Structural model for T-cell recognition of HLA class II-associated alloepitopes.

In an effort to investigate the structure-function relationship of HLA class II molecules vis-à-vis alloepitope expression, cloned T-cell reagents were used to define polymorphic epitopes associated with DR and DQ molecules. DNA sequences of genes encoding allelic or isotypic DR or DQ molecules that appear to express the same T-cell-defined epitopes were compared in an attempt to identify association of shared sequences with shared epitopes. When sequence sharing is associated with shared epitope expression, we suggest that it is the shared sequence that encodes the epitope in question. Based on the hypothetical three-dimensional structure of the class II molecule, an approximation is made as to which parts of the HLA class II molecule are involved in alloepitope expression. T-cell clones were generated from cells primed against HLA-DR2 haplotypes representing the cellularly defined subgroups Dw2 or Dw21 (previously designated MN2, FJ0, or Tb24). Those clones determined to be DR- or DQ-directed based on monoclonal antibody inhibition assays were tested by panel cell analysis utilizing DR2-positive and DR2-negative target cells. The data support the concept that amino acids 67, 70, 71, and 74 for DR molecules and amino acids 57, 70, and 71 for DQ molecules, which appear to comprise one face of the alpha helix, are of primary importance in T-cell recognition. In other cases, sharing of both the second hypervariable region (amino acids 25-33) and the third hypervariable region (amino acids 67-74) appears necessary to explain epitope sharing for DR molecules. We emphasize that the involvement of these two hypervariable regions may indicate that alloepitope expression involves the complex of class II molecule plus peptide, with the second HVR primarily involved in determining which peptides are bound and the third in T-cell receptor (TcR) recognition and/or peptide binding; we do not rule out that conformational changes of the second HVR can induce conformational changes in the third HVR. Finally, shared alloepitopes detected by some clones could not be explained based on shared primary sequences.     

The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, we found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons, as the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics.     

创伤病人的手术与焦虑状态调查

采用状态——特质焦虑问卷及１０项躯体性焦虑测试题，对４０例外科创伤病人手术前后的焦虑状态调查显示：术前状态焦虑量表评分显著高于术后；术前躯体性焦虑评分显著高于术后；高特质焦虑评分亚组与低特质焦虑评分亚组术前状态焦虑评分无显著差异，但术后则前者显著高于后者；急诊手术者手术前后状态焦虑评分显著高于择期手术者。     

Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.     

Strain differences in mouse cellular responses to Mycobacterium lepraemurium and BCG subcutaneous infections. I. Analysis of cell surface phenotype in local granulomas.

C57BL/6, BALB/c and CBA mice were subcutaneously infected with either Mycobacterium lepraemurium (MLM) or BCG, and studied for bacillary growth, granuloma size of infected footpads and draining lymph nodes (DLN), and DLN cell surface phenotype. Whereas, BCG-infected mice controlled the infection and developed early and large granulomas, MLM-infected mice exhibited major strain variations in their resistance to the infection, as well as in the granuloma size and kinetics. C57BL/6 mice, highly resistant, displayed early and regressive granulomas; BALB/c mice showed lower resistance and early granulomas that grew continuously; CBA mice, highly susceptible, developed late, soft, phagocyte-rich granulomas. Important strain differences in lymph node lymphocyte subset distribution could be observed prior to any infection: C57BL/6 mice displayed higher B cell percentages than both of the other strains and BALB/c mice showed the highest CD4/CD8 ratios, followed by CBA and C57BL/6 mice. BCG and MLM infections both induced similar changes of these parameters in all three strains: that is a decrease of the B cell percentage and a decrease of the CD4/CD8 ratio, and the strain differences observed in uninfected mice persisted. On the other hand, DLN cells stimulated by the infecting bacillus and interleukin 2 also displayed an increase of the CD8 T cell percentage as compared with normal lymph node cells, but this phenomenon was much less pronounced in BALB/c mice, whether infected by MLM or BCG, and in MLM-infected CBA mice, than in BCG- or MLM-infected C57BL/6 (B6) mice. Thus the ability of C57BL/6 mice to generate an early and persistent CD8 T cell response to mycobacteria may contribute to their resistance to MLM.     

Study of blood group B antigen with a specific monoclonal antibody (anti-B, b-183).

A murine anti-B monoclonal antibody was obtained by the hybridoma technique. This antibody called anti-B (b-183) is of IgM nature; it is capable of agglutinating normal B, B3, Bx, cis AB and some acquired B red cells. Its association constant is 1.1 X 10(8) l/mol, and appears high compared to those of the monoclonal anti-A. This monoclonal anti-B was used to determine the number of B sites on B3 and Bx red cells.     

Diagnosis of arylsulfatase A deficiency in intact cultured cells using a fluorescent derivative of cerebroside sulfate

A fluorescent derivative of cerebroside sulfate (12-(1-pyrene)dodecanoyl-sphingosylgalactosyl-0-3-sulfate (P12-sulfatide) has been synthesized as a potential substrate for the determination of cerebroside sulfatidase (or arylsulfatase A) activity. It was administered into cultured human skin fibroblasts and thereby utilized for the diagnosis of arylsulfatase A deficiency. Cultured skin fibroblasts from normal individuals and healthy persons suffering from a pseudoarylsulfatase A deficiency (PD) degraded the P12-sulfatide, while in cells derived from a metachromatic leukodystrophy (MLD) patient it remained essentially intact. This contrasts with in vitro determinations of enzymatic activity, where the MLD or PD-derived arylsulfatase A exhibit similar deficiency, in spite of a profoundly different clinical course. Administration of the fluorescent sulfatide into the intact cells permitted a sensitive and rapid diagnosis of MLD and its distinction from the PD-phenomenon. This might be of particular importance for cases in which a rapid diagnosis is required and for prenatal diagnosis of fetuses from families afflicted with both MLD and pseudo-deficiency mutant genes.     

Extensive study of DRB, DQA, and DQB gene polymorphism in 23 DR2-positive, insulin-dependent diabetes mellitus patients.

To gain insight into the HLA subregions involved in protection against insulin-dependent diabetes mellitus (IDDM) we investigated the polymorphism of HLA-DR and -DQ genes in 23 DR2 IDDM patients. Results show the following. (1) Fourteen patients (61%) possess the DRB1, DRB5, and DQB1 alleles found in DRw16/DQw5 healthy people. These data contrast with the 5% of DRw16 normally found in DR2 populations and are in agreement with former observations supporting that the DRw16 haplotype is not protective. (2) Nine DR2 patients, i.e., 39% versus 95% in published DR2 controls, possess the DRB alleles found in DRw15 unaffected people. Among them, six patients have also DQA1 and DQB1 alleles identical to those found in DRw15/DQw6 healthy individuals. These data confirm that the DRw15/DQw6 haplotype is protective but indicate that none of the DR or DQ alleles, alone or in association, confers an absolute protection. (3) Our most striking results concern the very high frequency of recombinant haplotypes among the DRw15 patients: 3 of 9. In these three patients recombinations led to the elimination of both DQB1 and DQA1 alleles usually associated with DRw15. This strongly suggests that the occurrence of IDDM in these DRw15 patients is due to the absence of the usual DQ product and thus reinforces the assumption that DQ rather than DR region is involved in the protection conferred by the DRw15/DQw6 haplotype. Finally, analysis of the non-DRw15 haplotypes in heterozygous patients showed that IDDM can occur in the absence of any DQ alpha beta heterodimer of susceptibility.     

Detection of N-(3-oxohexanoyl)-L-homoserine lactone in mice infected with Yersinia enterocolitica serotype O8

Yersinia enterocolitica synthesizes N-acyl-L-homoserine lactone (AHL) signal molecules via the LuxR-LuxI homologues YenR-YenI. In this study we checked two prototypes of mouse-virulent Y. enterocolitica serotype O8 strains WA-314 and 8081 for AHL production in vitro and in vivo (mouse infection model). We used thin-layer chromatography in combination with the Escherichia coli AHL biosensor to identify the AHL species produced. We detected only OHHL [N-(3-oxohexanoyl)-L-homoserine lactone] and not HHL (N-hexanoyl-L-homoserine lactone) produced by Y. enterocolitica O8 in culture supernatant or infected mouse tissue. This is the first report demonstrating AHL production by yersiniae during infection.