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A value of significantly less than 0

A value of significantly less than 0.05 was considered significant in every statistical tests. RESULTS Success of infected mice. proteins (CRP) boosts by many hundredfold in human beings and rabbits (13), whereas in mice CRP boosts just modestly (39). CRP is normally a pentameric proteins exhibiting Ca2+-reliant binding specificity for phosphocholine (PCh) (37), phosphoethanolamine (Family pet) (31), and specific various other ligands (analyzed in guide 1). A number of activities have already been seen in vitro that are in keeping with a job of CRP in web host defense. For instance, CRP binds several pathogens, including bacterias (18) and fungi (28, 29), and promotes their phagocytosis by individual leukocytes. CRP can be a powerful activator from the traditional pathway of supplement (17), and for that reason it could mediate opsonization of pathogens by supplement activation items. Probably due to the presence of PCh moieties in its cell wall C-polysaccharide, CRP reacts ORY-1001(trans) in vivo with the gram-positive bacterium (16, 20, 40). In contrast, protection was not observed after administration of human serum ORY-1001(trans) amyloid P-component (SAP) (16), an acute-phase protein in mice but not humans. SAP is usually structurally similar to CRP and has lectin-like binding specificity for galactose derivatives (14). SAP also binds PEt Adamts5 but not PCh (31). Recently, using CRP transgenic (CRPtg) mice capable of expressing human CRP in an acute-phase manner (9), we confirmed that CRP plays a significant role in vivo in host defense against pneumococcal infections (33). Subsequently we showed that although its protective effect was more pronounced in mice with an intact complement system, CRP offered significant protection even to mice that were decomplemented by cobra venom factor (34). The gram-negative pathogen serovar Typhimurium induces a disease in mice that is a model for human typhoid fever (6, 10, 12, 24). Like all gram-negative bacteria, serovar Typhimurium has phosphatidylethanolamine in its lipid bilayers; however, it does not bind CRP in vitro (21). Thus, based on these in vitro observations, CRP should not be expected to opsonize the bacterium. Nevertheless, in the present study we show that human CRP expressed by transgenic mice is usually protective against low-dose contamination with serovar Typhimurium. These data for the first time extend to gram-negative bacteria previous observations (33, 34) of CRP-mediated protection against pathogens. MATERIALS AND METHODS Mice. We previously described (33) CRPtg C57BL/6J congenic mice. These mice carry a 31-kb gene, 17 kb of 5-flanking sequence, and 11.3 kb of 3-flanking sequence (9), and they express high levels of human CRP in serum in response to injected endotoxin or after infection with pneumococci (33). C57BL/6J mice are allele (3, 15). To generate CRPtg mice resistant to serovar Typhimurium, we crossed female C57BL/6J CRPtg mice with DBA/2J males (Charles River Laboratories, Boston, Mass.) to produce CRPtg and non-tg F1 hybrids. F1s were backcrossed to DBA/2J to generate F2s. Since the allele is usually dominant, all F2 mice have ORY-1001(trans) the transgene using a previously described PCR method (23). In addition, since DBA/2J mice are C5 deficient (C5D), F2 mice were also screened for inheritance of the mutant allele by PCR (38). Finally, due to sexual dimorphism of CRP transgene expression, which results in higher levels of serum CRP in males (33, 35), only male mice were used. Mice were housed in groups of four, fed and watered ad libitum, maintained according to protocols established by the Animal Resources Program at this institution, and 10 to 12 weeks aged when used in experiments. Non-tg littermates served as controls. Bacteria. Wild-type (virulent) serovar Typhimurium strain LT2L (live oral vaccine strain (8). The Typhimurium strains used for infecting mice were collected by centrifugation from stationary-phase broth cultures (produced overnight at 37C) and washed and resuspended in Ringer’s lactate answer at 4C. Concentrations of bacteria were estimated from absorbance at 420 nm (antibody titers were determined by ELISA as previously described (22) using microtiter plates coated with whole-cell lysates of the virulent strain LT2L. Goat anti-mouse immunoglobulin G (IgG)-biotin or goat anti-mouse IgG2a-biotin followed by avidin-peroxidase (all from Bio-Rad Laboratories, Richmond, Calif.) and ABTS [2,2-azinobis(3-ethylbenzthiazolinesulfonic acid)] were used to develop the plates. The reported titer of each specimen is the reciprocal of the serum dilution giving five occasions higher absorbance than undiluted (pooled) preimmune serum. Statistical analyses. The ORY-1001(trans) data presented are from analyses of results pooled from three ORY-1001(trans) individual survival experiments, two bacterial clearance experiments, four organ.