Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). CONCLUSIONS: Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

Early appearance of functional plasma cells (CD138+CD98+) in non-immunized neonate mice.

Plasma cells (PCs) are terminally differentiated antibody-secreting cells, derived from activated B-lymphocytes in response to either T-independent or T-dependent antigens. The plasma cell population is scarce in circulation in non-immunized individuals. It is established that neonates are incapable of mounting an efficient immune response due to the immaturity of the immune system. However, this disadvantage is well overcome through the antibodies neonates receive from breastmilk. This implies that neonates will be only protected against antigens the mother had previously encountered. Thus, the child might be potentially susceptible to new antigens. This issue prompted us to seek for the presence of PCs in non-immunized neonate mice. We found a PC population identified as CD138+/CD98+ cells since day one after birth. These PCs were positive for Ki67 and expressed Blimp-1, B220, and CD19, which suggests the populations are plasmablasts and PCs with heterogeneous phenotype. These PCs were also determined to secrete antibodies, although mainly isotype IgM. Altogether, the results indicated that neonate PCs can produce antibodies against antigens they encounter in the first weeks of life, most likely coming from food, colonizing microbiota, or the environment.

Effects of Vitamin D Supplementation during the Induction and Progression of Osteoarthritis in a Rat Model.

Epidemiological studies correlate low levels of vitamin D with the osteoarthritis (OA) progression. Cytokines and metalloproteases play a major role in OA promoting the inflammation and degradation of the cartilage and can be induced through the Toll-like receptor (TLR) pathway. The aim of this study was to evaluate the protective effect of vitamin D supplementation on the development of osteoarthritis (OA) through examining the genetic regulation of TLRs, cytokines, and metalloproteases in chondrocytes as well as the wideness of cartilage in rats with OA. Our results demonstrate that the signaling through TLR-4 is a proinflammatory mechanism in osteoarthritis that drives the upregulation of MMP-3, IL-1ß, and TNF-α gene expression, leading to cartilage degradation and inflammation. Vitamin D supplementation had a protective effect during the onset but not during the chronic stage of OA in the rat model.

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.

14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.

Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation.

CRTAM is negatively regulated by ZEB1 in T cells.

T cell activation leads to the induction of genes that are required for appropriate immune responses. This includes CRTAM (Class-I MHC-restricted T cell associated molecule), a protein that plays a key role in T cell development, proliferation, and generating cell polarity during activation. We previously characterized the CRTAM promoter and described how AP-1 family members are important for inducing CRTAM expression upon antigenic activation. Here, we show that CRTAM is a molecular target for ZEB1 (zinc finger E-box-binding protein), a homeodomain/Zn finger transcription factor. Overexpression of ZEB1 repressed CRTAM promoter activity, as well as endogenous CRTAM levels in human T cells. ZEB1-mediated transcriptional repression was abolished when E-box-like elements in the CRTAM promoter are mutated. In summary, ZEB1 functions as a transcriptional repressor for the CRTAM gene in both non-stimulated and stimulated T cells, thereby modulating adaptive immune responses.

An Entamoeba histolytica rab-like encoding gene and protein: function and cellular location.

We identified here a 576 bp rab-like gene (EhrabB) in Entamoeba histolytica. EhrabB is located 332 bp upstream from the start codon of the Ehcp112 encoding gene, but is transcribed from the complementary strand. The EhrabB open reading frame predicts a 192 amino acid polypeptide (EhRabB) with 40-42% identity to Rab proteins, involved in vesicle docking regulation in endo and exocytic pathways of eukaryotic cells. Transcripts of 0.6 and 0.97 kb were detected by the EhrabB probe in northern blot assays. Using specific antibodies, EhRabB was located in small cytoplasmic vesicles by confocal microscopy. During phagocytosis, EhRabB was initially translocated to the plasma membrane and to the phagocytic mouths. The protein diminished after 10 min phagocytosis, suggesting that EhRabB could be participating in the regulation of the endocytosis process.

Identification of peptides presented by HLA class I molecules on cervical cancer cells with HPV-18 infection.

In this work we eluted peptides from purified class I MHC molecules, isolated from a novel human cervical carcinoma cell line (INBL), generated in our laboratory and positive for HPV-18 infection. A fraction of these peptides was capable of stimulating T lymphocytes obtained from a donor matched for HLA-Cw4 and who was also HPV-18+. Direct N-terminal Edman degradation of these peptides, revealed the sequence (XQFPIFLQF) that matched 85% with the sequence NVFPIFLQM localized in between the 54 and 62 residues of the HPV-18 L1 protein. After stimulation with the synthetic peptide NVFPIFLQM, T lymphocytes from the donor were capable to lyse INBL cells. Our results provide evidence of the existence of naturally occurring viral epitopes presented on cervical cancer cells by the HLA-Cw4 allele, that could be useful for immunotherapy on this type of patient.

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.

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.

Detection of a factor released by L5178Y lymphoblasts that inhibits mouse macrophage-activation induced by lipopolysaccharides.

BACKGROUND: Several factors inhibit cellular immune response by deactivating macrophages, but very few, such as those described here, prevent macrophage activation. METHODS: Ascites liquid from 12-day-old BALB/c mice bearing 5178Y lymphoma tumors was collected, and cell-free ascites liquid (CFAL) was separated from lymphoblasts. The supernatant (S1) was obtained from the homogenized and centrifuged lymphoblasts. Then, macrophage cultures containing 0.2 x 10(6) cells from lymphoma-bearing or healthy mice were added to 10 microL of CFAL or S1, plus 5 micrograms of lipopolysaccharides (LPS)/mL, 40 U interferon-gamma or a blend of both. Macrophages were incubated with CFAL or S1 prior to or after adding the activators to investigate whether any of the previously mentioned lymphoma fractions inhibited macrophage activation or whether they deactivated them. The effect of CFAL or S1 was estimated as the diminution of the amount of nitric oxide released by the experimental macrophage cultures with respect to controls (activated macrophages treated with none of the lymphoma fractions). RESULTS: LPS, IFN-gamma, and the LPS/gamma blend activated macrophages from both lymphoma-bearing and healthy mice. None of the lymphoma fractions deactivated macrophages. CFAL, but not S1, inhibited the macrophage activation, i.e., the percentage of inhibition of nitric oxide releasing 76.7% and 78.1% in macrophages from healthy and lymphoma-bearing mice, respectively. In addition, CFAL was unable to inhibit the macrophage-activation effect of IFN-gamma or the LPS/IFN-gamma blend. CONCLUSIONS: Mouse L5178Y lymphoma releases a factor that in vitro inhibits the macrophage activation induced by LPS, but not by IFN-gamma controls.

Non-typhi Salmonella serovars found in Mexican zoo animals.

The aim of the present study was to determine the bacteriological prevalence of subclinical non-typhi Salmonella infections in zoo animals and to determine the most frequently isolated serovars of the bacteria. A total of 267 samples were analyzed, including fecal samples from zoo animals and rodents, insects (Musca domestica and Periplaneta americana) and samples of the zoo animal's food. Salmonella was detected in 11.6% of the samples analyzed. Characterization of the isolates was performed with serotyping and pulsed-field gel electrophoresis. The following serovars were isolated: S. San Diego, S. Oranienburg, S. Weltevreden, S. Braenderup, S. Derby, S. 6,7, H:en x:- and S. 3,10, H:r:-. The isolates showed seven pulsed-field gel electrophoresis patterns with a Jaccard coefficient≥0.75 indicating a possible common origin. The prevalence of asymptomatic infections caused by Salmonella spp. in zoo animals was high. These findings demonstrate the diversity of Salmonella serovars in several captive wild animal species.

A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope.

Attenuated Salmonella strains are used widely as live carriers of antigens because they elicit both mucosal and systemic immunity against passenger antigens. However, they generally evoke poor cytotoxic T cell (CTL) responses because Salmonella resides within vacuolar compartments and the passenger antigens must travel to the cytosol and be processed through the MHC class I-dependent pathway to simulate CTLs. To address this problem, we designed a fusion protein to destabilize the phagosome membrane and allow a dengue epitope to reach the cytosol. The fusion protein was displayed on the bacterial surface of Salmonella enterica serovar Typhimurium SL3261 through the beta domain of the autotransporter MisL. The passenger alpha domain contained, from the N-terminus, a fusogenic sequence, the NS3 protein 298-306-amino acid CTL epitope from the dengue virus type 2, a molecular tag, and a recognition site for the protease OmpT to release it to the milieu. Display of the fusion protein on the bacterial surface was demonstrated by IFA and flow cytometry using antibodies against the molecular tag. Cleavage of the fusogenic protein-dengue peptide was demonstrated by flow cytometry using OmpT+ Escherichia coli strains. The recombinant Salmonella strains displaying the fusogenic-dengue peptide were able to lyse erythrocytes, induced specific proliferative responses, and elicited CTL responses. These results suggest that the recombinant fusion proteins containing fusogenic sequences provide a promising system to induce CTLs by live vector vaccines.

Surface appearance and instability of empty H-2 class I molecules under physiological conditions.

Recent evidence suggests that endogenously produced antigenic peptides are required for assembly of major histocompatibility complex class I chains with beta 2-microglobulin and transport to the cell surface. The RMA-S mutant cells are thought to be defective in intracellular peptide loading to class I molecules and, therefore, devoid of class I surface expression. Here we report that at physiological temperature (37 degrees C) "empty" class I molecules appear at the cell surface of RMA-S cells where they can be trapped with H-2 antibodies. In the absence of the stabilizing ligand, the class I molecules rapidly alter their conformation but remain at the cell surface as demonstrated with a rabbit antiserum. Such denatured H-2 molecules can also be found on normal wild-type RMA cells. However, their amount is strongly reduced after culture of RMA cells with a class I binding peptide. These findings indicate that empty class I molecules appear at the surface not only on mutant but also on normal cells, suggesting that in normal cells the supply with peptides is limited.

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.

Preformed purified peptide/major histocompatibility class I complexes are potent stimulators of class I-restricted T cell hybridomas.

A panel of antigen-specific, major histocompatibility complex class I-restricted T cell hybridomas has been generated to examine the capacity of peptide/class I complexes to stimulate T cells at the molecular level. Peptide/class I complexes were generated in detergent solution, purified and quantitated. Latex particles were subsequently coated with known amounts of preformed complexes and used to stimulate the T cell hybridomas. Stimulation was specific, i.e. only the appropriate peptide/class I combination were stimulatory, and quite sensitive, i.e. as little as 300 complexes per bead could be detected by the T cells. Preformed complexes were about 500,000 times more potent than free peptide in terms of T cell stimulation, demonstrating the physiological relevancy of the biochemically generated complexes. Surprisingly, the majority (including the most sensitive of the hybridomas) had lost CD8 expression, suggesting that antigen-specific stimulation of class I-restricted T cell hybridomas, as assessed by IL-2 release, does not depend on CD8.

Identification of CD4+ T cell epitopes of Taenia solium paramyosin.

T cell mediated response is involved in a protective immune response against experimental cysticercosis conferred by immunization with Taenia solium paramyosin (TPmy) to BALB/c mice. In this study, we analysed the TPmy amino acid sequence for predicted CD4+ T cells epitopes. Five different regions of this protein showed that the residues anchor to bind the I-Ad molecule, synthetic peptides containing these epitopes were evaluated for their ability to induce lymphoproliferative responses of spleen cells from TPmy immunized mice. Among them, Tp176 (amino acids 176-192 sequence DDLQRQMADANSAKSRL) was the immunodominant T cell epitope of TPmy. Delineation of this epitope should facilitate analysis of the role of CD4+ T cell response in experimental cysticercosis.