Salmonella secreted factor L deubiquitinase of Salmonella typhimurium inhibits NF-kappaB, suppresses IkappaBalpha ubiquitination and modulates innate immune responses

Salmonella enterica translocates virulent factors into host cells using type III secretion systems to promote host colonization, intracellular bacterial replication and survival, and disease pathogenesis. Among many effectors, the type III secretion system encoded in Salmonella pathogenicity island 2 translocates a Salmonella-specific protein, designated Salmonella secreted factor L (SseL), a putative virulence factor possessing deubiquitinase activity. In this study, we attempt to elucidate the mechanism and the function of SseL in vitro, in primary host macrophages and in vivo in infected mice. Expression of SseL in mammalian cells suppresses NF-kappaB activation downstream of IkappaBalpha kinases and impairs IkappaBalpha ubiquitination and degradation, but not IkappaBalpha phosphorylation. Disruption of the gene encoding SseL in S. enterica serovar typhimurium increases IkappaBalpha degradation and ubiquitination, as well as NF-kappaB activation in infected macrophages, compared with wild-type bacteria. Mice infected with SseL-deficient bacteria mount stronger inflammatory responses, associated with increased production of NF-kappaB-dependent cytokines. Thus, SseL represents one of the first bacterial deubiquitinases demonstrated to modulate the host inflammatory response in vivo.     

The plasma concentration of advanced oxidation protein products and arterial stiffness in apparently healthy adults

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of atherosclerosis. Advanced oxidation protein products (AOPP) are markers of oxidative stress and mediators of inflammation. Increased arterial stiffness is associated with increased risk of cardiovascular mortality and morbidity. The aim of this study was to evaluate the relationship between an indirect marker of arterial stiffness and the AOPP level in apparently healthy individuals. METHODS AND RESULTS: Arterial stiffness was estimated with the use of the stiffness index (SI(DVP)) which significantly correlated with age, mean blood pressure, body fat content and AOPP. The SI(DVP) was associated with AOPP concentration in both single (R = 0.22, p = 0.03) and multiple regression models adjusted for age, sex, mean blood pressure and body fat content (R(2) = 42%, p < 0.0001). CONCLUSIONS: The AOPP concentration is elevated in healthy people with increased values of stiffness index. This finding supports the concept that oxidative stress may contribute to arterial stiffening in humans.     

An hour after immunization peritoneal B-1 cells are activated to migrate to lymphoid organs where within 1 day they produce IgM antibodies that initiate elicitation of contact sensitivity

Elicitation of contact sensitivity (CS), a classic example of T cell-mediated immunity, requires Ag-specific IgM Abs to trigger an initiation process. This early process leads to local recruitment of CS-effector T cells after secondary Ag challenge. These Abs are produced by the B-1 subset of B cells within 1 day after primary skin immunization. In this study we report the surprising observation that B-1 cells in the peritoneal cavity are activated as early as 1 h after naive mice are painted with a contact-sensitizing Ag on the skin of the trunk and feet to begin the initiation of CS. B-1 cells in the spleen and draining lymph nodes produce the initiating Abs by 1 day after immunization, when we found increased numbers of Ag-specific IgM Ab-producing cells in these tissues by ELISPOT assay. Importantly, we show that contact-activated peritoneal B-1 cells migrate to these lymphoid tissues and then differentiate into Ag-specific IgM Ab-producing cells, resulting in specific CS-initiating IgM Abs in the serum by 1 day. Furthermore, pertussis toxin, which is known to inhibit signaling via G protein-coupled chemokines, inhibited the migration of contact-activated peritoneal B-1 cells to the lymphoid tissues, probably due to BLR-1 (Burkitt lymphoma receptor-1). These findings indicate that within 1 h after contact skin immunization, B-1 cells in the peritoneal cavity are activated to migrate to the lymphoid tissues by chemokine-dependent mechanisms to produce serum Ag-specific IgM Abs within 1 day after immunization, leading to local recruitment of CS-effector T cells.     

tRNAPhe cleavage by aminoglycosides is triggered off by formation of an abasic site

This communication reports the characteristics of the mechanism of highly specific tRNA(Phe) cleavage, which occurs in the anticodon loop in the presence of aminoglycoside antibiotic-neomycin B. The data prove that the cleavage requires previous depurination of the polynucleotide chain at position 37, which is occupied by a hypermodified guanine base-wybutine. The results suggest that the phenomenon, previously considered as selective with respect to the presence of tRNA hypermodification, may concern far more RNA molecules, namely the ones carrying abasic sites.     

Experimental inflammatory bowel disease--role of T cells.

BACKGROUND: Our experiments were aimed to test: 1. which lymphocyte subpopulations participate in mouse colitis, produced by intrarectal (i.r.) deposition of trinitrobenzene sulphonic acid (TNBSA, TNP hapten); 2. the expression of cell adhesion molecules on lymphocytes draining the site of reaction; 3. the influence of mouse haplotype on the development of colitis. METHODS: CBA/J, BALB/c and C57BI/6 inbred and outbred Swiss Webster strains were used. Mesentheric lymph node (MLN) cells of immunized animals, unseparated or separated into CD4+, CD8+ or gammadelta+ and alphabeta+ T cell subpopulations or depleted of B lymphocytes, were transferred into recipients which were challenged i.r. with TNBSA. Inflammatory reaction in the colon was confirmed macro- and microscopically and by myeloperoxidase (MPO) level. MLN lymphocyte surface markers were tested cytofluorimetrically using appropriate antibodies. RESULTS: Sensitization with TNP results in chronic colitis (hapten dose-dependent colon weight gain and cellular infiltrate, significant increase of MPO level) only in CBA/J and BALB/c strains and can be adoptively transferred in a cell-dose dependent manner into syngeneic recipients by T alphabeta+ cells of both CD4+ and CD8+ subpopulations. T gammadelta+ cells were ineffective and B lymphocytes do not participate in the passive transfer reaction. In MLN the number of T lymphocytes positive for cell adhesion molecules particularly LPAM-1 (V-CAM1) and LPAM-2 increases significantly. CONCLUSIONS: Both CD4+ and CD8+ lymphocytes participate in the development of TNP-induced colitis. High MPO level may suggests that both Th1 and Th2 cells are involved. Colitis is accompanied by a significant accumulation in MLN of T lymphocytes positive for several cell surface adhesion molecules characteristic for memory T cells. Significant differences in susceptibility to develop colitis were found between different strains of mice.     

Invariant NKT cells rapidly activated via immunization with diverse contact antigens collaborate in vitro with B-1 cells to initiate contact sensitivity

In cutaneous contact sensitivity there is an early elicited innate cascade of complement, mast cells, and platelets activated via IgM Abs. This response is required to initiate the elicitation of acquired classical contact sensitivity by leading to local recruitment of effector T cells. We recently performed in vivo experiments showing that collaboration is required between innate-like invariant Valpha14+ NKT cells (iNKT) and the innate-like B-1 B cell subset to induce this initiation process. Contact sensitization triggers iNKT cells to produce IL-4 to coactivate the B-1 cells along with specific Ag for production of the initiating IgM Abs. We now describe in vitro collaboration of iNKT and B-1 cells. Normal peritoneal B-1 cells, incubated in vitro with soluble Ag, and with 1-h in vivo immune iNKT cells producing IL-4, are activated to mediate the contact sensitivity-initiation cascade. The three components of this process can be activated by different Ag. Thus, 1-h iNKT cell activation, B-1 cell stimulation, and generation of immune effector T cells can be induced by sensitization with three different Ag to respectively generate IL-4 and Ag-specific IgM Abs, to recruit the Ag-specific effector T cells. These findings have relevance to allergic and autoimmune diseases in which infections can trigger exacerbation of T cell responses to allergens or to autoantigens.     

The investigation of Staphylococcus aureus and coagulase-negative staphylococci nasal carriage among patients undergoing haemodialysis

The frequency of nasal staphylococcal colonization among haemodialysed patients was investigated. The swabs were collected in 1998 and 2004 from 28 and 43 patients, respectively.

Staphylococcus aureus colonization rates were 57.1% and 27.9% in 1998 and 2004, respectively. Twenty-six coagulase-negative staphylococci (CNS) isolates were cultured: S. epidermidis (21), S. lugdunensis (2), single S. haemolyticus, S. warneri, and S. capitits isolates. One S. aureus and 10 CNS isolates were methicillin resistant. The methicillin-resistant S. aureus (MRSA) was resistant to β-lactams, tetracycline, and harbored the pvl gene encoding the Panton-Valentine leukocidin.

The decrease in S. aureus colonization at 6-year interval was observed. The presence of the pvl gene and a favorable antibiotic susceptibility pattern of the MRSA suggest that the isolate was a member of community-acquired MRSA (CA-MRSA). Concluding, screening of haemodialysed patients for staphylococcal colonization accompanied by characterization of cultured isolates is important to understand its epidemiology and to develop infection prevention measures and treatment strategies.     

Physiological Characterization of Polar Tn5-Induced Isoleucine-Valine Auxotrophs in ESCHERICHIA COLI K-12: Evidence for an Internal Promoter in the ilvOGEDA OPERON

The properties of 22 isoleucine-valine auxotrophs induced in Escherichia coli K-12 by the transposable element, Tn5, were characterized on the basis of growth requirements, cross-feeding behavior, and enzyme activity. Mutants defective in ilvA, ilvC, ilvD and ilvE were found. Mutation in ilvE were not completely polar on ilvD and ilvA enzyme activities (that is, ilvE mutants possessed a low constitutive level of expression of the enzymes coded by ilvD and ilvA), while mutations in ilvD were completely polar on ilvA enzyme activity. The data suggest that there is an internal promoter between the sites of Tn5 insertion in ilvE and ilvD.     

Epicutaneous immunization with DNP-BSA induces CD4(+) CD25(+) Treg cells that inhibit Tc1-mediated CS

As we have shown previously that protein antigen applied epicutaneously (EC) in mice inhibits TNP-specific Th1-mediated contact sensitivity (CS), we postulated that the maneuver of EC immunization might also suppress Tc1-dependent CS response. Here we showed that EC immunization of normal mice with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) applied on the skin in the form of a patch induces a state of subsequent unresponsiveness due to regulatory T cells (Treg) that inhibited sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by TCRαβ(+) CD4(+) CD25(+) lymphocytes harvested from lymph nodes (LNs) of skin-patched animals. Flow cytometry revealed that EC immunization with DNP-BSA increased TCRαβ(+) CD4(+) CD25(+) FoxP3(+) lymphocytes in subcutaneous LNs, suggesting that observed suppression was mediated by Treg cells. Further, in vitro experiments showed that EC immunization with DNP-BSA prior to 1-fluoro-2,4-dinitrobenzen sensitization suppressed LN cell proliferation and inhibited production of TNF-α, IL-12 and IFN-γ. Using a transwell system or anti-CTLA-4 mAb, we found that EC induced suppression required direct Treg-effector cell contact and is CTLA-4-dependent.