DR3 (Ws1, Apo3, LARD, TRAMP, TNFSFR12) is a member of the death domain-containing tumor necrosis factor receptor (TNFR) superfamily, members of which mediate a variety of developmental events including the regulation of cell proliferation, differentiation, and apoptosis. We have investigated the in vivo role(s) of DR3 by generating mice congenitally deficient in the expression of the DR3 gene. We show that negative selection and anti-CD3-induced apoptosis are significantly impaired in DR3-null mice. In contrast, both superantigeninduced negative selection and positive selection are normal. The pre-T-cell receptor-mediated checkpoint, which is dependent on TNFR signaling, is also unaffected in DR3-deficient mice. These data reveal a nonredundant in vivo role for this TNF receptor family member in the removal of self-reactive T cells in the thymus.The tumor necrosis factor receptor (TNFR) superfamily comprise a growing family of type I membrane bound glycoproteins which interact with the TNF family of soluble mediators and type II transmembrane proteins. At least 23 TNFR superfamily members and 17 known ligands have been identified in mammals (reviewed in references 3, 35, and 44). These receptors trigger pleiotropic responses, ranging from apoptosis and differentiation to proliferation, and have been implicated in immune regulation, host defense and lymphoid organ development.Members of the TNFR family are characterized by the presence of varying numbers (three to six) of cysteine-rich repeats in their cytoplasmic domains (52). TNFRs are subdivided based on the presence or absence of a 70-to 80-amino-acid region of homology in the cytoplasmic region called the death domain, through which these receptors trigger apoptosis (20,48). DR3 (also called Ws1, Apo3, TRAMP, LARD, TR3, and TNFRSF12) is one of six death domain-containing TNFR family members (the others are TNFR1, CD95/FAS, DR4, DR5, and DR6) and is the one most closely related to TNFR1. Studies on the TNFR1 crystal structure suggest that ligand binding or receptor overexpression results in receptor trimerization and recruitment of trimeric intracellular signaling molecules (4, 36). DR3, like TNFR1, recruits TNFR1-associated death domain protein (TRADD) and Fas-associated death domain-containing protein (FADD) (5,6,11,12,24) as downstream effectors of apoptosis. These, in turn, interact with caspase 8 (FLICE/MACH) (7, 31), and a cascade of interleukin-1␤-converting enzyme-like cysteine proteases which trigger cell death (13,17,27). DR3 also recruits TRAF2 via TRADD (18,24,29,39) and thus activates the transcription factor, NF-B, that induces the transcription of a number of immune genes (19). In this respect, DR3 (like TNFR1) is capable of inducing both apoptosis and expression of survival/activation genes and is likely to have multiple functions depending on the context of its expression.DR3 was first reported as the only death domain-containing TNFR family member with lymphoid organ-restricted expression (11,24). More recent studies have, however, shown DR3...

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Our previous studies have indicated that the IgG-binding M-family proteins (IgGBP) of group A streptococci may be involved in eliciting experimental acute poststreptococcal glomerulonephritis (APSGN) in the rabbit. These surface proteins were also found to trigger production of anti-IgG, which might conceivably act to enhance renal deposition of immune complexes (IC). In the present study, a clinical isolate of serotype M22 (strain AL168), an isogenic double mutant deficient for both the IgGBPs Mrp and Emm, as well as mutants deficient in only one of the proteins were tested for capacity to induce glomerulonephritis. Streptococci to be used for injecting rabbits were heat-killed. Surface-bound IgG was removed by 1 M KSCN and cells were then repeatedly washed in PBS before use. Rabbits were injected intravenously with 109 cells three times a week for 8 weeks and, following one month of rest, for another 6 weeks. Deposits of IgG and C3 as well as induced chemokines TNF-alpha, IL-1beta and IL-6 were traced in cryostat sections using specific antibodies and appropriate peroxidase-labelled anti-antibodies. In four rabbits immunized with the double mutant strain, no deposits were found, and as examined by TEM, only subtle and transient renal changes were observed. In contrast, the original strain AL168 induced pronounced inflammatory and degenerative glomerular changes in all four rabbits injected, and deposits of TNF-alpha, IL-1beta and IL-6 were found in mesangial and endothelial cells. Similar deposits and glomerular changes were seen in all eight rabbits injected with the mrp-emm+ mutant and in four out of seven animals receiving the mrp+emm- mutant. There was a highly significant correlation between high levels of circulating anti-IgG and development of APSGN. These results confirm an important role of streptococcal IgGBP in triggering experimental APSGN as earlier proposed by our group.

Group A streptococcal M proteins are type-specific virulence factors that inhibit phagocytosis. We used two M proteins, M5 and Emm22, to analyze the influence of genetic background on the properties of M proteins. Mutant strains, engineered to lack these M proteins, were complemented with genes encoding the homologous or heterologous M protein, and the complemented strains were analyzed for phagocytosis resistance. Neither the M5 nor the Emm22 protein conferred phagocytosis resistance in the heterologous background, but they did do so in the homologous background. This was not due to lack of surface expression in the heterologous background. Moreover, the M5 and Emm22 proteins expressed in heterologous background appeared to have normal structure, since they were not affected in their ability to bind different human plasma proteins. In particular, M5 or Emm22 had normal ability to bind human complement inhibitors, a property that has been implicated in phagocytosis resistance. Results similar to those obtained with M5 and Emm22 were obtained in experiments with the M6 and Emm4 proteins. Together, these data suggest that the surface expression of M protein alone may not be sufficient to confer phagocytosis resistance and consequently that strain-specific factors other than M and Emm proteins may contribute to the ability of group A streptococci to resist phagocytosis.Group A streptococcus (Streptococcus pyogenes) is the cause of tonsillitis and impetigo and also causes severe diseases, including necrotizing fasciitis and the streptococcal toxic shock syndrome (2). In addition, group A streptococcal infections are sometimes complicated by one of the postinfectious sequelae, rheumatic fever and glomerulonephritis.The M proteins of group A streptococci are major virulence factors that confer resistance to phagocytosis (14). The Nterminal sequences of the M proteins are highly variable, giving rise to the existing ϳ100 serotypes. Protection against phagocytosis is usually serotype specific. It should be noted that a large fraction of all group A streptococcal strains encode three proteins with structural features typical for M proteins (12, 18). These proteins, designated Mrp, Emm, and Enn, are encoded by adjacent genes on the chromosome and are regulated by a common positive regulatory gene element now designated mga (7,20). At least two of the gene products (Mrp and Emm) from a single strain can contribute to resistance against phagocytosis, although Emm appears to be more important (22, 32). Strains with three genes encoding M-like proteins differ from those with a single gene in that they usually express opacity factor (OF), a lipoproteinase that is both surface bound and secreted (25,26). Recent evidence indicates that OF contributes to group A streptococcal virulence (4).Streptococcal mutants lacking M protein(s) are readily phagocytosed. However, the antiphagocytic property can be restored by complementation with the homologous M protein (19). Moreover, there is evidence that introduction of DNA encoding heter...

Antibody-secreting cell (ASC) and antibodies in lymphocyte supernatant (ALS) assays are used to assess intestinal mucosal responses to enteric infections and vaccines. The ALS assay, performed on cell supernatants, may represent a convenient alternative to the more established ASC assay. The two methods, measuring immunoglobulin A to Salmonella enterica serovar Typhi lipopolysaccharide, were compared in volunteers vaccinated with a live-attenuated typhoid vaccine M01ZH09. The specificity of the ALS assay compared to the ASC assay was excellent (100%), as was sensitivity (82%). The ALS assay was less sensitive than the ASC assay at <42 spots/10 6 peripheral blood lymphocytes.