A. Ruth Foxwell

Microbial pattern recognition receptors mediate M-cell uptake of a gram negative bacterium

ABSTRACT The receptors involved in the sampling of particulate microbial antigens by the gut are largely unknown. Here we demonstrate for the first time in an in vitro M-cell model and in situ in isolated murine intestinal segments that the receptors TLR-4, PAF-R, and α5β1 integrin are all involved in mediating bacterial uptake associated with transcytosis. The pattern of expression of TLR-4 and α5β1 integrin differed between M cells and enterocytes. There was increased apical expression of TLR-4 in M-cell cultures, and it was present on the apical surface of murine M cells but not enterocytes in situ. In contrast, PAF-R was expressed equally by both cell types in vitro and was abundantly expressed throughout the intestinal epithelium. Inhibition of TLR-4 and PAF-R, but not TLR-2, reduced gram-negative bacterial uptake by both cell types, whereas inhibition of the apically expressed α5β1 integrin significantly reduced the ability of M cells to translocate bacteria. Hence, the involvement of each receptor was dependent not only on differences in the level of receptor expression but the cellular localization. Using bacteria that had mutations that affected the bacterial lipooligosaccharide structure indicated that the oligosaccharide moiety was important in bacterial uptake. Taken together, the data suggest that pathogen-associated molecular pattern interactions with pattern recognition receptors are key factors in M-cell recognition of intestinal antigens for mucosal immune priming.

Validation and quantitation of an in vitro M-cell model

This study has evaluated an in vitro model of the follicle-associated epithelia that overlie Peyers patches of the small intestine. The model shares many phenotypic characteristics of M cells in vivo. Co-cultures of the human adenocarcinoma cell line Caco-2 and freshly isolated Peyers patch cells were established. Fluorescence microscopy and quantitative image analysis were used to validate the model against known markers of M-cell phenotype. Apical expression of alkaline phosphatase was down-regulated in co-cultures and villin was re-distributed from the apical membrane to the cytoplasm. alpha5beta1 integrin was found on the apical surfaces of the monolayers and B and T lymphocytes integrated into the monolayers. Particle transport was temperature-dependent in co-cultures, indicating that a transcytotic route was responsible. This model provides opportunities to study factors that influence M-cell development, assess putative Peyers patch targeting in oral vaccine technologies, and study intestinal uptake in vitro.

Mucosal immunity in the lung and upper airway

The mucosal surfaces of the lungs and upper airways are common sites for infection. Extensive studies of the mechanisms associated with immune responses in the respiratory tract have found that understanding the system is challenging and involves many complex interactions to prevent and eliminate infection. Immune protection against diseases transmitted through the respiratory tract requires an understanding of the important aspects associated with beneficial, detrimental or ineffective immune responses. Two critical aspects of an immune response against a pathogen are that of the inductive stage, either induced by vaccination or primary infection, and the effector stage, the ability to recognise, respond to and eliminate the infection without detriment to the host. An immunisation strategy must not only have a measure of the induced antigen specific response, but this response must also be protective.

An action research protocol to strengthen system-wide inter-professional learning and practice [LP0775514]

BackgroundInter-professional learning (IPL) and inter-professional practice (IPP) are thought to be critical determinants of effective care, improved quality and safety and enhanced provider morale, yet few empirical studies have demonstrated this. Whole-of-system research is even less prevalent. We aim to provide a four year, multi-method, multi-collaborator action research program of IPL and IPP in defined, bounded health and education systems located in the Australian Capital Territory (ACT). The project is funded by the Australian Research Council under its industry Linkage Program.Methods/DesignThe program of research will examine in four inter-related, prospective studies, progress with IPL and IPP across tertiary education providers, professional education, regulatory and registration bodies, the ACT health systems streams of care activities and teams, units and wards of the provider facilities of the ACT health system. One key focus will be on push-pull mechanisms, ie, how the education sector creates student-enabled IPP and the health sector demands IPL-oriented practitioners. The studies will examine four research aims and meet 20 research project objectives in a comprehensive evaluation of ongoing progress with IPL and IPP.DiscussionIPP and IPL are said to be cornerstones of health system reforms. We will measure progress across an entire health system and the clinical and professional education systems that feed into it. The value of multi-methods, partnership research and a bi-directional push-pull model of IPL and IPP will be tested. Widespread dissemination of results to practitioners, policymakers, managers and researchers will be a key project goal.

A four-year, systems-wide intervention promoting interprofessional collaboration

BackgroundA four-year action research study was conducted across the Australian Capital Territory health system to strengthen interprofessional collaboration (IPC) though multiple intervention activities.MethodsWe developed 272 substantial IPC intervention activities involving 2,407 face-to-face encounters with health system personnel. Staff attitudes toward IPC were surveyed yearly using Heinemann et als Attitudes toward Health Care Teams and Parsell and Blighs Readiness for Interprofessional Learning scales (RIPLS). At studys end staff assessed whether project goals were achieved.ResultsOf the improvement projects, 76 exhibited progress, and 57 made considerable gains in IPC. Educational workshops and feedback sessions were well received and stimulated interprofessional activities. Over time staff scores on Heinemanns Quality of Interprofessional Care subscale did not change significantly and scores on the Doctor Centrality subscale increased, contrary to predictions. Scores on the RIPLS subscales of Teamwork & Collaboration and Professional Identity did not alter. On average for the assessment items 33% of staff agreed that goals had been achieved, 10% disagreed, and 57% checked neutral. There was most agreement that the study had resulted in increased sharing of knowledge between professions and improved quality of patient care, and least agreement that between-professional rivalries had lessened and communication and trust between professions improved.ConclusionsOur longitudinal interventional study of IPC involving multiple activities supporting increased IPC achieved many project-specific goals. However, improvements in attitudes over time were not demonstrated and neutral assessments predominated, highlighting the difficulties faced by studies targeting change at the systems level and over extended periods.

Characterization of a Novel Porin Protein from Moraxella catarrhalis and Identification of an Immunodominant Surface Loop

Moraxella catarrhalis is a gram-negative bacterium that is mainly responsible for respiratory tract infections. In this study we report a novel outer membrane protein (OMP), designated M35, with a molecular mass of 36.1 kDa. This protein was structurally homologous to classic gram-negative porins, such as OMP C from Escherichia coli and OMP K36 from Klebsiella pneumoniae, with a predicted structure of 8 surface loops and 16 antiparallel beta-sheets. The DNA sequences of the genes from 18 diverse clinical isolates showed that the gene was highly conserved (99.6 to 100% of nucleotides), with only one isolate (ID78LN266) having base variations that resulted in amino acid substitutions. Electrophoresis and analysis of recognition of the protein using mouse anti-M35 sera showed that M35 was expressed on the bacterial surface and constitutively expressed across M. catarrhalis isolates, with only ID78LN266 showing poor antibody recognition. Our results showed that the single amino acid mutation in loop 3 significantly affected antibody recognition, indicating that loop 3 appeared to contain an immunodominant B-cell epitope. The antibody specificity to loop 3 may be a potential mechanism for evasion of host immune responses targeted to M35, since loop 3 should theoretically orientate into the porin channel. Thus, M35 is a highly conserved, surface-expressed protein that is of significance for its potential functional role as an M. catarrhalis porin and is of interest as a vaccine candidate.

Mucosal immunization against respiratory bacterial pathogens

Bacterial respiratory diseases remain a major cause of morbidity and mortality throughout the world. The young and the elderly are particularly susceptible to the pathogens that cause these diseases. Therapeutic approaches remain dependent upon antibiotics contributing to the persistent increases in antibiotic resistance. The main causes of respiratory disease discussed in this review are Mycobacterium tuberculosis, Corynebacterium diphtheriae, Bordatella pertussis, Streptococcus pneumoniae, nontypeable Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa. All these organisms initiate disease at the mucosal surface of the respiratory tract and thus the efficacy of the host’s response to infection needs to be optimal at this site. Vaccines available for diseases caused by many of these pathogens have limitations in accessibility or efficacy, highlighting the need for improvements in approaches and products. The most significant challenges in both therapy and prevention of disease induced by bacteria in the respiratory tract remain the development of noninjectable vaccines and delivery systems/immunization regimens that improve mucosal immunity.

Moraxella catarrhalis M35 is a general porin that is important for growth under nutrient-limiting conditions and in the nasopharynges of mice.

Moraxella catarrhalis is a gram-negative respiratory pathogen that is an important causative agent for otitis media and exacerbations of chronic obstructive pulmonary disease. We have previously predicted the outer membrane protein M35 to be a general porin, and in the current study, we have investigated the function of M35 and its importance for survival of M. catarrhalis in vivo. Lipid bilayer experiments reveal that refolded M35 functions as a channel that is typical of gram-negative bacterial porins. M35 forms wide and water-filled channels with a single-channel conductance of about 1.25 nS in 1 M KCl solution and has only a small selectivity for cations over anions. When the in vitro growth characteristics of two M35 deletion mutant strains of M. catarrhalis were compared to the wild-type parent isolates, the growth of the mutant strains was inhibited only under nutrient-poor conditions. This growth defect could be eliminated by additional glutamic acid, but not additional aspartic acid, glycine, sucrose, or glucose. The mutant strains compensated for the lack of M35 by enhancing their uptake of glutamic acid, and this enhanced rate of glutamic acid uptake was attributed to the compensatory upregulation of a protein of approximately 40 kDa. M35 was also found to be essential for nasal colonization of mice, demonstrating that its presence is essential for survival of M. catarrhalis in vivo. These results suggest that M35 is a general porin that is necessary for the uptake of important energy sources by M. catarrhalis and that it is likely that M35 is an essential functional protein for in vivo colonization.

Kinetics of inflammatory cytokines in the clearance of non-typeable Haemophilus influenzae from the lung.

Levels of the pro‐inflammatory cytokines TNF‐α and IFN‐γ were measured from the time of infection to the time of complete clearance of non‐typeable Haemophilus influenzae (NTHi) from the lung in immune and non‐immune rats. Mucosal immunization facilitated production of significant levels of TNF‐α as early as 30 min post‐pulmonary challenge with NTHi in immune animals. Following the peak at 2 h, rapid decline of TNF‐α levels occurred from the alveolar spaces. Levels of TNF‐α in non‐immunized animals increased at a slower rate, peaked at a lower concentration and were slower to decline. The significantly larger number of macrophages seen in the immune animals at 1 h after bacterial challenge could partially account for the higher levels of TNF‐α. Interferon‐γ was not detected in immune or non‐immune rats at any time point before NTHi clearance after pulmonary challenge. Study of the kinetics of TNF‐α release demonstrates that immunized animals control the release of pro‐inflammatory cytokines more effectively than non‐immunized animals for enhanced clearance of bacterial infection from the lungs.

CHARACTERISTICS OF THE IMMUNOLOGICAL RESPONSE IN THE CLEARANCE OF NON-TYPEABLE HAEMOPHILUS INFLUENZAE FROM THE LUNG

Clearance of non‐typeable Haemophilus influenzae (NTHi) from the respiratory tract was investigated, over time, in immune and non‐immune rats. A triphasic pattern characterized the clearance of bacteria from the lungs. Mucosal immunization enhanced bacterial clearance from the lungs in each of the three phases compared with clearance from non‐immunized animals. Total clearance of bacteria was observed from lung tissue by 12 h in immune animals and 24 h in non‐immune animals. Polymorphonuclear leucocytes not only arrived earlier and initially in greater numbers, but disappeared earlier in immune animals (peaking at 8 h post‐challenge), compared with non‐immune animals (peaking at 12 h post‐challenge). Systemically derived and locally produced NTHi‐specific IgA and IgG correlated with enhanced bacterial clearance during the secondary phase. This model demonstrates that immunized animals up‐regulate and resolve inflammatory responses to pulmonary infection more rapidly than the non‐immunized controls.