Nimesulide inhibits pathogenic fungi: PGE2-dependent mechanisms

Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 μg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 μg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.     

Profile of Central and Effector Memory T Cells in the Progression of Chronic Human Chagas Disease

Background

Chronic Chagas disease presents several different clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive clinical forms. Several studies have demonstrated that immunoregulatory mechanisms are important processes for the control of the intense immune activity observed in the chronic phase. T cells play a critical role in parasite specific and non-specific immune response elicited by the host against Trypanosoma cruzi. Specifically, memory T cells, which are basically classified as central and effector memory cells, might have a distinct migratory activity, role and function during the human Chagas disease.

Methodology/Principal Findings

Based on the hypothesis that the disease severity in humans is correlated to the quality of immune responses against T. cruzi, we evaluated the memory profile of peripheral CD4⁺ and CD8⁺ T lymphocytes as well as its cytokine secretion before and after in vitro antigenic stimulation. We evaluated cellular response from non-infected individuals (NI), patients with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas disease. The expression of CD45RA, CD45RO and CCR7 surface molecules was determined on CD4⁺ and CD8⁺ T lymphocytes; the pattern of intracellular cytokines (IFN-γ, IL-10) synthesized by naive and memory cells was determined by flow cytometry. Our results revealed that IND and CARD patients have relatively lower percentages of naive (CD45RA^high) CD4⁺ and CD8⁺ T cells. However, statistical analysis of ex-vivo profiles of CD4⁺ T cells showed that IND have lower percentage of CD45RA^high in relation to non-infected individuals, but not in relation to CARD. Elevated percentages of memory (CD45RO^high) CD4⁺ T cells were also demonstrated in infected individuals, although statistically significant differences were only observed between IND and NI groups. Furthermore, when we analyzed the profile of secreted cytokines, we observed that CARD patients presented a significantly higher percentage of CD8⁺CD45RA^high IFN-γ-producing cells in control cultures and after antigen pulsing with soluble epimastigote antigens.

Conclusions

Based on a correlation between the frequency of IFN-γ producing CD8+ T cells in the T cell memory compartment and the chronic chagasic myocarditis, we propose that memory T cells can be involved in the induction of the development of the severe clinical forms of the Chagas disease by mechanisms modulated by IFN-γ. Furthermore, we showed that individuals from IND group presented more T_CM CD4⁺ T cells, which may induce a regulatory mechanism to protect the host against the exacerbated inflammatory response elicited by the infection.     

Physiological and production responses of plant growth forms to increases in limiting resources in alpine tundra: implications for differential community response to environmental change

Physiological and growth measurements were made on forbs and graminoids following additions of water and N+water in a graminoid-dominated dry meadow and a forb-dominated moist meadow, to determine if the community-level response was related to differential responses between the growth forms. Graminoids had higher photosynthetic rates and lower transpiration rates and foliar N concentrations than forbs, and consequently maintained higher photosynthetic N- and water-use efficiencies. Photosynthetic rates, stomatal conductance, and transpiration rates increased significantly only in response to N fertilization and only in moist meadow species. The increase in photosynthetic rates was unrelated to variation in foliar N concentration, but instead correlated with variation in stomatal conductance. Growth based N-use efficiency was higher in moist meadow graminoids than in moist meadow forbs, but did not differ between the growth forms in the dry meadow. The moist meadow community had higher biomass and N standing crops, but the relative increase in these factors in response to N fertilization was greater in the dry meadow. Graminoids had a greater relative increase in biomass and N accumulation than forbs following N fertilization, but moist meadow graminoids exhibited a greater response than dry meadow graminoids. The difference in the growth response between the dry meadow and moist meadow graminoids to N fertilization was correlated with more conservative leaf gas exchange responses in dry meadow species, presumably related to a higher frequency of soil water deficits in this community. Community-level response to the resource additions was therefore mediated by the plant growth form response, corresponding with differences between the growth forms in physiological factors related to resource acquisition and use.     

Effect of gluten diet on blood innate immune gene expressions and stool consistency in Spix’s Saddleback Tamarin (Leontocebus fuscicollis) raised in captivity

The callitrichids are non-human primates that feed on insects and plant matter in nature, but in captivity, they are fed mostly an artificial diet containing amounts of gluten, in their toxic forms in items such as wheat, barley and rye. The aim of this research was to estimate the blood β-defensin and Toll like receptor 5 (TLR5) gene expressions and to analyze the stool consistency (firm, soft, diarrheic) in Leontocebus fuscicollis raised in captivity. Blood samples of animals under gluten-free and gluten diets were collected and their fecal output quality was periodically monitored and classified during the course of the study. Gene expression was evaluated using real-time PCR. The stool consistencies of individuals fed a gluten diet were most frequently soft or diarrheic, while it was mostly normal in individuals fed a gluten-free diet. β-Defensin expression increased in individuals fed a gluten diet, but decreased after 15 days. Expression normalized between 30 and 45 days on a gluten-free diet. However, expression of the TLR5 gene did not change under a gluten diet. A gluten diet affects stool quality, and brings about an immediate increase in blood β-defensin expression in the beginning but decreases after 15 days.

     

Distribution of Dengue Virus Types 1 and 4 in Blood Components from Infected Blood Donors from Puerto Rico

Background

Dengue is a mosquito-borne viral disease caused by the four dengue viruses (DENV-1 to 4) that can also be transmitted by blood transfusion and organ transplantation. The distribution of DENV in the components of blood from infected donors is poorly understood.

Methods

We used an in-house TaqMan qRT-PCR assay to test residual samples of plasma, cellular components of whole blood (CCWB), serum and clot specimens from the same collection from blood donors who were DENV-RNA-reactive in a parallel blood safety study. To assess whether DENV RNA detected by TaqMan was associated with infectious virus, DENV infectivity in available samples was determined by culture in mosquito cells.

Results

DENV RNA was detected by TaqMan in all tested blood components, albeit more consistently in the cellular components; 78.8% of CCWB, 73.3% of clots, 86.7% of sera and 41.8% of plasma samples. DENV-1 was detected in 48 plasma and 97 CCWB samples while DENV-4 was detected in 21 plasma and 31 CCWB samples. In mosquito cell cultures, 29/111 (26.1%) plasma and 32/97 (32.7%) CCWB samples were infectious. A subset of samples from 29 donors was separately analyzed to compare DENV viral loads in the available blood components. DENV viral loads did not differ significantly between components and ranged from 3–8 log₁₀ PCR-detectable units/ml.

Conclusions

DENV was present in all tested components from most donors, and viral RNA was not preferentially distributed in any of the tested components. Infectious DENV was also present in similar proportions in cultured plasma, clot and CCWB samples, indicating that these components may serve as a resource when sample sizes are limited. However, these results suggest that the sensitivity of the nucleic acid tests (NAT) for these viruses would not be improved by testing whole blood or components other than plasma.     

Microclimate and substrate quality controls on nitrogen mineralization in a New England high salt marsh

New England high salt marsh primary productivity is limited by N, but variation in plant N availability across salt marsh vegetation zones has not been quantified. To investigate this, we measured in situ net N mineralization rates throughout the growing season in three zones of a Maine high salt marsh, Juncus gerardii, Spartina patens, and mixed perennial forb. We also measured microclimate factors (soil temperatures and moistures) and substrate quality parameters (soil organic matter, soil total nitrogen, soil C:N ratio) to see if either related to differences in net N mineralization. To determine the relative importance of substrate quality and microclimate, we measured N mineralization of the different soil types in the laboratory, holding microclimate parameters constant. We also investigated the relative importance of microclimate and substrate statistically, with principal components analysis and multiple regression. In situ net N mineralization rates were significantly higher in the forb zone than in graminoid zones, but graminoid zone N mineralization rates did not vary significantly from each other. Soil temperatures, moistures, carbon, and nitrogen were all significantly higher in the forb zone than in graminoid zones, but C:N ratio did not vary significantly across zones. Principal components analysis and multiple regression revealed that microclimate was a more significant predictor of total N mineralized over the course of the growing season than was substrate quality. In contrast, when microclimate conditions were held constant, forb zone N mineralization was still significantly higher than that of graminoid zones, suggesting that substrate quality does exert some control on this process. Thus, both microclimate and substrate quality appear to influence N mineralization rates across vegetation zones of this Maine salt marsh.     

Serovar Diversity of Pathogenic Leptospira Circulating in the French West Indies

Background

Leptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012.

Methods and Findings

Strains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and secY. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchi, and L. santarosai. We also identified L. kmetyi in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with L. interrogans serovars Icterohaemorrhagiae and Copenhageni, L. kirschneri serovar Bogvere, and L. borgpetersenii serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new secY alleles.

Conclusions

The overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars.     

Comparison of shoot tip culture and cryotherapy for eradication of ampeloviruses associated with Pineapple mealybug wilt in wild varieties

The Pineapple mealybug wilt-associated virus (PMWaV) provokes a disease that causes considerable losses to pineapple growers due to the reduced leaf tissue turgescence, resulting in leaf necrosis and death in severe cases. In this work, we describe the comparison of shoot tip culture and cryotherapy methods to eradicate the pineapple wilt disease-associated ampeloviruses. Plants from the accessions Ananas comosus var. comosus (AGB-009), var. bracteatus (AGB-119), and var. parguazensis (AGB-376) were indexed by RT-PCR, confirming mixed infections of PMWaV-1, PMWaV-2, and PMWaV-3. The accessions were cultured in vitro and their shoot tips treated by cryotherapy following a droplet vitrification protocol. The regeneration rate from shoot tip culture was 93% for AGB-376 and 100% for the other two accessions. After freezing, AGB-376 had 100% regeneration subsequent to exposure to PVS2 for 45 min, followed by 95% for AGB-009, while for AGB-119 the optimal exposure time was 60 min, with plant regeneration from nearly 80% of the shoot tips. For the accessions AGB-009 and AGB-376, all the recovered plants were virus free by the two methods, while 50% of the plants from accession AGB-119 remained infected. These results indicate that shoot tip culture alone or in association with cryotherapy is a promising routine method for virus removal from pineapple plant tissues and is useful to ensure that backup reserves of pineapple germplasm, conserved by in vitro bank and cryobank, are formed with virus-free plants.

     

Variation in nutrient availability and plant species diversity across forb and graminoid zones of a Northern New England high salt marsh

Plant zonation patterns across New England salt marshes have been investigated for years, but how nutrient availability differs between zones has received little attention. We investigated how N availability, P availability, and plant N status varied across Juncus gerardii, Spartina patens, and mixed forb zones of a Northern New England high salt marsh. We also investigated relationships between several edaphic factors and community production and diversity across the high marsh. P availability, soil salinity, and soil moisture were higher in the mixed forb zone than in the two graminoid zones. NH⁺ ₄-N availability was highest in the J. gerardii zone, but NO^– ₃-N availability and mid season net N mineralization rates did not vary among zones. Plant tissue N concentrations were highest in the mixed forb zone and lowest in the S. patens zone, reflecting plant physiologies more so than soil N availability. Community production was highest in the J. gerardii zone and was positively correlated with N availability and negatively correlated with soil moisture. Plant species diversity was highest in the mixed forb zone and was positively correlated with P availability and soil salinity. Thus, nutrient availability, plant N status, and plant species diversity varied across zones of this high marsh. Further investigation is needed to ascertain if soil nutrient availability influences or is a result of the production and diversity differences that exist between vegetation zones of New England high salt marshes.     

Deregulation of the Protocadherin Gene FAT1 Alters Muscle Shapes: Implications for the Pathogenesis of Facioscapulohumeral Dystrophy

Generation of skeletal muscles with forms adapted to their function is essential for normal movement. Muscle shape is patterned by the coordinated polarity of collectively migrating myoblasts. Constitutive inactivation of the protocadherin gene Fat1 uncoupled individual myoblast polarity within chains, altering the shape of selective groups of muscles in the shoulder and face. These shape abnormalities were followed by early onset regionalised muscle defects in adult Fat1-deficient mice. Tissue-specific ablation of Fat1 driven by Pax3-cre reproduced muscle shape defects in limb but not face muscles, indicating a cell-autonomous contribution of Fat1 in migrating muscle precursors. Strikingly, the topography of muscle abnormalities caused by Fat1 loss-of-function resembles that of human patients with facioscapulohumeral dystrophy (FSHD). FAT1 lies near the critical locus involved in causing FSHD, and Fat1 mutant mice also show retinal vasculopathy, mimicking another symptom of FSHD, and showed abnormal inner ear patterning, predictive of deafness, reminiscent of another burden of FSHD. Muscle-specific reduction of FAT1 expression and promoter silencing was observed in foetal FSHD1 cases. CGH array-based studies identified deletion polymorphisms within a putative regulatory enhancer of FAT1, predictive of tissue-specific depletion of FAT1 expression, which preferentially segregate with FSHD. Our study identifies FAT1 as a critical determinant of muscle form, misregulation of which associates with FSHD.