Guillain-Barré Syndrome-Related Campylobacter jejuni in Bangladesh: Ganglioside Mimicry and Cross-Reactive Antibodies

Background

Campylobacter jejuni is the predominant antecedent infection in Guillain-Barré syndrome (GBS). Molecular mimicry and cross-reactive immune responses to C. jejuni lipo-oligosaccharides (LOS) precipitate the development of GBS, although this mechanism has not been established in patients from developing countries. We determined the carbohydrate mimicry between C. jejuni LOS and gangliosides, and the cross-reactive antibody response in patients with GBS in Bangladesh.

Methodology

Sera from 97 GBS patients, and 120 neurological and family controls were tested for antibody reactivity against LOS from C. jejuni isolates from GBS patients in Bangladesh (BD-07, BD-39, BD-10, BD-67 and BD-94) by enzyme-linked immunosorbent assay (ELISA). Cross-reactivity to LOS was determined by ELISA. The LOS outer core structures of C. jejuni strains associated with GBS/MFS were determined by mass spectrometry.

Principle Findings

IgG antibodies to LOS from C. jejuni BD-07, BD-39, BD-10, and BD-67 IgG antibodies were found in serum from 56%, 58%, 14% and 15% of GBS patients respectively, as compared to very low frequency (<3%) in controls (p<0.001). Monoclonal antibodies specific for GM1 and GD1a reacted strongly with LOS from the C. jejuni strains (BD-07 and BD-39). Mass spectrometry analysis confirmed the presence of GM1 and GD1a carbohydrate mimics in the LOS from C. jejuni BD-07 and BD-39. Both BD-10 and BD-67 express the same LOS outer core, which appears to be a novel structure displaying GA2 and GD3 mimicry. Up to 90–100% of serum reactivity to gangliosides in two patients (DK-07 and DK-39) was inhibited by 50 µg/ml of LOS from the autologous C. jejuni isolates. However, patient DK-07 developed an anti-GD1a immune response while patient DK-39 developed an anti-GM1 immune response.

Conclusion

Carbohydrate mimicry between C. jejuni LOS and gangliosides, and cross-reactive serum antibody precipitate the majority of GBS cases in Bangladesh.     

Host Factors Determine Anti-GM1 Response Following Oral Challenge of Chickens with Guillain-Barré Syndrome Derived Campylobacter jejuni Strain GB11

Background

Anti-ganglioside antibodies with a pathogenic potential are present in C. jejuni-associated Guillain-Barré syndrome (GBS) patients and are probably induced by molecular mimicry. Immunization studies in rabbits and mice have demonstrated that these anti-ganglioside antibodies can be induced using purified lipo-oligosaccharides (LOS) from C. jejuni in a strong adjuvant.

Methodology/Principal Findings

To investigate whether natural colonization of chickens with a ganglioside-mimicking C. jejuni strain induces an anti-ganglioside response, and to investigate the diversity in anti-ganglioside response between and within genetically different chicken lines, we orally challenged chickens with different C. jejuni strains. Oral challenge of chickens with a C. jejuni strain from a GBS patient, containing a LOS that mimics ganglioside GM1, induced specific IgM and IgG anti-LOS and anti-GM1 antibodies. Inoculation of chickens with the Penner HS:3 serostrain, without a GM1-like structure, induced anti-LOS but no anti-ganglioside antibodies. We observed different patterns of anti-LOS/ganglioside response between and within the five strains of chickens.

Conclusions

Natural infection of chickens with C. jejuni induces anti-ganglioside antibodies. The production of antibodies is governed by both microbial and host factors.     

Comparative Genotyping of Campylobacter jejuni Strains from Patients with Guillain-Barré Syndrome in Bangladesh

Background

Campylobacter jejuni is a common cause of acute gastroenteritis and is associated with post-infectious neuropathies such as the Guillain-Barré syndrome (GBS) and the Miller Fisher syndrome (MFS). We here present comparative genotyping of 49 C. jejuni strains from Bangladesh that were recovered from patients with enteritis or GBS. All strains were serotyped and analyzed by lipo-oligosaccharide (LOS) genotyping, amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

C. jejuni HS:23 was a predominant serotype among GBS patients (50%), and no specific serotype was significantly associated with GBS compared to enteritis. PCR screening showed that 38/49 (78%) of strains could be assigned to LOS classes A, B, C, or E. The class A locus (4/7 vs 3/39; p<0.01) was significantly associated in the GBS-related strains as compared to enteritis strains. All GBS/oculomotor related strains contained the class B locus; which was also detected in 46% of control strains. Overlapping clonal groups were defined by MLST, AFLP and PFGE for strains from patients with gastroenteritis and GBS. MLST defined 22 sequence types (STs) and 7 clonal complexes including 7 STs not previously identified (ST-3742, ST-3741, ST-3743, ST-3748, ST-3968, ST-3969 and ST-3970). C. jejuni HS:23 strains from patients with GBS or enteritis were clonal and all strains belonged to ST-403 complex. Concordance between LOS class B and ST-403 complex was revealed. AFLP defined 25 different types at 90% similarity. The predominant AFLP type AF-20 coincided with the C. jejuni HS:23 and ST-403 complex.

Conclusion/Significance

LOS genotyping, MLST, AFLP and PFGE helped to identify the HS:23 strains from GBS or enteritis patients as clonal. Overall, genotypes exclusive for enteritis or for GBS-related strains were not obtained although LOS class A was significantly associated with GBS strains. Particularly, the presence of a clonal and putative neuropathogenic C. jejuni HS:23 serotype may contribute to the high prevalence of C. jejuni related GBS in Bangladesh.     

Campylobacter jejuni induces acute enterocolitis in gnotobiotic IL-10-/- mice via Toll-like-receptor-2 and -4 signaling

Background

Campylobacter jejuni is a leading cause of foodborne bacterial enterocolitis worldwide. Investigation of immunopathology is hampered by a lack of suitable vertebrate models. We have recently shown that gnotobiotic mice as well as conventional IL-10^−/− animals are susceptible to C. jejuni infection and develop intestinal immune responses. However, clinical symptoms of C. jejuni infection were rather subtle and did not reflect acute bloody diarrhea seen in human campylobacteriosis.

Methodology/Principal Findings

In order to overcome these limitations we generated gnotobiotic IL-10^−/− mice by quintuple antibiotic treatment starting right after weaning. The early treatment was essential to prevent these animals from chronic colitis. Following oral infection C. jejuni colonized the gastrointestinal tract at high levels and induced acute enterocolitis within 7 days as indicated by bloody diarrhea and pronounced histopathological changes of the colonic mucosa. Immunopathology was further characterized by increased numbers of apoptotic cells, regulatory T-cells, T- and B-lymphocytes as well as elevated TNF-α, IFN-γ, and MCP-1 concentrations in the inflamed colon. The induction of enterocolitis was specific for C. jejuni given that control animals infected with a commensal E. coli strain did not display any signs of disease. Most strikingly, intestinal immunopathology was ameliorated in mice lacking Toll-like-receptors-2 or -4 indicating that C. jejuni lipoproteins and lipooligosaccharide are essential for induction and progression of immunopathology.

Conclusion/Significance

Gnotobiotic IL-10^−/− mice develop acute enterocolitis following C. jejuni infection mimicking severe episodes of human campylobacteriosis and are thus well suited to further dissect mechanisms underlying Campylobacter infections in vivo.     

Oral Administration of Polymyxin B Modulates the Activity of Lipooligosaccharide E. coli B against Lung Metastases in Murine Tumor Models

Introduction

Polymyxin B (PmB) belongs to the group of cyclic peptide antibiotics, which neutralize the activity of LPS by binding to lipid A. The aim of this study was to analyze the effect of PmB on the biological activity of lipooligosaccharide (LOS E. coli B,rough form of LPS) in vitro and in experimental metastasis models.

Results

Cultures of murine macrophage J774A.1 cells and murine bone marrow-derived dendritic cells (BM-DC) stimulated in vitro with LOS and supplemented with PmB demonstrated a decrease in inflammatory cytokine production (IL-6, IL-10, TNF-α) and down-regulation of CD40, CD80, CD86 and MHC class II molecule expression. Additionally, PmB suspended in drinking water was given to the C57BL/6 mice seven or five days prior to the intravenous injection of B16 or LLC cells and intraperitoneal application of LOS. This strategy of PmB administration was continued throughout the duration of the experiments (29 or 21 days). In B16 model, statistically significant decrease in the number of metastases in mice treated with PmB and LOS (p<0.01) was found on the 14th day of the experiments, whereas the most intensive changes in surface-antigen expression and ex vivo production of IL-6, IL-1β and TNF-α by peritoneal cells were observed 7 days earlier. By contrast, antigen expression and ex vivo production of IL-6, IL-10, IFN-γ by splenocytes remained relatively high and stable. Statistically significant decrease in LLC metastases number was observed after the application of LOS (p<0.01) and in the group of mice preconditioned by PmB and subsequently treated with LOS (LOS + PmB, p<0.01).

Conclusions

In conclusion, prolonged in vivo application of PmB was not able to neutralize the LOS-induced immune cell activity but its presence in the organism of treated mice was important in modulation of the LOS-mediated response against the development of metastases.     

Complex of GM1- and GD1a-Like Lipo-Oligosaccharide Mimics GM1b,Inducing Anti-GM1b Antibodies

Objective

Molecular mimicry between Campylobacter jejuni lipo-oligosaccharides (LOSs) and human gangliosides GM1 and GD1a induces the production of anti-GM1 and anti-GD1a antibodies, and the development of Guillain-Barré syndrome. Complexes of two different gangliosides form new molecular shapes capable of enhancing recognition by anti-ganglioside antibodies. To test the hypothesis that the complex of GM1-like and GD1a-like LOSs of C. jejuni induces the development of anti-GM1b antibodies in Guillain-Barré syndrome patients.

Methods

Mass spectrometry analysis determined the LOS outer core structures, with which mice were immunized. IgG antibodies to single gangliosides and complex of gangliosides were tested in sera from Guillain-Barré syndrome patients from whom C. jejuni LOS had been isolated.

Results

Two isolates from GBS patients who had anti-GM1b antibodies, but neither anti-GM1 nor -GD1a antibodies, expressed both GM1-like and GD1a-like LOSs, but not GM1b-like LOS. Anti-GM1b antibodies were induced in one of the mice immunized with the C. jejuni bearing GM1-like and GD1a-like LOS. Sera from 20 patients had antibodies to the complex of GM1 and GD1a, all of which carried anti-GM1b reactivity. Five of these sera harbored neither anti-GM1 nor anti-GD1a antibodies. IgG antibodies to the complex were absorbed by GM1b, but by neither GM1 nor GD1a.

Conclusions

GM1-like and GD1a-like LOSs form a GM1b epitope, inducing the development of anti-GM1b antibodies in patients with Guillain-Barré syndrome subsequent to C. jejuni enteritis. Here, we present a new paradigm that the complex of two different structures forms a new molecular mimicry, inducing the production of autoantibodies.     

Delineation of the innate and adaptive T-cell immune outcome in the human host in response to Campylobacter jejuni infection

Background

Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Despite the significant health burden this infection presents, molecular understanding of C. jejuni-mediated disease pathogenesis remains poorly defined. Here, we report the characterisation of the early, innate immune response to C. jejuni using an ex-vivo human gut model of infection. Secondly, impact of bacterial-driven dendritic cell activation on T-cell mediated immunity was also sought.

Methodology

Healthy, control paediatric terminal ileum or colonic biopsy tissue was infected with C. jejuni for 8–12 hours. Bacterial colonisation was followed by confocal microscopy and mucosal innate immune responses measured by ELISA. Marked induction of IFNγ with modest increase in IL-22 and IL-17A was noted. Increased mucosal IL-12, IL-23, IL-1β and IL-6 were indicative of a cytokine milieu that may modulate subsequent T-cell mediated immunity. C. jejuni-driven human monocyte-derived dendritic cell activation was followed by analyses of T cell immune responses utilising flow cytometry and ELISA. Significant increase in Th-17, Th-1 and Th-17/Th-1 double-positive cells and corresponding cytokines was observed. The ability of IFNγ, IL-22 and IL-17 cytokines to exert host defence via modulation of C. jejuni adhesion and invasion to intestinal epithelia was measured by standard gentamicin protection assay.

Conclusions

Both innate and adaptive T cell-immunity to C. jejuni infection led to the release of IFNγ, IL-22 and IL-17A; suggesting a critical role for this cytokine triad in establishing host anti-microbial immunity during the acute and effectors phase of infection. In addition, to their known anti-microbial functions; IL-17A and IL-17F reduced the number of intracellular C. jejuni in intestinal epithelia, highlighting a novel aspect of how IL-17 family members may contribute to protective immunity against C. jejuni.     

Correlation between Genotypic Diversity,Lipooligosaccharide Gene Locus Class Variation,and Caco-2 Cell Invasion Potential of Campylobacter jejuni Isolates from Chicken Meat and Humans: Contribution to Virulotyping

Significant interest in studying the lipooligosaccharide (LOS) of Campylobacter jejuni has stemmed from its potential role in postinfection paralytic disorders. In this study we present the results of PCR screening of five LOS locus classes (A, B, C, D, and E) for a collection of 116 C. jejuni isolates from chicken meat (n = 76) and sporadic human cases of diarrhea (n = 40). We correlated LOS classes with clonal complexes (CC) assigned by multilocus sequence typing (MLST). Finally, we evaluated the invasion potential of a panel of 52 of these C. jejuni isolates for Caco-2 cells. PCR screening showed that 87.1% (101/116) of isolates could be assigned to LOS class A, B, C, D, or E. Concordance between LOS classes and certain MLST CC was revealed. The majority (85.7% [24/28]) of C. jejuni isolates grouped in CC-21 were shown to express LOS locus class C. The invasion potential of C. jejuni isolates possessing sialylated LOS (n = 29; classes A, B, and C) for Caco-2 cells was significantly higher (P < 0.0001) than that of C. jejuni isolates with nonsialylated LOS (n = 23; classes D and E). There was no significant difference in invasiveness between chicken meat and human isolates. However, C. jejuni isolates assigned to CC-206 (correlated with LOS class B) or CC-21 (correlated with LOS class C) showed statistically significantly higher levels of invasion than isolates from other CC. Correlation between LOS classes and CC was further confirmed by pulsed-field gel electrophoresis. The present study reveals a correlation between genotypic diversity and LOS locus classes of C. jejuni. We showed that simple PCR screening for C. jejuni LOS classes could reliably predict certain MLST CC and add to the interpretation of molecular-typing results. Our study corroborates that sialylation of LOS is advantageous for C. jejuni fitness and virulence in different hosts. The modulation of cell surface carbohydrate structure could enhance the ability of C. jejuni to adapt to or survive in a host.Campylobacter jejuni is an important human enteric pathogen worldwide (3, 7, 26). Infected humans exhibit a range of clinical spectra, from mild, watery diarrhea to severe inflammatory diarrhea (28). Factors influencing the virulence of C. jejuni include motility, chemotaxis, the ability to adhere to and invade intestinal cells, intracellular survival, and toxin production (28, 30, 52). Besides its role in human enteric illnesses, C. jejuni is a predominant infectious trigger of acute postinfectious neuropathies, such as Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) (1). Significant interest in studying the structure and biosynthesis of the core lipooligosaccharide (LOS) of C. jejuni has resulted from its potential role in these paralytic disorders. Many studies have now provided convincing evidence that molecular mimicry between C. jejuni LOS and gangliosides in human peripheral nerve tissue plays an important causal role in the pathogenesis of GBS/MFS (16, 17, 19, 21).Initial comparative studies of C. jejuni LOS structure and the corresponding DNA sequences of the LOS biosynthesis loci identified eight different LOS locus classes. Three of these classes, A, B, and C, harbor sialyltransferase genes involved in incorporating sialic acid into the LOS (42). Sialylation of the LOS core was found to be associated with ganglioside mimicry and also to affect immunogenicity and serum resistance (21). Recently, Parker et al. (43) identified 11 additional LOS classes on the basis of the sequence at the LOS biosynthesis locus. Their investigation also suggested that the LOS loci of C. jejuni strains are hot spots for genetic exchange, which can lead to mosaicism.Despite evidence on locus variation within C. jejuni LOS classes, PCR-based screening of a collection of 123 clinical and environmental strains showed that almost 60% of C. jejuni strains belong to class A, B, or C (42). Additionally, Godschalk et al. (16) found that 53% (9/17) of GBS-associated C. jejuni strains possessed LOS of class A, while 64% (35/55) of the non-GBS-associated isolates possessed LOS of class A, B, or C, and 62% (13/21) of enteritis-associated Campylobacter strains expressed LOS of class A, B, or C, as well. This relative representation of sialylated LOS classes A, B, and C was hypothesized to be advantageous for C. jejuni in the colonization and infection of various hosts (42, 49). Recently, Louwen et al. (34) demonstrated that C. jejuni strains possessing sialylated LOS (class A, B, or C) invade Caco-2 cells significantly better than nonsialylated strains (with class D or E). Knockout mutagenesis of the LOS sialyltransferase Cst-II in three C. jejuni strains revealed a significant reduction in the invasion potentials of the mutant strains (34). The possible role of LOS in adhesion and invasion was previously highlighted in the work of Perera et al. (44) and Kanipes et al. (29), where a C. jejuni waaF mutant strain showed significant reductions in levels of adherence to and invasion of INT-407 cells.LOS class diversity in C. jejuni strains isolated from chicken meat, an important source of human campylobacteriosis (6, 7, 26), has hardly been studied at all. In addition, the role of LOS class variation in the invasion potential of C. jejuni strains from chicken meat still needs to be explored. The epidemiological relevance of C. jejuni LOS gene screening can be further elaborated by correlating its results with results from other molecular-typing tools (e.g., multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]). In the present study, we screened a diverse collection of C. jejuni isolates, from consumer-packaged chicken meats and from sporadic human cases of diarrhea, by PCR for five LOS classes (A, B, C, D, and E). Then we correlated the LOS classes assigned by PCR screening with the genotypes assigned by PFGE and MLST. Finally, we tested the invasion potentials of a representative subset of C. jejuni isolates in relation to their LOS classes and genotypic diversity.     

The Natural Antimicrobial Carvacrol Inhibits Campylobacter jejuni Motility and Infection of Epithelial Cells

Background

Natural compounds with anti-microbial properties are attractive reagents to reduce the use of conventional antibiotics. Carvacrol, the main constituent of oregano oil, inhibits the growth of a variety of bacterial foodborne pathogens. As concentrations of carvacrol may vary in vivo or when used in animal feed, we here investigated the effect of subinhibitory concentrations of the compound on major virulence traits of the principal bacterial foodborne pathogen Campylobacter jejuni.

Methods/Principal Findings

Motility assays revealed that subinhibitory concentrations of carvacrol inhibited the motility of C. jejuni without affecting bacterial growth. Immunoblotting and electron microscopy showed that carvacrol-treated C. jejuni still expressed flagella. The loss of motility was not caused by reduced intracellular ATP levels. In vitro infection assays demonstrated that subinhibitory concentrations of carvacrol also abolished C. jejuni invasion of human epithelial cells. Bacterial uptake of invasive Escherichia coli was not blocked by carvacrol. Exposure of C. jejuni to carvacrol prior to infection also inhibited cellular infection, indicating that the inhibition of invasion was likely caused by an effect on the bacteria rather than inhibition of epithelial cell function.

Conclusions/Significance

Bacterial motility and invasion of eukaryotic cells are considered key steps in C. jejuni infection. Our results indicate that subinhibitory concentrations of carvacrol effectively block these virulence traits by interfering with flagella function without disturbing intracellular ATP levels. These results broaden the spectrum of anti-microbial activity of carvacrol and support the potential of the compound for use in novel infection prevention strategies.     

Genome analysis of Campylobacter jejuni strains isolated from a waterborne outbreak

Background

Waterborne Campylobacter jejuni outbreaks are common in the Nordic countries, and PFGE (pulsed field gel electrophoresis) remains the genotyping method of choice in outbreak investigations. However, PFGE cannot assess the clonal relationship between isolates, leading to difficulties in molecular epidemiological investigations. Here, we explored the applicability of whole genome sequencing to outbreak investigation by re-analysing three C. jejuni strains (one isolated from water and two from patients) from an earlier resolved Finnish waterborne outbreak from the year 2000.

Results

One of the patient strains had the same PFGE profile, as well as an identical overall gene synteny and three polymorphisms in comparison with the water strain. However, the other patient isolate, which showed only minor differences in the PFGE pattern relative to the water strain, harboured several polymorphisms as well as rearrangements in the integrated element CJIE2. We reconstructed the genealogy of these strains with ClonalFrame including in the analysis four C. jejuni isolated from chicken in 2012 having the same PFGE profile and sequence type as the outbreak strains. The three outbreak strains exhibited a paraphyletic relationship, implying that the drinking water from 2000 was probably contaminated with at least two different, but related, C. jejuni strains.

Conclusions

Our results emphasize the capability of whole genome sequencing to unambiguously resolve the clonal relationship between isolates of C. jejuni in an outbreak situation and evaluate the diversity of the C. jejuni population.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-768) contains supplementary material, which is available to authorized users.     

Campylobacter Infection as a Trigger for Guillain-Barré Syndrome in Egypt

Background

Most studies of Campylobacter infection triggering Guillain-Barré Syndrome (GBS) are conducted in western nations were Campylobacter infection and immunity is relatively rare. In this study, we explored Campylobacter infections, Campylobacter serotypes, autoantibodies to gangliosides, and GBS in Egypt, a country where Campylobacter exposure is common.

Methods

GBS cases (n = 133) were compared to age- and hospital-matched patient controls (n = 374). A nerve conduction study was performed on cases and a clinical history, serum sample, and stool specimen obtained for all subjects.

Results

Most (63.3%) cases were demyelinating type; median age four years. Cases were more likely than controls to have diarrhea (29.5% vs. 22.5%, Adjusted Odds Ratio (ORa) = 1.69, P = 0.03), to have higher geometric mean IgM anti-Campylobacter antibody titers (8.18 vs. 7.25 P<0.001), and to produce antiganglioside antibodies (e.g., anti-Gd1a, 35.3 vs. 11.5, ORa = 4.39, P<0.0001). Of 26 Penner:Lior Campylobacter serotypes isolated, only one (41:27, C. jejuni, P = 0.02) was associated with GBS.

Conclusions

Unlike results from western nations, data suggested that GBS cases were primarily in the young and cases and many controls had a history of infection to a variety of Campylobacter serotypes. Still, the higher rates of diarrhea and greater antibody production against Campylobacter and gangliosides in GBS patients were consistent with findings from western countries.     

Intranasal immunization of the combined lipooligosaccharide conjugates protects mice from the challenges with three serotypes of Moraxella catarrhalis

Background

There are no licensed vaccines available against Moraxella catarrhalis, a significant human respiratory pathogen. Lipooligosaccharide (LOS) based conjugate vaccines derived from individual serotype M. catarrhalis only showed partial protection coverage. A vaccine combining LOS conjugates of two or three serotypes might provide a broader protection.

Methods

Mice were immunized intranasally with the combined conjugates consisting of LOS from serotype A and B or serotype A, B, and C followed by challenge with different M. catarrhalis strains of three serotypes. Mouse lungs, nasal washes, and sera were collected after each challenge for bacterial counts, histological evaluation, cytokine profiles, antibody level and binding activity determinations.

Results

Intranasal administration of the combined LOS conjugates not only enhanced pulmonary bacterial clearance of all three serotypes of M. catarrhalis strains in vaccinated mice, but also elevated serotype-specific anti-LOS immunoglobulin (Ig)A and IgG titers in nasal wash and serum respectively. Mice vaccinated with the combined LOS conjugates also showed increased interferon (IFN)-γ, interleukin (IL)-12, and IL-4 in the lungs after challenges. Compared to the control group, mice immunized with the combined LOS conjugates also showed reduced lung inflammation after M. catarrhalis infections. The hyperimmune sera induced by the combined conjugates exhibited a broad cross-reactivity toward all three serotypes of M. catarrhalis under transmission electron microscopy.

Conclusions

The combined vaccine of serotype A and B LOS conjugates provides protection against most M. catarrhalis strains by eliciting humoral and cellular immune responses.     

Control of post-translational modifications in antithrombin during murine post-natal development by miR-200a

Background

Developmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant.

Results

In this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control.

Conclusions

These data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.     

Finnish Campylobacter jejuni strains of multilocus sequence type ST-22 complex have two lineages with different characteristics

Background

Campylobacter jejuni is the major cause of human bacterial gastroenteritis worldwide, and in a minority of cases, post-infectious complications may occur. ST-22 complex (usually Penner serotype 19) strains have been overrepresented among patients with postinfectious complications of campylobacteriosis. We here present a characterization of a collection of 27 Finnish C. jejuni strains of ST-22 complex, from humans (22 strains) and animal sources (five strains), with the aim of contributing to our knowledge of the pathogenesis of C. jejuni infections.

Methodology/Principal Findings

All strains were analyzed by pulsed-field gel electrophoresis (PFGE) genotyping, lipo-oligosaccharide (LOS) locus class, Y-glutamyl transpeptidase (GGT) activity, in vitro biofilm formation ability, invasion and adhesion in HeLa cells and induction of IL-8 production. ST-22 complex contained five STs (ST-22; ST-1947; ST-1966; ST-3892; ST-3996) which were homogeneous in having sialylated LOS class A₁ but on the other hand were distinguished into two major lineages according to the major STs (ST-22 and ST-1947) by different PFGE genotypes and certain other characteristics. All ST-22 strains had similar SmaI PFGE profiles, were GGT positive, and formed biofilms, except one strain, while ST-1947 strains were all GGT negative, did not form biofilm, had significantly higher motility than ST-22 (p<0.05) and had their SmaI PFGE profile. Invasion and adhesion as well as induction of IL-8 production on HeLa cells were strain-dependent characteristics.

Conclusions/Significance

ST-22 complex strains, reveal potential for molecular mimicry in host interactions upon infection as they all express sialylated LOS class A₁. The two major STs, ST-22 and ST-1947 formed two homogeneous lineages, which differed from each other both phenotypically and genetically, suggesting that the strains may have evolved separately, perhaps by interacting with different spectra of hosts. Further studies are needed in order to understand if these two lineages are associated with different disease outcomes.     

Gamma-irradiated influenza virus uniquely induces IFN-I mediated lymphocyte activation independent of the TLR7/MyD88 pathway

Background

We have shown previously in mice, that infection with live viruses, including influenza/A and Semliki Forest virus (SFV), induces systemic partial activation of lymphocytes, characterized by cell surface expression of CD69 and CD86, but not CD25. This partial lymphocytes activation is mediated by type-I interferons (IFN-I). Importantly, we have shown that γ-irradiated SFV does not induce IFN-I and the associated lymphocyte activation.

Principal Findings

Here we report that, in contrast to SFV, γ-irradiated influenza A virus elicits partial lymphocyte activation in vivo. Furthermore, we show that when using influenza viruses inactivated by a variety of methods (UV, ionising radiation and formalin treatment), as well as commercially available influenza vaccines, only γ-irradiated influenza virus is able to trigger IFN-I-dependent partial lymphocyte activation in the absence of the TLR7/MyD88 signalling pathways.

Conclusions

Our data suggest an important mechanism for the recognition of γ-irradiated influenza vaccine by cytosolic receptors, which correspond with the ability of γ-irradiated influenza virus to induce cross-reactive and cross-protective cytotoxic T cell responses.     

Pattern of infection and antibiotic activity among Streptococcus agalactiae isolates from adults in Mashhad,Iran

Background:

One of the main causes of sexually transmitted diseases is group B β- hemolytic streptococci (GBS) multiplying in the genital tracts. Penicillin is the most common drug for the treatment of infections caused by these bacteria, but in patients suffering from Penicillin allergy, Erythromycin and Clindamycin are used as alternative therapeutic drugs against GBS. Recently, resistance to these drugs has been reported more often. In this study, efforts have been made to determine the prevalence and antibiotic resistance of GBS.

Methods:

Modified Christie Atkins Munch-Petersen (CAMP) test was conducted on over 2400 samples of urine and discharge taken from vagina, urethra and prostate. The drug sensitivity was performed by double disk sensitivity tests to Bacitracin, Trimethoprim, and Sulfamethoxazole and then the resistant samples were investigated by E-test to determine the minimal inhibitory concentrations (MICs) value.

Results:

Twenty-three vaginal and 10 urethral discharge, 27urine and 6 prostatic secretion samples were GBS positive. The most symbiotic microorganisms with GBS were strains of Enterococci (90%), Staphylococcus saprophyticus (25%) and Candida albicans (6%). The disk diffusion method showed 18 cases with Penicillin resistance (MIC: 1.5 mg/ml).

Conclusion:

Taken together, GBS carriers’ rate in this study was found 20.65% (8.24% men and 12.4% women). Furthermore, findings showed high-level resistance to Erythromycin and Clindamycin.Key Words: Antibiotic resistance, Genitourinary system, Streptococcus agalactiae, minimal inhibitory concentration (MIC)     

Cell Surface Sialylation and Fucosylation Are Regulated by L1 via Phospholipase Cγ and Cooperate to Modulate Neurite Outgrowth,Cell Survival and Migration

Background

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown.

Methodology/Principal Findings

Using a panel of carbohydrate surface markers, we have shown that cell surface sialylation and fucosylation were downregulated in L1^−/y neurons versus L1^+/y neurons. Consistently, mRNA levels of sialyltransferase ST6Gal1, and fucosyltransferase FUT9 were significantly reduced in L1^−/y neurons. Moreover, treatment of L1^+/y neurons with L1 antibodies, triggering signal transduction downstream of L1, led to an increase in cell surface sialylation and fucosylation compared to rat IgG-treated cells. ShRNAs for both ST6Gal1 and FUT9 blocked L1 antibody-mediated enhancement of neurite outgrowth, cell survival and migration. A phospholipase Cγ (PLCγ) inhibitor and shRNA, as well as an Erk inhibitor, reduced ST6Gal1 and FUT9 mRNA levels and inhibited effects of L1 on neurite outgrowth and cell survival.

Conclusions

Neuronal surface sialylation and fucosylation are regulated via PLCγ by L1, modulating neurite outgrowth, cell survival and migration.     

Ras inhibition induces insulin sensitivity and glucose uptake

Background

Reduced glucose uptake due to insulin resistance is a pivotal mechanism in the pathogenesis of type 2 diabetes. It is also associated with increased inflammation. Ras inhibition downregulates inflammation in various experimental models. The aim of this study was to examine the effect of Ras inhibition on insulin sensitivity and glucose uptake, as well as its influence on type 2 diabetes development.

Methods and Findings

The effect of Ras inhibition on glucose uptake was examined both in vitro and in vivo. Ras was inhibited in cells transfected with a dominant-negative form of Ras or by 5-fluoro-farnesylthiosalicylic acid (F-FTS), a small-molecule Ras inhibitor. The involvement of IκB and NF-κB in Ras-inhibited glucose uptake was investigated by immunoblotting. High fat (HF)-induced diabetic mice were treated with F-FTS to test the effect of Ras inhibition on induction of hyperglycemia. Each of the Ras-inhibitory modes resulted in increased glucose uptake, whether in insulin-resistant C2C12 myotubes in vitro or in HF-induced diabetic mice in vivo. Ras inhibition also caused increased IκB expression accompanied by decreased expression of NF-κB . In fat-induced diabetic mice treated daily with F-FTS, both the incidence of hyperglycemia and the levels of serum insulin were significantly decreased.

Conclusions

Inhibition of Ras apparently induces a state of heightened insulin sensitization both in vitro and in vivo. Ras inhibition should therefore be considered as an approach worth testing for the treatment of type 2 diabetes.     

Targeting a Cross-Reactive Gly m 5 Soy Peptide as Responsible for Hypersensitivity Reactions in a Milk Allergy Mouse Model

Background

Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity.

Methods

Cow''s milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy.

Results

Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice.

Conclusions

Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.