylation status of haemoglobin.

Coagulation abnormalities in systemic lupus erythematosus.

Coagulation profile was studied in 55 patients of systemic lupus erythematosus (SLE). Abnormal kaolin clotting time (KCT) was observed in fewer patients (12.9%) as compared to abnormal Russel's viper venom time (RVVT, 20.4%) or activated partial thromboplastin time (APTT, 32.7%). Prolonged prothrombin time (PT), observed in 7.3 per cent patients was not found to be a sensitive test for lupus anticoagulant (LAC). The correction of RVVT and KCT on addition of inosithin suggested a deficiency of platelet lipid factor in these patients. The initial value of uncorrected KCT in patient's plasma did not correlate with the amount of inosithin required for neutralisation. Occurrence of thromboembolic events was significantly associated with prolonged KCT. No other clinical feature showed significant association with any coagulation abnormality.

Lupus anticoagulant and anticardiolipin antibodies in systemic lupus erythematosus.

BACKGROUND: Lupus anticoagulant and anticardiolipin antibodies are antiphospholipid antibodies which have been independently associated with a high incidence of thrombotic diseases. However, the importance of their combined occurrence has not yet been examined. METHODS: We investigated 70 patients with systemic lupus erythematosus for the presence of anticardiolipin antibodies paying particular attention to a history of thrombosis and abortion. Lupus anticoagulant was detected using the kaolin clotting time, its mixing tests with normal plasma and the inosithin neutralization test. Anticardiolipin antibodies were detected using the ELISA technique. RESULTS: Lupus anticoagulant was detected in 11 patients (16%) and anticardiolipin antibodies in 13 (19%). Six patients were positive for both lupus anticoagulant and anticardiolipin antibodies. These patients had a higher incidence of thrombosis or recurrent abortion (5 of 6) compared to those with lupus anticoagulant (2 of 5) or anticardiolipin antibodies alone (1 of 7). The amount of inosithin required to neutralize lupus anticoagulant was greater [mean (SD) 27.5 (20.5) micrograms] in patients with both lupus anticoagulant and anticardiolipin antibodies than in patients with lupus anticoagulant alone [mean (SD) 4.0 (5.4) micrograms]. CONCLUSION: The presence of lupus anticoagulant is associated with thrombosis and recurrent abortion which are more frequent when both lupus anticoagulant and anticardiolipin antibodies are present and these patients probably have more severe disease as the amount of inosithin required to neutralize the lupus anticoagulant was greater.

Platelet function disorders in north India.

BACKGROUND: Platelet function disorders are a fairly common cause of bleeding manifestations. Although their relative types and incidence are well documented, data from India are lacking. METHODS: Between 1970 and 1991, we studied the clinical and laboratory features of 144 north Indian patients who presented to our hospital with a bleeding diathesis in whom coagulation disorders, von Willebrand's disease and a history of drug ingestion were absent. RESULTS: Isolated platelet factor 3 availability defect was the commonest (56 cases) followed by the thrombasthenias (49 cases), arachidonic acid pathway defect (26 cases), storage pool disease (8 cases) and the Bernard-Soulier syndrome (3 cases). Isolated platelet factor 3 deficiency was rare (2 cases). Two varieties of thrombasthenia were seen--the classical Glanzmann's (13 cases) and thrombopathic (36 cases). The latter was characterized by absent or sub-normal platelet aggregation with agonists along with a reduced (to less than 50% of normal) total platelet factor 3 content. This has not been reported from western countries. Patients with classical Glanzmann's thrombopathic thrombasthenia with absent platelet aggregation and isolated platelet factor 3 deficiency were severe bleeders. Their family history suggested an autosomal recessive transmission in Glanzmann's and thrombopathic thrombasthenia and a possible autosomal dominant transmission in isolated platelet factor 3 availability defect and isolated platelet factor 3 deficiency. CONCLUSION: The frequency of various types of platelet function disorders in Indians is similar to that in western populations except that the incidence of thrombopathic thrombasthenias is higher in India.

Evaluation of factors predisposing to arterial thrombosis in coronary artery disease.

Estimation of antithrombin III, alpha 2 macroglobulin and alpha 1 antitrypsin in patients with stable and unstable angina and acute myocardial infarction (15 cases each) were carried out. Twenty age, sex and weight matched healthy subjects were included as controls. Mean platelet factor 4(PF4) levels measured in 10 cases of each subgroup were significantly elevated in myocardial infarction (MI) (48.4 +/- 15.16 ng/ml) and III unstable angina patients (44.7 +/- 15.9 ng/ml) as compared to controls (25.42 +/- 12.47 ng/ml; P less than 0.01). Mean antithrombin III (AT III) levels were markedly reduced in all patients with MI (39.65 +/- 12.8% of normal pooled plasma) and unstable angina (37.9 +/- 16.6% of normal pooled plasma) and in 9 patients with stable angina. Alpha I antitrypsin and alpha 2 macroglobulin levels in these cases showed no significant difference compared to normals. Reduced AT III in coronary artery disease suggests a prethrombotic tendency in these patients. Raised PF4 levels in acute phase of the disease suggests heightened platelet activation.

A pancreaticographic study of malnutrition-related diabetes mellitus.

Pancreatic involvement is considered to be the hallmark of malnutrition-related diabetes mellitus (MRDM). Of the 2 subgroups of the disease, fibrocalculous pancreatic diabetes (FCPD) is characterized by pancreatic calcification. The nature of pancreatic abnormalities in MRDM have not been studied extensively in Indian patients. The present study was designed to compare pancreatic abnormalities (exocrine and endocrine) including endoscopic retrograde pancreaticography in patients with FCPD and protein deficient pancreatic diabetes (PDPD), in relation to controls. Ten patients each of FCPD and PDPD were studied with regard to clinical features, biochemical exocrine and endocrine pancreatic responses, C-peptide response, islet cell antibody, and pancreatographic changes. Five normal pancreatograms were taken as control. Clinical and biochemical features in patient with FCPD and PDPD were as follows: pain in 8 and 2 patients, respectively; the mean duration of diabetes was similar in both groups (62.28 +/- 71.92 months V. 72 +/- 50.9 months); and faecal fat excretion and insulin requirements were comparable in both groups. The main pancreatic duct was dilated in 6 of 10 patient with FCPD and only 1 of 10 with PDPD on ultrasonography. On pancreatography the duct was dilated in 9 of 10 patients with FCPD and only 1 of 10 patients with PDPD. The number of side branches was reduced in all cases with MRDM; in those with FCPD, these were stunted and dilated while in PDPD side branches are thin and spastic. We conclude that pancreatic ductal changes involving the main duct and side branches are more frequent in patients with FCPD as compared to those with PDPD.

Plasma lipid profile in gallstone patients from North India.

One hundred and thirty five patients with gallstones along with eighty nine matched controls were studied ultrasonographically to look for any association with hyperlipidemias. Plasma cholesterol and triglycerides were estimated by colorimetric methods and lipoproteins were classified according to Beaumont's classification. Male to female ratio in gallstone patients was 1:3. Mean plasma cholesterol and triglyceride values were higher in male gallstones patients as compared to controls (166.40 +/- 54.21 vs 40.26 +/- 32.80 mg/dl, p <0.01 and 182.65 +/- 84.49 vs 133.18 +/- 52.37 mg/dl, p <0.01 respectively). In female gallstone patients, on the other hand, only plasma triglyceride levels were raised as compared to control (182.65 +/- 84.49 vs 133.18 +/- 52.32 mg/dl, p <0.01). Prevalence of type IIb and type IV was 24.32% and 29.72% in male gallstone patients and 13.2 and 39.70% respectively in female gallstone patients. Thus, more than half of our gallstone patients had hyperlipidemia, the commonest types amongst them being type IIb and type IV.

A pancreatography study of chronic calcific pancreatitis of the tropics.

Chronic Calcific Pancreatitis of Tropics is a disease of unknown aetiology and is characterised by chronic pancreatitis with calcification in young persons who present with pain, diabetes, and/or steatorrhoea. ERCP performed on 42 patients with this condition revealed changes compatible with chronic pancreatitis. These changes were however, more marked and somewhat different from those seen in the alcoholic chronic pancreatitis. Cystic dilatation, tortuosity, and obstruction of the main pancreatic duct were similar to that in alcoholic pancreatitis. The features of CCPT that were different from those of latter, were large pancreatic calculi, absence of strictures/stenosis and absence of irregularity of the ductal wall. The calculi were predominantly in the head region of the pancreas causing maximal dilatation of the main pancreatic duct in the head of pancreas. The secondary branches were stunted, short and scanty but revealed a lower grade of changes, than the changes documented in the main pancreatic duct. The pancreatic ductal changes in CCPT seems to be different from that seen in chronic alcoholic pancreatitis and may be due to the difference in the pathophysiology of the underlying disease.