Identification of a novel immunogenic HLA-A*0201-binding epitope of HER-2/neu with potent antitumor properties

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with aggressive disease. Immunogenic HER-2/neu CTL epitopes have been used as vaccines for the treatment of HER-2/neu positive malignancies with limited success. By applying prediction algorithms for MHC class I ligands and proteosomal cleavages, in this study, we describe the identification of HER-2/neu decamer LIAHNQVRQV spanning residues 85-94 (HER-2(10(85))). HER-2(10(85)) proved to bind with high affinity to HLA-A2.1 and was stable for 4 h in an off-kinetics assay. This peptide was immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded to tumor cell lines of various origin coexpressing human HER-2/neu and HLA-A2.1. This demonstrates that HER-2(10(85)) is naturally processed from endogenous HER-2/neu. Five of sixteen HER-2/neu+ HLA-A2.1+ breast cancer patients analyzed had HER-2(10(85))-reactive T cells ranging from 0.35-0.70% of CD8+ T cells. Depletion of T regulatory cells from PBMC enabled the rapid expansion of HLA-A2.1/HER-2(10(85))pentamer+/CD8+ cells (PENT+/CD8+), whereas significantly lower numbers of CTL could be generated from unfractionated PBMC. HER-2(10(85))-specific human CTL recognized the HER-2/neu+ HLA-A2.1+ tumor cell line SKBR3.A2, as determined by IFN-gamma intracellular staining and in the high sensitivity CD107alpha degranulation assay. Finally, HER-2(10(85)) significantly prolonged the survival of HHD mice inoculated with the transplantable ALC.A2.1.HER tumor both in prophylactic and therapeutic settings. These data demonstrate that HER-2(10(85)) is an immunogenic peptide, capable of eliciting CD8-mediated responses in vitro and in vivo, providing the platform for further exploitation of HER-2(10(85)) as a possible target for anticancer immunotherapy.     

EBAG9 adds a new layer of control on large dense-core vesicle exocytosis via interaction with Snapin

Regulated exocytosis is subject to several modulatory steps that include phosphorylation events and transient protein-protein interactions. The estrogen receptor-binding fragment-associated gene9 (EBAG9) gene product was recently identified as a modulator of tumor-associated O-linked glycan expression in nonneuronal cells; however, this molecule is expressed physiologically in essentially all mammalian tissues. Particular interest has developed toward this molecule because in some human tumor entities high expression levels correlated with clinical prognosis. To gain insight into the cellular function of EBAG9, we scored for interaction partners by using the yeast two-hybrid system. Here, we demonstrate that EBAG9 interacts with Snapin, which is likely to be a modulator of Synaptotagmin-associated regulated exocytosis. Strengthening of this interaction inhibited regulated secretion of neuropeptide Y from PC12 cells, whereas evoked neurotransmitter release from hippocampal neurons remained unaltered. Mechanistically, EBAG9 decreased phosphorylation of Snapin; subsequently, association of Snapin with synaptosome-associated protein of 25 kDa (SNAP25) and SNAP23 was diminished. We suggest that the occurrence of SNAP23, Snapin, and EBAG9 also in nonneuronal cells might extend the modulatory role of EBAG9 to a broad range of secretory cells. The conjunction between EBAG9 and Snapin adds an additional layer of control on exocytosis processes; in addition, mechanistic evidence is provided that inhibition of phosphorylation has a regulatory function in exocytosis.     

Discovery of (2S)-N-[(1R)-2-[4-cyclohexyl-4-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperidinyl]-1-[(4-fluorophenyl)methyl]-2-oxoethyl]-4-methyl-2-piperazinecarboxamide (MB243), a potent and selective melanocortin subtype-4 receptor agonist

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. Further in vivo development of lead agonist, MB243, is disclosed.     

Autoantibodies to fibrillin-1 activate normal human fibroblasts in culture through the TGF-beta pathway to recapitulate the "scleroderma phenotype"

Fibroblasts from patients with systemic sclerosis (SSc) are activated producing excessive amounts of extracellular matrix (ECM) components. Recently, we identified a new SSc-specific autoantibody against portions of fibrillin-1, a major component of ECM microfibrils and regulator of TGF-beta1 signaling. To examine a potential pathogenic role of anti-fibrillin-1 autoantibodies, normal human fibroblasts were treated with affinity-purified autoantibodies isolated from SSc sera and then examined for alterations in gene and protein expression levels using microarrays, quantitative RT-PCR, immunoblots, and immunofluorescence. Compared with fibroblasts cultured in normal medium or in medium containing normal human IgG, anti-fibrillin-1 autoantibody-treated normal dermal fibroblasts showed increased expression of COL and several other ECM components characteristically overexpressed in SSc fibroblasts. This was accompanied by phosphorylation and nuclear translocation of Smad3. Neutralization of TGF-beta1 with anti-TGF-beta1 Abs significantly diminished the activation of fibroblasts by anti-fibrillin-1 autoantibodies. These data indicate that anti-fibrillin-1 autoantibodies can induce the activation of normal dermal fibroblasts into a profibrotic phenotype resembling that of SSc by potentially causing the release of sequestered TGF-beta1 from fibrillin-1-containing microfibrils in the ECM.     

Substantial Genetic Effects Involved in Determination of Circulating Levels of Calciotropic Hormones in Human Pedigrees

This paper reports the results of a series of univariate and bivariate statistical genetic analyses that were performed on a sample of nuclear and more complex pedigrees (N = 567 individuals) of an ethnically homogenous White population. Our major objectives were: (1) To quantitatively evaluate the extent and pattern of the putative genetic effects on plasma level variation and covariation of the intact parathyroid hormone (PTH) and 25-hydroxyvitamin D [25(OH)D]; (2) To evaluate the extent of the possible genetic covariation between each of the two calciotropic hormones and two important bone mass traits, namely radiographic hands bone mineral density (BMD) and cortical index (CI). Variance component analysis, as implemented in the statistical package FISHER unambigously demonstrated that in addition to age, genetic factors contribute significantly to interindividual variation of both calciotropic hormones (37.5% for PTH and 53.3% for 25(OH)D). Complex segregation analysis strongly suggested the involvement of major gene effects into the determination of 25(OH)D levels, but was not clear cut with respect to PTH. Significant correlations between circulating levels of study hormones were found (-0.146, P < 0.05 in men and -0.194, P < 0.01 in women). However, no genetic correlation was revealed between PTH and 25(OH)D plasma concentrations. Bivariate analyses showed that familial cross correlations between PTH and BMD and CI measured at the bones of the hand were consistently statistically significant, suggesting moderate, but detectable pleiotropic genetic effects. The corresponding genetic correlations were -0.461 +/- 0.153 and -0.223 +/- 0.113, respectively. Circulating levels of 25(OH)D showed neither phenotypic nor genetic correlation with any of the BMD or CI variation.     

Chloroplast biogenesis 88. Protochlorophyllide b occurs in green but not in etiolated plants

It has recently been reported that protochlorophyllide (Pchlide) b is an abundant pigment in barley etioplasts but is rather unstable, as it is rapidly converted to Pchlide a by 7-formyl reductase during pigment extraction with conventional 80% acetone (Reinbothe, S., Pollmann, S., and Reinbothe, C. (2003) J. Biol. Chem. 278, 800-806). It has also been claimed that extraction of barley etioplasts with 100% acetone containing 0.1% diethyl pyrocarbonate prevents the conversion of Pchlide b to Pchlide a and leads to the detection of large amounts of Pchlide b in the isolated etioplasts. In this work the extraction protocol of Reinbothe et al. is compared with the more conventional 80% aqueous acetone extraction method. No Pchlide b was detected either in etiolated barley leaves or isolated barley etioplasts irrespective of the extraction protocol. On the other hands, small amounts of Pchlide b were detected in green barley leaves and isolated chloroplasts, extracted either with 80% acetone or 100% acetone containing 0.1% diethyl pyrocarbonate. It is concluded that the proposed occurrence of a light-harvesting POR-Pchlide-a,b complex in etiolated plant tissues is untenable, and its ensuing consequences and implications, for the greening process, are irrelevant.     

Discovery of thiophene-2-carboxylic acids as potent inhibitors of HCV NS5B polymerase and HCV subgenomic RNA replication. Part 1: Sulfonamides

The discovery of a novel class of HCV NS5B polymerase inhibitors, 3-arylsulfonylamino-5-phenyl-thiophene-2-carboxylic acids is described. SAR studies have yielded several potent inhibitors of HCV polymerase as well as of HCV subgenomic RNA replication in Huh-7 cells.     

Unstable angina is accompanied by immune cells dysfunction

Inflammatory process has been found to play an important role in the pathogenesis of coronary heart disease (CHD) and in the prognosis of coronary artery disease (CAD) patients. The purpose of our study was to investigate some cellular immune parameters during the development of angina in the stable and the unstable stage. We have investigated the proliferative capacity of lymphocytes isolated from the peripheral blood of stable and unstable angina patients. The proliferative capacity of peripheral lymphocytes was evaluated with the radioisotopic method of tritiated thymidine incorporation. The peripheral lymphocytes present an enhanced basal proliferation of cells and lectine induced stimulation (P = 0.02/ P = 0.001), especially in the unstable angina patients, correlated with an increased population of CD4+ peripheral T-lymphocytes (P = 0.0006). The cellular immune parameters announce the development of an acute coronary syndrome. The unstable angina presents alteration of some cellular immune parameters that indicate an inflammatory syndrome associated with an increased risk of CHD, having also a prediction value for the plaque instability.