Humoral immune response in breeding hens and protective immunity provided by administration of purified Salmonella Gallinarum porins.

The current studies were undertaken to assess the ability of humoral immune response in breeding hens to provide protective maternal antibody in the progeny. A highly purified outer membrane protein, 34 kDa, was isolated from a virulent strain of Salmonella Gallinarum. Cross-reactivity was observed between this protein and Salmonella Typhi porins; thus we consider this outer membrane protein as a Salmonella Gallinarum porin. To evaluate passive immunity against Salmonella Gallinarum, 200 broiler breeder hens were immunized with either 10 microg of Salmonella Gallinarum porins, 30 microg of Salmonella Gallinarum porins, or PBS without porins as a control group. Anti-Salmonella Gallinarum porin antibodies were detected in broiler breeder serum and in fertile eggs (P < 0.05). Consequently, chickens from immunized broiler breeder hens were protected between 53 to 70% against challenges of 20 to 500 half-maximal lethal dose of Salmonella Gallinarum (P < 0.001) when compared with control hens that were injected with PBS. These results suggest that Salmonella Gallinarum porins, as those of other Salmonella species, participate in the induction of the passive protective immunity, and the humoral immune response may be one of the mechanisms involved in the establishment of this protection.

Lipopopeptidephosphoglycan from Entamoeba histolytica activates human macrophages and dendritic cells and reaches their late endosomes.

Lipopopeptidephosphoglycan (LPPG) is a complex macromolecule from the surface of Entamoeba histolytica trophozoites. We analysed the interaction between LPPG and human macrophages and dendritic cells (DCs) and found that LPPG is internalized by these cells and activates them. The internalization process involves intracellular traffic from the cell membrane to late endosomes, as shown by co-localization of LPPG with late endosomes marked with FITC-dextran and LAMP-1. LPPG-activated DCs have increased expression of co-stimulatory molecules CD80, CD86 and CD40 and produce pro-inflammatory cytokines TNF-alpha, IL-8 and IL-12. Taken together, these results show that LPPG activates antigen-presenting cells and reaches intracellular compartments that are involved in antigen presentation.

Triggering receptor expressed on myeloid cells (TREM-1) is regulated post-transcriptionally and its ligand is present in the sera of some septic patients.

Inflammation is necessary for survival, but it is also an important cause of human morbidity and mortality, as exemplified by sepsis. During inflammation, cells of the innate immune system are recruited and activated in response to infection, trauma or injury. These cells are activated through receptors, such as Toll-like receptors (TLRs), which recognize microbial ligands such as lipopolysaccharide (LPS). Triggering receptor expressed on myeloid cells (TREM)-1 amplifies the inflammatory response initiated by TLRs, and its expression on the surface of monocytes increases in the presence of TLR ligands. Here we have shown that in monocytes TREM-1 mRNA levels, measured by reverse transcription-polymerase chain reaction (RT-PCR), remained unchanged and TREM-1 protein levels, measured by flow cytometry, increased, indicating that LPS increases TREM-1 expression by a post-transcriptional mechanism. We also showed that TREM-1/Fc fusion protein decreased the ability of the sera of some patients with sepsis to activate monocytes, indicating that the TREM-1 ligand, whose identity is unknown, may be present in the sera of some of these patients. We describe a mechanism for the regulation of TREM-1 expression on monocytes and the possible presence of its ligand in serum; these findings help to explain the contribution of TREM-1 during systemic inflammation.

The innate immune response to Entamoeba histolytica lipopeptidophosphoglycan is mediated by toll-like receptors 2 and 4.

Entamoeba histolytica is a human pathogen that may invade the intestinal mucosa, causing amoebic colitis or hepatic abscesses when the trophozoites travel through the portal circulation to the liver. Lipopeptidophosphoglycan (LPPG) is a molecular pattern of E. histolytica recognized by the human immune system. Here we report that LPPG is exposed on the cell surface of E. histolytica trophozoites, and is recognized by the host through toll-like receptor (TLR) 2 and TLR4. Correspondingly, human embryonic kidney (HEK)-293 cells were rendered LPPG responsive through overexpression of TLR2 or TLR4/MD2. Moreover, co-expression of CD14 enhanced LPPG signal transmission through TLR2 and TLR4. The interaction of LPPG with TLR2 and TLR4 resulted in activation of NF-kappaB and release of interleukin (IL)-10, IL-12p40, tumour necrosis factor (TNF)-alpha, and IL-8 from human monocytes. Consistent with these findings, responsiveness of mouse macrophages lacking TLR2 expression (TLR2-/-) or functional TLR4 (TLR4d/d) to E. histolytica LPPG challenge was impaired while double deficient macrophages were unresponsive. In contrast to wild-type control and TLR2-/- animals succumbing to lethal shock syndrome, TLR4d/d mice were resistant to systemic LPPG challenge-induced pathology.

Macrophages present exogenous antigens by class I major histocompatibility complex molecules via a secretory pathway as a consequence of interferon-gamma activation.

Macrophages can process and present exogenous antigens on major histocompatibility complex (MHC) class I molecules through an alternative mechanism involving the internalization of antigens and the secretion of peptides loading MHC class I molecules at the cell surface. In this paper, we found that interferon-gamma (IFN-gamma) -activated macrophages infected with Salmonella typhimurum secreted peptides able to load empty MHC Kb molecules on co-cultured TAP-2-deficient RMA-S cells, added as targets for peptide loading. The increase in class I Kb on the RMA-S cells, resulting from the macrophage-derived peptides, exhibited a comparable stability as the direct addition of an exogenous Kb-binding peptide (OVA257-264) to the RMA-S cells. In both cases, the Kb complexes were stable for at least 3 hr after separating the RMA-S cells from the macrophages. The endosomal inhibitors, leupeptin and ammonium chloride, did not inhibit the release of peptides and the increase in Kb staining on the RMA-S cells in the co-culture systems. Brefeldin A also had no effect. P815 cells previously co-cultured with Salmonella-infected macrophages became targets for cytotoxic T lymphocytes isolated from Salmonella-infected BALB/c mice. Taken together, our data suggest that IFN-gamma-activated macrophages process exogenous antigens in an intracellular compartment where serine proteases generate peptides released to the external environment for loading empty MHC class I molecules at the cell surface. This TAP-independent mechanism for the MHC class I presentation may be involved in priming cytotoxic T lymphocytes against intracellular pathogens in vivo.

A cloning vector for efficient generation of cholera toxin B gene fusions for epitope screening.

Gene fusion proteins with epitopes attached to the amino end of cholera toxin B subunit (CTB) are useful to raise immunological responses. We describe a cloning vector, designated pCTBtet, carrying a tetracycline resistance gene (TetR) between the leader peptide and mature CTB. Removal of TetR to insert oligonucleotides encoding fusion epitopes allowed for screening of tetracycline-sensitive clones. Restoration of the correct CTB reading phase was subsequently used to choose gene fusion candidate colonies. The use of pCTBtet permitted the rapid construction of 8 fusion proteins carrying 9-24 aa from Salmonella typhi OmpC and 6 hybrids with 7-31 aa from Escherichia coli colonization factor CFAI.

Immunogenicity of a Salmonella typhi CVD 908 candidate vaccine strain expressing the major surface protein gp63 of Leishmania mexicana mexicana.

Attenuated Salmonella typhi are attractive for use as live vector vaccines to express protozoal antigens and deliver them to the human immune system. The gene encoding the mature form of Leishmania mexicana mexicana gp63 under control of tac promoter was integrated into the delta aroC locus of the chromosome of attenuated delta aroC, delta aroD S. typhi strain CVD 908. After oral immunization of BALB/c mice with two 1 x 10(9) colony forming unit doses given 21 days apart, CVD 908 omega (delta aroC::Ptac-gp63) elicited a broad T cell-mediated immune response against L. m. mexicana gp63 as demonstrated by: (1) lymphoproliferative response to fixed whole L. m. mexicana promastigotes; (2) activation of IL-2 (but not IL-4)-producing lymphocytes; (3) appearance of cytotoxic T cells against mouse mastocytoma cells expressing gp63. This T-cell mediated immune response was associated with significant protection in F1 (BALB/cXC57Bl/6) mice challenged in their footpads with a wild type strain of L. m. mexicana.

Evaluation of Salmonella enteritidis-immune lymphokines on host resistance to Salmonella enterica ser. gallinarum infection in broiler chicks.

The prophylactic treatment of neonatal broiler chicks with lymphokines derived from S. enteritidis-immurazed chickens (SE-ILK) was evaluated for its effect on the birds' resistance to an experimental infection S. enterica ser. gallinarum (SG). On the day of hatch, chicks were injected intraperitoneally with either SE-ILK, control non-immune lymphokines (NILK), or were left untreated. Thirty minutes later, all chicks were orally gavaged with either 10(4) colony forming units (CFU) or 10(6) CFU SG. The chicks were observed twice daily for 10 days for morbidity and mortality. Chicks that died during the experiment had their livers cultured for SG. The survivors were killed and their livers, spleens and caecal tonsils cultured for SG. The prophylactic treatment of chickens with SE-ILK induced significant protection against extraintestinal SG infection when compared to the NILK-treated or non-treated controls as evidenced by: (1) a significant reduction (P< 0.005) in the mortality of chicks challenged with either 10(4) and 10(6) CFU SG; (2) an increased average weight gains of chicks challenged with either 10(4) and 10(6) CFU SG; and (3) a significant (P< 0.001) reduction in the number of chicks with organs culture-positive for SG. The results suggest that the prophylactic administration of SE-ILK can confer non-specific protection to chicks against a pathogenic species of Salmonella resulting in reduced morbidity, mortality, and organ infectivity caused by SG infections of broiler chicks, while enhancing performance during the first 10 days of Ufe.

Identification of a 35 kDa glycoprotein from Entamoeba histolytica by sera from patients with amoebic liver abscess and with mouse monoclonal antibody.

In this work, we explored the relevance of a 35 kDa glycoprotein (Gm) of the outer membrane from E. histolytica in the diagnosis of the amoebic liver abscess (ALA) through ELISA and immunoblotting. We were interested in defining the relevance of this antigen in the immune response in patients with amoebic liver abscess and in exploring whether the mouse monoclonal antibody against this 35 kDa glycoprotein recognises the same epitope. We found that 87% of ALA patients had raised antibody levels to Gm antigen, whereas none of the healthy control subjects presented this same increase. We also found 90% sensitivity, 100% specificity, 100% positive predictive value, 90% negative predictive value, and 90% prevalence value for this Gm antigen. Nonetheless, we did not find any statistically significant differences in the levels of immunoglobulins against Gm, although IgG showed a tendency to increase, probably because we are dealing with a secondary immune response. Using electroimmunotransfer blot assay, we found that sera from ALA patients recognise the 35 kDa Gm protein in the same way as it is recognised by the mouse monoclonal antibody, suggesting that is a relevant molecule for the diagnosis of amebiasis, and eventually could lead to its use as protection against the disease.

Construction of CTB fusion proteins for screening of monoclonal antibodies against Salmonella typhi OmpC peptide loops.

Mice were immunized with resin-bound peptides whose sequences have been proposed to be part of exposed loops in Salmonella typhi outer membrane protein OmpC. To screen hybridomas for monoclonal antibodies against those epitopes, we designed fusion proteins where the candidate peptide sequence was attached to the amino end of cholera toxin B-subunit (CTB). The constructed fusion proteins allowed the efficient selection of positive clones by GM1-ELISA. Selected antibodies recognized purified OmpC and whole Salmonella bacteria. This suggests a native structure of our genetically attached peptides in agreement with immunological properties reported for previous CTB recombinant fusion proteins. In a more general context, CTB hybrids could be used to screen for antibodies towards immunogenic epitopes in other systems. This might turn out to be particularly useful when producing antibodies against peptide sequences in microorganisms whose handling is difficult or that pose inherent health risks.

Influence of the pituitary gland on the immune response in young rats.

The response of hypophysectomized (HYPOX) and sham-operated (S-HYPOX) female and male Wistar young rats (8 weeks old) to antigenic stimulation was compared. Humoral antigenic responses against hemocyanin were measured by ELISA. [3H]thymidine incorporation into cultured spleen cells was used to determine proliferative response to concanavalin A (ConA) or antigenic stimulation. Anti-hemocyanin serum titers in the HYPOX animals was about half of that observed in control S-HYPOX rats. Similarly, the cellular proliferative response was significantly decreased in HYPOX animals when compared to S-HYPOX rats; the blastogenic response to hemocyanin in UC rats (which did not receive the antigen injection) was close to zero. S-HYPOX control rats responded to direct ConA stimulation as UC controls. Body weight and the weight of pituitary target organs (adrenal, thyroid, ovary and testes) was about 1/4 of that of controls. Hypophysectomy also resulted in a striking reduction in spleen weight. These results indicate that the pituitary gland is involved in cellular and humoral immune regulation in young rats.

Prevalence of antibodies against Entamoeba histolytica in Mexico measured by ELISA.

The prevalence of antibodies against Entamoeba histolytica was studied in the Mexican population using an immunoenzyme assay in solid phase (ELISA) and semiautomatic equipment. The antigen was a mixture of membrane proteins obtained by Triton X-100 extraction from an axenic culture of Entamoeba histolytica HM1-IMSS. The method was standardized by comparing serum samples from amoebic liver abscess patients with healthy volunteers. From the 60,538 samples supplied by the National Seroepidemiology Survey, antibodies were found in 4.49% (4.32-4.65% at 95% confidence limit). More significant titres occurred in the central region of the country. The ratio female to male was 1.25:1. The population living in metropolitan areas had probably been infected at a younger age than those living in the country. Important differences were found in the seroprevalence obtained by ELISA compared with a study which used indirect haemagglutination (IHA) in the same sample frame.

Immune response to porins isolated from Salmonella typhi in different mouse strains.

Outer membrane proteins (OMPs) are able to induce protection against the challenge with S. typhi in a murine model. Both humoral and cellular immunity are involved in the protective mechanisms. In order to determine whether the responsiveness to S. typhi porins is genetically controlled in mice, different strains were immunized i.p. with 30 micrograms of OMPs isolated from S. typhi 9,12,Vi:d at days 0 and 7. On day 21, spleen cells were recovered and the lymphoproliferative response to porins was assessed. The highest responses were found in mice with H-2k and H-2a haplotypes (C3H/HeJ and A/J), intermediate responses were found in mice with H-2b haplotype (C57Bl/6) and the lowest responses in H-2d mice (Balb/c). These results demonstrate that the responsiveness to S. typhi porins is in part controlled by the major histocompatibility complex class II molecules and will help to further study the mechanisms of the immune response to these proteins.

Salmonella typhi vaccine strain CVD 908 expressing the circumsporozoite protein of Plasmodium falciparum: strain construction and safety and immunogenicity in humans.

rcsp, encoding amino acids 21-398 of Plasmodium falciparum circumsporozoite protein (CSP), under control of tacP was integrated into the chromosomal delta aroC locus of attenuated delta aroC, delta aroD Salmonella typhi CVD 908. By immunoblot and ELISA, rCSP expression was greater from a multicopy plasmid than from the single chromosomal gene. CVD 908 omega (delta aroC1019::tacP-rcsp) was well tolerated by 10 volunteers who were fed two doses of 5 x 10(7) organisms 8 days apart. Seven subjects excreted the vaccine strain for 1-3 days. All subjects developed serologic responses to O and H antigens of the live vector, whereas 3 vaccinees responded to the foreign antigen: 1 developed an 80-fold rise in serum anti-sporozoite antibody, another had a 4-fold rise in antibody to a recombinant portion of CSP (residues 309-345), while a third vaccinee developed CSP-specific CD8+ cytotoxic T lymphocyte activity. This is the first report of attenuated S. typhi eliciting a human serologic or a cytotoxic T lymphocyte response to a foreign protein. Improved foreign gene expression should enhance immunogenicity.