Oral curcumin ameliorates acute murine campylobacteriosis.

Introduction: Human infections with the food-borne enteropathogen Campylobacter jejuni are responsible for increasing incidences of acute campylobacteriosis cases worldwide. Since antibiotic treatment is usually not indicated and the severity of the enteritis directly correlates with the risk of developing serious autoimmune disease later-on, novel antibiotics-independent intervention strategies with non-toxic compounds to ameliorate and even prevent campylobacteriosis are utmost wanted. Given its known pleiotropic health-promoting properties, curcumin constitutes such a promising candidate molecule. In our actual preclinical placebo-controlled intervention trial, we tested the anti-microbial and anti-inflammatory effects of oral curcumin pretreatment during acute experimental campylobacteriosis. Methods: Therefore, secondary abiotic IL-10-/- mice were challenged with synthetic curcumin via the drinking water starting a week prior oral C. jejuni infection. To assess anti-pathogenic, clinical, immune-modulatory, and functional effects of curcumin prophylaxis, gastrointestinal C. jejuni bacteria were cultured, clinical signs and colonic histopathological changes quantitated, pro-inflammatory immune cell responses determined by in situ immunohistochemistry and intestinal, extra-intestinal and systemic pro-inflammatory mediator measurements, and finally, intestinal epithelial barrier function tested by electrophysiological resistance analysis of colonic ex vivo biopsies in the Ussing chamber. Results and discussion: Whereas placebo counterparts were suffering from severe enterocolitis characterized by wasting symptoms and bloody diarrhea on day 6 post-infection, curcumin pretreated mice, however, were clinically far less compromised and displayed less severe microscopic inflammatory sequelae such as histopathological changes and epithelial cell apoptosis in the colon. In addition, curcumin pretreatment could mitigate pro-inflammatory innate and adaptive immune responses in the intestinal tract and importantly, rescue colonic epithelial barrier integrity upon C. jejuni infection. Remarkably, the disease-mitigating effects of exogenous curcumin was also observed in organs beyond the infected intestines and strikingly, even systemically given basal hepatic, renal, and serum concentrations of pro-inflammatory mediators measured in curcumin pretreated mice on day 6 post-infection. In conclusion, the anti-Campylobacter and disease-mitigating including anti-inflammatory effects upon oral curcumin application observed here highlight the polyphenolic compound as a promising antibiotics-independent option for the prevention from severe acute campylobacteriosis and its potential post-infectious complications.

The ALPK1 pathway drives the inflammatory response to Campylobacter jejuni in human intestinal epithelial cells.

The Gram-negative bacterium Campylobacter jejuni is a major cause of foodborne disease in humans. After infection, C. jejuni rapidly colonizes the mucus layer of the small and large intestine and induces a potent pro-inflammatory response characterized by the production of a large repertoire of cytokines, chemokines, and innate effector molecules, resulting in (bloody) diarrhea. The virulence mechanisms by which C. jejuni causes this intestinal response are still largely unknown. Here we show that C. jejuni releases a potent pro-inflammatory compound into its environment, which activates an NF-κB-mediated pro-inflammatory response including the induction of CXCL8, CXCL2, TNFAIP2 and PTGS2. This response was dependent on a functional ALPK1 receptor and independent of Toll-like Receptor and Nod-like Receptor signaling. Chemical characterization, inactivation of the heptose-biosynthesis pathway by the deletion of the hldE gene and in vitro engineering identified the released factor as the LOS-intermediate ADP-heptose and/or related heptose phosphates. During C. jejuni infection of intestinal cells, the ALPK1-NF-κB axis was potently activated by released heptose metabolites without the need for a type III or type IV injection machinery. Our results classify ADP-heptose and/or related heptose phosphates as a major virulence factor of C. jejuni that may play an important role during Campylobacter infection in humans.

Campylobacter fetus Induced Proinflammatory Response in Bovine Endometrial Epithelial Cells.

Campylobacter fetus subsp. fetus is the causal agent of sporadic abortion in bovines and infertility that produces economic losses in livestock. In many infectious diseases, the immune response has an important role in limiting the invasion and proliferation of bacterial pathogens. Innate immune sensing of microorganisms is mediated by pattern-recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) and induces the secretion of several proinflammatory cytokines, like IL-1ß, TNF-α, and IL-8. In this study, the expression of IL-1ß, TNF-α, IL-8, and IFN-γ in bovine endometrial epithelial cells infected with C. fetus and Salmonella Typhimurium (a bacterial invasion control) was analyzed. The results showed that expression levels of IL-1ß and IL-8 were high at the beginning of the infection and decreased throughout the intracellular period. Unlike in this same assay, the expression levels of IFN-γ increased through time and reached the highest peak at 4 hours post infection. In cells infected with S. Typhimurium, the results showed that IL8 expression levels were highly induced by infection but not IFN-γ. In cells infected with S. Typhimurium or C. fetus subsp. fetus, the results showed that TNF-α expression did not show any change during infection. A cytoskeleton inhibition assay was performed to determine if cytokine expression was modified by C. fetus subsp. fetus intracellular invasion. IL-1ß and IL-8 expression were downregulated when an intracellular invasion was avoided. The results obtained in this study suggest that bovine endometrial epithelial cells could recognize C. fetus subsp. fetus resulting in early proinflammatory response.

A porcine ligated loop model reveals new insight into the host immune response against Campylobacter jejuni.

The symptoms of infectious diarrheal disease are mediated by a combination of a pathogen's virulence factors and the host immune system. Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide due to its near-ubiquitous zoonotic association with poultry. One of the outstanding questions is to what extent the bacteria are responsible for the diarrheal symptoms via intestinal cell necrosis versus immune cell initiated tissue damage. To determine the stepwise process of inflammation that leads to diarrhea, we used a piglet ligated intestinal loop model to study the intestinal response to C. jejuni. Pigs were chosen due to the anatomical similarity between the porcine and the human intestine. We found that the abundance of neutrophil related proteins increased in the intestinal lumen during C. jejuni infection, including proteins related to neutrophil migration (neutrophil elastase and MMP9), actin reorganization (Arp2/3), and antimicrobial proteins (lipocalin-2, myeloperoxidase, S100A8, and S100A9). The appearance of neutrophil proteins also corresponded with increases of the inflammatory cytokines IL-8 and TNF-α. Compared to infection with the C. jejuni wild-type strain, infection with the noninvasive C. jejuni ∆ciaD mutant resulted in a blunted inflammatory response, with less inflammatory cytokines and neutrophil markers. These findings indicate that intestinal inflammation is driven by C. jejuni virulence and that neutrophils are the predominant cell type responding to C. jejuni infection. We propose that this model can be used as a platform to study the early immune events during infection with intestinal pathogens.

Curcumin Mitigates Immune-Induced Epithelial Barrier Dysfunction by Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa, a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni-induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10-/- mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni-induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response-represented by TNF-α, IL-1ß, and IL-6 secretion-was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni-triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients.

Bacteriemia por Campylobacter jejuni en un paciente con infección por VIH en etapa SIDA / Campylobacter jejuni bacteremia in a patient with HIV infection in AIDS stage

Resumen La bacteriemia es una presentación atípica de la infección por Campylobacter jejuni, y es más frecuente en pacientes con inmunodepresión avanzada debido al VIH u otras enfermedades sistémicas. Debido a la terapia anti-retroviral, en las últimas décadas el número de casos ha disminuido. Presentamos el caso de una mujer en situación de calle, con VIH en etapa C3, que cursó con una bacteriemia por C. jejuni durante su hospitalización por una tuberculosis pulmonar. Realizamos una breve revisión de bacteriemia por C. jejuni en pacientes con VIH.

Bacteremia is an atypical presentation of Campylobacter jejuni infection and it is more frequent in patients with advanced inmunodepression due to HIV or other sistemic diseases. Because of the highly active antiretroviral therapy, in the last decades the number of cases had declined. We report a case of a homeless woman with HIV in C3 stage who was diagnosed with the bacteremia during her hospitalization for pulmonary tuberculosis, and a brief review of C. jejuni bacteremia in HIV patients.

In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter.

Campylobacteriosis is a widespread infectious disease, leading to a major health and economic burden. Chickens are considered as the most common infection source for humans. Campylobacter mainly multiplies in the mucus layer of their caeca. No effective control measures are currently available, but passive immunisation of chickens with pathogen-specific maternal IgY antibodies, present in egg yolk of immunised chickens, reduces Campylobacter colonisation. To explore this strategy further, anti-Campylobacter nanobodies, directed against the flagella and major outer membrane proteins, were fused to the constant domains of chicken IgA and IgY, combining the benefits of nanobodies and the effector functions of the Fc-domains. The designer chimeric antibodies were effectively produced in leaves of Nicotiana benthamiana and seeds of Arabidopsis thaliana. Stable expression of the chimeric antibodies in seeds resulted in production levels between 1% and 8% of the total soluble protein. These in planta produced antibodies do not only bind to their purified antigens but also to Campylobacter bacterial cells. In addition, the anti-flagellin chimeric antibodies are reducing the motility of Campylobacter bacteria. These antibody-containing Arabidopsis seeds can be tested for oral passive immunisation of chickens and, if effective, the chimeric antibodies can be produced in crop seeds.

CapC, a Novel Autotransporter and Virulence Factor of Campylobacter jejuni.

Campylobacter jejuni is recognized as an important causative agent of bacterial gastroenteritis in the developed world. Despite the identification of several factors contributing to infection, characterization of the virulence strategies employed by C. jejuni remains a significant challenge. Bacterial autotransporter proteins are a major class of secretory proteins in Gram-negative bacteria, and notably, many autotransporter proteins contribute to bacterial virulence. The aim of this study was to characterize the C. jejuni 81116 C8J_1278 gene (capC), predicted to encode an autotransporter protein, and examine the contribution of this factor to virulence of C. jejuni The predicted CapC protein has a number of features that are consistent with autotransporters, including the N-terminal signal sequence and the C-terminal ß-barrel domain and was determined to localize to the outer membrane. Inactivation of the capC gene in C. jejuni 81116 and C. jejuni M1 resulted in reduced insecticidal activity in Galleria mellonella larvae. Furthermore, C. jejuni capC mutants displayed significantly reduced adherence to and invasion of nonpolarized, partially differentiated Caco-2 and T84 intestinal epithelial cells. Gentamicin treatment showed that the reduced invasion of the capC mutant is primarily caused by reduced adherence to intestinal epithelial cells, not by reduced invasion capability. C. jejuni capC mutants caused reduced interleukin 8 (IL-8) secretion from intestinal epithelial cells and elicited a significantly diminished immune reaction in Galleria larvae, indicating that CapC functions as an immunogen. In conclusion, CapC is a new virulence determinant of C. jejuni that contributes to the integral infection process of adhesion to human intestinal epithelial cells.IMPORTANCECampylobacter jejuni is a major causative agent of human gastroenteritis, making this zoonotic pathogen of significant importance to human and veterinary public health worldwide. The mechanisms by which C. jejuni interacts with intestinal epithelial cells and causes disease are still poorly understood due, in part, to the heterogeneity of C. jejuni infection biology. Given the importance of C. jejuni to public health, the need to characterize novel and existing virulence mechanisms is apparent. The significance of our research is in demonstrating the role of CapC, a novel virulence factor in C. jejuni that contributes to adhesion and invasion of the intestinal epithelium, thereby in part, addressing the dearth of knowledge concerning the factors involved in Campylobacter pathogenesis and the variation observed in the severity of human infection.

Selection for pro-inflammatory mediators produces chickens more resistant to Campylobacter jejuni.

Campylobacter spp. are the second leading cause of bacterial-induced foodborne illnesses with an estimated economic burden of nearly $2B USD per year. Most human illness associated with campylobacteriosis is due to infection by C. jejuni and chickens are recognized as a reservoir that could lead to foodborne illness in humans resulting from handling or consuming raw or undercooked chicken. We recently developed a novel breeding strategy based on identification and selection of chickens with an inherently high and low phenotype of pro-inflammatory mediators including IL-6, CXCLi2, and CCLi2, hereafter referred to as the high and low lines, respectively. We have shown the high line chickens are more resistant to the foodborne and poultry pathogens Salmonella enterica serovar Enteritidis, Eimeria tenella, and Clostridium perfringens-induced necrotic enteritis compared to the low line. The objective of this study was to determine whether the same trend of enhanced resistance in the high line birds was observed for C. jejuni. Birds were challenged at 2 d of age by oral gavage (0.5 mL) with 5 × 106 colony forming units (cfu) of C. jejuni/mL, necropsied 4 d post challenge, and cecal content collected to determine if there was a difference in C. jejuni resistance between the high and low line chickens. There were fewer (P = 0.01) chickens from the high line (28/40 = 71.8%) that were colonized by C. jejuni compared to the low line (37/39 = 94.9%). The amount of C. jejuni recovered from the ceca of infected birds was quantified; however, no differences were observed (P = 0.10). Since the high line birds were also more resistant to C. jejuni, it provides additional validation of selection based on pro-inflammatory mediators producing a line of chickens with increased natural resistance against diverse foodborne and poultry pathogens. The poultry industry is moving towards reduced therapeutics and, as such, our breeding strategy would be a viable method to incorporate into traditional poultry breeding programs.

Interleukin-18 Mediates Immune Responses to Campylobacter jejuni Infection in Gnotobiotic Mice.

BACKGROUND: Human Campylobacter jejuni infections are progressively rising worldwide. Information about the molecular mechanisms underlying campylobacteriosis, however, are limited. In the present study we investigated whether cytokines such as IL-23, IL-22 and IL-18, which share pivotal functions in host immunity, were involved in mediating intestinal and systemic immunopathological responses upon C. jejuni infection. METHODOLOGY/PRINCIPAL FINDINGS: To assure stable infection, gnotobiotic (i.e. secondary abiotic) IL-23p19-/-, IL-22-/- and IL-18-/- mice were generated by broad-spectrum antibiotic treatment. Following peroral C. jejuni strain 81-176 infection, mice of all genotypes harbored comparably high pathogenic loads in their intestines. As compared to wildtype controls, however, IL-18-/- mice displayed less distinct C. jejuni induced sequelae as indicated by less pronounced large intestinal shrinkage and lower numbers of apoptotic cells in the colonic epithelial layer at day 8 postinfection (p.i.). Furthermore, lower colonic numbers of adaptive immune cells including regulatory T cells and B lymphocytes were accompanied by less distinct secretion of pro-inflammatory cytokines such as TNF and IFN-γ and lower IL-17A mRNA expression levels in colonic ex vivo biopsies of infected IL-18-/- as compared to wildtype mice. Upon C. jejuni infection, colonic IL-23p19 expression was up-regulated in IL-18-/- mice only, whereas IL-22 mRNA levels were lower in uninfected and infected IL-23p19-/- as well as infected IL-18-/- as compared to respective wildtype control mice. Remarkably, not only intestinal, but also systemic infection-induced immune responses were less pronounced in IL-18-/- mice as indicated by lower TNF, IFN-γ and IL-6 serum levels as compared to wildtype mice. CONCLUSION/SIGNIFICANCE: We here show for the first time that IL-18 is essentially involved in mediating C. jejuni infection in the gnotobiotic mouse model. Future studies need to further unravel the underlying regulatory mechanisms orchestrating pathogen-host interaction.

Psychological comorbidity increases the risk for postinfectious IBS partly by enhanced susceptibility to develop infectious gastroenteritis.

OBJECTIVE: Psychological factors increase the risk to develop postinfectious IBS (PI-IBS), but the mechanisms involved are unclear. As stress affects the immune system, we investigated the potential interaction between psychological factors, the immune response against infectious gastroenteritis (IGE) and the development of IGE and PI-IBS in a large cohort exposed to contaminated drinking water. DESIGN: 18 620 people exposed to contaminated drinking water (norovirus, Giardia lamblia, Campylobacter jejuni) were invited to participate in a prospective controlled cohort study. They were asked to complete questionnaires assessing demographic, psychological and clinical data during the outbreak and 1 year later. At both time points, in-depth immune function (peripheral blood and rectal biopsies) was studied in a subgroup of subjects. RESULTS: 1379 subjects completed the questionnaires during the outbreak, of which 271 developed IGE. Risk factors for IGE included younger age, pre-existing dyspepsia-like symptoms, anxiety and drinking contaminated tap water. Anxiety scores before the outbreak inversely correlated with interleukin-2-expressing CD4+ T cells (r=0.6, p=0.01, n=23). At follow-up, 34 of 172 (20%) IGE subjects developed IBS compared with 24/366 exposed participants (7%, p<0.0001, χ(2) test). A Th2 cytokine phenotype at time of infection was associated with increased risk for PI-IBS 1 year later. Except for increased B cell numbers, no evidence for systemic or rectal mucosal immune activation in PI-IBS was demonstrated at follow-up. CONCLUSIONS: Our study shows that the increased risk of patients with psychological comorbidity to develop PI-IBS may partly result from an increased susceptibility to develop IGE, possibly resulting from a Th2-immune bias. TRIAL REGISTRATION NUMBER: (ClinicalTrials.gov NCT01497847).

Occupational Exposure to Swine, Poultry, and Cattle and Antibody Biomarkers of Campylobacter jejuni Exposure and Autoimmune Peripheral Neuropathy.

INTRODUCTION: Foodborne Campylobacter jejuni infection has been associated with an increased risk of autoimmune peripheral neuropathy, but risks of occupational exposure to C. jejuni have received less attention. This study compared anti-C. jejuni IgA, IgG, and IgM antibody levels, as well as the likelihood of testing positive for any of five anti-ganglioside autoantibodies, between animal farmers and non-farmers. Anti-C. jejuni antibody levels were also compared between farmers with different animal herd or flock sizes. The relationship between anti-C. jejuni antibody levels and detection of anti-ganglioside autoantibodies was also assessed. METHODS: Serum samples from 129 Agricultural Health Study swine farmers (some of whom also worked with other animals) and 46 non-farmers, all from Iowa, were analyzed for anti-C. jejuni antibodies and anti-ganglioside autoantibodies using ELISA. Information on animal exposures was assessed using questionnaire data. Anti-C. jejuni antibody levels were compared using Mann-Whitney tests and linear regression on log-transformed outcomes. Fisher's Exact Tests and logistic regression were used to compare likelihood of positivity for anti-ganglioside autoantibodies. RESULTS: Farmers had significantly higher levels of anti-C. jejuni IgA (p < 0.0001) and IgG (p = 0.02) antibodies compared to non-farmers. There was no consistent pattern of anti-C. jejuni antibody levels based on animal herd or flock size. A higher percentage of farmers (21%) tested positive for anti-ganglioside autoantibodies compared to non-farmers (9%), but this difference was not statistically significant (p = 0.11). There was no significant association between anti-C. jejuni antibody levels and anti-ganglioside autoantibodies. CONCLUSIONS: The findings provide evidence that farmers who work with animals may be at increased risk of exposure to C. jejuni. Future research should include longitudinal studies of exposures and outcomes, as well as studies of interventions to reduce exposure. Policies to reduce occupational exposure to C. jejuni should be considered.

Virulence genes and cytokine profile in systemic murine Campylobacter coli infection.

Campylobacter coli are one of the most common bacteria in bacterial gastroenteritis and acute enterocolitis in humans. However, relatively little is known regarding the mechanisms of pathogenesis and host response to C. coli infections. To investigate the influence of genetic changes, we first used PCR to demonstrate the presence of the known virulence genes cadF, virB11, cdtB, cdtC and ceuE in the clinical isolate C. coli 26536, which was isolated from the liver of infected BALB/c mice. Sequence analyses of the cadF, virB11, cdtB and ceuE genes in C. coli 26536 confirmed the stability in these virulence genes during their transmission through the host. We further investigated C. coli infection for the bacterial clearance from the liver and spleen of infected mice, and for their immune response. C. coli persisted well in both organs, with better survival in the liver. We also determined the levels of several pro-inflammatory cytokines (i.e., interleukin [IL]-6, IL-12, interferon-γ, tumor necrosis factor-α) and the anti-inflammatory cytokine IL-10 in plasma and in liver homogenates from the infected mice, using enzyme-linked immunosorbent assays. The lowest levels among these cytokines were for tumor necrosis factor-α in the plasma and IL-6 in the liver on days 1, 3 and 8 post-infection. The most pronounced production was for IL-10, in both plasma (days 1 and 8 post-infection) and liver (day 8 post-infection), which suggests that it has a role in healing of the organ inflammation. Our findings showed dynamic relationships between pro- and anti-inflammatory cytokines and thus contribute toward clarification of the healing processes involved in the resolution of C. coli infections.

Transcriptomic and proteomic analyses reveal key innate immune signatures in the host response to the gastrointestinal pathogen Campylobacter concisus.

Pathogenic species within the genus Campylobacter are responsible for a considerable burden on global health. Campylobacter concisus is an emergent pathogen that plays a role in acute and chronic gastrointestinal disease. Despite ongoing research on Campylobacter virulence mechanisms, little is known regarding the immunological profile of the host response to Campylobacter infection. In this study, we describe a comprehensive global profile of innate immune responses to C. concisus infection in differentiated THP-1 macrophages infected with an adherent and invasive strain of C. concisus. Using RNA sequencing (RNA-seq), quantitative PCR (qPCR), mass spectrometry, and confocal microscopy, we observed differential expression of pattern recognition receptors and robust upregulation of DNA- and RNA-sensing molecules. In particular, we observed IFI16 inflammasome assembly in C. concisus-infected macrophages. Global profiling of the transcriptome revealed the significant regulation of a total of 8,343 transcripts upon infection with C. concisus, which included the activation of key inflammatory pathways involving CREB1, NF-κB, STAT, and interferon regulatory factor signaling. Thirteen microRNAs and 333 noncoding RNAs were significantly regulated upon infection, including MIR221, which has been associated with colorectal carcinogenesis. This study represents a major advance in our understanding of host recognition and innate immune responses to infection by C. concisus.

Inflammasome activation by Campylobacter jejuni.

The Gram-negative pathogen Campylobacter jejuni is the most common cause of bacterial foodborne disease worldwide. The mechanisms that lead to bacterial invasion of eukaryotic cells and massive intestinal inflammation are still unknown. In this study, we report that C. jejuni infection of mouse macrophages induces upregulation of pro-IL-1ß transcript and secretion of IL-1ß without eliciting cell death. Immunoblotting indicated cleavage of caspase-1 and IL-1ß in infected cells. In bone marrow-derived macrophages from different knockout mice, IL-1ß secretion was found to require NLRP3, ASC, and caspase-1/11 but not NLRC4. In contrast to NLRP3 activation by ATP, C. jejuni activation did not require priming of these macrophages. C. jejuni also activated the NLRP3 inflammasome in human macrophages as indicated by the presence of ASC foci and caspase-1-positive cells. Analysis of a vast array of C. jejuni mutants with defects in capsule formation, LPS biosynthesis, chemotaxis, flagella synthesis and flagellin (-like) secretion, type 6 secretion system needle protein, or cytolethal distending toxin revealed a direct correlation between the number of intracellular bacteria and NLRP3 inflammasome activation. The C. jejuni invasion-related activation of the NLRP3 inflammasome without cytotoxicity and even in nonprimed cells extends the known repertoire of bacterial inflammasome activation and likely contributes to C. jejuni-induced intestinal inflammation.

Interaction between Campylobacter and intestinal epithelial cells leads to a different proinflammatory response in human and porcine host.

Campylobacter jejuni and Campylobacter coli are recognized as the leading causes of human diarrheal disease throughout the development world. Unlike human beings, gastrointestinal tract of pigs are frequently colonized by Campylobacter to a high level in a commensal manner. The aim of this study was to identify the differences underlying the divergent outcome following Campylobacter challenge in porcine versus human host. In order to address this, a comparative in vitro infection model was combined with microscopy, gentamicin protection assay, ELISA and quantitative PCR techniques. Invasion assays revealed that Campylobacter invaded human cells up to 10-fold more than porcine cells (p<0.05). In addition, gene expression of proinflammatory genes encoding for IL1α, IL6, IL8, CXCL2 and CCL20 were strongly up-regulated by Campylobacter in human epithelial cell at early times of infection, whereas a very reduced cytokine gene expression was detected in porcine epithelial cells. These data indicate that Campylobacter fails to invade porcine cells compared to human cells, and this leads to a lack of proinflammatory response induction, probably due to its pathogenic or commensal behavior in human and porcine host, respectively.

Case of acute pancreatitis associated with Campylobacter enteritis.

A 25-year-old man was admitted with the chief complaints of right flank pain, watery diarrhea, and fever. Blood tests revealed high levels of inflammatory markers, and infectious enteritis was diagnosed. A stool culture obtained on admission revealed no growth of any significant pathogens. Conservative therapy was undertaken with fasting and fluid replacement. On day 2 of admission, the fever resolved, the frequency of defecation reduced, the right flank pain began to subside, and the white blood cell count started to decrease. On hospital day 4, the frequency of diarrhea decreased to approximately 5 times per day, and the right flank pain resolved. However, the patient developed epigastric pain and increased blood levels of the pancreatic enzymes. Abdominal computed tomography revealed mild pancreatic enlargement. Acute pancreatitis was diagnosed, and conservative therapy with fasting and fluid replacement was continued. A day later, the blood levels of the pancreatic enzymes peaked out. On hospital day 7, the patient passed stools with fresh blood, and Campylobacter jejuni/coli was detected by culture. Lower gastrointestinal endoscopy performed on hospital day 8 revealed diffuse aphthae extending from the terminal ileum to the entire colon. Based on the findings, pancreatitis associated with Campylobacter enteritis was diagnosed. In the present case, a possible mechanism of onset of pancreatitis was invasion of the pancreatic duct by Campylobacter and the host immune responses to Campylobacter.

Functional characterization of exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) of Campylobacter jejuni.

The inorganic polyphosphate (poly-P) is a key regulator of stress responses and virulence in many bacterial pathogens including Campylobacter jejuni. The role of exopolyphosphatases/guanosine pentaphosphate (pppGpp) phosphohydrolases (PPX/GPPA) in poly-P homeostasis and C. jejuni pathobiology remains unexplored. Here, we analyzed deletion mutants (∆ppx1, ∆ppx2) and the double knockout mutant (dkppx), all ∆ppx mutants exhibited increased capacity to accumulate poly-P; however only ∆ppx1 and dkppx mutants showed decreased accumulation of ppGpp, an alarmone molecule that regulates stringent response in bacteria, suggesting potential dual role for PPX1/GPPA. Nutrient survival defect of ∆ppx mutants was rescued by the supplementation of specific amino acids implying that survival defect may be associated with decreased ppGpp and/ or increased poly-P in ∆ppx mutants. The ppk1 and spoT were upregulated in both ∆ppx1 and ∆ppx2 suggesting a compensatory role for SpoT and Ppk1 in poly-P and ppGpp homeostasis. The lack of ppx genes resulted in defects in motility, biofilm formation, nutrient stress survival, invasion and intracellular survival indicating that maintaining a certain level of poly-P is critical for ppx genes in C. jejuni pathophysiology. Both ppx1 and ppx2 mutants were resistant to human complement-mediated killing; however, the dkppx mutant was sensitive. The serum susceptibility did not occur in the presence of MgCl 2 and EGTA suggesting an involvement of the classical or lectin pathway of complement mediated killing. Interestingly, the chicken serum did not have any effect on the ∆ppx mutants' survival. The observed serum susceptibility was not related to C. jejuni surface capsule and lipooligosaccharide structures. Our study underscores the importance of PPX/GPPA proteins in poly-P and ppGpp homeostasis, two critical molecules that modulate environmental stress responses and virulence in C. jejuni.

Sialylation of Campylobacter jejuni endotoxin promotes dendritic cell-mediated B cell responses through CD14-dependent production of IFN-ß and TNF-α.

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis and often precedes development of Guillain-Barré syndrome (GBS), a life-threatening paralytic disease. The incorporation of the carbohydrate sialic acid into C. jejuni lipooligosaccharides (LOS) is associated with increased severity of gastroenteritis and with induction of GBS; however, the underlying mechanisms remain completely unknown. In this study, we demonstrate that sialic acids in C. jejuni endotoxin enhance the rapid production of IFN-ß and TNF-α by human dendritic cells (DCs). Using neutralizing Abs and receptors it was shown that these DC-derived cytokines promote the proliferation of human mucosal B cells in a T cell-independent manner. The production of both IFN-ß and TNF-α by DCs in response to LOS requires CD14, and the amplified response of DCs to sialylated C. jejuni LOS is CD14 dependent. Together, these results indicate that sialylation of C. jejuni LOS increases DC activation and promotes subsequent B cell responses through CD14-driven production of IFN-ß and TNF-α. This enhanced DC/B cell response may explain the increased pathogenicity of sialylated C. jejuni and may be key to the initiation of B cell-mediated autoimmunity in GBS.

Production and evaluation of chicken egg-yolk-derived antibodies against Campylobacter jejuni colonization-associated proteins.

Campylobacter jejuni is one of the most important causes of foodborne gastroenteritis. Chickens are considered a reservoir host of C. jejuni, and epidemiological studies have shown that contaminated chicken meat is a primary source of human infection. The objective of this study was to produce chicken egg-yolk-derived antibody (IgY) against the five C. jejuni colonization-associated proteins or CAPs (CadF, FlaA, MOMP, FlpA, and CmeC). Recombinant C. jejuni CAPs were expressed in Escherichia coli and were purified by affinity chromatography. Specific-pathogen-free laying hens were hyperimmunized with each recombinant CAP to induce production of α-CAP-specific IgY. Egg yolks were collected from immunized and nonimmunized hens and were lyophilized to obtain egg-yolk powder (EYP) with or without α-C. jejuni CAP-specific IgY. IgY was purified from EYP, and the antibody response in serum and egg yolk was tested by indirect enzyme-linked immunosorbent assay. The α-C. jejuni CAP-specific IgY levels were significantly (p<0.05) higher in both serum and EYP obtained from immunized hens as compared with the nonimmunized hens. Each α-C. jejuni CAP-specific IgY reacted with the C. jejuni cells and recombinant CAPs as detected by immunofluorescence microscopy and Western blot assays, respectively. We also show that α-CadF, α-MOMP, and α-CmeC IgY significantly reduced adherence of C. jejuni to the chicken hepatocellular carcinoma (LMH) cells, suggesting that these α-C. jejuni CAP-specific IgY may be useful as a passive immunotherapeutic to reduce C. jejuni colonization in chickens.