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Summary Dietary emulsifiers carboxymethylcellulose (CMC) and polysorbate-80 (P80) disturb gut microbiota, promoting chronic inflammation. Mice with minimal microbiota are protected against emulsifiers’ effects, leading us to hypothesize that these compounds might provoke select pathobionts to promote inflammation. Gnotobiotic wild-type (WT) and interleukin-10 (IL-10)−/− mice were colonized with Crohn’s-disease-associated adherent-invasive E. coli (AIEC) and subsequently administered CMC or P80. AIEC colonization of GF and altered Schaedler flora (ASF) mice results in chronic intestinal inflammation and metabolism dysregulations when consuming the emulsifier. In IL-10−/− mice, AIEC mono-colonization results in severe intestinal inflammation in response to emulsifiers. Exposure of AIEC to emulsifiers in vitro increases its motility and ability to adhere to intestinal epithelial cells. Transcriptomic analysis reveals that emulsifiers directly induce expression of clusters of genes that mediate AIEC virulence and promotion of inflammation. To conclude, emulsifiers promote virulence and encroachment of pathobionts, providing a means by which these compounds may drive inflammation in hosts carrying such bacteria., Graphical Abstract, Highlights • Dietary emulsifiers alter the intestinal microbiota, promoting chronic inflammation • Select pathobionts are required to mediate the detrimental effects of emulsifiers • Emulsifiers directly induce the expression of bacterial virulence genes • Microbiota-based dietary intervention appears warranted, Through gnotobiotic and molecular approaches, Viennois et al. demonstrate that in mice, the ability of a dietary emulsifier to promote chronic intestinal inflammation and colitis-associated cancer is mediated by the ability of these compounds to directly induce the expression of clusters of genes that mediate virulence of a select pathobiont.

Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni (CJ), and Shigella sp. are major causes of bacterial diarrhea worldwide, but there are no licensed vaccines against any of these pathogens. Most current approaches to ETEC vaccines are based on recombinant proteins that are involved in virulence, particularly adhesins. In contrast, approaches to Shigella and CJ vaccines have included conjugate vaccines in which Shigella lipopolysaccharides (LPS) or CJ capsule polysaccharides are chemically conjugated to proteins. We have explored the feasibility of developing a multi-pathogen vaccine by using ETEC proteins as conjugating partners for CJ and Shigella polysaccharides. We synthesized three vaccines in which two CJ polysaccharides were conjugated to two recombinant ETEC adhesins based on CFA/I (CfaEB) and CS6 (CssBA), and LPS from Shigella flexneri was also conjugated to CfaEB. The vaccines were immunogenic in mice as monovalent, bivalent and trivalent formulations. Importantly, functional antibodies capable of inducing hemaglutination inhibition (HAI) of a CFA/I expressing ETEC strain were induced in all vaccines containing CfaEB. These data suggest that conjugate vaccines could be a platform for a multi-pathogen, multi-serotype vaccine against the three major causes of diarrheal disease worldwide.

Campylobacter jejuni infections are a leading cause of bacterial food-borne diarrhoeal illness worldwide, and Campylobacter infections in children are associated with stunted growth and therefore long-term deficits into adulthood. Despite this global impact on health and human capital, how zoonotic C. jejuni responds to the human host remains unclear. Unlike other intestinal pathogens, C. jejuni does not harbour pathogen-defining toxins that explicitly contribute to disease in humans. This makes understanding Campylobacter pathogenesis challenging and supports a broad examination of bacterial factors that contribute to C. jejuni infection. Here, we use a controlled human infection model to characterize C. jejuni transcriptional and genetic adaptations in vivo, along with a non-human primate infection model to validate our approach. We found that variation in 11 genes is associated with either acute or persistent human infections and includes products involved in host cell invasion, bile sensing and flagella modification, plus additional potential therapeutic targets. In particular, a functional version of the cell invasion protein A (cipA) gene product is strongly associated with persistently infecting bacteria and we identified its biochemical role in flagella modification. These data characterize the adaptive C. jejuni response to primate infections and suggest therapy design should consider the intrinsic differences between acute and persistently infecting bacteria. In addition, RNA sequencing revealed conserved responses during natural host commensalism and human infections. Thirty-nine genes were differentially regulated in vivo across hosts, lifestyles and C. jejuni strains. This conserved in vivo response highlights important C. jejuni survival mechanisms such as iron acquisition and evasion of the host mucosal immune response. These advances highlight pathogen adaptability across host species and demonstrate the utility of multidisciplinary collaborations in future clinical trials to study pathogens in vivo.

Background Campylobacter species are a leading cause of diarrheal disease globally with significant morbidity. Primary prevention efforts have yielded limited results. Rifaximin chemoprophylaxis decreases rates of travelers' diarrhea and may be suitable for high-risk persons. We assessed the efficacy of rifaximin in the controlled human infection model for Campylobacter jejuni. Methods Twenty-eight subjects were admitted to an inpatient facility and randomized to a twice-daily dose of 550 mg rifaximin or placebo. The following day, subjects ingested 1.7 × 105 colony-forming units of C. jejuni strain CG8421. Subjects continued prophylaxis for 3 additional days, were followed for campylobacteriosis for 144 hours, and were subsequently treated with azithromycin and ciprofloxacin. Samples were collected to assess immunologic responses to CG8421. Results There was no difference (P = 1.0) in the frequency of campylobacteriosis in those receiving rifaximin (86.7%) or placebo (84.6%). Additionally, there were no differences in the clinical signs and symptoms of C. jejuni infection to include abdominal pain/cramps (P = 1.0), nausea (P = 1.0), vomiting (P = .2), or fever (P = 1.0) across study groups. Immune responses to the CG8421 strain were comparable across treatment groups. Conclusions Rifaximin did not prevent campylobacteriosis in this controlled human infection model. Given the morbidity associated with Campylobacter infection, primary prevention efforts remain a significant need. Clinical trials registration NCT02280044.

Introduction Endemic Burkitt's lymphoma (eBL) is associated with Epstein–Barr virus and repeated malaria infections. A defining feature of eBL is the translocation of the c‐myc oncogene to the control of the immunoglobulin promoter. Activation‐induced cytidine deaminase (AID) has been shown to be critical for this translocation. Malaria infection induces AID in germinal center B cells, but whether malaria infection more broadly affects AID activation in extrafollicular B cells is unknown. Methods We either stimulated purified B cells from AID‐green fluorescence protein (GFP) reporter mice or infected AID‐GFP mice with Plasmodium chabaudi, AID fluorescence was monitored in B cell subsets by flow cytometry. Results In vitro analysis of B cells from these mice revealed that CpG (a Toll‐like receptor 9 ligand) was a potent inducer of AID in both mature and immature B cell subsets. Infection of AID‐GFP mice with Plasmodium chabaudi demonstrated that AID expression occurs in transitional and marginal zone B cells during acute malaria infection. Transitional B cells were also capable of differentiating into antibody secreting cells when stimulated in vitro with CpG when isolated from a P. chabaudi‐infected mouse. Conclusions These data suggest that P. chabaudi is capable of inducing AID expression in B cell subsets that do not participate in the germinal center reaction, suggesting an alternative role for malaria in the etiology of eBL.

SummaryBackground Patients with Crohn's disease (CD) have serologic responses to various microbial antigens. Serologic markers are associated with aggressive forms of disease and can be detected before onset of symptoms. Their utility in pre-clinical disease or prediction of complicated disease course before diagnosis is unclear. Aim To evaluate the pattern of serologic anti-microbial antibodies long prior to diagnosis and the subsequent risk of complicated Crohn's disease at diagnosis. Methods Sera from 100 US military personnel with Crohn's disease were obtained from the Department of Defense Serum Repository. For each patient, four samples were obtained at different time points before and around diagnosis, and were tested for 6 microbiota-directed antibodies (ASCA-IgA, ASCA-IgG, anti-OmpC, anti-CBir1, anti-A4-Fla2 and anti-FlaX). Associations between the presence and accumulation of Crohn's disease anti-microbial antibodies before diagnosis and with the later development of complications were evaluated. Results Overall, 65 patients were positive for at least one Crohn's disease associated anti-microbial antibody in the earliest available sample, at a median of 6 years before Crohn's disease diagnosis (interquartile range, 5.6–8.2). The number of positive anti-microbial antibodies increased up to the time of Crohn's disease diagnosis. Complicated disease developed around the time of diagnosis in 24 patients. The proportion of positive antimicrobial antibodies before diagnosis was higher in patients with complicated vs. noncomplicated Crohn's disease. There was an inverse relationship between the time to first complication and the magnitude of serologic response before diagnosis. Conclusion The presence and accumulation of circulating anti-microbial antibodies years before Crohn's disease diagnosis was associated with complicated Crohn's disease at or shortly after diagnosis.

Background There is a recognized need for biological markers to facilitate diagnoses of irritable bowel syndrome (IBS) and to distinguish it from other functional and organic disorders. As postinfectious IBS (PI-IBS) is believed to account for as many as one third of all IBS cases, here we sought to identify differences in specific cytokines and serologic responses across patients with idiopathic IBS and PI-IBS and healthy controls. Methods At total of 120 US military personnel were identified from the Defense Medical Surveillance System-based International Classification of Diseases, 9th Revision, Clinical Modification (ICD9-CM) codes recorded during medical encounters and were grouped based on infectious gastroenteritis (IGE) episode (Shigella, Campylobacter, Salmonella, or an unspecified pathogen) followed by IBS, IBS without antecedent IGE, or IGE without subsequent IBS within 2 years of the IGE exposure. Sera from subjects were assayed for cytokine levels and antibodies against a panel of microbiome antigens. Results In total, 10 of 118 markers considered were shown to differ between IBS patients and healthy controls, including cytokines interleukin-6 (IL-6), IL-8, IL-1β, and macrophage inflammatory protein-1β (MIP-1β), as well as antibody responses to microbial antigens. Antimicrobial antibody response profiles also differed between PI-IBS cases compared with IBS cases without an antecedent episode of acute IGE. Comparisons also suggest that immunoglobulin A (IgA) and IgG profiles may point to pathogen-specific origins among PI-IBS cases. Conclusion Taken together, these results provide further evidence as to the molecular distinctness of classes of IBS cases and that serum biomarkers may prove useful in elucidating their pathobiological pathways.

High rates of coinfection occur in malaria endemic regions, leading to more severe disease outcomes. Understanding how coinfecting pathogens influence the immune system is important in the development of treatment strategies that reduce morbidity and mortality. Using the Plasmodium chabaudi mouse model of malaria and immunization with model Ags that are either T-dependent (4-hydroxy-3-nitrophenyl [NP]-OVA) or T-independent (NP-Ficoll), we analyzed the effects of acute malaria on the development of humoral immunity to secondary Ags. Total Ig and IgG1 NP–specific Ab responses to NP-OVA were significantly decreased in the P. chabaudi–infected group compared with the uninfected group, whereas NP-specific IgG2c Ab was significantly increased in the P. chabaudi–infected group. In contrast, following injection with T-independent NP-Ficoll, the P. chabaudi–infected group had significantly increased NP-specific total Ig, IgM, and IgG2c Ab titers compared with controls. Treatment with anti–IFN-γ led to an abrogation of the NP-specific IgG2c Ab induced by P. chabaudi infection but did not affect other NP-specific Ab isotypes or titers. IFN-γ depletion also increased the percentage of plasma cells in both P. chabaudi–infected and uninfected groups but decreased the percentage of B cells with a germinal center (GC) phenotype. Using immunofluorescent microscopy, we were able to detect NP+ GCs in the spleens of noninfected mice, but there were no detectible NP+ GCs in mice infected with P. chabaudi. These data suggest that during P. chabaudi infection, there is a shift toward an extrafollicular Ab response that could be responsible for decreased Ab responses to secondary T-dependent Ags.

CD4 T cells, and especially T follicular helper cells, are critical for the generation of a robust humoral response to an infection or vaccination. Importantly, immunosenescence affects CD4 T cell function and the accumulation of intrinsic defects decreases the cognate helper functions of these cells. However, much less is known about the contribution of the aged microenvironment to this impaired CD4 T cell response. In the current study, we have employed a preclinical model to determine whether the aged environment contributes to the defects in CD4 T cell functions with aging. Using an adoptive transfer model in mice, we demonstrate for the first time that the aged microenvironment negatively impacts at least three steps of the CD4 T cell response to antigenic stimulation. First, the recruitment of CD4 T cells to the spleen is reduced in aged compared to young hosts, which correlates with dysregulated chemokine expression in the aged organ. Second, the priming of CD4 T cells by DCs is reduced in aged compared to young mice. Finally, naive CD4 T cells show a reduced transition to a T follicular helper cell phenotype in the aged environment, which impairs the subsequent generation of germinal centers. These studies have provided new insights into how aging impacts the immune system and how these changes influence the development of immunity to infections or vaccinations.

Immunosenescence influences many components of the immune system. Most importantly, profound changes in T cell function are evident in older individuals. The impact of aging on specific T cell subsets has been difficult to examine, but recent advances in murine model systems and new insights into T cell function have allowed for the more precise examination of how T cell responses change with aging. Importantly, recent studies have shown that age-related enhancement of both Th17 generation and regulatory T cell function may contribute to significant changes in immune function. In this review, we summarize the current views on how aging influences the factors that impact T cell function and how this can affect the immune response to infections, vaccinations, and tumors.

This study showed no significant difference in the development of the primary endpoint, Campylobacteriosis, between subjects treated with rifaximin or placebo. However, the difference in mean total loose stool output (mL) is significantly reduced in the rifaximin group, and within this group a reduction in the maximum volume of loose stools in 24 hours was also observed. Therefore, whilst rifaximin does not protect against Campylobacteriosis, this objective data relating to stool volume suggests that fluid loss is reduced, and therefore should be considered when evaluating the efficacy of rifaximin in reducing the severity of the burden of disease in all cause bacterial travelers’ diarrhea. Based on the high diarrhea attack rates noted in this human challenge model, the efficacy of rifaximin chemoprophylaxis against campylobacteriosis in a field setting requires further evaluation. Clayton D. Harro1, Joanna E. Rimmer2,3,4, David A. Sack1, Kawsar R. Talaat1, Ramiro L. Gutierrez2, Barbara DeNearing1, Chad K. Porter2, Jessica Brubaker1, Alexander C. Maue2, Renee M. Laird2, Frederic Poly2, Patricia Guerry2, Kayla Jaep2, Ashley Alcala2, David R. Tribble5 and Mark S. Riddle2. 1Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, 2Enteric Diseases Department, Naval Medical Research Center, Silver Spring, MD, 3School of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom, 4Academic Department of Military Medicine, Royal Centre for Defence Medicine (Academia and Research), Medical Directorate, Joint Medical Command, ICT Centre, Birmingham Research Park, B15 2SQ Birmingham, UK, 5Infectious Disease Clinical Research Program (IDCRP), Uniformed Services University of the Health Sciences, Bethesda, MD Does Rifaximin Chemoprophylaxis Prevent Campylobacteriosis in the Human Challenge Model?

DNA vaccination is known to elicit robust cellular and humoral responses to encoded antigen. The co-administration of costimulatory molecules CD80 (B7-1), CD86 (B7-2) and CD154 (CD40L) has been shown to enhance immune responses in several murine models. The role of specific costimulatory molecules in non-rodent species remains incompletely characterized. In these studies, we demonstrate that the co-administration of CD80 and CD86, but not CD154, to an existing candidate subunit DNA vaccine (ESAT-6) against bovine tuberculosis, enhances protection after aerosol challenge with virulent Mycobacterium bovis. Additionally, we have shown that vaccination with M. bovis BCG is protective against tuberculosis following aerosol challenge in cattle. Two independent trials were conducted in cattle to determine the adjuvant effect of encoded antigen + CD80/CD86 and directly compare the adjuvant activities of CD80/CD86 to those of CD154. Co-administration of either CD80/CD86 or CD154 enhanced ESAT-6-specific IFN-gamma responses as compared to animals vaccinated with ESAT-6 DNA alone. However, following aerosol challenge, only animals vaccinated with CD80/CD86 possessed decreased pathology of the lungs and associated lymph nodes, as measured by gross examination, radiographic lesion morphometry and bacterial recovery. Collectively, these results demonstrate that the co-administration of costimulatory molecules with a protective antigen target enhances bovine immune responses to DNA vaccination, and that CD80/CD86 is superior to CD154 in augmenting DNA vaccine-induced protection in experimental bovine tuberculosis.

Granulysin and NK-lysin are antimicrobial proteins found in the granules of human and swine cytotoxic lymphocytes. A murine counterpart to granulysin has not been identified to date, indicating the importance of additional models to fully characterize the role of granulysin-like molecules in the immune response to infectious disease. Two partial nucleotide sequences corresponding to the complete functional domain of granulysin and NK-lysin were amplified from bovine PBMC mRNA. Following stimulation with phorbol ester and calcium ionophore, expression of the bovine gene was detected in CD3+ T cells, CD4+ T cells, CD8+ T cells, WC1+ γδ T cells, and PBMC depleted of CD3+ T cells, but was absent in CD21+ cells and CD14+ cells. Intracellular flow cytometry and immunoblotting confirmed the presence of protein corresponding to the bovine granulysin homologue in activated T lymphocytes and PBMC. Synthetic human, bovine, and swine peptides corresponding to the C terminus of helix 2 through helix 3 region of granulysin displayed potent antimicrobial activity against Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, and Mycobacterium bovis bacillus Calmette-Guérin. Human and bovine peptides corresponding to helix 2 displayed antimycobacterial activity against M. bovis bacillus Calmette-Guérin. Expression of the bovine gene was detected in laser microscopy-dissected lymph node lesions from an M. bovis-infected animal. The identification of a biologically active bovine homologue to granulysin demonstrates the potential of the bovine model in characterizing the role of granulysin in the immune response to a variety of infectious agents.

Campylobacter jejuni is a major cause of diarrheal disease and results in high levels of morbidity and economic loss in both industrialized and developing regions of the world. To date, prior vaccine approaches have failed to confer protection against this enteric pathogen. Key challenges to the development of a practical Campylobacter vaccine for human use include a lack of understanding of Campylobacter pathogenesis and well-defined immune correlates of protection. With the discovery that C. jejuni expresses a capsule polysaccharide associated with virulence, a conjugate vaccine approach is currently being evaluated. Conjugate vaccines have been successfully developed and implemented against other invasive mucosal pathogens including Streptococcus pneumoniae, Neisseria meningitidis, and Hemophilus influenzae. Furthermore, Shigella-based conjugate vaccines based on lipopolysaccharide have shown promising results in field trials. A prototype C. jejuni conjugate vaccine is currently entering human testing.

Campylobacter jejuni is a major cause of bacterial diarrheal disease worldwide. The organism is characterized by a diversity of polysaccharide structures, including a polysaccharide capsule. Most C. jejuni capsules are known to be decorated nonstoichiometrically with methyl phosphoramidate (MeOPN). The capsule of C. jejuni 81-176 has been shown to be required for serum resistance, but here we show that an encapsulated mutant lacking the MeOPN modification, an mpnC mutant, was equally as sensitive to serum killing as the nonencapsulated mutant. A nonencapsulated mutant, a kpsM mutant, exhibited significantly reduced colonization compared to that of wild-type 81-176 in a mouse intestinal colonization model, and the mpnC mutant showed an intermediate level of colonization. Both mutants were associated with higher levels of interleukin 17 (IL-17) expression from lamina propria CD4 + cells than from cells from animals infected with 81-176. In addition, reduced levels of Toll-like receptor 4 (TLR4) and TLR2 activation were observed following in vitro stimulation of human reporter cell lines with the kpsM and mpnC mutants compared to those with wild-type 81-176. The data suggest that the capsule polysaccharide of C. jejuni and the MeOPN modification modulate the host immune response.

Campylobacter jejuni remains a major cause of bacterial diarrhea worldwide and is associated with numerous sequelae, including Guillain Barre Syndrome, inflammatory bowel disease, reactive arthritis, and irritable bowel syndrome. C. jejuni is unusual for an intestinal pathogen in its ability to coat its surface with a polysaccharide capsule (CPS). These capsular polysaccharides vary in sugar composition and linkage, especially those involving heptoses of unusual configuration and O-methyl phosphoramidate linkages. This structural diversity is consistent with CPS being the major serodeterminant of the Penner scheme, of which there are 47 C. jejuni serotypes. Both CPS expression and expression of modifications are subject to phase variation by slip strand mismatch repair. Although capsules are virulence factors for other pathogens, the role of CPS in C. jejuni disease has not been well defined beyond descriptive studies demonstrating a role in serum resistance and for diarrhea in a ferret model of disease. However, perhaps the most compelling evidence for a role in pathogenesis are data that CPS conjugate vaccines protect against diarrheal disease in non-human primates. A CPS conjugate vaccine approach against this pathogen is intriguing, but several questions need to be addressed, including the valency of CPS types required for an effective vaccine. There have been numerous studies of prevalence of CPS serotypes in the developed world, but few studies from developing countries where the disease incidence is higher. The complexity and cost of Penner serotyping has limited its usefulness, and a recently developed multiplex PCR method for determination of capsule type offers the potential of a more rapid and affordable method. Comparative studies have shown a strong correlation of the two methods and studies are beginning to ascertain CPS-type distribution worldwide, as well as examination of correlation of severity of illness with specific CPS types.

Age-related declines in humoral responses contribute to the reduced efficacy of vaccines in older populations. Using an adoptive transfer model, we have shown that age-related intrinsic declines in CD4 T cell function contribute significantly to the reduced humoral responses observed with aging, resulting in reduced B cell expansion and differentiation as well as reduced IgG production. In this current study, we show that the helper function of aged CD4 T cells can be enhanced using a TLR-binding adjuvant or an adjuvant containing proinflammatory (PI) cytokines. The helper function of aged CD4 T cells was also enhanced when PI cytokines were added during in vitro CD4 effector generation. Enhanced helper activity resulted in improved expansion and differentiation of B cells and affinity maturation of IgG. PI cytokines also induced significant production of effector cytokines, including IL-4, IFN-γ, IL-17, and IL-21, by both young and aged CD4 T cells. Importantly, we also show that proinflammatory adjuvants can significantly enhance the humoral response in intact aged animals. We propose that one of the mechanisms involved in the ability of adjuvants to enhance both young and aged T cell responses includes driving multifaceted T cell differentiation and production of multiple cytokines by responding CD4 T cells.

It is well established that increasing age is associated with a decreased capacity of the immune system to mediate effective immune responses to vaccination and invading pathogens. Because of the inherent limitations of conducting experiments in humans, much of what we have learned is owed to the utility of experimental mouse models of aging. Recent studies performed in the mouse have demonstrated mechanisms responsible for age-related declines in the function of CD4(+) and CD8(+) cells. This review describes key findings regarding age-related defects in T-cell function and discusses the impact these defects have on vaccine efficacy and immunity.

Age-related declines in immune function are associated with reduced humoral responses following vaccination or infection. Central to this defect is a decline in naïve CD4+ T cell function. In this review, we discuss factors intrinsic and extrinsic to the CD4+ T cell compartment that affect host immunity and propose means by which deficient CD4+ T cell function can be fully restored in the aged.

Understanding how aging impacts the function of memory CD4 T cells is critical for designing effective vaccines. Our studies show that immunological memory generated during youth functions well into old age, whereas that generated later in life functions poorly. This is the result of declines in the function of naive CD4 T cells from aged individuals and contributes to reduced efficacy of vaccines in the elderly. To begin to identify the cause of this defect, we examined the function of memory T cells generated from bone marrow precursor cells (BMPC) from young or aged mice in young hosts. In two different models, memory cells derived from young and aged BMPC exhibit good ex vivo and in vivo function. Importantly, memory CD4 T cells generated from aged BMPC exhibit potent cognate helper function for humoral responses, which are critical for effective immunization. These results indicate that there are no apparent age-related intrinsic defects in BMPC with regards to generation of functional memory T cells.

The potency of genetic immunization observed in the mouse has demonstrated the utility of DNA vaccines to induce cell-mediated and humoral immune responses. However, it has been relatively difficult to generate comparable responses in non-rodent species. The use of molecular adjuvants may increase the magnitude of these suboptimal responses. In this study, we demonstrate that the co-administration of plasmid-encoded GM-CSF and CD80/CD86 with a novel ESAT-6:CFP10 DNA vaccine against bovine tuberculosis enhances antigen-specific cell-mediated immune responses. ESAT-6:CFP10+GM-CSF+CD80/CD86 DNA vaccinated animals exhibited significant (p

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide; however, our understanding of the human immune response to C. jejuni infection is limited. A previous human challenge model has shown that C. jejuni elicits IFNγ production by peripheral blood mononuclear cells, a response associated with protection from clinical disease following re-infection. In this study, we investigate T lymphocyte profiles associated with campylobacteriosis using specimens from a new human challenge model in which C. jejuni-naïve subjects were challenged and re-challenged with C. jejuni CG8421. Multiparameter flow cytometry was used to investigate T lymphocytes as a source of cytokines, including IFNγ, and to identify cytokine patterns associated with either campylobacteriosis or protection from disease. Unexpectedly, all but one subject evaluated re-experienced campylobacteriosis after re-challenge. We show that CD4+ T cells make IFNγ and other pro-inflammatory cytokines in response to infection; however, multifunctional cytokine response patterns were not found. Cytokine production from peripheral CD4+ T cells was not enhanced following re-challenge, which may suggest deletion or tolerance. Evaluation of alternative paradigms or models is needed to better understand the immune components of protection from campylobacteriosis.

Cell-mediated immune responses are critical for protective immunity to mycobacterial infections. Recent progress in defining mycobacterial antigens has determined that region of difference 1 (RD1) gene products induce strong T-cell responses, particularly the early secretory antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP10). However, comprehensive analysis of the immune response towards these antigens is incompletely characterized. To evaluate recall responses to ESAT-6 and CFP10, peripheral blood mononuclear cells fromM. bovis-infected cattle were stimulated in vitro with a recombinant ESAT-6 (rESAT-6)-CFP10 fusion protein and compared to responses induced byM. bovis-derived purified protein derivative. Following antigenic stimulation, activation marker expression was evaluated. Significant proliferative responses (P< 0.05) were evident in CD4+, CD8+, immunoglobulin M-positive, and CD172a+cell fractions after 6 days of culture. Expression of CD25 and CD26 was increased (P< 0.05) on CD4+, CD8+, and γδ T-cell-receptor-positive cells. CD4+and CD8+cells also exhibited significant changes (P< 0.05) in expression of CD45 isoforms. Using a flow cytometry-based proliferation assay, it was determined that CD45R expression is downregulated (P< 0.05) and that CD45RO expression is upregulated (P< 0.05) on proliferating (i.e., activated) CD4+cells. Collectively, data indicate that recall immune responses directed toward the rESAT-6-CFP10 fusion protein or purified protein derivative are comparable and that recall to mycobacterial antigens correlates with a CD45RO+phenotype.

Setting : 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) is a potent modulator of immune responses and may be beneficial in the treatment of tuberculosis. Recent evidence suggest that 1,25(OH) 2 D 3 may affect T-dependent responses in cattle; however, mechanisms by which this vitamin modulates activation of bovine T cells are unclear. Objective : Determine the effects of 1,25(OH) 2 D 3 on the expression of CD25, CD44, and CD62L by bovine T cell subsets proliferating in response to antigen stimulation. Design : Antigen-specific recall responses of Mycobacterium bovis bacille Calmette–Guerin (BCG) vaccinated cattle were used as a model system to evaluate effects of 1,25(OH) 2 D 3 on the proliferation and activation of bovine T cell subsets. Results : CD4 + and γδ TCR + cells were the predominant T cell subsets responding to soluble crude M. bovis -derived antigens (i.e., purified protein derivative and a BCG whole cell sonicate) by proliferation and activation-induced alterations in phenotype. These subsets exhibited increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi upon antigen stimulation. Addition of 1,25(OH) 2 D 3 inhibited proliferation of CD4 + cells and decreased the expression of CD44 on responding (i.e., proliferating) CD4 + and γδ TCR + cells. Conclusion : These findings suggest that the production of 1,25(OH) 2 D 3 by macrophages within tuberculous lesions would inhibit proliferation and CD44 expression by co-localized CD4 + and γδ TCR + cells.

The humoral response in aged individuals is significantly reduced compared to young, thus contributing to the reduced efficacy of vaccines commonly seen in older populations. Using an adoptive transfer model, we have shown that age-related intrinsic declines in the function of naïve CD4 T cells from aged donors contribute significantly to reduced humoral responses. This decline leads to reduced B cell expansion and differentiation as well as reduced IgG production. In this study, we show that the helper function of aged CD4 T cells can be enhanced using a Toll-like receptor (TLR) binding adjuvant or an adjuvant containing proinflammatory (PI) cytokines. In addition, we show that the helper function of aged CD4 T cells can be enhanced by adding PI cytokines during in vitro effector generation. We observed enhanced expansion and differentiation of B cells and affinity maturation of IgG. Importantly, we found that PI cytokines also induced significant production of a variety of effector cytokines, including IL-4, IFNg, IL-17, and IL-21, by both young and aged CD4 T cells. The use of PI adjuvants can also improve humoral responses in intact aged animals. We propose that one of the mechanisms involved in the ability of adjuvants to enhance both young and aged T cell responses includes driving multifaceted T cell differentiation and production of multiple cytokines by responding T cells. This work funded by NIH grants AG21054 and AG02160 to LH.

In vivo, age-related defects in CD4+ T cell function lead to reduced expansion, IL-2 production, NF-κB activation and B cell helper activity. These defects are significant as they result in reduced vaccine efficacy in the elderly. To date, relatively little is known about what factors contribute to these age-related defects in vivo. By using real time-PCR (RT-PCR) and intracellular flow cytometry, we have identified candidate cell subsets and molecules that may contribute to the observed immunosuppression. Spleens of young (4 months) and aged (24 months) mice displayed significantly higher expression of interleukin-10 (IL-10) as determined by RT-PCR. Analysis of cell subsets isolated by sorting revealed a significant increase in IL-10 expression in aged CD4+ T cells compared to young. Following intracellular cytokine staining, a statistically significant increase in CD4+IL-10+ cells (~15 fold) was observed in aged (26 months) compared to young (2 months) mice. Aged CD4+ T cells exhibited significantly higher expression of CD25 (~3 fold), GITR (~5 fold) and CTLA-4 (~4 fold) compared to young CD4+ T cells, as well as greater overall numbers of cells expressing each marker. Following intracellular staining, both intact and transgenic CD4+ T cell populations in aged mice displayed greater numbers of Foxp3-expressing cells compared to young populations. Our results demonstrate that in aged mice, several molecules and cells associated with immune regulation are present in significantly higher amounts than in young mice and that future experiments are required to determine the effects of these elements on naïve CD4+ T cell and aged immune responses. This research was supported by NIH grants AG02160 and AG21054.

The elderly exhibit reduced responses to vaccinations, leaving them more susceptible to preventable infectious diseases such as influenza and pneumonia. When the aged are vaccinated, the resulting antibody titers are lower than those seen in younger individuals and the function of the antibodies produced is also diminished. We have examined how reductions in CD4 T cell function contribute to reduced humoral responses, using an adoptive transfer model which allows us to compare identical numbers of antigen-specific naive T cells from young and aged TCR transgenic (Tg) mice. Naive CD4 T cells from aged Tg mice exhibit significantly reduced cognate helper function, leading to reduced B cell expansion and differentiation and reduced antibody production, even in young hosts. The cognate helper function of these aged Tg CD4 T cells can be enhanced significantly by prior effector generation with a cytokine cocktail containing TNFalpha, IL-1 and IL-6 (T16) or by using this cocktail as an adjuvant for immunization. Using this cocktail, enhanced B cell responses, including significantly higher antibody titers, are observed. Gene expression analysis shows that T16 enhances CD40L expression by the aged CD4 T cells, possibly accounting for the enhanced cognate function. Importantly, this cocktail also enhances humoral responses in intact aged animals. These results suggest that the efficacy of vaccines for the elderly can be enhanced by use of appropriate adjuvants. Supported by NIH grants AG21054 and AG02160 to LH.