Protease-activated receptor 2, dipeptidyl peptidase I, and proteases mediate Clostridium difficile toxin A enteritis

BACKGROUND & AIMS: We studied the role of protease-activated receptor 2 (PAR(2)) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. METHODS: We injected TxA into ileal loops in PAR(2) or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR(2) and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. RESULTS: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR(2) deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%-28% and tissue and fluid myeloperoxidase by 31%-71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR(2) and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca(2+) responses to PAR(2) AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. CONCLUSIONS: PAR(2) and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR(2) and up-regulates PAR(2) and activating proteases, and PAR(2) causes inflammation by neurogenic mechanisms.

Voltage-gated potassium currents within the dorsal vagal nucleus: inhibition by BDS toxin

Voltage-gated potassium (Kv) channels are essential components of neuronal excitability. The Kv3.4 channel protein is widely distributed throughout the central nervous system (CNS), where it can form heteromeric or homomeric Kv3 channels. Electrophysiological studies reported here highlight a functional role for this channel protein within neurons of the dorsal vagal nucleus (DVN). Current clamp experiments revealed that blood depressing substance (BDS) and intracellular dialysis of an anti-Kv3.4 antibody prolonged the action potential duration. In addition, a BDS sensitive, voltage-dependent, slowly inactivating outward current was observed in voltage clamp recordings from DVN neurons. Electrical stimulation of the solitary tract evoked EPSPs and IPSPs in DVN neurons and BDS increased the average amplitude and decreased the paired pulse ratio, consistent with a presynaptic site of action. This presynaptic modulation was action potential dependent as revealed by ongoing synaptic activity. Given the role of the Kv3 proteins in shaping neuronal excitability, these data highlight a role for homomeric Kv3.4 channels in spike timing and neurotransmitter release in low frequency firing neurons of the DVN.

Nutrient-limited toxin production and the dynamics of two phytoplankton in culture media: a mathematical model

In this paper we have proposed and analyzed a simple mathematical model consisting of four variables, viz., nutrient concentration, toxin producing phytoplankton (TPP), non-toxic phytoplankton (NTP), and toxin concentration. Limitation in the concentration of the extracellular nutrient has been incorporated as an environmental stress condition for the plankton population, and the liberation of toxic chemicals has been described by a monotonic function of extracellular nutrient. The model is analyzed and simulated to reproduce the experimental findings of Graneli and Johansson [Graneli, E., Johansson, N., 2003. Increase in the production of allelopathic Prymnesium parvum cells grown under N- or P-deficient conditions. Harmful Algae 2, 135–145]. The robustness of the numerical experiments are tested by a formal parameter sensitivity analysis. As the first theoretical model consistent with the experiment of Graneli and Johansson (2003), our results demonstrate that, when nutrient-deficient conditions are favorable for the TPP population to release toxic chemicals, the TPP species control the bloom of other phytoplankton species which are non-toxic. Consistent with the observations made by Graneli and Johansson (2003), our model overcomes the limitation of not incorporating the effect of nutrient-limited toxic production in several other models developed on plankton dynamics.

Toxin-allelopathy among phytoplankton species prevents competitive exclusion

Toxic or allelopathic compounds liberated by toxin-producing phytoplankton (TPP) acts as a strong mediator in plankton dynamics. On an analysis of a set of phytoplankton biomass data that have been collected by our group in the northwest part of the Bay of Bengal, and by analysis of a three-component mathematical model under a constant as well as a stochastic environment, we explore the role of toxin-allelopathy in determining the dynamic behavior of the competing phytoplankton species. The overall results, based on analytical and numerical wings, demonstrate that toxin-allelopathy due to the TPP promotes a stable co-existence of those competitive phytoplankton that would otherwise exhibit competitive exclusion of the weak species. Our study suggests that TPP might be a potential candidate for maintaining the co-existence and diversity of competing phytoplankton species.

Competing effects of toxin-producing phytoplankton on overall plankton populations in the Bay of Bengal

The coexistence of a large number of phytoplankton species on a seemingly limited variety of resources is a classical problem in ecology, known as ‘the paradox of the plankton’. Strong fluctuations in species abundance due to the external factors or competitive interactions leading to oscillations, chaos and short-term equilibria have been cited so far to explain multi-species coexistence and biodiversity of phytoplankton. However, none of the explanations has been universally accepted. The qualitative view and statistical analysis of our field data establish two distinct roles of toxin-producing phytoplankton (TPP): toxin allelopathy weakens the interspecific competition among phytoplankton groups and the inhibition due to ingestion of toxic substances reduces the abundance of the grazer zooplankton. Structuring the overall plankton population as a combination of nontoxic phytoplankton (NTP), toxic phytoplankton, and zooplankton, here we offer a novel solution to the plankton paradox governed by the activity of TPP. We demonstrate our findings through qualitative analysis of our sample data followed by analysis of a mathematical model.

Modelling the growth and movement of cyanobacteria in river systems

Bloom-forming and toxin-producing cyanobacteria remain a persistent nuisance across the world. Modelling of cyanobacteria in freshwaters is an important tool for understanding their population dynamics and predicting the location and timing of the bloom events in lakes and rivers. In this article, a new deterministic model is introduced which simulates the growth and movement of cyanobacterial blooms in river systems. The model focuses on the mathematical description of the bloom formation, vertical migration and lateral transport of colonies within river environments by taking into account the four major factors that affect the cyanobacterial bloom formation in freshwaters: light, nutrients, temperature and river flow. The model consists of two sub-models: a vertical migration model with respect to growth of cyanobacteria in relation to light, nutrients and temperature; and a hydraulic model to simulate the horizontal movement of the bloom. This article presents the model algorithms and highlights some important model results. The effects of nutrient limitation, varying illumination and river flow characteristics on cyanobacterial movement are simulated. The results indicate that under high light intensities and in nutrient-rich waters colonies sink further as a result of carbohydrate accumulation in the cells. In turbulent environments, vertical migration is retarded by vertical velocity component generated by turbulent shear stress. (c) 2006 Elsevier B.V. All rights reserved.

A review and classification of the existing models of cyanobacteria

Bloom-forming and toxin-producing cyanobacteria remain a persistent nuisance across the world. Modelling of cyanobacteria in freshwaters is an important tool for understanding their population dynamics and predicting bloom occurrence in lakes and rivers. In this paper existing key models of cyanobacteria are reviewed, evaluated and classified. Two major groups emerge: deterministic mathematical and artificial neural network models. Mathematical models can be further subcategorized into those models concerned with impounded water bodies and those concerned with rivers. Most existing models focus on a single aspect such as the growth of transport mechanisms, but there are a few models which couple both.

Identifying the critical parameters of a cyanobacterial growth and movement model by using generalised sensitivity analysis

Bloom-forming and toxin-producing cyanobacteria remain a persistent nuisance across the world. Modelling of cyanobacteria in freshwaters is an important tool for understanding their population dynamics and predicting the location and timing of the bloom events in lakes and rivers. A new deterministic-mathematical model was developed, which simulates the growth and movement of cyanobacterial blooms in river systems. The model focuses on the mathematical description of the bloom formation, vertical migration and lateral transport of colonies within river environments by taking into account the major factors that affect the cyanobacterial bloom formation in rivers including, light, nutrients and temperature. A technique called generalised sensitivity analysis was applied to the model to identify the critical parameter uncertainties in the model and investigates the interaction between the chosen parameters of the model. The result of the analysis suggested that 8 out of 12 parameters were significant in obtaining the observed cyanobacterial behaviour in a simulation. It was found that there was a high degree of correlation between the half-saturation rate constants used in the model.

Biological control of black vine weevil (Otiorhynchus sulcatus, Coleoptera: Curculionidae) by entomopathogenic bacteria and their cell-free toxic metabolites

Biocontrol agents such as Xeiwrhabduf, nemalophilci and X. nematophila ssp. bovienii and their cell-free protein toxin complexes were lethal to larvae of O. sulcatus when applied to potting compost in the absence of plants. Similarly, strawberry plants infected with 0. sulcaitfi larvae were protected from damage by applications of both cell suspensions of the bacteria and solutions of their cell-free toxic metabolites, indicating that it is the protein toxins, which are responsible for the lethal effects observed. These toxic metabolites were found more effective against 0. sulccitus larvae when treated in soil microflora. Insect mortality is increased by increasing temperature and bacterial concentration. The toxins remained pathogenic for several months when stored in potting soil either at 15 or 20°C, however, bacterial cells were not as persistent as the toxins. It is therefore suggested that these bacteria and their toxic metabolites can he applied in soil for insect pest control.

Mechanisms of internalization and recycling of the chemokine receptor, CCR5

CCR5 is a G protein-coupled receptor that binds several natural chemokines but it is also a coreceptor for the entry of M tropic strains of HIV-1 into cells. Levels of CCR5 on the cell surface are important for the rate of HIV-1 infection and are determined by a number of factors including the rates of CCR5 internalization and recycling. Here we investigated the involvement of the actin cytoskeleton in the control of ligand-induced internalization and recycling of CCR5. Cytochalasin D, an actin depolymerizing agent, inhibited chemokine-induced internalization of CCR5 and recycling of the receptor in stably transfected CHO cells and in the monocytic cell line, THP-1. CCR5 internalization and recycling were inhibited by Toxin B and C-3 exoenzyme treatment in CHO and THP-1 cells, confirming activation of members of the RhoGTPase family by CCR5. The specific Rho kinase inhibitor Y27632, however, had no effect on CCR5 internalization or recycling. Ligand-induced activation of CCR5 leads to Rho kinase-dependent formation of focal adhesion complexes. These data indicate that CCR5 internalization and recycling are regulated by actin polymerization and activation of small G proteins in a Rho-dependent manner.

GM crops: a potential for pest mismanagement

In the absence of equivalent research on genetically modified (GM) pest-resistant crops, their impact in pest management can be predicted from experience with traditionally bred varieties which share with GM crops the characteristic that the resistance is based on high expression of a single toxin. Such varieties lead to the rapid selection of tolerant pest strains, damage biological control and induce tolerance to synthetic pesticides. By contrast, polygenic and more broadly based resistant varieties will maintain their resistance for longer, and often synergise beneficially with biological control. The pests also become more susceptible to insecticides, giving the opportunity for applications which are selective in favour of natural enemies. However, although GM crops compare badly with traditional pest-resistant varieties, they compare favourably with insecticides, the technology they are most likely to replace.

Microbiology of the human intestinal tract and approaches for its dietary modulation

Gut bacteria can be categorised as being either beneficial or potentially pathogenic due to their metabolic activities and fermentation end-products. Health-promoting effects of the microflora may include immunostimulation, improved digestion and absorption, vitamin synthesis, inhibition of the growth of potential pathogens and lowering of gas distension. Detrimental effects are carcinogen production, intestinal putrefaction, toxin production, diarrhoea/constipation and intestinal infections. Certain indigenous bacteria such as bifidobacteria and lactobacilli are considered to be examples of health-promoting constituents of the microflora. They may aid digestion of lactose in lactose-intolerant individuals, reduce diarrhoea, help resist infections and assist in inflammatory conditions. Probiotics, prebiotics and synbiotics are functional foods that fortify the lactate producing microflora of the human or animal gut.

Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children

Children with autistic spectrum disorders (ASDs) tend to suffer from severe gastrointestinal problems. Such symptoms may be due to a disruption of the indigenous gut flora promoting the overgrowth of potentially pathogenic micro-organisms. The faecal flora of patients with ASDs was studied and compared with those of two control groups (healthy siblings and unrelated healthy children). Faecal bacterial populations were assessed through the use of a culture-independent technique, fluorescence in situ hybridization, using oligonucleotide probes targeting predominant components of the gut flora. The faecal flora of ASD patients contained a higher incidence of the Clostridium histolyticum group (Clostridium clusters I and 11) of bacteria than that of healthy children. However, the non-autistic sibling group had an intermediate level of the C. histolyticum group, which was not significantly different from either of the other subject groups. Members of the C. histolyticum group are recognized toxin-producers and may contribute towards gut dysfunction, with their metabolic products also exerting systemic effects. Strategies to reduce clostridial population levels harboured by ASD patients or to improve their gut microflora profile through dietary modulation may help to alleviate gut disorders common in such patients.

Prebiotics and resistance to gastrointestinal infections

Acute gut disorder is a cause for significant medicinal and economic concern. Certain individual pathogens of the gut, often transmitted in food or water, have the ability to cause severe discomfort. There is a need to manage such conditions more effectively. The route of reducing the risk of intestinal infections through diet remains largely unexplored. Antibiotics are effective at inhibiting pathogens; however, these should not be prescribed in the absence of disease and therefore cannot be used prophylactically. Moreover, their indiscriminate use has reduced effectiveness. Evidence has accumulated to suggest that some of the health-promoting bacteria in the gut (probiotics) can elicit a multiplicity of inhibitory effects against pathogens. Hence, an increase in their numbers should prove effective at repressing pathogen colonisation if/when infectious agents enter the gut. As such, fortification of indigenous bifidobacteria/lactobacilli by using prebiotics should improve protection. There are a number of potential mechanisms for lactic acid bacteria to reduce intestinal infections. Firstly, metabolic endproducts such as acids excreted by these micro-organisms may lower the gut pH to levels below those at which pathogens are able to effectively compete. Also, many lactobacilli and bifidobacteria species are able to excrete natural antibiotics, which can have a broad spectrum of activity. Other mechanisms include an improved immune stimulation, competition for nutrients and blocking of pathogen adhesion sites in the gut. Many intestinal pathogens like type 1 fimbriated Escherichia coli, salmonellae and campylobacters utilise oligosaccharide receptor sites in the gut. Once established, they can then cause gastroenteritis through invasive and/or toxin forming properties. One extrapolation of the prebiotic concept is to simulate such receptor sites in the gut lumen. Hence, the pathogen is 'decoyed' into not binding at the host mucosal interface. The combined effects of prebiotics upon the lactic acid flora and anti-adhesive strategies may lead towards new dietary interventions against food safety agents.

Sensitivity analysis of a cyanobacterial growth and movement model under two different flow regimes

Bloom-forming and toxin-producing cyanobacteria remain a persistent nuisance across the world. Modelling cyanobacterial behaviour in freshwaters is an important tool for understanding their population dynamics and predicting the location and timing of the bloom events in lakes, reservoirs and rivers. A new deterministic–mathematical model was developed, which simulates the growth and movement of cyanobacterial blooms in river systems. The model focuses on the mathematical description of the bloom formation, vertical migration and lateral transport of colonies within river environments by taking into account the major factors that affect the cyanobacterial bloom formation in rivers including light, nutrients and temperature. A parameter sensitivity analysis using a one-at-a-time approach was carried out. There were two objectives of the sensitivity analysis presented in this paper: to identify the key parameters controlling the growth and movement patterns of cyanobacteria and to provide a means for model validation. The result of the analysis suggested that maximum growth rate and day length period were the most significant parameters in determining the population growth and colony depth, respectively.