Commensal-induced regulatory T cells mediate protection against pathogen-stimulated NF-kappaB activation

Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-kappaB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-kappaB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to na?ve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-kappaB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis-fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-kappaB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-kappaB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.     

Probiotics: from myth to reality. Demonstration of functionality in animal models of disease and in human clinical trials

The enteric flora comprise approximately 95% of the total number of cells in the human body and are capable of eliciting immune responses while also protecting against microbial pathogens. However, the resident bacterial flora of the gastrointestinal tract (GIT) may also be implicated in the pathogenesis of several chronic conditions such as inflammatory bowel disease (IBD). The University College Cork-based Probiotic Research Group has successfully isolated and identified lactic acid bacteria (LAB) which exhibit beneficial probiotic traits. These characteristics include the demonstration of bile tolerance; acid resistance; adherence to host epithelial tissue; and in vitro antagonism of potentially-pathogenic micro-organisms or those which have been implicated in promoting inflammation. The primary objective of this report is to describe the strategy adopted for the selection of potentially effective probiotic bacteria. The study further describes the evaluation of two m embers of the resulting panel of micro-organisms (Lactobacillus salivarius subsp. salivarius UCC118 and Bifidobacterium longum infantis 35624) under in vitro conditions and throughout in vivo murine and human feeding trials. Specifically, an initial feeding study completed in Balb/c mice focused upon (i) effective delivery of the probiotic micro-organisms to the GIT and evaluation of the ability of the introduced strains to survive transit through, and possibly colonise, the murine GIT; (ii) accepting the complexity of the hostile GIT and faecal environments, development of a method of enumerating the introduced bacterial strains using conventional microbiological techniques; and (iii) assessment of the effects of administered bacterial strains on the numbers of specific recoverable indigenous bacteria in the murine GIT and faeces. Additional research, exploiting the availability of murine models of inflammatory bowel disease, demonstrated the beneficial effects of administering probi otic combinations of Lactobacillus salivarius UCC118 and Bifidobacterium longum infantis 35624 in prevention of illness-related weight loss. A further ethically-approved feeding trial, successfully conducted in 80 healthy volunteers, demonstrated that yoghurt can be used as a vehicle for delivery of Lactobacillus salivarius strain UCC118 to the human GIT with considerable efficacy in influencing gut flora and colonisation.     

Amplitude reduction and phase shifts of melatonin, cortisol and other circadian rhythms after a gradual advance of sleep and light exposure in humans

Background

The phase and amplitude of rhythms in physiology and behavior are generated by circadian oscillators and entrained to the 24-h day by exposure to the light-dark cycle and feedback from the sleep-wake cycle. The extent to which the phase and amplitude of multiple rhythms are similarly affected during altered timing of light exposure and the sleep-wake cycle has not been fully characterized.

Methodology/Principal Findings

We assessed the phase and amplitude of the rhythms of melatonin, core body temperature, cortisol, alertness, performance and sleep after a perturbation of entrainment by a gradual advance of the sleep-wake schedule (10 h in 5 days) and associated light-dark cycle in 14 healthy men. The light-dark cycle consisted either of moderate intensity ‘room’ light (∼90–150 lux) or moderate light supplemented with bright light (∼10,000 lux) for 5 to 8 hours following sleep. After the advance of the sleep-wake schedule in moderate light, no significant advance of the melatonin rhythm was observed whereas, after bright light supplementation the phase advance was 8.1 h (SEM 0.7 h). Individual differences in phase shifts correlated across variables. The amplitude of the melatonin rhythm assessed under constant conditions was reduced after moderate light by 54% (17–94%) and after bright light by 52% (range 12–84%), as compared to the amplitude at baseline in the presence of a sleep-wake cycle. Individual differences in amplitude reduction of the melatonin rhythm correlated with the amplitude of body temperature, cortisol and alertness.

Conclusions/Significance

Alterations in the timing of the sleep-wake cycle and associated bright or moderate light exposure can lead to changes in phase and reduction of circadian amplitude which are consistent across multiple variables but differ between individuals. These data have implications for our understanding of circadian organization and the negative health outcomes associated with shift-work, jet-lag and exposure to artificial light.     

Multiple novel nesprin-1 and nesprin-2 variants act as versatile tissue-specific intracellular scaffolds

Background

Nesprins (Nuclear envelope spectrin-repeat proteins) are a novel family of giant spectrin-repeat containing proteins. The nesprin-1 and nesprin-2 genes consist of 146 and 116 exons which encode proteins of ∼1mDa and ∼800 kDa is size respectively when all the exons are utilised in translation. However emerging data suggests that the nesprins have multiple alternative start and termination sites throughout their genes allowing the generation of smaller isoforms.

Results

In this study we set out to identify novel alternatively transcribed nesprin variants by screening the EST database and by using RACE analysis to identify cDNA ends. These two methods provided potential hits for alternative start and termination sites that were validated by PCR and DNA sequencing. We show that these alternative sites are not only expressed in a tissue specific manner but by combining different sites together it is possible to create a wide array of nesprin variants. By cloning and expressing small novel nesprin variants into human fibroblasts and U2OS cells we show localization to actin stress-fibres, focal adhesions, microtubules, the nucleolus, nuclear matrix and the nuclear envelope (NE). Furthermore we show that the sub-cellular localization of individual nesprin variants can vary depending on the cell type, suggesting any single nesprin variant may have different functions in different cell types.

Conclusions

These studies suggest nesprins act as highly versatile tissue specific intracellular protein scaffolds and identify potential novel functions for nesprins beyond cytoplasmic-nuclear coupling. These alternate functions may also account for the diverse range of disease phenotypes observed when these genes are mutated.     

The effects of organic carbon, ammoniacal-nitrogen, and oxygen partial pressure on the stratification of membrane-aerated biofilms

The purpose of this study was to examine the effects of different nutrient (carbon, nitrogen, oxygen) concentrations on the microbial activity and community structure in membrane-aerated biofilms (MABs). MABs were grown under well-defined conditions of fluid flow, substrate concentration, and membrane oxygen partial pressure. Biofilms were then removed and thin-sliced using a cryostat/microtome parallel to the membrane. Individual slices were analyzed for changes with depth in biomass density, respiratory activity, and the population densities of ammonia-oxidizing and denitrifying bacteria populations. Oxygen-sensing microelectrodes were used to determine the depth of oxygen penetration into each biofilm. Our results demonstrated that ammonia-oxidizing bacteria grow near the membrane, while denitrifying bacteria grow a substantial distance from the membrane. However, nitrifying and denitrifying bacteria did not grow simultaneously when organic concentrations became too high or ammonia concentrations became too low. In conclusion, membrane-aerated biofilms exhibit substantial stratification with respect to community structure and activity. A fundamental understanding of the factors that control this stratification will help optimize the performance of full-scale membrane-aerated biofilm reactors for wastewater treatment.     

A Bifidobacterial pilus‐associated protein promotes colonic epithelial proliferation

Development of the human gut microbiota commences at birth, with certain bifidobacterial species representing dominant and early colonisers of the newborn gastrointestinal tract. The molecular basis of Bifidobacterium colonisation, persistence and presumed communication with the host has remained obscure. We previously identified tight adherence (Tad) pili from Bifidobacterium breve UCC2003 as an essential colonisation factor. Here, we demonstrate that bifidobacterial Tad pili also promote in vivo colonic epithelial proliferation. A significant increase in cell proliferation was detectable 5 days postadministration of B. breve UCC2003. Using advanced functional genomic approaches, bacterial strains either (a) producing the Tad₂₀₀₃ pili or (b) lacking the TadE or TadF pseudopilins were created. Analysis of the ability of these mutant strains to promote epithelial cell proliferation in vivo demonstrated that the pilin subunit, TadE, is the bifidobacterial molecule responsible for this proliferation response. These findings were confirmed in vitro using purified TadE protein. Our data imply that bifidobacterial Tad pili may contribute to the maturation of the naïve gut in early life through the production of a specific scaffold of extracellular protein structures, which stimulate growth of the neonatal mucosa.     

Expression of IL-20 in synovium and lesional skin of patients with psoriatic arthritis: differential response to alefacept treatment

Introduction

Psoriatic arthritis (PsA) is an inflammatory joint disease associated with psoriasis. Alefacept (a lymphocyte function-associated antigen (LFA)-3 Ig fusion protein that binds to CD2 and functions as an antagonist to T-cell activation) has been shown to result in improvement in psoriasis but has limited effectiveness in PsA. Interleukin-20 (IL-20) is a key proinflammatory cytokine involved in the pathogenesis of psoriasis. The effects of alefacept treatment on IL-20 expression in the synovium of patients with psoriasis and PsA are currently unknown.

Methods

Eleven patients with active PsA and chronic plaque psoriasis were treated with alefacept (7.5 mg per week for 12 weeks) in an open-label study. Skin biopsies were taken before and after 1 and 6 weeks, whereas synovial biopsies were obtained before and 4 and 12 weeks after treatment. Synovial biopsies from patients with rheumatoid arthritis (RA) (n = 10) were used as disease controls. Immunohistochemical analysis was performed to detect IL-20 expression, and stained synovial tissue sections were evaluated with digital image analysis. Double staining was performed with IL-20 and CD68 (macrophages), and conversely with CD55 (fibroblast-like synoviocytes, FLSs) to determine the phenotype of IL-20-positive cells in PsA synovium. IL-20 expression in skin sections (n = 6) was analyzed semiquantitatively.

Results

IL-20 was abundantly expressed in both PsA and RA synovial tissues. In inflamed PsA synovium, CD68⁺ macrophages and CD55⁺ FLSs coexpressed IL-20, and its expression correlated with the numbers of FLSs. IL-20 expression in lesional skin of PsA patients decreased significantly (P = 0.04) 6 weeks after treatment and correlated positively with the Psoriasis Area and Severity Index (PASI). IL-20 expression in PsA synovium was not affected by alefacept.

Conclusions

Conceivably, the relatively limited effectiveness of alefacept in PsA patients (compared with anti-tumor necrosis factor (TNF) therapy) might be explained in part by persistent FLS-derived IL-20 expression.     

Photic resetting of the human circadian pacemaker in the absence of conscious vision

Ocular light exposure patterns are the primary stimuli for entraining the human circadian system to the local 24-h day. Many totally blind persons cannot use these stimuli and, therefore, have circadian rhythms that are not entrained. However, a few otherwise totally blind persons retain the ability to suppress plasma melatonin concentrations after ocular light exposure, probably using a neural pathway that includes the site of the human circadian pacemaker, suggesting that light information is reaching this site. To test definitively whether ocular light exposure could affect the circadian pacemaker of some blind persons and whether melatonin suppression in response to bright light correlates with light-induced phase shifts of thecircadian system, the authorsperformed experiments with 5 totally blind volunteers using a protocol known to induce phase shifts of the circadian pacemaker in sighted individuals. In the 2 blind individuals who maintained light-induced melatonin suppression, the circadian system was shifted by appropriately timed bright-light stimuli. These data demonstrate that light can affect the circadian pacemaker of some totally blind individuals--either by altering the phase of the circadian pacemaker or by affecting its amplitude. They are consistent with data from animal studies demonstrating that there are different neural pathways and retinal cells that relay photic information to the brain: one for conscious light perception and the other for non-image-forming functions.