Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis.

Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237+/-13 versus 279+/-3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.

Transmitted drug resistant HIV-1 and association with virologic and CD4 cell count response to combination antiretroviral therapy in the EuroSIDA Study.

OBJECTIVES: To investigate prevalence of transmitted drug-resistant human immunodeficiency virus (TDR) and factors associated with TDR and to compare virological and CD4 count response to combination antiretroviral therapy. METHODS: In this study, 525 mostly chronically infected EuroSIDA patients were included who had genotypic resistance tests performed on plasma samples collected while antiretroviral therapy naive. TDR was defined as at least one resistance mutation from a list proposed for genotypic TDR surveillance. Multivariable logistic regression was used to analyze factors associated with detection of TDR, with virological (viral load<500 copies/mL) and CD4 count response (>or=50% increase) to combination antiretroviral therapy at months 6-12. RESULTS: The overall prevalence of TDR was 11.4%, which was stable over 1996-2004. There were no significant differences in virological suppression (those resistant to at least one drug prescribed versus susceptible), adjusted odds ratio: 0.68 (95% confidence interval: 0.27 to 1.71; P=0.408) or CD4 count response, adjusted odds ratio: 1.65 (95% confidence interval: 0.73 to 3.73; P=0.231). CONCLUSIONS: Prevalence of TDR in antiretroviral-naive patients was found to be in line with other European studies. No significant differences were found in virological and CD4 count response after initiation of first-line combination antiretroviral therapy between resistant and susceptible patients, possibly due to the small number of patients with resistance and consequently low power.

Characterization and detection of naturally occurring antibodies against IL-1 alpha and IL-1 beta in normal human plasma.

During the development and testing of a radioreceptor assay (RRA) for human IL-1, we have detected and identified the presence of auto-antibodies to IL-1 in normal human plasma (NHP). The RRA is based on the competition between human 125I-labeled rIL-1 alpha and standard or unknown quantities of IL-1 alpha or IL-1 beta for binding to a limited amounts of IL-1 receptor (IL-1R) isolated from the EL4 mouse thymoma cell line. NHP from 20 out of 100 unselected blood donors were found to completely inhibit the binding of 125I-labeled IL-1 alpha to its receptor, suggesting the presence in these NHP samples of either abnormal amounts of IL-1 or of a factor binding to the 125I-labeled IL-1 alpha. Special care was taken to ascertain that the inhibitory factors were antibodies and not soluble IL-1 receptor antagonist. When plasma samples with inhibiting activity were incubated with labeled IL-1 alpha and chromatographed on a Sephadex G200 column, they were found to contain 125I-labeled complexes with an apparent molecular weight of 150-200kD. The IL-1 binding factor could be eliminated from plasma by incubation with protein A-Sepharose, suggesting that it consisted in IgG antibodies directed against IL-1. Furthermore, the antibody nature of the inhibiting factor was confirmed by its binding to purified rIL-1 coupled to Sepharose. Screening of 200 NHP samples by incubation with 100 pg of 125I-labeled IL-1 followed by precipitation with 12% of polyethylene glycol (PEG) confirmed that about 25% of NHP contain detectable IgG antibodies to IL-1 alpha, while only 2% of NHP contain antibodies to IL-1 beta. No correlation between the presence of these anti-IL-1 antibodies and any particular major histocompatibility complex or any pathological conditions was detected. We suggest that all serum samples assayed for IL-1 alpha or IL-1 beta content should be pretested with the PEG precipitation assay described here.

Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A.

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.

Cloning of a cDNA encoding a plasma membrane-associated, uronide binding phosphoprotein with physical properties similar to viral movement proteins.

Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication.

Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity.

Malignant cells are frequently recognized and destroyed by T cells, hence the development of T cell vaccines against established tumors. The challenge is to induce protective type 1 immune responses, with efficient Th1 and CTL activation, and long-term immunological memory. These goals are similar as in many infectious diseases, where successful immune protection is ideally induced with live vaccines. However, large-scale development of live vaccines is prevented by their very limited availability and vector immunogenicity. Synthetic vaccines have multiple advantages. Each of their components (antigens, adjuvants, delivery systems) contributes specifically to induction and maintenance of T cell responses. Here we summarize current experience with vaccines based on proteins and peptide antigens, and discuss approaches for the molecular characterization of clonotypic T cell responses. With carefully designed step-by-step modifications of innovative vaccine formulations, T cell vaccination can be optimized towards the goal of inducing therapeutic immune responses in humans.

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC

The dexamethasone-induced inhibition of proliferation, migration, and invasion in glioma cell lines is antagonized by macrophage migration inhibitory factor (MIF) and can be enhanced by specific MIF inhibitors.

Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.

Radiothérapie externe accélérée postopératoire des carcinomes épidermoïdes localement évolués de la sphère ORL: étude prospective de phase II [Prospective study of accelerated postoperative radiation therapy in patients with squamous-cell carcinoma of the head and neck]

PURPOSE: To assess the feasibility and efficacy of accelerated postoperative radiation therapy (RT) in patients with squamous-cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: Between December 1997 and July 2001, 68 patients (male to female ratio: 52/16; median age: 60-years (range: 43-81) with pT1-pT4 and/or pN0-pN3 SCCHN (24 oropharynx, 19 oral cavity, 13 hypopharynx, 5 larynx, 3 unknown primary, 2 maxillary sinus, and 2 salivary gland) were included in this prospective study. Postoperative RT was indicated because extracapsular infiltration (ECI) was observed in 20 (29%), positive surgical margins (PSM) in 20 (29%) or both in 23 patients (34%). Treatment consisted of external beam RT 66 Gy in 5 weeks and 3 days. Median follow-up was 15 months. RESULTS: According to CTC 2.0, acute morbidity was acceptable: grade 3 mucositis was observed in 15 (22%) patients, grade 3 dysphagia in 19 (28%) patients, grade 3 skin erythema in 21 (31%) patients with a median weight loss of 3.1 kg (range: 0-16). No grade 4 toxicity was observed. Median time to relapse was 13 months; we observed only three (4%) local and four (6%) regional relapses, whereas eight (12%) patients developed distant metastases without any evidence of locoregional recurrence. The 2 years overall-, disease-free survival, and actuarial locoregional control rates were 85, 73 and 83% respectively. CONCLUSION: The reduction of the overall treatment time using postoperative accelerated RT with weekly concomitant boost (six fractions per week) is feasible with local control rates comparable to that of published data. Acute RT-related morbidity is acceptable.

Respective roles of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) in cell surface expression of CRLR/RAMP heterodimeric receptors.

Receptor activity modifying proteins RAMP1, RAMP2, and RAMP3 are responsible for defining affinity to ligands of the calcitonin receptor-like receptor (CRLR). It has also been proposed that receptor activity-modifying proteins (RAMP) are molecular chaperones required for CRLR transport to the cell surface. Here, we have studied the respective roles of CRLR and RAMP in transporting CRLR/RAMP heterodimers to the plasma membrane by using a highly specific binding assay that allows quantitative detection of cell surface-expressed CRLR or RAMP in the Xenopus oocytes expression system. We show that: (i) heterodimer assembly is not a prerequisite for efficient cell surface expression of CRLR, (ii) N-glycosylated RAMP2 and RAMP3 are expressed at the cell surface and their transport to the plasma membrane requires N-glycans, (iii) RAMP1 is not N-glycosylated and is transported to the plasma membrane only upon formation of heterodimers with CRLR, and (iv) introduction of N-glycosylation sites in the RAMP1 sequence (D58N/G60S, Y71N, and K103N/P105S) allows cell surface expression of these mutants at levels similar to that of wild-type RAMP1 co-expressed with CRLR. Our data argue against a chaperone function for RAMP and identify the role of N-glycosylation in targeting these molecules to the cell surface.

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness.

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ∼1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ∼1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.

Cis association of Ly49A with MHC class I restricts natural killer cell inhibition.

Natural killer (NK) cell function is negatively regulated by inhibitory receptors interacting with major histocompatibility complex class I molecules expressed on target cells. Here we show that the inhibitory Ly49A NK cell receptor not only binds to its H-2D(d) ligand expressed on potential target cells (in trans) but also is constitutively associated with H-2D(d) in cis (on the same cell). Cis association and trans interaction occur through the same binding site. Consequently, cis association restricts the number of Ly49A receptors available for binding of H-2D(d) on target cells and reduces NK cell inhibition through Ly49A. By lowering the threshold at which NK cell activation exceeds NK cell inhibition, cis interaction allows optimal discrimination of normal and abnormal host cells.

HLA-C cell surface expression and control of HIV/AIDS correlate with a variant upstream of HLA-C.

A variant 35 kb upstream of the HLA-C gene (-35C/T) was previously shown to associate with HLA-C mRNA expression level and steady-state plasma HIV RNA levels. We genotyped this variant in 1,698 patients of European ancestry with HIV. Individuals with known seroconversion dates were used for disease progression analysis and those with longitudinal viral load data were used for viral load analysis. We further tested cell surface expression of HLA-C in normal donors using an HLA-C-specific antibody. We show that the -35C allele is a proxy for high HLA-C cell surface expression, and that individuals with high-expressing HLA-C alleles progress more slowly to AIDS and control viremia significantly better than individuals with low HLA-C expressing alleles. These data strongly implicate high HLA-C expression levels in more effective control of HIV-1, potentially through better antigen presentation to cytotoxic T lymphocytes or recognition and killing of infected cells by natural killer cells.

In vivo imaging of T cell delivery to tumors after adoptive transfer therapy.

Adoptive transfer therapy of in vitro-expanded tumor-specific cytolytic T lymphocytes (CTLs) can mediate objective cancer regression in patients. Yet, technical limitations hamper precise monitoring of posttherapy T cell responses. Here we show in a mouse model that fused single photon emission computed tomography and x-ray computed tomography allows quantitative whole-body imaging of (111)In-oxine-labeled CTLs at tumor sites. Assessment of CTL localization is rapid, noninvasive, three-dimensional, and can be repeated for longitudinal analyses. We compared the effects of lymphodepletion before adoptive transfer on CTL recruitment and report that combined treatment increased intratumoral delivery of CTLs and improved antitumor efficacy. Because (111)In-oxine is a Food and Drug Administration-approved clinical agent, and human SPECT-CT systems are available, this approach should be clinically translatable, insofar as it may assess the efficacy of immunization procedures in individual patients and lead to development of more effective therapies.

Association of the hemochromatosis gene with pazopanib-induced transaminase elevation in renal cell carcinoma.

BACKGROUND & AIMS: Pazopanib has demonstrated clinical benefit in patients with advanced renal cell carcinoma (RCC) and is generally well tolerated. However, transaminase elevations have commonly been observed. This 2-stage study sought to identify genetic determinants of alanine transaminase (ALT) elevations in pazopanib-treated white patients with RCC.¦METHODS: Data from two separate clinical studies were used to examine the association of genetic polymorphisms with maximum on-treatment ALT levels.¦RESULTS: Of 6852 polymorphisms in 282 candidate genes examined in an exploratory dataset of 115 patients, 92 polymorphisms in 40 genes were significantly associated with ALT elevation (p<0.01). Two markers (rs2858996 and rs707889) in the HFE gene, which are not yet known to be associated with hemochromatosis, showed evidence for replication. Because of multiple comparisons, there was a 12% likelihood the replication occurred by chance. These two markers demonstrated strong linkage disequilibrium (r(2)=0.99). In the combined dataset, median (25-75th percentile) maximum ALT values were 1.2 (0.7-1.9), 1.1 (0.8-2.5), and 5.4 (1.9-7.6)×ULN for rs2858996 GG (n=148), GT (n=82), and TT (n=1 2) genotypes, respectively. All 12 TT patients had a maximum ALT>ULN, and 8 (67%) had ALT≥3×ULN. The odds ratio (95% CI) for ALT≥3×ULN for TT genotype was 39.7 (2.2-703.7) compared with other genotypes. As a predictor of ALT≥3×ULN, the TT genotype had a negative predictive value of 0.83 and positive predictive value of 0.67. No TT patients developed liver failure.¦CONCLUSIONS: The rs2858996/rs707889 polymorphisms in the HFE gene may be associated with reversible ALT elevation in pazo-panib-treated patients with RCC.