Reference limits for GAD65 and IA-2 autoantibodies by chemiluminescence immunoassay in Northern European adults and children

The GAD65 and IA-2 antibodies (Abs) are biomarkers of the development of type 1 diabetes mellitus (T1DM) in both children and adults. The upper reference limit for the autoantibodies made by the manufacture was established on an adult Chinese population. Here, we established upper reference limits for Northern European adults and children in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Serum samples from healthy Danish children (0–18 years) and adults (18–70 years) were analysed for GAD65Ab and IA-2Ab using MAGLUMI 800 Chemiluminescence Immunoassay (CLIA). The Kruskal–Wallis test was used for evaluating differences between gender and age groups. No gender or age differences were found for neither GAD65Ab nor IA-2Ab, and a combined upper reference limit for both children and adults could be established. An upper reference limit of 5.1?IU/mL was defined for GAD65Ab and 11.5?U/mL for IA-2Ab. Our results showed a substantial discrepancy with the reference limits established by the manufacturer.     

Physicochemical Changes in HDL3 after Bezafibrate Treatment: Influence on Free Cholesterol Efflux from Human Fibroblasts

The effects of bezafibrate, a well-used fibric acid hypolipidemic agent, were investigated in 10 moderately hypertriglyceridemic patients. The aim was to quantify the physico-chemical modifications to high-density lipoprotein subfraction 3 (HDL₃) induced by treatment and to assess, in vitro, the alterations in its principal physiological function, the efflux of intracellular free cholesterol. Treatment (200mg/thrice/d for 3 months) resulted in a 48% decrease in plasma triglycerides, with an increase in the HDL cholesterol, due mainly to an increase in the HDL₃ (P < 0.01). Composition analysis of HDL₃indicated an increase in cholesterol esters (P < 0.01), free cholesterol (P < 0.01), and phospholipids (P < 0.01), coupled with a decrease in the protein content of the molecule compared with pretreatment values. Fluorescense anisotropy at 24°C was significantly higher post-treatment than pretreatment (P < 0.01). The cholesterol effluxing capacity of pretreatment HDL₃ was 28%, and post-treatment this increased to 50% (P < 0.01). Multivariate analyses indicated that the increased capacity of HDL₃ to promote free cholesterol efflux was, in part, due to increased HDL₃ phospholipid content and a more adequate fluidity of the molecule. These findings suggest that bezafibrate induces a lowering of plasma triglycerides and that the resultant physico-chemical alterations of the HDL₃ molecule make it more efficient as an acceptor of intracellular free cholesterol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Impact of TCR status and genotype on outcome in adult T-cell acute lymphoblastic leukemia: a LALA-94 study

Patients with T-cell acute lymphoblastic leukemias (T-ALLs) within the Leucemies Aigues Lymphoblastiques de l'Adulte-94 (LALA-94) prospective trial were treated with a 4-drug per 4-week induction, with intermediate-dose cytarabine and mitoxantrone salvage treatment for patients not achieving complete remission (CR) in 1 course. Only the latter received allografts, if possible, thus providing an informative setting for assessing early response. Representative patients with T-ALL (91 patients) were classified into surface T-cell receptor (TCR)-expressing T-ALL patients (TCRalphabeta+ or TCRgammadelta+), pre-alphabeta T-ALL patients (cTCRbeta+, TCR-), and immature (IM) cTCRbeta-, TCR- T-ALL patients; 81 patients underwent genotyping for SIL-TAL1, CALM-AF10, HOX11, and HOX11L2. Overall, CR was obtained in 81 (89%) patients; relapse rate was 62% at 4 years and overall survival (OS) rate was 38%. CR rate was significantly lower in IM T-ALL patients after 1 course (45% vs 87%; P < .001) and after salvage (74% vs 97%; P = .002), with the latter inducing a higher rate of CR (9 [64%] of 14) than initial induction. Once CR was obtained, cumulative relapse rates were similar for IM, pre-alphabeta, and TCR+ T-ALL patients (P = .51), but were higher in HOX11L2 (83%) and SIL-TAL1 (82%) T-ALL patients compared with other genetic subgroups (48%; P = .05). This was associated with an inferior OS for HOX11L2 T-ALLs (13% vs 47% in HOX11L2-T-ALLs; P = .009). The majority of patients with HOX11 T-ALL underwent allografting, predominantly in second CR, but were not associated with a superior OS. Both TCR and genotypic stratification can therefore contribute to risk-adapted management of adult T-ALLs.     

Familial components of the multiple metabolic syndrome: the ARIC Study

Summary The association of a parental history of diabetes mellitus and hypertension with the multiple metabolic syndrome (MMS) was studied in a population survey of middle-aged adults. The eligible population was drawn from the baseline examination of the Atherosclerosis Risk in Communities Study, a population-based, bi-ethnic, multi-centre cohort study. The MMS was defined as a multivariate, categorical phenotype of co-occurring diabetes, hypertension, and dyslipidaemia. MMS cases (n = 356) were compared to disorder-free control subjects (n = 6797) with respect to their parental history of diabetes and hypertension. MMS cases were more likely to report a history of diabetes in both parents (odds ratio [OR] 4.7, 95 % confidence interval (CI) 1.5–14.7) or a history of hypertension in both parents (OR 1.9, 95 % CI 1.1–3.0) than control subjects, adjusting for BMI, waist-to-hip ratio, age, gender, and ethnicity/centre. A parental history of diabetes and hypertension in both parents was associated with the greatest increase in odds of MMS (OR 8.3, 95 % CI 3.0–22.8). A dose-response relationship between the number of parental disorders (one; two; three to four) and the odds of MMS was observed (OR 1.2, 95 % CI 0.9–1.7; OR 2.0, 95 % CI 1.4–2.8; OR 4.0, 95 % CI 2.5–6.2). Based on the marked associations observed between a parental history of MMS components and the clustering of these metabolic disorders in the offspring generation, we conclude that genetic and/or non-genetic familial influences play a role in the development of the multiple metabolic syndrome. [Diabetologia (1997) 40: 963–970] Received: 20 February 1997 and in revised form: 2 May 1997     

Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis

Unlike previously reported cases with total protein 4.2 deficiency due to mutations in the EPB42 gene, we describe a total deficiency in protein 4.2 with normal EPB42 alleles. Hereditary spherocytosis (HS) was observed in a Japanese woman (unsplenectomized) and her daughter (splenectomized). The mother showed a partial deficiency in band 3 and a proportional reduction in protein 4.2. She was heterozygous for a novel allele of the EPB3 gene, allele Okinawa, which contains the two mutations that define the Memphis II polymorphism (K56E, AAG → GAG, and P854L, CCG → CTG) and, additionally, the mutation: G714R, GGG → AGG, located in a highly conserved position of transmembrane segment 9. The latter change was responsible for HS. In trans to allele Okinawa, the daughter displayed allele Fukuoka: G130R, GGA → AGA, an allele known to alter the binding of protein 4.2 to band 3. The daughter presented with a more pronounced decrease of band 3, and lacked protein 4.2, resulting in aggravated haemolytic features. Although the father was not available for study, heterozygosity for allele Fukuoka has been documented in another individual who showed no clinical or haematological signs, and a normal content of band 3. We suggest that band 3 Okinawa binds virtually all the protein 4.2 in red cell precursors, band 3 Fukuoka being unable to do so, and that the impossibility of band 3 Okinawa incorporation into the membrane leads to degradation of the band 3 Okinawa protein 4.2 complex. In contrast, band 3 Fukuoka, free of bound protein 4.2, could then incorporate normally into the bilayer. Thus, protein 4.2 would not appear in the daughter's red cell membrane.     

The haemostatic plug in haemophilia A: a morphological study of haemostatic plug formation in bleeding time skin wounds of patients with severe haemophilia A

Haemostatic plug formation in four patients with severe haemophilia A (VIII:C less than 1%) was studied in skin biopsies taken at 3, 10 and 30 min and 2 h after a template bleeding time wound had been made. The primary haemostatic plug showed relatively minor changes, consisting of a delay in platelet degranulation and interdigitation. Some platelet aggregates not attached to vessels were encountered in the wound. Subsequently the primary haemostatic plug changed into a firm stable degranulated mass of interdigitated platelets. The major abnormality occurred during the fibrinous transformation. At 2 h many haemostatic plugs consisted of a thin peripheral layer of fibrin and platelet remnants around a central area containing red and white blood cells with a varying amount of plasma and only relatively few fibrin fibres. These observations suggest that fibrin formation in the periphery of the plug is less dependent of factor VIII than in central areas. The lack of fibrin formation in the centre of the plug compensating for the platelet lysis at 2 h may have caused the central erosion of the plug.     

Incidence and outcomes of catheter related thrombosis (CRT) in patients with acute leukemia using a platelet-adjusted low molecular weight heparin regimen

The purpose of this study is to evaluate the efficacy and safety of novel oral anticoagulant (NOAC) versus warfarin therapy in patients undergoing different operations. We performed a systematic review of MEDLINE, EMBASE, Cochrane Controlled Trials Register, and reports presented at scientific meetings. The efficacy and safety of NOACs during the perioperative period was compared to that using warfarin. Of the 2652 studies initially reviewed, we identified 9 that included 15,880 patients for the meta-analysis. Compared to warfarin, dabigatran increased the risk of major bleeding (RR 1.37, 95% CI 1.06–1.78, P?=?0.02). Apixaban (RR 0.63, 95% CI 0.40–0.99, P?=?0.04) reduced thrombotic events. NOAC therapy decreased thrombotic events in patients undergoing non-cardiac surgery (RR 0.68, 95% CI 0.50–0.92, P?=?0.02). Compared to warfarin, the administration of NOACs in the perioperative period has the same risk of thromboembolism and major bleeding. But patients undergoing non-cardiac surgery may benefit more from perioperative NOAC therapy. Apixaban may reduce thrombotic events and dabigatran increases the risk of major bleeding during the perioperative period.     

Results of a prospective phase 2 study combining imatinib mesylate and cytarabine for the treatment of Philadelphia-positive patients with chronic myelogenous leukemia in chronic phase

In chronic myelogenous leukemia (CML) imatinib mesylate has been shown to selectively inhibit the tyrosine kinase domain of the oncogenic bcr-abl fusion protein. Using this agent alone high rates of cytogenetic responses were recorded. However, several mechanisms of resistance have been described. In vitro studies examining the effects of imatinib mesylate plus cytarabine have shown synergistic antiproliferative effects of this combination. Thus, the CML French Group decided to perform a phase 2 trial testing a combination of imatinib mesylate and low-dose cytarabine in 30 previously untreated patients in chronic phase. Treatment was administered on 28-day cycles. Patients were treated continuously with imatinib mesylate orally at a dose of 400 mg daily. Cytarabine was given on days 15 to 28 of each cycle at an initial dose of 20 mg/m2/d via subcutaneous injection. Adverse events were frequently observed with grade 3 or 4 hematologic toxicities and nonhematologic toxicities in 53% (n = 16) and 23% (n = 7) of patients, respectively. The cumulative incidence of complete cytogenetic response (CCR) at 12 months was 83% and at 6 months 100% of the patients achieved complete hematologic response (CHR). We concluded that the combination was safe and promising given the rates of response.     

Functions of vasopressin and oxytocin in bone mass regulation

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr^−/− mice have osteopenia, and Avpr1α^−/− mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr^−/−:Avpr1α^−/− double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α^−/− cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.Over the past decade, we have described direct actions of anterior and posterior pituitary hormones on the skeleton (1–8). We have shown that these actions are exerted via G protein-coupled receptors resident on both osteoblasts and osteoclasts. We also find that the skeleton is highly sensitive to the action of posterior pituitary hormones; for example, mice haploinsufficient in oxytocin (Oxt) have osteopenic bones, but lactation is normal; lactation is impaired only in Oxt^−/− mice (2). Likewise, Tshr haploinsufficient mice are completely euthyroid with normal thyroid follicles but display significant osteopenia (4). The exquisite sensitivity of the skeleton to pituitary hormones comes as no surprise, considering that the pituitary gland and the skeleton are both evolutionarily more primitive than target endocrine organs (7).Apart from the known actions of growth hormone on the skeleton, Tsh, Fsh, Acth, Oxt, and vasopressin (Avp) have all been shown to regulate the formation and/or function of both osteoblasts and osteoclasts and thus to control bone remodeling in vivo (2–4, 6–8). The two neurohypophyseal hormones Oxt and Avp have opposing functions (2, 3). Oxt stimulates and Avp inhibits osteoblast formation. Consequently, the genetic deletion of the Oxt receptor (Oxtr) and Avp receptor 1α (Avpr1α) yields opposing phenotypes, notably osteopenia in Oxtr^−/− mice and high bone mass in Avpr1α^−/− mice (2, 3). These findings may explain the rapid recovery of bone loss at weaning when plasma Oxt levels are high (9) and also the profound loss of bone noted in chronic hyponatremic states, such as the syndrome of inappropriate antidiuretic hormone secretion (SIADH), in which serum Avp levels are elevated (3).We find high levels of Oxtr expression on both osteoclasts and osteoblasts (2, 10), in addition to their abundant expression in breast and uterine tissue, where they regulate lactation and parturition, respectively (11). Avpr1αs, in contrast, are distributed more ubiquitously, whereas Avpr2s are localized mainly in the kidney, where they regulate free water excretion (12). Osteoblasts express both Avpr1α and Avrpr2 (3). The only other known isoform, Avpr1β, is expressed predominantly in the pancreas and pituitary; it regulates ACTH secretion from pituitary corticotrophs (13). Sequence alignment shows that the binding sites of the Oxtr and Avprs are highly conserved, with specific amino acids within the predicted binding pocket providing ligand selectivity (14–16). The respective ligands Oxt and Avp also are homologous nonapeptides, differing in only two amino acids, and are known to interact with the other’s receptor with different affinities (17).To our knowledge, osteoblasts and osteoclasts are the only cells in which Oxtr, Avpr1α, and Avpr2 are coexpressed. We also have shown that osteoblastic Oxtrs undergo internalization and nuclear translocation upon binding to Oxt and that this action is independent of cytosolic Erk phosphorylation (18). Avpr1α activation by Avp also activates Erk phosphorylation within minutes (3). The homology between the ligands and their respective receptors and converging downstream signals suggest that Avp and Oxtr may share receptors with opposing or convergent signals. Here, we have explored these interactions in the regulation of osteoblastic bone formation by using mice lacking one or both receptors, chemical inhibitors, and physiological models of high bone turnover.