Analysis of the interaction of Aeromonas caviae, A. hydrophila and A. sobria with mucins

Aeromonas species are known to be involved in human gastrointestinal diseases. These organisms colonize the gastrointestinal tract. Aeromonas hydrophila, A. caviae, and A. sobria have been demonstrated microscopically to adhere to animal cell lines that express mucous receptors, but quantitative studies of adherence to mucosal components such as mucin have not been published to date. Purified bovine submaxillary gland, hog gastric mucin, and fish skin mucin were used as a model to study mucin-binding activity among A. caviae, A. hydrophila, and A. sobria strains. Our findings revealed that binding of radiolabeled and enzyme-conjugated mucins to Aeromonas cells varied depending on the labeling procedure. The highest binding was observed when the three mucin preparations were labeled with horseradish peroxidase. Binding of the various horseradish peroxidase-labeled mucins by A. caviae, A. hydrophila, and A. sobria cells is a common property among Aeromonas species isolated from human infections, diseased fish, and from environmental sources. The proportion of Aeromonas strains which bind the various horseradish peroxidase-labeled mucins was significantly higher for A. hydrophila than for A. caviae and A. sobria. Bacterial cell-surface extracts containing active mucin-binding components recognized the horseradish peroxidase-labeled mucins. The molecular masses of the mucin-binding proteins were estimated by SDS-PAGE and Western blot as follows: A. caviae strain A4812 (95 and 44 kDa); A. hydrophila strain 48748 (97, 45, 33 and 22 kDa); and A. sobria strain 48739 (95 and 43 kDa). Mucin interaction with Aeromonas cells was also studied in terms of growth in mucin-rich media. The culture conditions greatly influence the expression of A. hydrophila mucin-binding activity.     

Inhibition of heparan sulphate and other glycosaminoglycans binding to Helicobacter pylori by various polysulphated carbohydrates

Abstract Heparan sulphate binding to Helicobacter pylori at pH 4 to 5 was inhibited with various sulphated polysaccharides (heparin and chondroitin sulphates, fucoidan, carrageenans and some others), but not by carboxylated or nonsulphated compounds. Heparin binding proteins are exposed on the cell surface.     

Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.     

The humoral immuneresponse to Helicobacter pylori infection in children with gastrointestinal symptoms

The prevalence of Helicobacter pylori is high in Eastern Europe. The purpose of this study was to estimate the prevalence of H. pylori in symptomatic Lithuanian children and to identify the infection by clinicopathological and serological analyses. One hundred sixteen symptomatic children (age 8-16) with gastritis and duodenal ulcer were included. Biopsies were histologically assessed according to the Sydney-System. Serum IgG antibodies against H. pylori were detected by an enzyme-linked immunosorbent assay (ELISA), using low molecular mass antigen. The western blot technique was used to detect serum antibodies against the cytotoxin-associated protein (CagA) using whole cell antigen. Histologically the prevalence of H. pylori infection was 79% and not influenced by demographic factors. Mucosal inflammation and atrophy were associated with a H. pylori infection. Intestinal metaplasia was found in eight children, suggesting early H. pylori acquisition in life. Increased levels of IgG antibodies were detected in 57% of children. The prevalence of IgG antibodies was significantly higher in patients with duodenal ulcer compared to children with gastritis. Forty-four (67%) H. pylori-seropositive children had antibodies against CagA. Low molecular weight-ELISA and whole cell-western blot results were significantly associated with histopathology, the presence of duodenal ulcer and the CagA status. A high number of false seronegative cases were due to poor immunological responses in children and poor locally validated tests. The prevalence of H. pylori infection in Lithuanian children is higher compared to Western Europe. The infection is acquired in early life. Diagnosing H. pylori infection, serology is helpful, but endoscopy/histology remains as gold standard.     

Immunogenic proteins of Helicobacter pullorum,Helicobacter bilis and Helicobacter hepaticus identified by two-dimensional gel electrophoresis and immunoblotting

The ecological niches occupied by various species of Helicobacter are not yet known and the full spectrum of diseases associated with Helicobacter infections are not yet defined. Since these fastidious microaerofilic bacteria require special growth conditions new and improved molecular and serologic diagnostic methods have been developed to increase our understanding of their pathogenesis and virulence characteristics. Immunogenic cell surface proteins of Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus were characterised by proteomic techniques using two-dimensional electrophoresis and immunoblotting with antisera from immunised rabbits. Cross-reactivity between the three Helicobacter species were analysed after a four-step cross-absorption experiment. For H. pullorum, H. bilis and H. hepaticus 21, 13 and 27 specific immunogenic proteins, respectively, were identified. These proteins could be of important sero-diagnostic value for analyses of sera from humans, laboratory animals and for the veterinarian field.     

Fibronectin binding to Salmonella strains

Abstract Recent investigations by many workers have elucidated the mechanisms which may explain the clinical observation that an excessive intake of dietary carbohydrates can aggravate oral candidosis. These include the enhancement of growth, multiplication and adherence of Candida species to oral surfaces and the production of excessive quantities of short-chain carboxylic acids as byproducts of sugar metabolism. The resultant acidic milieu, in addition to provoking a mucosal inflammatory reaction, could also activate the highly potent phospholipases and acid proteases of Candida and suppress the growth of normal commensal flora. A unifying hypothesis encompassing the above is proposed to explain the exacerbation of mucosal candidoses observed in environments replete with carbohydrates such as glucose and sucrose.     

Capitalizing on Synergies—A Discourse Analysis of the Process of Collaboration Among Providers of Integrative Health Care

Background

Integrative health care (IHC) combines therapies and providers from complementary and conventional health care. Previous studies on IHC have shown power relations between providers but few studies have explored how the interaction develops over time. The objective of this study was to explore the development of IHC collaboration and interaction among participating providers during a series of consensus case conferences for managing patients with back and neck pain.

Methods

This qualitative study was conducted within a pragmatic randomized controlled clinical trial in primary care. Patients'' treatment plans were developed based on IHC provider consensus conferences (n = 26) of which 15 (5 of the first, 5 in the middle, and 5 of the last in the clinical trial) were selected for analysis. Findings were derived by means of discourse analysis, focusing on the participants’ use of subject positions during the conferences.

Findings

The IHC team in this study gradually formed a group identity, moving their subject positions from individual treating subjects to members of a team and were able to make consensus-based decisions about patients’ individual treatment plans. In the discourse, the IHC team identified collaborative shortcomings and problematized the provision of IHC. They were able to capitalize on the synergies in their collaboration and developed a shared vision of IHC provision.

Conclusions

The process of IHC collaboration involved the gradual formation of an IHC team identity, which facilitated interdisciplinary, non-hierarchical consensus-based decision-making in the team. The discourse further suggests that a reform of some legal and organizational health sector barriers might be needed to realize sustainable implementation of IHC services in Sweden.     

The single-molecule mechanics of the latent TGF-β1 complex

Background

TGF-β1 controls many pathophysiological processes including tissue homeostasis, fibrosis, and cancer progression. Together with its latency-associated peptide (LAP), TGF-β1 binds to the latent TGF-β1-binding protein-1 (LTBP-1), which is part of the extracellular matrix (ECM). Transmission of cell force via integrins is one major mechanism to activate latent TGF-β1 from ECM stores. Latent TGF-β1 mechanical activation is more efficient with higher cell forces and ECM stiffening. However, little is known about the molecular events involved in this mechanical activation mechanism.

Results

By using single-molecule force spectroscopy and magnetic microbeads, we analyzed how forces exerted on the LAP lead to conformational changes in the latent complex that can ultimately result in TGF-β1 release. We demonstrate the unfolding of two LAP key domains for mechanical TGF-β1 activation: the α1 helix and the latency lasso, which together have been referred to as the “straitjacket” that keeps TGF-β1 associated with LAP. The simultaneous unfolding of both domains, leading to full opening of the straitjacket at a force of ∼40 pN, was achieved only when TGF-β1 was bound to the LTBP-1 in the ECM.

Conclusions

Our results directly demonstrate opening of the TGF-β1 straitjacket by application of mechanical force in the order of magnitude of what can be transmitted by single integrins. For this mechanism to be in place, binding of latent TGF-β1 to LTBP-1 is mandatory. Interfering with mechanical activation of latent TGF-β1 by reducing integrin affinity, cell contractility, and binding of latent TGF-β1 to the ECM provides new possibilities to therapeutically modulate TGF-β1 actions.     

T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells

To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution signaling map. Compared with effector/memory T cells, naive T cells displayed stronger activation of proximal signaling molecules after TCR triggering alone. Conversely, distal phosphorylation events, like pErk and pS6-ribosomal protein, were stronger in effector/memory subsets. CD28 costimulation specifically induced signaling necessary for proper NF-κB activation, whereas CD2 signaled more strongly to S6-ribosomal protein. Analysis of resting regulatory T cells (rTregs; CD4(+)CD45RA(+)FOXP3(+)) and activated regulatory T cells (actTregs; CD4(+)CD45RA(-)FOXP3(++)) revealed that, although rTregs had low basal, but inducible, Erk activity, actTregs displayed high basal Erk phosphorylation and little or no Akt activation. Interestingly, the use of Mek inhibitors to block Erk activation inhibited activation-dependent FOXP3 upregulation in rTregs, their transition to actTregs, and the resulting increase in suppressive capacity. In summary, our systems approach unraveled distinct differences in signaling elicited by CD28 and CD2 costimulation and between rTregs and actTregs. Blocking rTreg transition to highly suppressive actTregs by Mek inhibitors might have future therapeutic applications.