Utilization of ELISA Using Thioredoxin Peroxidase-1 and Tandem Repeat Proteins for Diagnosis of Schistosoma japonicum Infection among Water Buffaloes

Background

The presence of animal reservoirs in Schistosoma japonicum infection has been a major obstacle in the control of schistosomiasis. Previous studies have proven that the inclusion of control measures on animal reservoir hosts for schistosomiasis contributed to the decrease of human cases. Animal surveillance should therefore be included to strengthen and improve the capabilities of current serological tests.

Methodology/Principal Findings

Thioredoxin peroxidase-1 (SjTPx-1) and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) were initially evaluated against human sera. The previous test showed high sensitivity and specificity for antibody detection against SjTPx-1 and Sj7TR. In this study, the immunodiagnostic potential of these recombinant proteins was evaluated using enzyme-linked immunoassay on 50 water buffalo serum samples collected in Cagayan, the Philippines as compared with the soluble egg antigen (SEA). For specificity, 3 goat serum samples positive with Fasciola hepatica were used and among the antigens used, only SEA showed cross-reaction. Stool PCR targeting the S. japonicum 82 bp mitochondrial NAD 1 gene was done to confirm the true positives and served as the standard test. Twenty three samples were positive for stool PCR. SjTPx-1 and Sj1TR gave the highest sensitivity among the recombinant proteins tested for water buffalo samples with 82.61% and 78.26% respectively which were higher than that of SEA (69.57%).

Conclusions/Significance

These results prove that SjTPx-1 works both for humans and water buffaloes making it a good candidate antigen for zoonotic diagnosis. Sj1TR showed good results for water buffaloes and therefore can also be used as a possible candidate for detecting animal schistosome infection.     

Regulatory T Cells Prevent Th2 Immune Responses and Pulmonary Eosinophilia during Respiratory Syncytial Virus Infection in Mice

During viral infection, inflammation and recovery are tightly controlled by competing proinflammatory and regulatory immune pathways. Respiratory syncytial virus (RSV) is the leading global cause of infantile bronchiolitis, which is associated with recurrent wheeze and asthma diagnosis in later life. Th2-driven disease has been well described under some conditions for RSV-infected mice. In the present studies, we used the Foxp3^DTR mice (which allow specific conditional depletion of Foxp3⁺ T cells) to investigate the functional effects of regulatory T cells (Tregs) during A2-strain RSV infection. Infected Treg-depleted mice lost significantly more weight than wild-type mice, indicating enhanced disease. This enhancement was characterized by increased cellularity in the bronchoalveolar lavage (BAL) fluid and notable lung eosinophilia not seen in control mice. This was accompanied by abundant CD4⁺ and CD8⁺ T cells exhibiting an activated phenotype and induction of interleukin 13 (IL-13)- and GATA3-expressing Th2-type CD4⁺ T cells that remained present in the airways even 14 days after infection. Therefore, Treg cells perform vital anti-inflammatory functions during RSV infection, suppressing pathogenic T cell responses and inhibiting lung eosinophilia. These findings provide additional evidence that dysregulation of normal immune responses to viral infection may contribute to severe RSV disease.     

Circadian rhythm does not affect responses to chemical cues in the red swamp crayfish,Procambarus clarkii

Studies of crayfish chemical ecology have been conducted in both day and night conditions. This variation may hinder the comparison of data among studies, if the responses by crayfish to chemical cues are dependent upon the time at which the cues are encountered. We tested the hypothesis that responses to chemical cues are dependent on observation time using the red swamp crayfish, Procambarus clarkii. Procambarus clarkii is known to exhibit a light-regulated circadian rhythm, with nocturnal activity peaks. Habitat use differed significantly between non-stimulated periods and periods of exposure to a food stimulus, but no effects of photoperiod (normal vs. reversed) or laboratory conditions (dark vs. light) were observed. The results suggest that, all else being equal, (1) studies of crayfish chemical ecology can be successfully conducted in a variety of experimental conditions, and (2) previous studies conducted at various times of the day should have comparable results.     

TNFalpha-induced cytoprotection requires the production of free radicals within mitochondria in C2C12 myotubes

We previously reported that tumour necrosis factor alpha (TNFalpha) can mimic classic ischemic preconditioning (IPC) in both cells and heart. However, the signalling pathways involved remain incompletely understood. One potential protective pathway could be TNFalpha-induced reactive oxygen species (ROS). We hypothesized that TNFalpha cytoprotection occurs through the generation of ROS which originate within the mitochondria. C(2)C(12) myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNFalpha (0.5 ng/ml) prior to a simulated ischemic insult. ROS generation was evaluated on cells stained with dichlorofluorescin diacetate (DCFH-DA) by flow cytometry. The source of TNFalpha-induced ROS was examined with Mitotracker Red CM-H(2)XRos. The bioenergetics of the mitochondria were evaluated by investigation of the respiratory parameters and the inner mitochondrial membrane potential. Pretreatment with TNFalpha improved cell viability compared with the simulated ischemic control (TNFalpha: 75 +/- 1% versus 34 +/- 1% for the control: p<0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNFalpha-stimulated cells to 15 +/- 1% (p<0.001 versus TNFalpha). Similar results were obtained with IPC. TNFalpha stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNFalpha to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNFalpha-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNFalpha.     

Potential relationship between glutathione metabolism and flocculation in the yeast Kluyveromyces lactis

Reduced glutathione (GSH) is involved in biochemical and physiological processes in cells. Flocculation is an important mechanism in microorganisms. The present study concerned the potential relationship between GSH metabolism and flocculation. Two yeast strains, a flocculent (Kluyveromyces lactis 5c) and a nonflocculent (Kluyveromyces lactis 5a) strain, were used. The level of intracellular GSH measured during the growth period was significantly higher in the nonflocculent than in the flocculent strain; in contrast, the flocculent strain exhibited brighter staining of vacuoles than the nonflocculent strain when observed using epifluorescence microscopy. Compounds acting either on flocculation (EDTA, galactose) or on GSH metabolism (buthionine sulfoximine, and N-acetylcysteine) were tested on the flocculent strain during the growth period. Both EDTA and galactose fully inhibited flocculation and induced GSH overproduction of 58% and 153%, respectively. Buthionine sulfoximine decreased GSH level by 76% but had no effect on flocculation; N-acetylcysteine increased the GSH level and flocculation by 106% and 41%, respectively. Combination of EDTA and N-acetylcysteine produced similar effects than with each of them. Combination of galactose and N-acetylcysteine increased the GSH level but decreased flocculation. These results demonstrated that GSH homeostasis is linked to the flocculation mechanism. A hypothesis related to stress is given.     

Proteolytic cleavage of covalently linked cell wall proteins by Candida albicans Sap9 and Sap10

The cell wall of the human-pathogenic fungus Candida albicans is a robust but also dynamic structure which mediates adaptation to changing environmental conditions during infection. Sap9 and Sap10 are cell surface-associated proteases which function in C. albicans cell wall integrity and interaction with human epithelial cells and neutrophils. In this study, we have analyzed the enzymatic properties of Sap9 and Sap10 and investigated whether these proteases cleave proteins on the fungal cell surface. We show that Sap9 and Sap10, in contrast to other aspartic proteases, exhibit a near-neutral pH optimum of proteolytic activity and prefer the processing of peptides containing basic or dibasic residues. However, both proteases also cleaved at nonbasic sites, and not all tested peptides with dibasic residues were processed. By digesting isolated cell walls with Sap9 or Sap10, we identified the covalently linked cell wall proteins (CWPs) Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 as in vitro protease substrates. Proteolytic cleavage of the chitinase Cht2 and the glucan-cross-linking protein Pir1 by Sap9 was verified using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that upon deletion of CHT2, suggesting a direct influence of Sap9 and Sap10 on Cht2 function. In contrast, cell surface changes elicited by SAP9 and SAP10 deletion had no major impact on the phagocytosis and killing of C. albicans by human macrophages. We propose that Sap9 and Sap10 influence distinct cell wall functions by proteolytic cleavage of covalently linked cell wall proteins.     

Laminin α1 Chain Synthesis in the Mouse Developing Lung: Requirement for Epithelial–Mesenchymal Contact and Possible Role in Bronchial Smooth muscle Development

Laminins, the main components of basement membranes, are heterotrimers consisting of α, β, and γ polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of β1 and γ1 chains and differ from each other on their α chain, which is α1 and α2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial–mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin α1 chain. Synthesis of laminin α1 chain however returns upon re-establishment of epithelial–mesenchymal contact. Cell–cell contact is critical, since laminin α1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial–mesenchymal cocultures in which heterotypic cell–cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicated both epithelial and mesenchymal cells synthesize laminin α1 chain upon heterotypic cell– cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin α1, α2, and β/γ chains. Lung explants exposed to monoclonal antibodies to laminin α1 chain exhibited alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle α actin and desmin. Taken together, our studies suggest that laminin α1 chain synthesis is regulated by epithelial–mesenchymal interaction and may play a role in airway smooth muscle development.     

Herbivore and pathogen effects on tree growth are additive,but mediated by tree diversity and plant traits

Herbivores and fungal pathogens are key drivers of plant community composition and functioning. The effects of herbivores and pathogens are mediated by the diversity and functional characteristics of their host plants. However, the combined effects of herbivory and pathogen damage, and their consequences for plant performance, have not yet been addressed in the context of biodiversity–ecosystem functioning research. We analyzed the relationships between herbivory, fungal pathogen damage and their effects on tree growth in a large‐scale forest‐biodiversity experiment. Moreover, we tested whether variation in leaf trait and climatic niche characteristics among tree species influenced these relationships. We found significant positive effects of herbivory on pathogen damage, and vice versa. These effects were attenuated by tree species richness—because herbivory increased and pathogen damage decreased with increasing richness—and were most pronounced for species with soft leaves and narrow climatic niches. However, herbivory and pathogens had contrasting, independent effects on tree growth, with pathogens decreasing and herbivory increasing growth. The positive herbivory effects indicate that trees might be able to (over‐)compensate for local damage at the level of the whole tree. Nevertheless, we found a dependence of these effects on richness, leaf traits and climatic niche characteristics of the tree species. This could mean that the ability for compensation is influenced by both biodiversity loss and tree species identity—including effects of larger‐scale climatic adaptations that have been rarely considered in this context. Our results suggest that herbivory and pathogens have additive but contrasting effects on tree growth. Considering effects of both herbivory and pathogens may thus help to better understand the net effects of damage on tree performance in communities differing in diversity. Moreover, our study shows how species richness and species characteristics (leaf traits and climatic niches) can modify tree growth responses to leaf damage under real‐world conditions.     

Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

BackgroundCirculating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients.MethodsWe measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival.ResultsThe average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005).ConclusionsCirculating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.