Dose-Dependent Circulating Immunoglobulin A Antibody-Secreting Cell and Serum Antibody Responses in Swedish Volunteers to an Oral Inactivated Enterotoxigenic Escherichia coli Vaccine

The immunogenicity of different preparations of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine was evaluated in Swedish volunteers previously unexposed to ETEC infection. The vaccine preparations consisted of recombinant cholera toxin B subunit (CTB) and various amounts of formalin-killed whole bacteria expressing the most prevalent colonization factor antigens (CFAs). Significant immunoglobulin A (IgA) antibody-secreting cell (ASC) responses against CTB and the various CFA components were seen in a majority of volunteers after two doses of ETEC vaccine independent of the vaccine lot given. The IgA ASC responses against CTB were significantly higher after the second than after the first immunization, whereas the CFA-specific IgA ASC responses were almost comparable after the first and second doses of ETEC vaccine. Two immunizations with one-third of a full dose of CFA-ETEC bacteria induced lower frequencies of IgA ASC responses against all the different CFAs than two full vaccine doses, i.e., 63 versus 80% for CFA/I, 56 versus 70% for CS1, 31 versus 65% for CS2, and 56 versus 75% for CS4. The proportion of vaccinees responding with rises in the titer of serum IgA antibody against the various CFA antigens was also lower after immunization with the reduced dose of CFA-ETEC bacteria. These findings suggest that measurements of circulating IgA ASCs can be used not only for qualitative but also for quantitative assessments of the immunogenicity of individual fimbrial antigens in various preparations of ETEC vaccine.     

Enterotoxigenic Escherichia coli Elicits Immune Responses to Multiple Surface Proteins

Enterotoxigenic Escherichia coli (ETEC) causes considerable morbidity and mortality due to diarrheal illness in developing countries, particularly in young children. Despite the global importance of these heterogeneous pathogens, a broadly protective vaccine is not yet available. While much is known regarding the immunology of well-characterized virulence proteins, in particular the heat-labile toxin (LT) and colonization factors (CFs), to date, evaluation of the immune response to other antigens has been limited. However, the availability of genomic DNA sequences for ETEC strains coupled with proteomics technology affords opportunities to examine novel uncharacterized antigens that might also serve as targets for vaccine development. Analysis of whole or fractionated bacterial proteomes with convalescent-phase sera can potentially accelerate identification of secreted or surface-expressed targets that are recognized during the course of infection. Here we report results of an immunoproteomics approach to antigen discovery with ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407. Immunoblotting of proteins separated by two-dimensional electrophoresis (2DE) with sera from mice infected with strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 or with convalescent human sera obtained following natural ETEC infections demonstrated multiple immunoreactive molecules in culture supernatant, outer membrane, and outer membrane vesicle preparations, suggesting that many antigens are recognized during the course of infection. Proteins identified by this approach included established virulence determinants, more recently identified putative virulence factors, as well as novel secreted and outer membrane proteins. Together, these studies suggest that existing and emerging proteomics technologies can provide a useful complement to ongoing approaches to ETEC vaccine development.Infectious diarrhea substantially impacts human health in the developing world, where hundreds of millions of infections occur each year. Several pathogens, rotavirus, Shigella, Vibrio cholerae, and enterotoxigenic Escherichia coli (ETEC), each contribute significantly to this disease burden and collectively result in an estimated 2 million deaths due to diarrheal illness annually (52). Therefore, ETEC remains a high priority for vaccine development.Enterotoxigenic E. coli strains constitute a phenotypically and genetically diverse pathotype that have in common the production of enterotoxin heat-labile toxin (LT) and/or heat-stable toxin (ST). In the classic paradigm for ETEC pathogenesis, organisms must colonize the small intestine via fimbrial colonization factor antigens (CFAs) for effective toxin delivery and subsequent diarrhea (18). Since the early identification of colonization factors (CFs) as important virulence determinants (15), these structures have been a central focus of ETEC vaccine development, and significant inroads have been made into the identification of a broad array of CFs (22, 43), with over 25 antigens identified thus far. ETEC vaccines currently in development are designed to target the most prevalent CFs (56). Moreover, recent elegant structural characterization of the colonization factor antigen I (CFA/I) pilus has provided additional molecular details of pilus tip adhesin molecules that might be exploited (33) as more highly conserved vaccine targets.However, the remarkable plasticity of E. coli genomes (45) and studies demonstrating that many ETEC strains do not produce an identifiable CF (40, 54) suggest that additional antigens would likely need to be considered to produce a broadly protective vaccine. While much is known about the immunology of the CFs and LT following infection (44, 46, 63), very little is known about the nature of immune responses to ETEC in general, and there is no information regarding immunogenicity of more recently discovered putative virulence factors.Furthermore, large-scale epidemiologic studies have suggested that additional plasmid or chromosomally encoded factors contribute to the development of an effective protective immune response attributable to prior natural infections with ETEC (55). However, the identity of other antigens that might be involved in the development of protective immune responses to ETEC remains largely unexplored.The advent of high-throughput sequencing of multiple genomes and advances in proteomics permit avenues for discovery of novel antigens which might be useful in ETEC vaccine development. Two complete ETEC genomes, ETEC {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 and E24377A (45), and one draft genome sequence, B7A (45), as well as several plasmid sequences (21) are now publicly available. While it is anticipated that dozens if not hundreds of ETEC genome sequences will ultimately be made available, these existing genomes permit some initial antigen discovery and validation efforts that were not previously possible.Recent studies of mice have demonstrated that mice exposed to ETEC are protected from subsequent intestinal colonization (47). Therefore, these studies were undertaken to characterize the nature of protective immune responses afforded by prior exposures to ETEC in this model and to validate immune responses to selected antigens using sera from patients naturally infected with ETEC.     

狂犬疫苗接种诱生细胞免疫的研究

狂犬疫苗对免疫接种志愿者和免疫实验小鼠能诱导产生显著的细胞介导免疫应答。免疫接种者的外周血淋巴细胞在体外经刀豆球蛋白Ａ刺激呈很强的增殖反应，增殖指数（ＳＩ）达８．５％－１５．４％；在ＨｐＢＬ的ＣｏｎＡ培养上清中可测及白细胞介素２（ＩＬ－２）活性明显升高。在接种者的血清中还可测及高水平干扰素。免疫试验小鼠其脾淋巴细胞在体外用不同狂犬病毒株抗原进行刺激亦呈显著的特异性增殖反应，ＳＩ达７．４％－１８．３     

Intestinal Secretory Immunoglobulin A Response to Enteroaggregative Escherichia coli in Travelers with Diarrhea

We examined stool samples from travelers for secretory immunoglobulin A (sIgA) to enteroaggregative Escherichia coli (EAEC) during episodes of acute diarrhea. Ten paired samples from 10 patients with diarrhea caused by EAEC were examined for the presence of specific sIgA by dot blot and Western blot immunoassays. Five samples were positive by dot blotting, and two samples were positive by Western blotting.     

EatA,an Immunogenic Protective Antigen of Enterotoxigenic Escherichia coli,Degrades Intestinal Mucin

Enterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality due to infectious diarrhea in developing countries for which there is presently no effective vaccine. A central challenge in ETEC vaccinology has been the identification of conserved surface antigens to formulate a broadly protective vaccine. Here, we demonstrate that EatA, an immunogenic secreted serine protease of ETEC, contributes to virulence by degrading MUC2, the major protein present in the small intestinal mucous layer, and that removal of this barrier in vitro accelerates toxin access to the enterocyte surface. In addition, we demonstrate that vaccination with the recombinant secreted passenger domain of EatA (rEatA_p) elicits high titers of antibody and is protective against intestinal infection with ETEC. These findings may have significant implications for development of both subunit and live-attenuated vaccines against ETEC and other enteric pathogens, including Shigella flexneri, that express similar proteins.     

Characterization of Mucosal Immune Responses to Enterotoxigenic Escherichia coli Vaccine Antigens in a Human Challenge Model: Response Profiles after Primary Infection and Homologous Rechallenge with Strain H10407

Enterotoxigenic Escherichia coli (ETEC) bacteria are the most common bacterial cause of diarrhea in children in resource-poor settings as well as in travelers. Although there are several approaches to develop an effective vaccine for ETEC, no licensed vaccines are currently available. A significant challenge to successful vaccine development is our poor understanding of the immune responses that correlate best with protection against ETEC illness. In this study, ETEC-specific mucosal immune responses were characterized and compared in subjects challenged with ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 and in subjects rechallenged with the homologous organism. IgA responses to lipopolysaccharide (LPS), heat-labile toxin B subunit (LTB), and colonization factor antigen I (CFA/I) in antibody in lymphocyte supernatant (ALS), feces, lavage fluid, and saliva samples were evaluated. In all assay comparisons, ALS was the most sensitive indicator of a local immune response, but serum IgA was also a useful indirect marker of immune response to oral antigens. Volunteers challenged and then rechallenged with strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 were protected from illness following rechallenge. Comparing mucosal antibody responses after primary and homologous rechallenge, protection against disease was reflected in reduced antibody responses to key ETEC antigens and in reduced fecal shedding of the {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 challenge strain. Subjects challenged with strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 mounted stronger antibody responses to LPS and LTB than subjects in the rechallenge group, while responses to CFA/I in the rechallenge group were higher than in the challenge group. We anticipate that this study will help provide an immunological benchmark for the evaluation of ETEC vaccines and immunization regimens in the future.     

Interleukin-8 Response in an Intestinal HCT-8 Cell Line Infected with Enteroaggregative and Enterotoxigenic Escherichia coli

This study examined the interleukin-8 (IL-8) response of the intestinal adenocarcinoma HCT-8 cell line to infection with enteroaggregative and enterotoxigenic Escherichia coli pathotypes isolated from patients with travelers' diarrhea. Individual diarrheagenic E. coli strains (enteroaggregative E. coli [EAEC]; n = 30), heat-stable enterotoxin (ST)-producing enterotoxigenic E. coli (ETEC ST; n = 11), heat-labile enterotoxin (LT)-producing enterotoxigenic E. coli (ETEC LT; n = 10), and ST- and LT-producing enterotoxigenic E. coli (ETEC ST:LT; n = 8) were coincubated with HCT-8 cells for 3 h. Tissue culture supernatants were assayed for IL-8 content by enzyme-linked immunosorbent assay. Fifty percent of EAEC (72% of those EAEC carrying the virulence factors aggR, aggA, and aspU and 40% of those EAEC not carrying virulence factors) and 64% of ETEC ST elicited IL-8 production. In contrast, 10% of ETEC LT elicited the production of IL-8 above baseline. These results suggest that (i) the HCT-8 cell line infection model can be used as a tool to differentiate proinflammatory E. coli from noninflammatory isolates; (ii) EAEC has a heterogeneous ability to induce the production of IL-8, and this may be associated with the presence of virulence factors; and (iii) ETEC ST can elicit an inflammatory response and helps explain our earlier findings of increased fecal IL-8 in patients with ETEC diarrhea.     

Antibody Responses in Humans against Coli Surface Antigen 6 of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains expressing only coli surface antigen 6 (CS6) have previously been isolated from patients with diarrhea, but the immunogenicity of CS6 has not been established in humans. We have detected CS6-specific immunoglobulin A responses in the feces and blood of patients convalescing from natural ETEC disease and of volunteers given an oral ETEC vaccine.     

Kinetics of Local and Systemic Immune Responses to an Oral Cholera Vaccine Given Alone or Together with Acetylcysteine

The possibility that a mucolytic drug, i.e., acetylcysteine, given orally may enhance the gut mucosal or systemic immune response to an oral B-subunit–whole-cell (B-WC) cholera vaccine was evaluated for 40 adult Swedish volunteers, and the kinetics of the immune responses were monitored for responding volunteers. Two doses of vaccine induced similar frequencies of immunoglobulin A (IgA) and IgG antitoxin responses (80 to 90%) and vibriocidal titer increases (60 to 65%) in serum irrespective of whether the vaccine was given alone or together with 2 g of acetylcysteine. In feces the frequencies of IgA antitoxin (67%) and antibacterial (33 to 40%) antibody responses were also comparable in the two immunization groups. Six months after vaccination, IgA and IgG antitoxin as well as vibriocidal antibody titer increases in serum could still be detected in approximately 80% of initially responding vaccinees. Significantly elevated fecal antitoxin and antibacterial IgA antibody levels were found in, respectively, 50 and 43% of those volunteers who initially had responded to the vaccine. Determination of IgA antibodies in feces does not seem to offer any advantages compared to determination in serum for assessment of immune responses after immunization with inactivated cholera vaccine.     

Antibody Avidity in Humoral Immune Responses in Bangladeshi Children and Adults following Administration of an Oral Killed Cholera Vaccine

Antibody avidity for antigens following disease or vaccination increases with affinity maturation and somatic hypermutation. In this study, we followed children and adults in Bangladesh for 1 year following oral cholera vaccination and measured the avidity of antibodies to the T cell-dependent antigen cholera toxin B subunit (CTB) and the T cell-independent antigen lipopolysaccharide (LPS) in comparison with responses in other immunological measurements. Children produced CTB-specific IgG and IgA antibodies of high avidity following vaccination, which persisted for several months; the magnitudes of responses were comparable to those seen in adult vaccinees. The avidity of LPS-specific IgG and IgA antibodies in vaccinees increased significantly shortly after the second dose of vaccine but waned rapidly to baseline levels thereafter. CTB-specific memory B cells were present for only a short time following vaccination, and we did not find significant memory B cell responses to LPS in any age group. For older children, there was a significant correlation between CTB-specific memory T cell responses after the second dose of vaccine and CTB-specific IgG antibody avidity indices over the subsequent year. These findings suggest that vaccination induces a longer-lasting increase in the avidity of antibodies to a T cell-dependent antigen than is measured by a memory B cell response to that antigen and that early memory T cell responses correlate well with the subsequent development of higher-avidity antibodies.     

Antibiotic resistance in Enterotoxigenic and non-enterotoxigenic Escherichia coli.

Antibiotic disk susceptibility tests were done on 220 strains of Escherichia coli belonging to serotypes reported in the literature to be associated with the production of enterotoxin. A total of 128 (58%) were resistant to one or more antibiotics, sulfa drugs, or chemotherapeutic agents. An analysis of these strains revealed primary, secondary, and tertiary drug resistance patterns that indicated a selective pattern in the formation of multiple drug resistance in E. coli. Resistances to certain antibiotics were more likely to occur in pairs and triads (secondary resistance patterns) that were often combined or coexisted in a single strain of E. coli to produce tertiary drug resistance patterns, conferring drug resistance to five or six different antibiotics. Among enterotoxin-associated serotypes, single and multiple drug resistance was less frequently associated with enterotoxin-produced strains than with strains from the same serotype that were not enterotoxigenic. Within the enterotoxigenic E. coli, single and multiple resistance to antibiotics was more frequent in strains producing only heat-stable enterotoxin (ST) than in strains producing only heat-labile enterotoxin (LT) or both. The number of resistances to different antibiotics per resistant strain averaged approximately 1.4 for LT plus ST or LT strains, and 3.9 for ST strains and nonenterotoxigenic strains. Phenotypic characterization of 170 strains for four usually plasmid-mediated characteristics showed that the number of antibiotics to which a strain was directly resistant varied with the type and number of plasmid-mediated characteristics present.     

Memory B Cell and Other Immune Responses in Children Receiving Two Doses of an Oral Killed Cholera Vaccine Compared to Responses following Natural Cholera Infection in Bangladesh

Current oral cholera vaccines induce lower protective efficacy and shorter duration of protection against cholera than wild-type infection provides, and this difference is most pronounced in young children. Despite this, there are limited data comparing immune responses in children following wild-type disease versus vaccination, especially with regard to memory responses associated with long-term immunity. Here, we report a comparison of immune responses in young children (2 to 5 years of age; n = 20) and older children (6 to 17 years of age; n = 20) given two doses of an oral killed cholera vaccine containing recombinant cholera toxin B subunit (CtxB) 14 days apart and compare these responses to those induced in similarly aged children recovering from infection with Vibrio cholerae O1 Ogawa in Bangladesh. We found that the two vaccine groups had comparable vibriocidal and lipopolysaccharide (LPS)-specific plasma antibody responses. Vaccinees developed lower levels of IgG memory B cell (MBC) responses against CtxB but no significant MBC responses against LPS. In contrast, children recovering from natural cholera infection developed prominent LPS IgG and IgA MBC responses, as well as CtxB IgG MBC responses. Plasma LPS IgG, IgA, and IgM responses, as well as vibriocidal responses, were also significantly higher in children following disease than after vaccination. Our findings suggest that acute and memory immune responses following oral cholera vaccination in children are significantly lower than those observed following wild-type disease, especially responses targeting LPS. These findings may explain, in part, the lower efficacy of oral cholera vaccination in children.     

Oral Immunization with a Salmonella typhimurium Vaccine Vector Expressing Recombinant Enterotoxigenic Escherichia coli K99 Fimbriae Elicits Elevated Antibody Titers for Protective Immunity

Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves. In an effort to develop a safe and effective vaccine for the prevention of F5⁺ ETEC infections, a balanced lethal asd⁺ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd⁺. Introduction of this plasmid into an attenuated Salmonella typhimurium Δaro Δasd strain, H683, resulted in strain AP112, which stably expresses E. coli K99 fimbriae. A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks. IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer’s patches (PP), and spleens of vaccinated and control BALB/c mice. This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen. These antibodies were important for protective immunity. One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector. These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.     

The Oral,Live Attenuated Enterotoxigenic Escherichia coli Vaccine ACE527 Reduces the Incidence and Severity of Diarrhea in a Human Challenge Model of Diarrheal Disease

An oral, live attenuated, three-strain recombinant bacterial vaccine, ACE527, was demonstrated to generate strong immune responses to colonization factor and toxin antigens of enterotoxigenic Escherichia coli (ETEC) in human volunteers. The vaccine was safe and well tolerated at doses of up to 10¹¹ CFU, administered in each of two doses given 21 days apart. These observations have now been extended in a phase 2b study with a total of 70 subjects. Fifty-six of these subjects were challenged 28 days after the second dose of vaccine with the highly virulent ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 to obtain preliminary indicators of efficacy against disease and to support further development of the vaccine for both travelers and infants in countries where ETEC is endemic. The vaccine had a significant impact on intestinal colonization by the challenge strain, as measured by quantitative fecal culture 2 days after challenge, demonstrating the induction of a functional immune response to the CFA/I antigen. The incidence and severity of diarrhea were also reduced in vaccinees as measured by a number of secondary and ad hoc endpoints, although the 27% reduction seen in the primary endpoint, moderate to severe diarrhea, was not statistically significant. Together, these observations support the hypothesis that the ACE527 vaccine has a dual mode of action, targeting both colonization factors and the heat-labile enterotoxin (LT), and suggest that it should be further developed for more advanced trials to evaluate its impact on the burden of ETEC disease in field settings.     

Human Leukocyte Antigens and Cellular Immune Responses to Anthrax Vaccine Adsorbed

Interindividual variations in vaccine-induced immune responses are in part due to host genetic polymorphisms in the human leukocyte antigen (HLA) and other gene families. This study examined associations between HLA genotypes, haplotypes, and homozygosity and protective antigen (PA)-specific cellular immune responses in healthy subjects following immunization with Anthrax Vaccine Adsorbed (AVA). While limited associations were observed between individual HLA alleles or haplotypes and variable lymphocyte proliferative (LP) responses to AVA, analyses of homozygosity supported the hypothesis of a “heterozygote advantage.” Individuals who were homozygous for any HLA locus demonstrated significantly lower PA-specific LP than subjects who were heterozygous at all eight loci (median stimulation indices [SI], 1.84 versus 2.95, P = 0.009). Similarly, we found that class I (HLA-A) and class II (HLA-DQA1 and HLA-DQB1) homozygosity was significantly associated with an overall decrease in LP compared with heterozygosity at those three loci. Specifically, individuals who were homozygous at these loci had significantly lower PA-specific LP than subjects heterozygous for HLA-A (median SI, 1.48 versus 2.13, P = 0.005), HLA-DQA1 (median SI, 1.75 versus 2.11, P = 0.007), and HLA-DQB1 (median SI, 1.48 versus 2.13, P = 0.002) loci, respectively. Finally, homozygosity at an increasing number (≥4) of HLA loci was significantly correlated with a reduction in LP response (P < 0.001) in a dose-dependent manner. Additional studies are needed to reproduce these findings and determine whether HLA-heterozygous individuals generate stronger cellular immune response to other virulence factors (Bacillus anthracis LF and EF) than HLA-homozygous subjects.     

Immune Responses to the O-Specific Polysaccharide Antigen in Children Who Received a Killed Oral Cholera Vaccine Compared to Responses following Natural Cholera Infection in Bangladesh

Current oral cholera vaccines induce lower levels of protective efficacy and shorter durations of protection in young children than in adults. Immunity against cholera is serogroup specific, and immune responses to Vibrio cholerae lipopolysaccharide (LPS), the antigen that mediates serogroup-specific responses, are associated with protection against disease. Despite this, responses against V. cholerae O-specific polysaccharide (OSP), a key component of the LPS responsible for specificity, have not been characterized in children. Here, we report a comparison of polysaccharide antibody responses in children from a region in Bangladesh where cholera is endemic, including infants (6 to 23 months, n = 15), young children (24 to 59 months, n = 14), and older children (5 to 15 years, n = 23) who received two doses of a killed oral cholera vaccine 14 days apart. We found that infants and young children receiving the vaccine did not mount an IgG, IgA, or IgM antibody response to V. cholerae OSP or LPS, whereas older children showed significant responses. In comparison to the vaccinees, young children with wild-type V. cholerae O1 Ogawa infection did mount significant antibody responses against OSP and LPS. We also demonstrated that OSP responses correlated with age in vaccinees, but not in cholera patients, reflecting the ability of even young children with wild-type cholera to develop OSP responses. These differences might contribute to the lower efficacy of protection rendered by vaccination than by wild-type disease in young children and suggest that efforts to improve lipopolysaccharide-specific responses might be critical for achieving optimal cholera vaccine efficacy in this younger age group.     

Escherichia coli and Staphylococcus aureus Elicit Differential Innate Immune Responses following Intramammary Infection

Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1β (IL-1β), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.     

Triggering of T Cell-Mediated Immune Responses by Allogenic Tumor Cell Vaccine in Patients with Oral Cancer

We investigated the immunomodulatory activity of allogenic whole tumor cell vaccine in oral cancer patients in vitro by two-color flow cytometry. Vaccine treatment significantly increased the expression of CD69 and HLA-DR in CD3⁺ T-cell subsets. The frequency of Interferon-gamma and Interleukin (IL)-2 expressing CD4⁺/CD8⁺ T-cell subsets was significantly higher with a concomitant reduction in IL-4 and IL-10 expressing T-cells in the vaccine treated group as compared with the untreated controls. Vaccine treatment significantly increased T-cell receptor (TCR), Vβ3, Vβ5 and Vβ8 usage. The results indicate that the allogenic whole tumor cell vaccine is able to trigger T-cell mediated immunity in patients with intraoral squamous cell carcinoma.