Signalling by means of toll-like receptors (TLRs) is essential for the development of innate and adaptive immune responses. UNC93B1, essential for signalling of TLR3, TLR7 and TLR9 in both humans and mice, physically interacts with these TLRs in the endoplasmic reticulum (ER). Here we show that the function of the polytopic membrane protein UNC93B1 is to deliver the nucleotide-sensing receptors TLR7 and TLR9 from the ER to endolysosomes. In dendritic cells of 3d mice, which express an UNC93B1 missense mutant (H412R) incapable of TLR binding, neither TLR7 nor TLR9 exits the ER. Furthermore, the trafficking and signalling defects of the nucleotide-sensing TLRs in 3d dendritic cells are corrected by expression of wild-type UNC93B1. However, UNC93B1 is dispensable for ligand recognition and signal initiation by TLRs. To our knowledge, UNC93B1 is the first protein to be identified as a molecule specifically involved in trafficking of nucleotide-sensing TLRs. By inhibiting the interaction between UNC93B1 and TLRs it should be possible to achieve specific regulation of the nucleotide-sensing TLRs without compromising signalling via the cell-surface-disposed TLRs.

Skeletal muscle is the primary tissue responsible for insulin-dependent glucose uptake in vivo; therefore, glucose uptake by this tissue plays an important role in determining glycemia. Glucose uptake in muscle occurs by a system of facilitated diffusion involving at least two distinct glucose transporters, GLUT-1 and GLUT-4. Both bind the fungal metabolite and inhibitor of glucose transport cytochalasin B. In human skeletal muscle, both types of transporters are detected immunologically, and corresponding mRNA transcripts of both transporter forms are detected. In human skeletal muscle cells in culture, in which contamination by other tissues is ruled out, a 50,000-Mr polypeptide is photolabeled with cytochalasin B. In rat skeletal muscle, acute treatment with insulin in vivo increases glucose-transport activity and the number of specific cytochalasin B-binding sites at the plasma membrane. In mildly diabetic (streptozocin-induced) rats, the number of cytochalasin B-binding sites is decreased in total membranes, and preferentially in the plasma membrane. In response to acute insulin treatment, however, there is still recruitment of glucose transporters to the plasma membrane from an intracellular membrane store. Hence, migration of transporters does occur in this form of diabetes. In L6 muscle cells in culture, acute treatment (1 h) with insulin causes recruitment of glucose transporters to the plasma membrane, and prolonged exposure to insulin or to glucose-deprived medium causes increased expression of GLUT-1 mRNA and GLUT-1 protein. Prolonged exposure (24 h) to high glucose in the medium causes a decrease in the number of glucose transporters in the plasma membrane. Hence, in those cells the expression of the GLUT-1 glucose transporter is modulated by insulin.

Human neutrophils have been shown recently to express both the beta and the gamma chains of the interleukin-2 receptor (IL-2R). IL-15, a cytokine that has recently been cloned and characterized, was found to share many of the biological functions of IL-2 and is known to mediate signals through IL-2R beta and IL-2R gamma. In recent studies, we observed that IL-2 exerts few effects on various neutrophil functions, but information on IL-15-neutrophil interactions is lacking. In this study, we observed that IL-15, in contrast to IL-2, induces important morphological cell shape changes that are typical of activated neutrophils. Furthermore, phagocytosis of opsonized sheep red blood cells was significantly increased by IL-15 but not by IL-2. However, similar to IL-2, IL-15 did not modulate the oxidative burst response. Furthermore, we observed that de novo RNA synthesis is increased in neutrophils by IL-15 along with de novo protein synthesis, whereas no significant effect of IL-2 was noted. Among the different proteins that were found to be upregulated by IL-15, one was identified by microsequencing as the cytoskeletal protein actin. Finally, we found that IL-15 delays apoptosis of neutrophils more efficiently than IL-2 when evaluated by both microscopic observations and flow cytometry procedures. Furthermore, this phenomenon was dose-dependent (10 to 500 ng/mL), and, at 500 ng/mL, IL-15 delayed apoptosis as strongly as granulocyte-macrophage colony-stimulating factor. This study is the first to show that IL-15 is a significant neutrophil agonist. Moreover, in view of the differential effects of IL-15 and IL-2 on this cell type, our results support the existence of a specific IL-15R component(s) on human neutrophils.

Endoglin is predominantly expressed on endothelium and is mutated in hereditary hemorrhagic telangiectasia (HHT) type 1 (HHT1). We report the analysis of endoglin in tissues of a newborn (family 2), who died of a cerebral arteriovenous malformation (CAVM), and in a lung specimen surgically resected from a 78-year-old patient (family 5), with a pulmonary AVM (PAVM). The clinically affected father of the newborn revealed a novel mutation that was absent in his parents and was identified as a duplication of exons 3 to 8, by quantitative multiplex polymerase chain reaction. The corresponding mutant protein (116-kd monomer) and the missense mutant protein (80-kd monomer) present in family 5 were detected only as transient intracellular species and were unreactive by Western blot analysis and immunostaining. Normal endoglin (90-kd monomer) was reduced by 50% on peripheral blood-activated monocytes of the HHT1 patients. When analyzed by immunostaining and densitometry, presumed normal blood vessels of the newborn lung and brain and vessels adjacent to the adult PAVM showed a 50% reduction in the endoglin/PECAM-1 ratio. A similar ratio was observed in the CAVM and PAVM, suggesting that all blood vessels of HHT1 patients express reduced endoglin in situ and that AVMs are not attributed to a focal loss of endoglin.

The effect of continuous insulin stimulation on the rates of turnover and on the total cellular contents of the glucose-transporter proteins GLUT1 and GLUT4 in 3T3-L1 adipocytes was investigated. Pulse-and-chase studies with [35S]methionine followed by immunoprecipitation of GLUT1 and GLUT4 with isoform-specific antibodies revealed the half-lives of these proteins to be 19 h and 50 h respectively. Inclusion of 100 nM insulin in the chase medium resulted in a decrease in the half-lives of both proteins to about 15.5 h. This effect of insulin was specific for the glucose-transporter proteins, as the average half-life of all proteins was found to be 55 h both with and without insulin stimulation. The effect of insulin on the rate of synthesis of the glucose transporters was determined by the rate of incorporation of [35S]methionine. After 24 h of insulin treatment, the rate of synthesis of GLUT1 and GLUT4 were elevated over control levels by 3.5-fold and 2-fold respectively. After 72 h of treatment under the same conditions, the rate of synthesis of GLUT1 remained elevated by 2.5-fold, whereas the GLUT4 synthesis rate was not different from control levels. Western-blot analysis of total cellular membranes revealed a 4.5-fold increase in total cellular GLUT1 content and a 50% decrease in total cellular GLUT4 after 72 h of insulin treatment. These observations suggest that the rates of synthesis and degradation of GLUT1 and GLUT4 in 3T3-L1 adipocytes are regulated independently and that these cells respond to prolonged insulin treatment by altering the metabolism of GLUT1 and GLUT4 proteins in a specific manner.

SUMMARY Left ventricular (LV) size and function were studied by echocardiography in 145 normal children. The LV end-diastolic diameter (EDD) and its percentage change with systole (%ALVD) were measured and mean velocity of circumferential fiber shortening (Vcf) calculated. The LV pre-ejection period (PEP) and ejection time (LVET) were determined from recordings of aortic valve motion.The EDD increased by approximately threefold during childhood and was best correlated with the log of body weight (r = 0. the log of body surface area (r = 0.96). The mean %lALVD was 36 ± 4 (SD), and this index of LV function was independent of age and heart rate. Mean Vcf was higher, and the absolute values of PEP and LVET shorter, in younger children with a faster heart rate. The mean ratio of PEP/LVET was 0.31 ± 0.003, and was relatively independent of age (r = -0.41) and heart rate (r = 0.37). The %ZALVD and PEP/LVET appear to be particularly useful indices of LV function because they remain constant during the course of childhood.casionally a shallow left lateral decubitus position was required to record the ventricular septum clearly. Left ventricular dimensions were measured in the standard manner ( fig. 1). End-diastolic diameter (EDD) was measured at the start of the QRS complex. End-systolic diameter (ESD) was measured at the point in late systole where the septum and LV posterior wall were in closest apposition. These measurements were made with the transducer angled slightly inferiorly and laterally from the point of maximal excursion of the mitral valve in subjects over one year of age. In younger children, as previously noted by Sahn et al.,7 the mitral leaflets appear to extend farther toward the apex, and the LV diameter decreases rapidly as the transducer is directed below the mitral valve. Therefore, in infants under one year of age the LV dimensions were measured at the point of maximal excursion of the mitral valve, from a position from which both leaflets could be visualized. Recordings satisfactory for determination of LV dimensions were obtained in 143 of the 145 subjects (99%).Left ventricular systolic time intervals were determined from recordings of the aortic valve at 100 mm/sec paper speed ( fig. 2)

A single-center cohort study of kidney and kidneypancreas recipients was conducted to evaluate the association between new immunosuppressive regimens and risk of thrombotic microangiopathy (TMA). From January 1st,1996 to December 31, 2002, 368 patients received a kidney or kidney-pancreas transplant at our center. Four immunosuppressive regimens were evaluated as potential risk factors of TMA: cyclosporin + mycophenolate mofetil (CsA + MMF), cyclosporin + sirolimus (CsA + SRL), tacrolimus + myophenolate mofetil (FK + MMF), and tacrolimus + sirolimus (FK + SRL). Thirteen patients developed biopsy-proven TMA in the absence of vascular rejection. The incidence of TMA was significantly different in the four immunosuppressive regimens studied (p < 0.001). The incidence of TMA was highest in the CsA + SRL group (20.7%). The relative risk of TMA was 16.1 [95% confidence interval (CI): 4.3-60.8] for patients in the CsA + SRL group as compared with those in the FK + MMF group. We also investigated in vitro the pathophysiological basis of this association. The CsA-SRL combination was found to be the only regimen that concomitantly displayed pro-necrotic and anti-angiogenic activities on arterial endothelial cells. We propose that this combination concurs to development of TMA through dual activities on endothelial cell death and repair.

In this study, we examine the role of the putative cargo receptor B cell-associated protein (Bap)29/31 in the export of MHC class I molecules out of the endoplasmic reticulum (ER). We show that Bap31 binds to two allotypes of mouse class I molecules, with the interaction initiated at the time of H chain association with β2-microglobulin and maintained until the class I molecule has left the ER. We also show that Bap31 is part of the peptide-loading complex, although is not required for its formation. Bap31 binds not only to class I molecules, but can bind to tapasin in the absence of class I. Consistent with an important role in recruiting class I molecules to transport vesicles, we show that in the absence of Bap29/31, there is a loss of class I colocalization with mSec31 (p137), a component of mammalian coat protein complex II coats. This observation is also associated with a delay in class I traffic from ER to Golgi. Our results are consistent with the view that class I molecules are largely recruited to ER exit sites by Bap29/31, and that Bap29/31 is a cargo receptor for MHC class I molecules.