Origin and spreading of canine morbillivirus in South America.

Canine distemper virus (CDV) is a Morbillivirus (Canine morbillivirus) that greatly impacts domestic and wildlife carnivores worldwide. The CDV RNA genome has high genetic variability, evidenced by several lineages that follow a global geographic pattern. The evolutionary trajectories and population dynamics of CDV lineages are still unclear and debatable, particularly in South America, where relatively few sequences are available. We performed phylogenetic and Bayesian analyses using an updated dataset of the highly variable hemagglutinin (H) gene, including seven South American countries. The time to the most recent common ancestor (tMRCA) of the current CDV lineages was dated to the early 1900s in North America. Maximum likelihood and Bayesian maximum clade credibility phylogenies showed similar topologies with two main branches (L1 and L2) corresponding to the NA1 lineage (L1) and the remaining lineages worldwide (L2). The four circulating lineages in South America (EU1/SA1, SA2, SA3, NA4/SA4) arose from independent migration events from North America and Europe. North American strains colonized most northern South American countries via Ecuador and then Colombia and Peru, originating the SA3 and NA4/SA4 lineages during their spread. The entry and expansion in the southern part of South America (Argentina, Brazil, Chile, and Uruguay) occurred through three independent migration events and gave rise to the EU1/SA1 and SA2 lineages. South American lineages have specific combinations of amino acids under positive selection that constitute signatures of taxonomic and evolutionary relevance. Our findings provide a comprehensive scenario for the origin and migration routes of Canine morbillivirus in South America and highlight the importance of phylodynamics in understanding the geographic patterns of modern genetic variability.

The effectiveness of treating irrigation water using ultraviolet radiation or sulphuric acid fertilizer for reducing generic Escherichia coli on fresh produce-a controlled intervention trial.

AIMS: The aims of this study were to: (i) estimate the effectiveness of ultraviolet radiation (UV) and sulphuric acid-based fertilizer (SA), at reducing levels of generic Escherichia coli in surface irrigation water and on produce and surface soil in open produce fields; and (ii) describe the population dynamics of generic E. coli in produce fields. METHODS AND RESULTS: Spinach and cantaloupe plots were randomly assigned to control, UV or SA treatment groups. Irrigation water was inoculated with Rifampicin-resistant E. coli prior to treatment. More than 75% of UV- and SA-treated tank water samples had counts below the detection limit, compared to a mean count of 3·3 Log10 CFU per ml before treatment. Levels of Rifampicin-resistant E. coli in soil and produce both increased and decreased over 10-15 days after irrigation, depending on the plot and time-period. CONCLUSIONS: UV and SA treatments effectively reduce the levels of E. coli in surface irrigation water. Their effectiveness at reducing contamination on produce was dependent on environmental conditions. Applying wait-times after irrigation and prior to harvest is not a reliable means of mitigating against contaminated produce. SIGNIFICANCE AND IMPACT OF THE STUDY: The results are of timely importance for the agricultural industry as new FSMA guidelines require producers to demonstrate a low microbial load in irrigation water or allow producers to apply a wait-time to mitigate the risk of contaminated produce.

Applying the comet assay to fresh vs frozen animal solid tissues: A technical approach.

The in vivo comet assay is usually performed in fresh tissues by processing cells immediately after collection, an approach that is not always possible from a logistical point of view. Although the comet assay has been applied to frozen rodent tissue samples on several occasions, there is currently no agreement on the best way to freeze and thaw them. We have tested two different thawing procedures and compared the levels of DNA strand breaks (SBs) and Fpg-sensitive sites in fresh and frozen (for up to year) liver, kidney and lung tissue samples, from untreated and methyl methanosulfonate treated rats. Tissues were snap frozen, stored at -80 °C and processed in such a way that the tissue remained frozen until the cells were in suspension. Our results showed that comparable levels of DNA SBs were detected in fresh and frozen liver and lung samples stored at -80 °C for up to 1 year and 3 months, respectively. In kidney, similar levels of SBs were detected either in fresh or in frozen tissues stored for up to 1 year. However, more studies are needed to control the variability observed in the Fpg-sensitive site levels in this tissue at the different freezing periods.

Liver elastography: what it is, how it is done, and how it is interpreted. / Elastografía hepática: ¿qué es, cómo se hace y cómo se interpreta?

Liver elastography is a noninvasive method for diagnosing fibrosis that has been developed over the last decade in response to the limitations of liver biopsies, blood markers, and traditional imaging modalities. There are different methods of measuring tissue stiffness through ultrasound; thus far, shear wave elastography has proven superior for diagnosing clinically significant liver fibrosis, where early detection modifies the approach to treatment and improves prognosis. This article aims to provide a brief review of the different methods for performing elastography with ultrasound, focusing especially on shear wave elastography and on technical aspects for carrying out the procedure and key points for interpreting the findings.

Hatching asynchrony vs. foraging efficiency: the response to food availability in specialist vs. generalist tit species.

Breeding mistiming is increasingly frequent in several ecosystems in the face of current climate change. Species belonging to higher trophic levels must employ mechanisms to reduce it. One of these mechanisms is hatching asynchrony, with the eggs in a clutch hatching over a period of several days. Some authors have suggested it to be adaptive when food is unpredictable. However, these birds can also suffer associated costs. We tested whether a species with higher foraging efficiency avoid hatching asynchrony compared to its sister species. We studied hatching asynchrony and nestling provisioning in relation to food availability in sympatric populations of blue and great tits. For the first time, we show that sister species respond to food availability with different strategies. Blue tit feeding rates readily responded to the abundance of their main prey, and also reduced the impact of nestling size hierarchy on mean nestling weight, consequently increasing fledging rate. Our results suggest that levels of hatching asynchrony seem to be influenced by species-specific life history traits, as generalist foragers rely less on it. They also highlight the importance of multi-species approaches when studying the response of organisms to environmental unpredictability.

The influence of landscape configuration and environment on population genetic structure in a sedentary passerine: insights from loci located in different genomic regions.

The study of the factors structuring genetic variation can help to infer the neutral and adaptive processes shaping the demographic and evolutionary trajectories of natural populations. Here, we analyse the role of isolation by distance (IBD), isolation by resistance (IBR, defined by landscape composition) and isolation by environment (IBE, estimated as habitat and elevation dissimilarity) in structuring genetic variation in 25 blue tit (Cyanistes caeruleus) populations. We typed 1385 individuals at 26 microsatellite loci classified into two groups by considering whether they are located into genomic regions that are actively (TL; 12 loci) or not (NTL; 14 loci) transcribed to RNA. Population genetic differentiation was mostly detected using the panel of NTL. Landscape genetic analyses showed a pattern of IBD for all loci and the panel of NTL, but genetic differentiation estimated at TL was only explained by IBR models considering high resistance for natural vegetation and low resistance for agricultural lands. Finally, the absence for IBE suggests a lack of divergent selection pressures associated with differences in habitat and elevation. Overall, our study shows that markers located in different genomic regions can yield contrasting inferences on landscape-level patterns of realized gene flow in natural populations.

Mycobacterium tuberculosis entry into mast cells through cholesterol-rich membrane microdomains.

Cholesterol-enriched membrane microdomains (lipid rafts) play a role in the uptake of many pathogens. Mycobacteria are one of the intracellular pathogens that utilize lipid rafts in order to invade both phagocytic and non-phagocytic cells. However, the mechanism of Mycobacterium tuberculosis uptake by mast cell is not known. To address this issue, we investigated the interaction of M. tuberculosis (H37Rv strain) with mast cells. Confocal microscopy showed that interaction of mycobacterium with mast cell resulted in changes in the mast cell surface, with formation of pseudopod-like structure and activation with visibly extruded granules. Moreover, infection of mast cells with Mycobacteria induced cholesterol accumulation at the site of bacterial entry and around intracellular mycobacteria. Disruption of mast cells lipid rafts by cholesterol depletion markedly inhibited the mycobacterium entry. Intracellular multiplication of M. tuberculosis within mast cells was also observed. Overall, our results indicate that M. tuberculosis employs a cholesterol-dependent pathway to infect mast cells, which leads to degranulation and mast cell morphological changes. These results suggest that although mast cells are capable to respond to M. tuberculosis infection, entry of mycobacterium through lipid rafts may allow replication within mast cells.

Nutrient regulation of tumor and vascular endothelial cell proliferation.

Specific bioactive dietary components, such as the steroid receptor superfamily ligands vitamins A and D, have been studied extensively as potential cancer preventive and therapeutic agents due to their ability to regulate key processes in a variety of cell types which are dysregulated in neoplastic transformation namely, proliferation and differentiation. Alteration of one or more factors that regulate cell cycle control has been described as a predisposing event for early tumor development. In addition to tumor cell proliferation, the viability, growth and metastasis of solid tumors are also dependent on the vascularization of the tumor and establishment of blood flow. Both vitamins A and D exhibit anti-angiogenic properties which further strengthen their role as potential targets for the prevention and treatment of cancer. This review focuses on the role of vitamins A and D in preventing early tumor initiation and progression via control of the cell cycle in both tumor and vascular endothelial cells.

Trichinella spiralis: histamine secretion induced by TSL-1 antigens from unsensitized mast cells.

Mast cells' hyperplasia and activation are prominent features in Trichinella spiralis infection. Recently, it was shown that TSL-1 antigens from T. spiralis muscle larvae induce IL-4 and TNF release by unsensitized, normal mast cells (MC) involving an Ig-independent mechanism. In this study, we characterized histamine secretion induced by TSL-1 antigens from normal, unsensitized rat peritoneal MC. Maximum histamine secretion (30+/-5.3% SEM, n=13) was achieved with 30 ng/mL TSL-1 antigens. However, TSL-1 did not induce an increase in beta-hexosaminidase release or NADPH oxidase activity by MC. Interestingly, histamine secretion by TSL-1 was completed at 10s, and was inhibited by both Bordetella pertussis toxin and neuraminidase V, characteristics similar to those involved in substance P-induced histamine secretion. However, in contrast to substance P, TSL-1 induced histamine secretion in the absence of detectable changes in intracellular Ca(2+). We are investigating the molecular pathways involved in MC activation by TSL-1.

Analysis of immune lesions in neurocysticercosis patients: central nervous system response to helminth appears Th1-like instead of Th2.

Neurocysticercosis (NCC) caused by the helminth Taenia solium is the most common parasitic infection of the human central nervous system (CNS) worldwide. Because clinical symptoms are associated with localized immunological responses in the brain, characterization of these responses are pivotal for understanding the pathogenesis of cysticercosis. Immunohistochemical analysis of brain specimens from several patients with cysticercosis revealed at least four types of immune responses, including: (i) an antibody response (IgM + plasma cells), (ii) a predominant NK response, (iii) an infiltrate with abundant macrophages and granulocytes, and (iv) an intense infiltrate with a predominance of macrophages and T cells. The intensity and type of immunity appeared to be associated somewhat with the parasite's viability and anatomical location. In most of the lesions, cell mediated responses were evident and proinflammatory cytokines including IL12 predominated. Moreover, IL4 was undetectable in the immune infiltrates. Thus, the CNS response to this helminth, unlike the systemic response, is predominately Th1-like.

Effects of toxin A from Clostridium difficile on mast cell activation and survival.

Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-alpha). In contrast, 1 microg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 microg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response to C. difficile infection by releasing TNF-alpha upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficile toxins.

TGF-beta1 inhibits the release of histamine and tumor necrosis factor-alpha from mast cells through an autocrine pathway.

Transforming growth factor beta1 (TGF-beta1) is a member of a gene superfamily involved in the regulation of cell growth and differentiation, tissue repair, fibrosis, and inflammatory responses. Given the role of the mast cell (MC) in inflammation and fibrosis, the effect of TGF-beta1 on MC mediator release was studied. In vitro treatment of rat peritoneal MC (PMC) with TGF-beta1 (10(-10) M) for 20 h followed by washes inhibited (23%) antigen stimulated histamine release. Similar pretreatment of PMC with TGF-beta1 (10(-10) M) inhibited (27%) tumor necrosis factor-alpha (TNF-alpha) dependent cytotoxicity and reduced (31%) mRNA levels of TNF-alpha, but did not inhibit nitric oxide (NO) release. By contrast, the presence of TGF-beta1 throughout the cytotoxic assay, but without pretreatment of PMC did not modulate TNF-alpha release. At least 2 h pretreatment with TGF-beta1 was required to inhibit MC TNF-alpha-dependent cytotoxicity. This inhibitory effect of TGF-beta1 was abrogated by antibody to TGF-beta1. Interestingly, the treatment of PMC with anti-TGF-beta1 antibody alone significantly increased the release of histamine and TNF-alpha. Furthermore, freshly isolated rat PMC (10(7)) contained 35 +/- 7 pg latent TGF-beta1 and 51 +/- 9 pg was spontaneously released within 30 min of culture. However, stimulation of PMC with antigen inhibited the spontaneous release of TGF-beta1 by 43%. The duration of pretreatment with TGF-beta1 required to inhibit MC TNF-alpha release was similar to that required for downregulation of MC TNF-alpha-dependent cytotoxicity by IFN-gamma. TGF-beta1 and IFN-gamma had an additive inhibition on TNF-alpha release by PMC. This inhibitory effect was abrogated and TNF-alpha-dependent cytotoxicity was enhanced by the addition of anti-TGF-beta1 antibody, but not by anti-IFN-gamma. These results suggest MC mediator release is regulated by TGF-beta1 in an autocrine manner.

Regulation of mRNA levels of TNF-alpha and the alpha chain of the high-affinity receptor for IgE in mast cells by IFN-gamma and alpha/beta.

Rat mast cell lines (hybrid rat mast cells, HRMC, and rat cultured mast cells, RCMC) and mast cells from the rat body cavity were used to test the hypothesis that IFN-alpha/beta and IFN-gamma inhibit tumor necrosis factor alpha (TNF-alpha)-mediated cytotoxicity by depressing the steady-state levels of mRNA for TNF-alpha. In vitro treatment of mast cells with IFN-gamma and IFN-alpha/beta depressed mRNA levels. By contrast, IFN pretreatment of mast cell lines induced an increase in levels of mRNA for the IFN-inducible gene, 2'5'-oligoadenylate synthetase and also for high-affinity IgE-receptor-alpha. The IFN-mediated regulation of mast cells may be an important mechanism in the control of inflammatory pathways characterized by Th1- and Th2-type responses.

Inhibitory effects of sulfasalazine and its metabolites on histamine release and TNF-alpha production by mast cells.

Sulfasalazine is an effective treatment in some inflammatory diseases that exhibit mast cell (MC) hyperplasia. However, its effect on MC has been incompletely studied. We have established that sulfasalazine inhibits the release of histamine and TNF-alpha from MC. Sulfasalazine and its metabolites, 5-aminosalicylic acid (5-ASA) and to a lesser extent sulfapyridine, inhibited Ag-stimulated histamine release from rat peritoneal MC in a concentration-dependent manner with a 50% inhibitory concentration of 6 x 10(6)M, 8 x 10(-6)M, and 3 x 10(-4)M, respectively. Similar results were observed with sulfapyridine and 5-ASA on Ag-stimulated histamine release of another population of MC, namely rat intestinal mucosal MC, but sulfasalazine was markedly less potent than its metabolites. Interestingly, sulfasalazine and sulfapyridine, but not 5-ASA, inhibited Ag-stimulated TNF-alpha released by MC. Similar results were observed with MC-mediated cytotoxic activity in which sulfasalazine and sulfapyridine, but nor 5-ASA, inhibited MC TNF-alpha-dependent cytotoxicity in a concentration-dependent manner. The addition of sulfasalazine to MC, up to 12 h after the cytotoxic assay (16 h) had started, significantly inhibited cytotoxic activity, suggesting that sulfasalazine inhibited the cytotoxic mediator, TNF-alpha. Indeed, affinity studies demonstrated that sulfasalazine binds TNF-alpha. Furthermore, the inhibition of MC cytotoxicity by sulfasalazine appeared to require new protein synthesis. Pretreatment of MC with sulfasalazine also inhibited the release of TNF-alpha and reduced the levels of TNF-alpha mRNA. Thus, sulfasalazine inhibits MC-mediated, TNF-alpha-dependent cytotoxicity by multiple mechanisms: competitive inhibition of soluble TNF-alpha, reduction of levels of TNF-alpha mRNA, and inhibition of TNF-alpha release.

Inhibition of tumour necrosis factor-alpha (TNF-alpha) release from mast cells by the anti-inflammatory drugs, sodium cromoglycate and nedocromil sodium.

TNF-alpha is a cytokine thought to be involved in the pathogenesis of asthma and in several other inflammatory conditions. Given recent evidence that mast cells (MC) are an important source of TNF-alpha, we investigated the effects of two anti-inflammatory drugs, nedocromil sodium (NED) and sodium cromoglycate (SCG), on rat MC-derived TNF-alpha. We established that at least 2 h pretreatment with NED or SCG followed by washing was required to inhibit TNF-alpha-dependent cytotoxicity by rat peritoneal MC (PMC). A maximum inhibition of TNF-alpha occurred after 6 h treatment. The inhibitory effect of NED and SCG (10(-5)-10(-3)M) was concentration-dependent (20-37% for NED and 16-37% for SCG). The time-course analysis and the use of cycloheximide, an inhibitor of protein synthesis, provided strong evidence that new protein synthesis by the MC is required for this inhibitory effect. Furthermore, 24 h treatment with 1 mM NED inhibited the levels of mRNA for TNF-alpha by 59-83%. In addition to the effect on TNF-alpha-dependent cytotoxicity by MC, 20 min pretreatment with 10(-4) M NED and SCG inhibited antigen-stimulated TNF-alpha release (6h) by 42% and 48%, respectively. Interestingly, the functionally distinct intestinal mucosal MC (IMMC) is unresponsive to these drugs with regard to histamine secretion. However, as with PMC, 2h pretreatment with NED or SCG inhibited TNF-alpha-dependent cytotoxicity by IMMC. These effects may be important in the action of these drugs in vivo in the late phase reaction in asthma or other inflammatory conditions.

Interferon and antiallergic drug regulation of histamine and tumor necrosis factor-alpha in rat mast cell subsets.

We have further characterized the heterogeneity of mast cells (MCs) by comparing the ability of rat peritoneal MCs (PMCs) and intestinal mucosal MCs (IMMCs) to produce tumor necrosis factor (TNF)-alpha and by investigating its regulation by interferon (IFN) and the antiallergic drugs nedocromil sodium (NED) and sodium cromoglycate (SCG). Although IMMCs store less TNF-alpha than PMCs, they produced comparable amounts of TNF-alpha in cytotoxic assays. Just as SCG and NED inhibit histamine secretion from PMCs but not IMMCs, IFN exhibited a similar differential effect on histamine release from these cells. However, SCG, NED, and IFN inhibit TNF-alpha-dependent cytotoxicity by both PMCs and IMMCs and reduce the steady-state levels of mRNA for TNF-alpha in PMCs. Thus, the modulation of MC mediator release depends upon the MC population and mediator studied. The inhibitory effect of SCG and NED on TNF-alpha release from MCs may explain some of their anti-inflammatory and therapeutic effects.