Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic rats

Summary The role of glucagon in diabetic hyperglycaemia has been a matter of controversy because of difficulties in the production of selective glucagon deficiency. We developed a high-capacity (40 nmol/ ml), high-affinity (0.6·10¹¹ l/mol) monoclonal glucagon antibody (Glu-mAb) and gave i.v. injections (4 ml/kg) to rats in order to study the effect of selective glucagon deficiency on blood glucose. Controls received a mAb against trinitrophenyl. Glu-mAb completely abolished the hyperglycaemic effect of 2.86 nmol/kg glucagon in normal rats (p<0.05, n=6). In moderately hyperglycaemic rats injected with streptozotocin as neonates (N-STZ), Glu-mAb abolished a postprandial increase in blood glucose (from 11.2±0.7 mmol/l to 17.3±1.8 mmol/l in controls vs 10.5±0.9 mmol/l to 9.3±1.0 mmol/l; cross-over: n=6, p<0.05). No significant effect of Glu-mAb treatment was observed in more hyperglycaemic N-STZ rats (cross-over, n=4) and in severely hyperglycaemic rats injected with STZ as adults (n=6), but after insulin treatment of the latter, at doses partially restoring blood glucose levels (12.7±4.3 mmol/l), Glu-mAb administration almost normalized blood glucose (maximal difference: 6.0±3.8 mmol/l; cross-over: n=5, p<0.05). In conclusion, our results provide strong additional evidence for the hypothesis that glucagon is involved in the pathogenesis of diabetes. The hormone plays an important role in the development of STZ-diabetic hyperglycaemia, but glucagon neutralization only leads to normoglycaemia in the presence of insulin.Abbreviations A-STZ rats Adult streptozotocin injected rats - AUC area under the curve - BW body weight - Con-mAb monoclonal control antibody - Glu-mAb monoclonal anti-glucagon antibody - K_a constant of association - NIDDM non-insulin-dependent diabetes mellitus - N-STZ rats neonatal streptozotocin-injected rats - OGTT oral glucose tolerance test - STZ streptozotocin     

Gender differences in systolic left ventricular function in hypertensive patients with electrocardiographic left ventricular hypertrophy (the LIFE study)

Echocardiography was performed in 944 untreated hypertensive patients (391 women and 553 men, mean age 66 years) who had electrocardiographic left ventricular (LV) hypertrophy at baseline in the Losartan Intervention For End point reduction in hypertension (LIFE) study to evaluate gender-associated differences in systolic LV function. Women had significantly lower diastolic blood pressure (175/97 vs 173/99 mm Hg) and body surface area and a higher body mass index (all p < 0.01). Women also had higher LV ejection fraction (EF), endocardial and midwall fractional shortening (63% vs 60%, 35% and 33%, and 16% vs 15%, respectively, all p < 0.01), higher stress-corrected midwall fractional shortening (98% vs 96%, p < 0.05), and lower circumferential end-systolic wall stress (178 vs 187 kdynes/cm(2), p < 0.01). There was no difference in age or LV mass indexed for height(2.7), but relative wall thickness was higher in women (0.42 vs 0.41, p < 0.05). In multiple regression analyses: (1) EF and endocardial fractional shortening were 2% to 3% higher in women than men, independent of the effects of LV stress, body mass index, and height (multiple r = 0.77 and 0.75, respectively, gender p < 0.02 in both models); (2) midwall fractional shortening was 0.5% higher in women, independent of the effects of age, body mass index, circumferential end-systolic stress, and absence of diabetes (multiple r = 0.36, p = 0.014 for gender); and (3) stress-corrected LV midwall fractional shortening was 2% higher (p = 0.004) in women, independent of the effects of age, height, heart rate, body mass index, and diabetes (multiple r = 0.33). Thus, female gender is an independent predictor of higher systolic LV function in hypertensive patients with electrocardiographic LV hypertrophy.     

Platelet-associated IgG in immune thrombocytopenic purpura

A method for the measurement of immunoglobulin G associated with gel- filtered platelets is described and finding in 70 control subjects and 37 patients with immune thrombocytopenic purpura (ITP) are reported. Control platelet-associated IgG (PAIgG) levels (nanograms IgG per 10(9) platelets) averaged (+/-SD) 1231+/-424; samples studied after 24 and 48 hr remained within the control range. PAIgG values of 19 adult and 12 childhood patients with chronic ITP averaged 4711+/-3025 and 4923+/- 3955, respectively, and differed significantly from controls (p less than 0.001). There was an inverse correlation between PAIgG values and the chronic ITP patient's platelet count. Six patients with childhood acute ITP had PAIgG levels ranging from 5588 to 56,250 and appeared to represent a different statistical population from those with chronic ITP. In chronic ITP patients responding to splenectomy, there was an immediate normalization of PAIgG levels; however, a certain percentage of patients studied several months after splenectomy evidenced elevated PAIgG levels in association with normal platelet counts. These data showed that the direct measurement of platelet associated antibody is a useful technique in the diagnosis and follow-up of patients with chronic ITP. Preliminary studies in patients with acute ITP have suggested that this method may be useful in differentiating acute and chronic childhood ITP.     

Clinical significance of p53 mutations in relapsed T-cell acute lymphoblastic leukemia

In T-cell acute lymphoblastic leukemia (T-ALL), p53 gene mutations were found in 12 of 51 patients in first relapse (24%). In a retrospective study, bone marrow samples at diagnosis were obtained from 9 of the 12 relapsed patients with p53 mutation; only one patient was found to harbor a p53 mutation at diagnosis. No further p53 mutations were identified in 18 unpaired diagnosis T-ALL samples. This is the first report of a p53 mutation in T-ALL at diagnosis. p53 mutations in relapsed T-ALL were clinically relevant. Patients with p53 mutations experience a shorter duration of survival than those patients without p53 mutations. Additionally, patients with p53 mutations were significantly less likely to have achieved a complete second remission from reinduction therapy than those patients without p53 mutations and experience a shorter duration of survival from relapse even when a second reinduction is obtained. Though primarily identified only at relapse, p53 mutations were also associated with a decreased duration of first remission and overall decrease in survival from diagnosis. Patients with p53 mutations had a 3.8-fold increase in risk of death than those patients without p53 mutations. These findings suggest that p53 mutation is associated with poor clinical outcome that is characterized by (1) a shortened duration of survival after first relapse; (2) a reduced response to reinduction therapy; (3) a shortened duration of first remission; and, hence, (4) an overall decreased duration of survival and increased risk of death.     

Mechanisms for Enlarging Lesion Size During Irrigated Tip Radiofrequency Ablation:

INTRODUCTION: Irrigated tip radiofrequency ablation of cardiac arrhythmias was developed to increase the size of the radiofrequency-induced lesion, since cooling of the electrode tip allows use of higher power settings. The purpose of this study was to determine if the increased lesion size during irrigated tip ablation is caused by the cooling effect solely or if increased electrical conductivity around the tip also contributes by increasing the "current-delivering size" of the tip: the so-called "virtual electrode effect." METHODS AND RESULTS: In vitro strips of left ventricular porcine myocardium and in vivo canine left ventricles were ablated. In vitro closed loop tip and showerhead irrigated tip catheters were compared. In vitro and in vivo showerhead tip catheters irrigated with solutions having different ionic content were compared. We found no difference in lesion size for closed loop and showerhead-type catheters (998 +/- 345 vs. 811 +/- 313 mm(3) during power-controlled ablation and 227 +/- 76 vs 318 +/- 127 mm(3) during temperature-controlled ablation). For irrigation with liquids having increasing ionic strength we found a decrease in lesion volume in vitro (361 +/- 249 vs. 812 +/- 229 mm(3) (P < 0.001) for power-controlled and 156 +/- 78 vs. 318 +/- 127 mm(3) (P < 0.05) for temperature-controlled ablation and nonsignificant differences in vivo. CONCLUSIONS: The mechanism for enlarging lesion size during radiofrequency irrigated-tip ablation is that higher power levels can be used. There is no virtual electrode effect caused by the highly conductive surroundings of the tip during irrigation. In vitro this effect is shown to be opposite: it decreases lesion size.     

A steady decline in pancreas transplantation rates

Background/objectives

After years of growth in many pancreas transplant programs, UNOS has reported declining transplant numbers in the USA. This precipitating trend urges for an evaluation of the transplant numbers and scientific productivity in the Eurotransplant region and the UK.

Gain of function in IKs secondary to a mutation in KCNE5 associated with atrial fibrillation

BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and a major cause of cardiovascular morbidity and mortality. Among the gene defects previously associated with AF is a gain of function of the slowly activating delayed rectifier potassium current IKs, secondary to mutations in KCNQ1. Coexpression of KCNE5, the gene encoding the MiRP4 beta-subunit, has been shown to reduce IKs. OBJECTIVE: The purpose of this study was to test the hypothesis that mutations in KCNE5 are associated with AF in a large cohort of patients with AF. METHODS: One-hundred fifty-eight patients with AF were screened for mutations in the coding region of KCNE5. RESULTS: A missense mutation involving substitution of a phenylalanine for leucine at position 65 (L65F) was identified in one patient. This patient did not have a history of familial AF, and neither KCNQ1 nor KCNE2 mutations were found. Transient transfection of Chinese hamster ovary (CHO) cells expressing IKs(KCNQ1+KCNE1) with KCNE5 suppressed the developing and tail currents of IKs in a concentration-dependent manner. Transient transfection with KCNE5-L65F failed to suppress IKs, yielding a current indistinguishable from that recorded in the absence of KCNE5. Developing currents recorded during a test pulse to +60 mV and tail currents recorded upon repolarization to -40 mV both showed a significant concentration-dependent gain of function in IKs with expression of KCNE5-L65F vs KCNE5-WT. CONCLUSION: The results of this study suggest that a missense mutation in KCNE5 may be associated with nonfamilial or acquired forms of AF. The arrhythmogenic mechanism most likely is a gain of function of IKs.     

Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways

Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4- sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.     

Shedding of transferrin receptor from rat reticulocytes during maturation in vitro: soluble transferrin receptor is derived from receptor shed in vesicles

Measurements of circulating transferrin (Tf) receptor are useful in assessing erythropoiesis; however, steps involved in the generation of soluble Tf receptor from cellular receptor are incompletely understood. To obtain a better understanding of this process, we investigated the loss of Tf receptor during terminal maturation of rat reticulocytes in vitro. Previous studies have identified Tf receptor-containing vesicles in the culture medium of maturing reticulocytes. In the present study, vesicle-free reticulocyte culture medium was found to contain functional and immunoreactive soluble Tf receptor, which increased over time. During a 44-hour incubation, Tf receptor on reticulocytes decreased by approximately 69%, while, of the Tf receptor shed to the medium, 65% was present in vesicles and 35% was in a soluble form. Isolated vesicles reincubated in fresh medium released soluble Tf receptor to the medium. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated soluble receptor protein was mainly 190 Kd and 95 Kd under nonreducing and reducing conditions, respectively, similar in size to the vesicular and cellular receptor. Our studies show that loss of Tf receptor from rat reticulocytes during maturation in vitro involves shedding of cellular Tf receptor in vesicles and release of soluble receptor from these vesicles.