The link between lymphocyte deficiency and autoimmunity: roles of endogenous T and B lymphocytes in tolerance

We demonstrate that transfer of OVA-specific DO11 CD4(+) T cells into mice that lack T and B cells and produce secreted OVA as an endogenous self-protein results in a severe systemic autoimmune reaction with skin inflammation, wasting, and death. The transferred DO11 T cells undergo massive expansion and produce IL-2 and IFN-gamma abundantly. Transfer of DO11 cells into OVA-expressing animals in which T cells are absent but B cells are present, leads to mild disease with no death. In this situation, the DO11 cells undergo similar expansion but show poor Th1 differentiation. This regulatory effect of B cells correlates with profound TCR down-regulation. If T cells are present, the DO11 cells fail to expand independent of B cells. These results suggest that both endogenous T and B lymphocytes control T cell tolerance induction and pathogenicity, but at different stages of an anti-self response. Although endogenous T cells prevent expansion and maintain homeostasis, endogenous B cells limit subsequent effector responses of autoreactive CD4(+) T cells.     

Oct4 is required for primordial germ cell survival

Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline.     

Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.     

Crystal structure of HslUV complexed with a vinyl sulfone inhibitor: corroboration of a proposed mechanism of allosteric activation of HslV by HslU

On the basis of the structure of a HslUV complex, a mechanism of allosteric activation of the HslV protease, wherein binding of the HslU chaperone propagates a conformational change to the active site cleft of the protease, has been proposed. Here, the 3.1 A X-ray crystallographic structure of Haemophilus influenzae HslUV complexed with a vinyl sulfone inhibitor is described. The inhibitor, which reacts to form a covalent linkage to Thr1 of HslV, binds in an "antiparallel beta" manner, with hydrogen-bond interactions between the peptide backbone of the protease and that of the inhibitor, and with two leucinyl side chains of the inhibitor binding in the S1 and S3 specificity pockets of the protease. Comparison of the structure of the HslUV-inhibitor complex with that of HslV without inhibitor and in the absence of HslU reveals that backbone interactions would correctly position a substrate for cleavage in the HslUV complex, but not in the HslV protease alone, corroborating the proposed mechanism of allosteric activation. This activation mechanism differs from that of the eukaryotic proteasome, for which binding of activators opens a gated channel that controls access of substrates to the protease, but does not perturb the active site environment.     

Endocytosis of adeno-associated virus type 5 leads to accumulation of virus particles in the Golgi compartment

Among the adeno-associated virus (AAV) serotypes which are discussed as vectors for gene therapy AAV type 5 (AAV5) represents a candidate with unique advantages. To further our knowledge on AAV5-specific characteristics, we studied the entry pathway of wild-type virus in HeLa cells in the absence of helper virus by immunofluorescence and electron microscopy and by Western blot analysis. We found virus binding at the apical cell surface, especially at microvilli and, with increasing incubation time, virus accumulation at cell-cell boundaries. The different binding kinetics suggest different binding properties at apical versus lateral plasma membranes. Endocytosis of viruses was predominantly by clathrin-coated vesicles from both membrane domains; however, particles were also detected in noncoated pits. AAV5 particles were mainly routed to the Golgi area, where they could be detected within cisternae of the trans-Golgi network and within vesicles associated with cisternae and with the dictyosomal stacks of the Golgi apparatus. These data suggest that AAV5 makes use of endocytic routes that have hitherto not been described as pathways for virus entry.     

Chromosome analysis in 31 cases of benign and malignant breast tumors: a study in Brazil

Cultures of 31 breast tumors, being 20 carcinomas and 11 benign lesions, were cytogenetically analysed. Clonal chromosome aberrations were detected in 16 carcinomas and in 4 benign lesions. Nine carcinomas and 2 benign lesions had multiple cytogenetically unrelated and related clones, whereas a single abnormal clone was observed in 7 carcinomas and in 2 benign lesions. Polyploid clones were found in 7 carcinomas and in 2 benign lesions. The presence of clonal chromosome aberrations and polyploid cells was not associated with the clinicopathologic parameters tested. Carcinomas had more clonal changes than benign lesions (p = 0.031), showing that cytogenetic features are of diagnostic value and that different chromosome anomalies might have different pathogenetic and prognostic significance.     

Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 4: synthesis and structure-activity relationships for 1-[N-(methyl)-N-(phenylsulfonyl)amino]-2-(phenyl)-4-(4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidin-1-yl)butanes

(2S)-2-(3-Chlorophenyl)-1-[N-(methyl)-N-(phenylsulfonyl)amino]-4-[spiro(2,3-dihydrobenzthiophene-3,4'-piperidin-1'-yl)]butane S-oxide (1b) has been identified as a potent CCR5 antagonist having an IC50=10 nM. Herein, structure-activity relationship studies of non-spiro piperidines are described, which led to the discovery of 4-(N-(alkyl)-N-(benzyloxycarbonyl)amino)piperidine derivatives (3-5) as potent CCR5 antagonists.