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Cell-mediated immune (CMI) response in 16 Indian kala-azar (KA) and 12 post-kala-azar dermal leishmaniasis (PKADL) patients was studied in detail by in vitro lymphocyte transformation experiments and by in vivo skin testing. Peripheral blood lymphocytes of active KA patients failed to be stimulated by leishmania antigen. On the other hand, lymphocytes from a majority of the active KA patients could be stimulated by phytohemagglutinin. Active KA patients also failed to show delayed type hypersensitivity reaction to leishmanin, although 72% of them showed delayed type hypersensitivity to a purified protein derivative of tuberculin. Longitudinal studies indicated that antigen-specific CMI response usually appeared in treated KA patients after 12 to 20 weeks of antileishmanial drug therapy, although individual variations were noted. CMI response in PKADL patients was variable as about two-thirds of them showed positive sensitization to leishmania antigen in either in vivo or in vitro tests. Usually, patients with newly acquired PKADL exhibited better CMI response than those with chronic PKADL. However, lymphocytes from all of these patients could be stimulated normally by phytohemagglutinin. Results presented in this study show an impairment of CMI response in active KA which appears to be more specific to leishmania than generalized in nature. Moreover, restoration of specific T-cell responsiveness was aided by antileishmanial drug therapy which resulted in the reduction of antigenic load by parasite destruction and a concomitant decrease in circulating antibody levels, particularly that of the immunoglobulin G class. We suggest that the protection afforded by specific CMI response against Leishmania donovani infection may not be absolute and probably depends on other host-related factors leading to parasite destruction and patient recovery.

ABSTRACTClostridium difficileis a spore-forming bacillus that produces toxin-mediated enteric disease. C. difficileexpresses two major virulence factors, toxin A (TcdA) and toxin B (TcdB). Human and animal studies demonstrate a clear association between humoral immunity to these toxins and protection against C. difficileinfection (CDI). The receptor binding-domains (RBDs) of TcdA and TcdB are known to be immunogenic. Here, we tested the immunoadjuvant properties of Salmonella entericaserovar Typhimurium flagellin (FliC) subunit D1 as an innate immune agonist expressed as a recombinant fusion vaccine targeting the RBDs of TcdA and TcdB in mice. Intraperitoneally immunized mice developed prominent anti-TcdA and anti-TcdB immunoglobulin G in serum. The protective efficacy of the recombinant vaccines, with or without an adjuvant, was tested in a mouse model of CDI that closely represents the human disease. Following intraperitoneal immunization equivalent to two doses of toxoid A and toxoid B vaccine adjuvanted with alum and oral challenge with C. difficileVPI 10463, C57BL/6 mice were able to mount a protective immune response that prevented diarrhea and death compared to mice immunzed with alum alone. These results are significantly different from those for control mice (P< 0.001). These results provide evidence that a recombinant protein-based vaccine targeting the RBDs of the C. difficiletoxins adjuvanted with S. Typhimurium flagellin can induce rapid, high-level protection in a mouse model of CDI when challenged with the homologous strain from which the vaccine antigens were derived and warrant further preclinical testing against clinically relevant C. difficilestrains in the mouse and hamster models of CDI.

ABSTRACTEhrlichia chaffeensisis an obligate intracellular bacterium that infects primarily monocytes and macrophages and causes potentially fatal human monocytic ehrlichiosis (HME) that mimics toxic-shock-like syndrome in immunocompetent hosts. Early recruitment of neutrophils to the sites of infection is critical for the control of bacterial infection and inflammatory responses. We recently observed rapid and sustained neutrophil recruitment at a primary site of infection (peritoneum) following lethal murine ehrlichial infection compared to innocuous ehrlichial infection. We examined here the contribution of neutrophils to protective immunity or immunopathology during infection with monocytic Ehrlichia. Unexpectedly, depletion of neutrophils from lethally infected mice enhanced bacterial elimination, decreased immune-mediated pathology, and prolonged survival. Furthermore, compared to lethally infected sham controls, neutrophil depletion in infected mice resulted in amelioration of pathogenic responses, as evidenced by a decreased number of tumor necrosis factor alpha (TNF-a)-producing CD8+T cells, which is known to mediate immunopathology and toxic shock in a murine model of fatal ehrlichiosis. Although neutrophil depletion did not influence the number of CD4+Th1 cells and NKT cells producing gamma interferon (IFN-?), it increased the ratio of IFN-?- to IL-10-producing NKT cells as well as the ratio of IFN-? to interleukin 10 (IL-10) transcripts in the liver. This may ameliorate the net suppressive effect of IL-10 on IFN-?-mediated activation of infected macrophages and thus may account for the enhanced bacterial elimination. Finally, transcriptional analysis of gene expression in the liver indicated that neutrophils contribute to overproduction of cytokines and chemokines during fatal ehrlichiosis. In conclusion, these results revealed an unexpected role of neutrophils in supporting bacterial replication indirectly and promoting immunopathology during severe infection with an intracellular bacterium.

Clostridium difficile is the leading cause of nosocomial infectious diarrhea. C. difficile produces two toxins (A and B), and systemic and mucosal anti-toxin A antibodies prevent or limit C. difficile-associated diarrhea. To evaluate whether transcutaneous immunization with formalin-treated C. difficile toxin A (CDA) induces systemic and mucosal anti-CDA immune responses, we transcutaneously immunized three cohorts of mice with CDA with or without immunoadjuvantative cholera toxin (CT) on days 0, 14, 28, and 42. Mice transcutaneously immunized with CDA and CT developed prominent anti-CDA and anti-CT immunoglobulin G (IgG) and IgA responses in serum and anti-CDA and anti-CT IgA responses in stool. Sera from immunized mice were able to neutralize C. difficile toxin A activity in an in vitro cell culture assay. CDA itself demonstrated adjuvant activity and enhanced both serum and stool anti-CT IgA responses. Our results suggest that transcutaneous immunization with CDA toxoid may be a feasible immunization strategy against C. difficile, an important cause of morbidity and mortality against which current preventative strategies are failing.

ABSTRACTClostridium difficileis the leading cause of nosocomial infectious diarrhea. C. difficileproduces two toxins (A and B), and systemic and mucosal anti-toxin A antibodies prevent or limit C. difficile-associated diarrhea. To evaluate whether transcutaneous immunization with formalin-treated C. difficiletoxin A (CDA) induces systemic and mucosal anti-CDA immune responses, we transcutaneously immunized three cohorts of mice with CDA with or without immunoadjuvantative cholera toxin (CT) on days 0, 14, 28, and 42. Mice transcutaneously immunized with CDA and CT developed prominent anti-CDA and anti-CT immunoglobulin G (IgG) and IgA responses in serum and anti-CDA and anti-CT IgA responses in stool. Sera from immunized mice were able to neutralize C. difficiletoxin A activity in an in vitro cell culture assay. CDA itself demonstrated adjuvant activity and enhanced both serum and stool anti-CT IgA responses. Our results suggest that transcutaneous immunization with CDA toxoid may be a feasible immunization strategy against C. difficile, an important cause of morbidity and mortality against which current preventative strategies are failing.

Progressive visceral infection of golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative responses of their splenic or peripheral blood mononuclear cells (SPMC or PBMC, respectively) to in vitro stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals (I-SPMC or I-PBMC) was earlier shown to restore almost completely their lymphoproliferative responses to PMA plus Io. The present study was directed to evaluate the status of protein kinase C (PKC), a molecule(s) known to play a key role in the lymphoproliferative process. Our results demonstrate that PKC activities (Ca(2+), phosphatidyl serine, and diacyl glycerol dependent) in the cytosolic fraction of untreated nonadherent I-SPMC or I-PBMC as well as in the membrane fraction of PMA-treated cells were decreased significantly relative to those for normal controls. However, removal of adherent cells from I-SPMC or I-PBMC and subsequent overnight in vitro cultivation of nonadherent cells (lymphocytes) resulted in significant restoration of PKC activity in the cytosolic or membrane fraction of untreated or PMA-treated cells, respectively. Partial, though significant, restoration of PKC activity could also be achieved in the membrane fraction of PMA-treated cells following overnight in vitro treatment of I-SPMC or I-PBMC with the Ser/Thr phosphatase inhibitor okadaic acid (OA) or an anti-transforming growth factor beta (anti-TGF-beta) neutralizing antibody. These results correlated well with the ability of OA or the anti-TGF-beta antibody to restore the lymphoproliferative response of I-SPMC or I-PBMC following stimulation with PMA plus Io. Interestingly enough, immunoblotting experiments failed to show any reduction in the level or translocation (following PMA treatment) of conventional PKC isoforms in the SPMC or PBMC of infected animals compared to those of normal controls. The results presented in this study suggest that the adherent cells generated in the SPMC or PBMC of infected animals exert a suppressive effect on the proliferative response of nonadherent cells (lymphocytes) which is likely to be mediated through the downregulation of the activation pathway involving PKC and its downstream molecules such as mitogen-activated protein kinases. Further, the observed suppression of PKC activity and subsequent lymphoproliferative responses can be attributed to alternations in the intracellular phosphorylation-dephosphorylation events. The relevance of these results is discussed in relation to the role of TGF-beta, levels of which are known to be elevated in visceral leishmaniasis.

ABSTRACTProgressive visceral infection of golden hamsters by Leishmania donovaniamastigotes led to gradual impairment of the proliferative responses of their splenic or peripheral blood mononuclear cells (SPMC or PBMC, respectively) to in vitro stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals (I-SPMC or I-PBMC) was earlier shown to restore almost completely their lymphoproliferative responses to PMA plus Io. The present study was directed to evaluate the status of protein kinase C (PKC), a molecule(s) known to play a key role in the lymphoproliferative process. Our results demonstrate that PKC activities (Ca2+, phosphatidyl serine, and diacyl glycerol dependent) in the cytosolic fraction of untreated nonadherent I-SPMC or I-PBMC as well as in the membrane fraction of PMA-treated cells were decreased significantly relative to those for normal controls. However, removal of adherent cells from I-SPMC or I-PBMC and subsequent overnight in vitro cultivation of nonadherent cells (lymphocytes) resulted in significant restoration of PKC activity in the cytosolic or membrane fraction of untreated or PMA-treated cells, respectively. Partial, though significant, restoration of PKC activity could also be achieved in the membrane fraction of PMA-treated cells following overnight in vitro treatment of I-SPMC or I-PBMC with the Ser/Thr phosphatase inhibitor okadaic acid (OA) or an anti-transforming growth factor β (anti-TGF-β) neutralizing antibody. These results correlated well with the ability of OA or the anti-TGF-β antibody to restore the lymphoproliferative response of I-SPMC or I-PBMC following stimulation with PMA plus Io. Interestingly enough, immunoblotting experiments failed to show any reduction in the level or translocation (following PMA treatment) of conventional PKC isoforms in the SPMC or PBMC of infected animals compared to those of normal controls. The results presented in this study suggest that the adherent cells generated in the SPMC or PBMC of infected animals exert a suppressive effect on the proliferative response of nonadherent cells (lymphocytes) which is likely to be mediated through the downregulation of the activation pathway involving PKC and its downstream molecules such as mitogen-activated protein kinases. Further, the observed suppression of PKC activity and subsequent lymphoproliferative responses can be attributed to alternations in the intracellular phosphorylation-dephosphorylation events. The relevance of these results is discussed in relation to the role of TGF-β, levels of which are known to be elevated in visceral leishmaniasis.

A non-O1 non-O139 Vibrio cholerae strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259 tcpA gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (aldA, tagA, tcpP/H, toxT, acfB/C, and int) and intergenic regions (tcpF to toxT and tcpH to tcpA) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30 degrees C and pH 6.5) led to transcriptional activation of tcpP/H, toxR, toxT, and tcpA genes, but not of ctxA, as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of ctxAB. Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a V. cholerae O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of toxT, ctxA, and tcpA when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly ( approximately 1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of toxT, ctxA, or tcpA genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of V. cholerae strains belonging to non-O1 serogroups.

A toxigenic non-O1/non-O139 strain of Vibrio cholerae (10259) was found to contain a new variant of the toxin-coregulated pilus (TCP) protein gene (tcpA) as determined by PCR and Southern hybridization experiments. Nucleotide sequence analysis data of the new tcpA gene in strain 10259 (O53) showed it to be about 74 and 72% identical to those of O1 classical and El Tor biotype strains, respectively. The predicted amino acid sequence of the 10259 TcpA protein shared about 81 and 78% identity with the corresponding sequences of classical and El Tor TcpA strains, respectively. An antiserum raised against the TCP of a classical strain, O395, although it recognized the TcpA protein of strain 10259 in an immunoblotting experiment, exhibited considerably less protection against 10259 challenge compared to that observed against the parent strain. Incidentally, the tcpA sequences of two other toxigenic non-O1/non-O139 strains (V2 and S7, both belonging to the serogroup O37) were determined to be almost identical to that of classical tcpA. Further, tcpA of another toxigenic non-O1/non-O139 strain V315-1 (O nontypeable) was closely related to that of El Tor tcpA. Analysis of these results with those already available in the literature suggests that there are at least four major variants of the tcpA gene in V. cholerae which probably evolved in parallel from a common ancestral gene. Existence of highly conserved as well as hypervariable regions within the sequence of the TcpA protein would also predict that such evolution is under the control of considerable selection pressure.

ABSTRACTA toxigenic non-O1/non-O139 strain of Vibrio cholerae(10259) was found to contain a new variant of the toxin-coregulated pilus (TCP) protein gene (tcpA) as determined by PCR and Southern hybridization experiments. Nucleotide sequence analysis data of the new tcpAgene in strain 10259 (O53) showed it to be about 74 and 72% identical to those of O1 classical and El Tor biotype strains, respectively. The predicted amino acid sequence of the 10259 TcpA protein shared about 81 and 78% identity with the corresponding sequences of classical and El Tor TcpA strains, respectively. An antiserum raised against the TCP of a classical strain, O395, although it recognized the TcpA protein of strain 10259 in an immunoblotting experiment, exhibited considerably less protection against 10259 challenge compared to that observed against the parent strain. Incidentally, the tcpAsequences of two other toxigenic non-O1/non-O139 strains (V2 and S7, both belonging to the serogroup O37) were determined to be almost identical to that of classicaltcpA. Further, tcpAof another toxigenic non-O1/non-O139 strain V315-1 (O nontypeable) was closely related to that of El Tor tcpA. Analysis of these results with those already available in the literature suggests that there are at least four major variants of the tcpAgene in V. choleraewhich probably evolved in parallel from a common ancestral gene. Existence of highly conserved as well as hypervariable regions within the sequence of the TcpA protein would also predict that such evolution is under the control of considerable selection pressure.

A lectin with strong hemagglutinating activity toward erythrocytes of several animal species was isolated from an 18-h culture supernatant of a diarrheagenic strain, V2, of non-O1 Vibrio cholerae. The hemagglutinin (HA) was purified free of lipopolysaccharide by salt fractionation followed by gel filtration, hydrophobic interaction chromatography, and, finally, gel filtration in the presence of urea and deoxycholate. The purification procedure resulted in an HA preparation with 80-fold enhancement of specific activity. The HA consisted of noncovalently bound subunits of Mr 62,000 and behaved essentially as a single component with pI 6.0. Nonpolar and acidic amino acids contributed 46 and 24%, respectively, to the total amino acid residues. Electron micrographs of the HA showed it to consist of large, nonstoichiometric aggregates' of disklike molecules of 10-nm diameter. Inhibition of the HA by the glycoproteins fetuin, asialofetuin, and mucin, but not by ovalbumin and simple sugars, suggested the specific requirement of complex carbohydrates for binding. Rabbit antisera to the purified HA inhibited the hemagglutinating activities of the crude cell-free HA preparations, but not cell-associated HA activities of the parent (V2) or of other O1 and non-O1 V. cholerae strains. This suggested that the released and cell-associated HA activities were mediated by antigenically distinct components. Immunoblotting experiments showed that the antisera recognized a polypeptide component of Mr 62,000 in the cell envelope preparations of the parent and several other V. cholerae O1 and non-O1 strains. These data suggested that the HA was a nonfimbrial lectin of somatic origin with no protease activity and was apparently distinct from V. cholerae HAs described so far.

A comparative immunological study between two colostrum pools of Indian and Swedish mothers was carried out to evaluate their protective properties against Vibrio cholerae. Antibacterial and antitoxin titers were significantly higher in the Indian colostrum pool (ICP) than in the Swedish colostrum pool (SCP). Antilipopolysaccharide as well as antitoxin antibodies belonged to secretory immunoglobulin A (IgA) and IgM classes as determined by the enzyme-linked immunosorbent assay. ICP could significantly inhibit the adherence of V. cholerae to intestinal slices in vitro, whereas such activity was virtually absent in SCP. Moreover, ICP could induce significant protection against live vibrio challenge in rabbit ileal loops, whereas only a weak protective activity was observed with SCP. A secretory IgA fraction was obtained from ICP by using gel filtration and immunoadsorbent techniques. Human secretory IgA thus obtained exhibited antiadherence as well as protective activities against V. cholerae.

Two Escherichia coli strains (B/M 369 and C-35) belonging to enteropathogenic serogroup O86 were isolated from patients with infantile diarrhea and studied with respect to their cellular adherence properties. Both strains exhibited adherence (Ad+) to HEp-2 and HeLa cell monolayers in vitro and expressed mannose-resistant hemagglutinating (MRHA+) activity towards human, chicken, and sheep (but not mouse, rabbit, or guinea pig) erythrocytes. Cellular adherence properties of both strains could be substantially reduced by pronase treatment and by heat treatment (100 degrees C for 5 min) of bacteria. Electron microscopic examination failed to reveal fimbria- or pilus-like structures on the bacterial cell surface. Conjugation experiments conducted with these strains suggested that both MRHA and HEp-2 and HeLa cell adherence factors were encoded by the same plasmid, with a size of 55 to 57 megadaltons (MDa). Further biochemical studies indicated that the cellular adherence factors were associated with cell surface structures of bacteria that were proteinaceous in nature. An antiserum, rendered specific for the 57-MDa plasmid (pRP201) products of B/M 369 by adsorption, reacted with both MRHA+ Ad+ strains, B/M 369 and C-35, but not with their 57- or 55-MDa plasmidless MRHA- Ad- transconjugants or with other MRHA- Ad- E. coli strains. Immunological studies showed that the absorbed antiserum recognized two proteins with subunit molecular sizes of 18 and 14.5 kDa that were present on the cell surfaces of both strains. Furthermore, the absorbed antiserum at subagglutinating dilutions did inhibit, although only partially, the MRHA and HEp-2 and HeLa cell adherence activities of both E. coli strains. All these results would indicate that some of the E. coli strains belonging to enteropathogenic serogroups express their adherence potential through factors that were hitherto unrecognized.

Different classes of immunoglobulins (immunoglobulin M [IgM], IgG, and secretory IgA) were purified from pooled serum and milk samples of convalescent cholera patients by gel filtration and immunoadsorbent techniques. The purity of these preparations was established by immunodiffusion and the enzyme-linked immunosorbent assay, using class-specific antisera. The biological properties of antibodies present in these crude and purified immunoglobulin preparations were evaluated by tests related to cholera. Purified human IgM and IgG exhibited both agglutinating and vibriocidal properties. On the other hand, human secretory IgA was not vibriocidal (even in the presence of lysozyme), although it showed agglutinating properties. Both IgG and secretory IgA could effectively neutralize cholera toxin action in vivo, whereas such activity was virtually absent in IgM. The toxin-neutralizing capacity of IgG was, however, higher than that of secretory IgA. All three classes of human antibodies could significantly inhibit the adherence of Vibrio cholerae to intestinal slices in vitro. These results are discussed in relation to the protective immune mechanism during cholera infection.

Antibacterial and antitoxin responses in the acute and convalescent (7 to 10 days) sera of 14 cholera patients were determined by various serological techniques. Similar studies were also carried out with corresponding milk samples of six of these patients who were lactating women. A significant rise in antibacterial titers was observed in all convalescent serum and milk samples. A similar rise in antitoxin titers was observable in all serum and four milk samples. Specificity of the antibacterial titers was further evaluated by the indirect hemagglutination test using lipopolysaccharide antigen, and close correlations were noted between these titers and vibrio agglutination (P<0.001) and vibriocidal (P<0.001) titers of sera. Serum and milk convalescent cholera patients could effectively neutralize cholera toxin action in vivo, although the neutralizing activity of serum was higher than that of milk. Determination of antibody titers by the enzyme-linked immunosorbent assay demonstrated that anti-lipopolysaccharide activity in sera belonged predominantly to immunoglobulin M (IgM) and, to a lesser extent, to IgG and IgA, whereas such activity in milk was mostly contributed by secretory IgA, although some IgM antibodies also could be detected. On the other hand, antitoxic activity in convalescent sera primarily belonged to IgG, whereas such activity in milk was almost exclusively contributed by secretory IgA. These results demonstrate that an antibody response in the mammary gland was stimulated due to the antigen exposure in the gut and are consistent with the idea of a common homing pattern of immunocytes within the secretory immune system. Moreover, some differences in the antibody production mechanism between the systemic and secretory immune systems are indicated.

Vibrio cholerae O1 organisms belonging to different biotypes and serotypes were shown to express major outer membrane proteins (MOMPs) with subunit molecular masses of 48 to 50, 40 to 43, 35 to 36, 27 to 28, and 20 kDa. Antisera raised against individual MOMPs of a V. cholerae O1 strain recognized MOMPs of corresponding molecular masses in other O1 and non-O1 strains. Serological data also suggested possible differences in the cell surface exposition of these MOMPs. However, no marked differences between V. cholerae cells grown in vitro and in vivo could be noted in respect to the expression or surface exposition of these MOMPs. Of five MOMPs studied in this work, 40- to 43- and 20-kDa cell surface proteins were shown to be of considerable importance, as antisera to these proteins induced significant protection against V. cholerae challenge in the suckling mouse model. Similar protection, although to a lesser extent, was demonstrable with the antiserum to the 27- to 28-kDa protein. These results were corroborated with the Fab (immunoglobulin G) [Fab(IgG)] fragments of the antisera, thereby suggesting that the observed protection induced by anti-MOMP antibodies did not arise as a result of bacterial clumping. Subsequent studies demonstrated that these antisera as well as their Fab (IgG) fragments induced significant inhibition of intestinal colonization of V. cholerae. The 40- to 43- and 27- to 28-kDa proteins appeared to be porinlike, while the 20-kDa protein was found to be antigenically related to TcpA (subunit A of toxin-coregulated pilus). All these results demonstrate the involvement of more than one cell surface antigen of V. cholerae in the induction of protective immunity through inhibition of intestinal colonization of vibrios.