Novel Campylobacter concisus lipooligosaccharide is a determinant of inflammatory potential and virulence

This work was supported in part by Department of Veterans Affairs Merit Review award BX000727 (to G.A.J.). The authors also acknowledge National Institutes of Health National Center for Research Resources Shared Instrumentation Grant S10 RR029446 (to H. E. Witkowska) for acquisition of the Synapt G2 high definition mass spectrometer, which is located at the University of California, San Francisco Sandler-Moore Mass Spectrometry Core Facility and supported by the Sandler Family Foundation, the Gordon and Betty Moore Foundation, National Institutes of Health/National Cancer Institute Cancer Center Support Grant P30 CA082103, and the Canary Foundation. G.A.J. is a recipient of the Senior Research Career Scientist award from the Department of Veterans Affairs. R.H. is funded by a Career Researcher Fellowship from NHS Research Scotland. The BISCUIT study was funded by a Clinical Academic Training Fellowship from the Chief Scientist Office (CAF/08/01). This is paper number 116 from the Center for Immunochemistry. The contents of this article do not represent the views of the Department of Veterans Affairs or the United States Government. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. K.B. acknowledges funding from the Child Health Research Charitable Incorporated Organisation and the Bogue Fellowship for travel. The authors declare that they have no conflicts of interest with the contents of this article.Peer reviewe

Enterohepatic Helicobacter in ulcerative colitis:Potential pathogenic entities?

Background: Changes in bacterial populations termed "dysbiosis" are thought central to ulcerative colitis (UC) pathogenesis. In particular, the possibility that novel Helicobacter organisms play a role in human UC has been debated but not comprehensively investigated. The aim of this study was to develop a molecular approach to investigate the presence of Helicobacter organisms in adults with and without UC.Methodology/Principal Findings: A dual molecular approach to detect Helicobacter was developed. Oligonucleotide probes against the genus Helicobacter were designed and optimised alongside a validation of published H. pylori probes. A comprehensive evaluation of Helicobacter genus and H. pylori PCR primers was also undertaken. The combined approach was then assessed in a range of gastrointestinal samples prior to assessment of a UC cohort. Archival colonic samples were available from 106 individuals for FISH analysis (57 with UC and 49 non-IBD controls). A further 118 individuals were collected prospectively for dual FISH and PCR analysis (86 UC and 32 non-IBD controls). An additional 27 non-IBD controls were available for PCR analysis. All Helicobacter PCR-positive samples were sequenced. The association between Helicobacter and each study group was statistically analysed using the Pearson Chi Squared 2 tailed test. Helicobacter genus PCR positivity was significantly higher in UC than controls (32 of 77 versus 11 of 59, p = 0.004). Sequence analysis indicated enterohepatic Helicobacter species prevalence was significantly higher in the UC group compared to the control group (30 of 77 versus 2 of 59, p&lt;0.0001). PCR and FISH results were concordant in 74 (67.9%) of subjects. The majority of discordant results were attributable to a higher positivity rate with FISH than PCR.Conclusions/Significance: Helicobacter organisms warrant consideration as potential pathogenic entities in UC. Isolation of these organisms from colonic tissue is needed to enable interrogation of pathogenicity against established criteria.</p

The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing

Peer reviewedPublisher PD

The inflammatory microenvironment in colorectal neoplasia

Peer reviewedPublisher PD

Novel Campylobacter concisus lipooligosaccharide is a determinant of inflammatory potential and virulence

The pathogenicity of Campylobacter concisus, increasingly found in the human gastrointestinal (GI) tract, is unclear. Some studies indicate that its role in GI conditions has been underestimated, whereas others suggest that the organism has a commensal-like phenotype. For the enteropathogen C. jejuni, the lipooligosaccharide (LOS) is a main driver of virulence. We investigated the LOS structure of four C. concisus clinical isolates and correlated the inflammatory potential of each isolate with bacterial virulence. Mass spectrometric analyses of lipid A revealed a novel hexa-acylated diglucosamine moiety with two or three phosphoryl substituents. Molecular and fragment ion analysis indicated that the oligosaccharide portion of the LOS had only a single phosphate and lacked phosphoethanolamine and sialic acid substitution, which are hallmarks of the C. jejuni LOS. Consistent with our structural findings, C. concisus LOS and live bacteria induced less TNF-α secretion in human monocytes than did C. jejuni Furthermore, the C. concisus bacteria were less virulent than C. jejuni in a Galleria mellonella infection model. The correlation of the novel lipid A structure, decreased phosphorylation, and lack of sialylation along with reduced inflammatory potential and virulence support the significance of the LOS as a determinant in the relative pathogenicity of C. concisus

Novel Campylobacter concisus lipooligosaccharide is a determinant of inflammatory potential and virulence

The pathogenicity of Campylobacter concisus, increasingly found in the human gastrointestinal (GI) tract, is unclear. Some studies indicate that its role in GI conditions has been underestimated, whereas others suggest that the organism has a commensal-like phenotype. For the enteropathogen C. jejuni, the lipooligosaccharide (LOS) is a main driver of virulence. We investigated the LOS structure of four C. concisus clinical isolates and correlated the inflammatory potential of each isolate with bacterial virulence. Mass spectrometric analyses of lipid A revealed a novel hexa-acylated diglucosamine moiety with two or three phosphoryl substituents. Molecular and fragment ion analysis indicated that the oligosaccharide portion of the LOS had only a single phosphate and lacked phosphoethanolamine and sialic acid substitution, which are hallmarks of the C. jejuni LOS. Consistent with our structural findings, C. concisus LOS and live bacteria induced less TNF-α secretion in human monocytes than did C. jejuni Furthermore, the C. concisus bacteria were less virulent than C. jejuni in a Galleria mellonella infection model. The correlation of the novel lipid A structure, decreased phosphorylation, and lack of sialylation along with reduced inflammatory potential and virulence support the significance of the LOS as a determinant in the relative pathogenicity of C. concisus

Growth of Pediococcus acidilactici on sugar cane blackstrap molasses

Pediococcus acidilactici (IL01) has grown in MRS (Man, Rogosa and Sharpe) broth modified by substitution of glucose by 2.0% (MRS-2), 3.0% (MRS-3), 4.0% (MRS-4) and 5.0% (MRS-5) sugar cane blackstrap molasses. The highest acid production was obtained in MRS-5 broth maintained at a constant pH of 5.0. The highest biomass production was obtained when P. acidilactici was grown in MRS-5 broth at initial pH 6.5, while productivity was higher in MRS-2 broth (28.16%). When the MRS-2 broth was utilized at initial pH 6.5 for a 20-hour fermentation period, the highest growth rate (dx/dt) was found in a period of 8 to 16 hours (0.290 g cells/L.h), while the specific growth rate (µ) was 0.175 (h-1) for that period, differently from the 0.441 (h-1) obtained for the period comprising the 4th to the 12th hour. The growth in MRS broth was 5.08% (2.95 g/l) higher than in MRS-2 broth (2.80 g/l). The data obtained have shown that P. acidilactici has had a significant growth in molasses as the main carbon source, and that it is possible to substitute MRS glucose by this carbon source with the purpose of obtaining a more economical growth medium for the potential large scale productions.<br>Pediococcus acidilactici (IL01) cresceu em caldo MRS (Man, Rogosa and Sharpe) modificado por adição de 2,0% (MRS-2), 3,0% (MRS-3), 4,0% (MRS-4) and 5,0% (MRS-5) de melaço de cana de açúcar, em substituição à glicose. A maior produção de ácido ocorreu em caldo MRS-5 com pH constante 5,0. A produção de biomassa foi mais acentuada em caldo MRS-5 com pH inicial de 6,5, embora a produtividade tenha sido maior em caldo MRS-2 (28,16%). Em caldo MRS-2 e em pH inicial de 6,5 durante uma fermentação de 20 horas, a velocidade de crescimento (dx/dt) foi maior entre a 8ª e 16ª hora (0,290 g celulas/L.h) enquanto a velocidade específica de crescimento µ foi 0,175 (h-1) para este período, diferente de 0,441 (h-1) obtido no período compreendido entre a 4ª e 12ª hora. O crescimento em caldo MRS foi 5,08% (2,95 g/l) maior que em caldo MRS-2 (2,80 g/l). Os dados obtidos mostraram que P. acidilactici cresceu bem em melaço como principal fonte de carbono e que é possível substituir a glicose do MRS por esta fonte de carbono, com o objetivo de obter um meio de crescimento mais econômico para eventuais produções em grande escala

The Chemical Synthesis of Knob Domain Antibody Fragments

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.</p

Prognosis following upper gastrointestinal bleeding: Record linkage study

Upper gastrointestinal (GI) bleeding is one of the most common, high risk emergency disorders in the western world. Almost nothing has been reported on longer term prognosis following upper GI bleeding. The aim of this study was to establish mortality up to three years following hospital admission with upper GI bleeding and its relationship with aetiology, co-morbidities and socio-demographic factors.Systematic record linkage of hospital inpatient and mortality data for 14 212 people in Wales, UK, hospitalised with upper GI bleeding between 1999 and 2004 with three year follow-up to 2007. The main outcome measures were mortality rates, standardised mortality ratios (SMRs) and relative survival.Mortality at three years was 36.7% overall, based on 5215 fatalities. It was highest for upper GI malignancy (95% died within three years) and varices (52%). Compared with the general population, mortality was increased 27-fold during the first month after admission. It fell to 4.3 by month four, but remained significantly elevated during every month throughout the three years following admission. The most important independent prognostic predictors of mortality at three years were older age (mortality increased 53 fold for people aged 85 years and over compared with those under 40 years); oesophageal and gastric/duodenal malignancy (48 and 32 respectively) and gastric varices aetiologies (2.8) when compared with other bleeds; non-upper GI malignancy, liver disease and renal failure co-morbidities (15, 7.9 and 3.9); social deprivation (29% increase for quintile V vs I); incident bleeds as an inpatient (31% vs admitted with bleeding) and male patients (25% vs female).Our study shows a high late as well as early mortality for upper GI bleeding, with very poor longer term prognosis following bleeding due to malignancies and varices. Aetiologies with the worst prognosis were often associated with high levels of social deprivation