Exposure of Bifidobacterium longum subsp. infantis to Milk Oligosaccharides Increases Adhesion to Epithelial Cells and Induces a Substantial Transcriptional Response

Devon Kavanaugh is in receipt of a Teagasc Walsh Fellowship. The authors would also like to acknowledge the support of Science Foundation Ireland under Grant No. 08/SRC/B1393 and the Alimentary Glycoscience Research Cluster (AGRC).peer-reviewedIn this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3′sialyllactose and 6′sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3′sialyllactose did not enhance adhesion. However, treatment with 6′sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3′- and 6′-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3′- and 6′-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6′sialyllactose, and 3′sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions.Science Foundation IrelandTeagasc Walsh Fellowship Programm

Bovine glycomacropeptide promotes the growth of Bifidobacterium longum ssp. infantis and modulates its gene expression

peer-reviewedBovine milk glycomacropeptide (GMP) is derived from κ-casein, with exclusively o-linked glycosylation. Glycomacropeptide promoted the growth of Bifidobacterium longum ssp. infantis in a concentration-dependent manner, and this activity was lost following periodate treatment of the GMP (GMP-P), which disables biological recognition of the conjugated oligosaccharides. Transcriptional analysis of B. longum ssp. infantis following exposure to GMP revealed a substantial response to GMP relative to bacteria treated with GMP-P, with a greater number of differentially expressed transcripts and larger fold changes versus the control. Therefore, stimulation of B. longum ssp. infantis growth by GMP is intrinsically linked to the peptide's O-linked glycosylation. The pool of differentially expressed transcripts included 2 glycoside hydrolase (family 25) genes, which were substantially upregulated following exposure to GMP, but not GMP-P. These GH25 genes were present in duplicated genomic islands that also contained genes encoding fibronectin type III binding domain proteins and numerous phage-related proteins, all of which were also upregulated. Homologs of this genomic arrangement were present in other Bifidobacterium species, which suggest it may be a conserved domain for the utilization of glycosylated peptides. This study provides insights into the molecular basis for the prebiotic effect of bovine milk GMP on B. longum ssp. infantis

Giant pulmonary artery aneurysm in a patient with vasoreactive pulmonary hypertension: a case report

<p>Abstract</p> <p>Background</p> <p>Pulmonary artery aneurysms are a rare condition, frequently associated with pulmonary hypertension. However, the evolution and treatment of this pathology is still not clear.</p> <p>Case Presentation</p> <p>The authors report a case of a 65-year old patient with pulmonary artery aneurysm associated with pulmonary arterial hypertension. Due to a positive vasoreactivity test, treatment with calcium channel blockers was started with near normalization of the right cardiac pressures. Nevertheless, after 20 months of treatment, the pulmonary artery aneurysm size remained unchanged with an associated severe pulmonary regurgitation and causing extrinsic compression of the main left coronary artery. Surgical correction was successfully performed.</p> <p>Conclusions</p> <p>This is the first case report of a pulmonary artery aneurysm described to be associated with vasoreactive pulmonary hypertension in a living patient. Although medical therapy for pulmonary hypertension was started, surgical correction of the aneurysm was executed in order to prevent its future complications.</p

Elevated apoptosis impairs epithelial cell turnover and shortens villi in TNF-driven intestinal inflammation

The intestinal epithelial monolayer, at the boundary between microbes and the host immune system, plays an important role in the development of inflammatory bowel disease (IBD), particularly as a target and producer of pro-inflammatory TNF. Chronic overexpression of TNF leads to IBD-like pathology over time, but the mechanisms driving early pathogenesis events are not clear. We studied the epithelial response to inflammation by combining mathematical models with in vivo experimental models resembling acute and chronic TNF-mediated injury. We found significant villus atrophy with increased epithelial cell death along the crypt-villus axis, most dramatically at the villus tips, in both acute and chronic inflammation. In the acute model, we observed overexpression of TNF receptor I in the villus tip rapidly after TNF injection and concurrent with elevated levels of intracellular TNF and rapid shedding at the tip. In the chronic model, sustained villus atrophy was accompanied by a reduction in absolute epithelial cell turnover. Mathematical modelling demonstrated that increased cell apoptosis on the villus body explains the reduction in epithelial cell turnover along the crypt-villus axis observed in chronic inflammation. Cell destruction in the villus was not accompanied by changes in proliferative cell number or division rate within the crypt. Epithelial morphology and immunological changes in the chronic setting suggest a repair response to cell damage although the villus length is not recovered. A better understanding of how this state is further destabilised and results in clinical pathology resembling IBD will help identify suitable pathways for therapeutic intervention

Immunomodulatory effects exerted by Lactobacillus salivarius strains on innate immune cells

Despite increased application of commensal bacteria for attempting to improve the symptoms of a variety of inflammatory conditions, including inflammatory bowel diseases, diarrhoea and irritable bowel syndrome, therapeutic approaches that involve live bacteria are hampered by a limited understanding of bacterium-host interactions. Lactobacilli are natural inhabitants of the mammalian gastrointestinal tract and many lactobacilli are regarded as probiotics meaning that they exert a beneficial influence on the health status of their consumers. Modulation of immune responses is a plausible mechanism underlying these beneficial effects. The aim of this thesis was to investigate the effect of 33 Lactobacillus salivarius strains on the production of inflammatory cytokines from a variety of human and mouse immune cells. Induction of immune responses in vitro was shown to be bacterial- and mouse strain-dependent, cell type-dependent, blood donor-dependent and bacterial cell number-dependent. Collectively, these data suggest the importance of a case-by-case selection of candidate strains for their potential therapeutic application. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs) and play a critical role in shaping microbial-specific innate and adaptive immune responses. Following ligand engagement, TLRs trigger a complex network of signalling that culminate in the production of inflammatory mediators. The investigation of the molecular mechanisms underlying the Lb. salivarius-host interaction resulted in the identification of a novel role for TLR2 in negatively regulating TLR4 signalling originated from subcellular compartments within macrophages. Notably, sustained activation of JAK/STAT cascade and M1-signature genes in TLR2-/- macrophages was ablated by selective TLR4 and JAK inhibitors and by absence of TLR4 in TLR2/4-/- cells. In addition, other negative regulators of TLR signalling triggered by Lb. salivarius strains were found to be the adapter molecules TIRAP and TRIF. Understanding negative regulation of TLR signalling may pave the way for the development of novel therapeutics to limit inflammation in multiple diseases

Candidate HIV-1 Tat vaccine development: from basic science to clinical trials.

Abstract not availabl

Macrophage cytokine responses to commensal Gram-positive Lactobacillus salivarius strains are TLR2-independent and Myd88-dependent.

The mechanisms through which cells of the host innate immune system distinguish commensal bacteria from pathogens are currently unclear. Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) expressed by host cells which recognize microbe-associated molecular patterns (MAMPs) common to both commensal and pathogenic bacteria. Of the different TLRs, TLR2/6 recognize bacterial lipopeptides and trigger cytokines responses, especially to Gram-positive and Gram-negative pathogens. We report here that TLR2 is dispensable for triggering macrophage cytokine responses to different strains of the Gram-positive commensal bacterial species Lactobacillus salivarius. The L. salivarius UCC118 strain strongly upregulated expression of the PRRs, Mincle (Clec4e), TLR1 and TLR2 in macrophages while downregulating other TLR pathways. Cytokine responses triggered by L. salivarius UCC118 were predominantly TLR2-independent but MyD88-dependent. However, macrophage cytokine responses triggered by another Gram-positive commensal bacteria, Bifidobacterium breve UCC2003 were predominantly TLR2-dependent. Thus, we report a differential requirement for TLR2-dependency in triggering macrophage cytokine responses to different commensal Gram-positive bacteria. Furthermore, TNF-α responses to the TLR2 ligand FSL-1 and L. salivarius UCC118 were partially Mincle-dependent suggesting that PRR pathways such as Mincle contribute to the recognition of MAMPs on distinct Gram-positive commensal bacteria. Ultimately, integration of signals from these different PRR pathways and other MyD88-dependent pathways may determine immune responses to commensal bacteria at the host-microbe interface.This work was supported by grants from Science Foundation Ireland—namely a research centre Grant (SFI-12/RC/2273) to APC Microbiome Ireland.S

Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 10 6 viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 �g) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of &lt;15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens