Acute blockade of endogenous melatonin by Luzindole, with or without peripheral LPS injection, induces jejunal inflammation and morphological alterations in Swiss mice.

This study investigated the acute blockade of endogenous melatonin (MLT) using Luzindole with or without systemic lipopolysaccharide (LPS) challenge and evaluated changes in inflammatory and oxidative stress markers in the mouse jejunum. Luzindole is an MT1/MT2 MLT receptor antagonist. Both receptors occur in the small intestine. Swiss mice were treated with either saline (0.35 mg/kg, ip), Luzindole (0.35 mg/kg, ip), LPS (1.25 mg/kg, ip), or Luzindole+LPS (0.35 and 1.25 mg/kg, ip, respectively). Jejunum samples were evaluated regarding intestinal morphometry, histopathological crypt scoring, and PAS-positive villus goblet cell counting. Inflammatory Iba-1, interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, nuclear factor (NF)-kB, myeloperoxidase (MPO), and oxidative stress (NP-SHs, catalase, MDA, nitrate/nitrite) markers were assessed. Mice treated with Luzindole, LPS, and Luzindole+LPS showed villus height shortening. Crypt damage was worse in the LPS group. Luzindole, LPS, and Luzindole+LPS reduced the PAS-goblet cell labeling and increased Iba-1-immunolabelled cells compared to the saline group. Immunoblotting for IL-1ß, TNF-α, and NF-kB was greater in the Luzindole group. The LPS-challenged group showed higher MPO activity than the saline and Luzindole groups. Catalase was reduced in the Luzindole and Luzindole+LPS groups compared to saline. The Luzindole group showed an increase in NP-SHs, an effect related to compensatory GSH activity. The acute blockade of endogenous MLT with Luzindole induced early changes in inflammatory markers with altered intestinal morphology. The other non-detectable deleterious effects of Luzindole may be balanced by the unopposed direct action of MLT in immune cells bypassing the MT1/MT2 receptors.

Acute blockade of endogenous melatonin by Luzindole, with or without peripheral LPS injection, induces jejunal inflammation and morphological alterations in Swiss mice

This study investigated the acute blockade of endogenous melatonin (MLT) using Luzindole with or without systemic lipopolysaccharide (LPS) challenge and evaluated changes in inflammatory and oxidative stress markers in the mouse jejunum. Luzindole is an MT1/MT2 MLT receptor antagonist. Both receptors occur in the small intestine. Swiss mice were treated with either saline (0.35 mg/kg, ip), Luzindole (0.35 mg/kg, ip), LPS (1.25 mg/kg, ip), or Luzindole+LPS (0.35 and 1.25 mg/kg, ip, respectively). Jejunum samples were evaluated regarding intestinal morphometry, histopathological crypt scoring, and PAS-positive villus goblet cell counting. Inflammatory Iba-1, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, nuclear factor (NF)-kB, myeloperoxidase (MPO), and oxidative stress (NP-SHs, catalase, MDA, nitrate/nitrite) markers were assessed. Mice treated with Luzindole, LPS, and Luzindole+LPS showed villus height shortening. Crypt damage was worse in the LPS group. Luzindole, LPS, and Luzindole+LPS reduced the PAS-goblet cell labeling and increased Iba-1-immunolabelled cells compared to the saline group. Immunoblotting for IL-1β, TNF-α, and NF-kB was greater in the Luzindole group. The LPS-challenged group showed higher MPO activity than the saline and Luzindole groups. Catalase was reduced in the Luzindole and Luzindole+LPS groups compared to saline. The Luzindole group showed an increase in NP-SHs, an effect related to compensatory GSH activity. The acute blockade of endogenous MLT with Luzindole induced early changes in inflammatory markers with altered intestinal morphology. The other non-detectable deleterious effects of Luzindole may be balanced by the unopposed direct action of MLT in immune cells bypassing the MT1/MT2 receptors.

Aeromonas piscicola AH-3 expresses an extracellular collagenase with cytotoxic properties.

UNLABELLED: The aim of this study was to investigate the presence and the phenotypic expression of a gene coding for a putative collagenase. This gene (AHA_0517) was identified in Aeromonas hydrophila ATCC 7966 genome and named colAh. We constructed and characterized an Aeromonas piscicola AH-3::colAh knockout mutant. Collagenolytic activity of the wild-type and mutant strains was determined, demonstrating that colAh encodes for a collagenase. ColAh-collagen interaction was assayed by Far-Western blot, and cytopathic effects were investigated in Vero cells. We demonstrated that ColAh is a gluzincin metallopeptidase (approx. 100 kDa), able to cleave and physically interact with collagen, that contributes for Aeromonas collagenolytic activity and cytotoxicity. ColAh possess the consensus HEXXH sequence and a glutamic acid as the third zinc binding positioned downstream the HEXXH motif, but has low sequence similarity and distinct domain architecture to the well-known clostridial collagenases. In addition, these results highlight the importance of exploring new microbial collagenases that may have significant relevance for the health and biotechnological industries. SIGNIFICANCE AND IMPACT OF THE STUDY: Collagenases play a central role in processes where collagen digestion is needed, for example host invasion by pathogenic micro-organisms. We identified a new collagenase from Aeromonas using an integrated in silico/in vitro strategy. This enzyme is able to bind and cleave collagen, contributes for AH-3 cytotoxicity and shares low similarity with known bacterial collagenases. This is the first report of an enzyme belonging to the gluzincin subfamily of the M9 family of peptidases in Aeromonas. This study increases the current knowledge on collagenolytic enzymes bringing new perspectives for biotechnology/medical purposes.

Impact of microbial count distributions on human health risk estimates.

Quantitative microbiological risk assessment (QMRA) is influenced by the choice of the probability distribution used to describe pathogen concentrations, as this may eventually have a large effect on the distribution of doses at exposure. When fitting a probability distribution to microbial enumeration data, several factors may have an impact on the accuracy of that fit. Analysis of the best statistical fits of different distributions alone does not provide a clear indication of the impact in terms of risk estimates. Thus, in this study we focus on the impact of fitting microbial distributions on risk estimates, at two different concentration scenarios and at a range of prevalence levels. By using five different parametric distributions, we investigate whether different characteristics of a good fit are crucial for an accurate risk estimate. Among the factors studied are the importance of accounting for the Poisson randomness in counts, the difference between treating "true" zeroes as such or as censored below a limit of quantification (LOQ) and the importance of making the correct assumption about the underlying distribution of concentrations. By running a simulation experiment with zero-inflated Poisson-lognormal distributed data and an existing QMRA model from retail to consumer level, it was possible to assess the difference between expected risk and the risk estimated with using a lognormal, a zero-inflated lognormal, a Poisson-gamma, a zero-inflated Poisson-gamma and a zero-inflated Poisson-lognormal distribution. We show that the impact of the choice of different probability distributions to describe concentrations at retail on risk estimates is dependent both on concentration and prevalence levels. We also show that the use of an LOQ should be done consciously, especially when zero-inflation is not used. In general, zero-inflation does not necessarily improve the absolute risk estimation, but performance of zero-inflated distributions in QMRA tends to be more robust to changes in prevalence and concentration levels, and to the use of an LOQ to interpret zero values, compared to that of their non-zero-inflated counterparts.

Fitting a distribution to microbial counts: making sense of zeroes.

The accurate estimation of true prevalence and concentration of microorganisms in foods is an important element of quantitative microbiological risk assessment (QMRA). This estimation is often based on microbial detection and enumeration data. Among such data are artificial zero counts, that originated by chance from contaminated food products. When these products are not differentiated from uncontaminated products that originate true zero counts, the estimates of true prevalence and concentration may be inaccurate. This inaccuracy is especially relevant in situations where highly pathogenic bacteria are involved and where growth can occur along the food pathway. Our aim was to develop a method that provides accurate estimates of concentration parameters and differentiates between artificial and true zeroes, thus also accurately estimating true prevalence. We first show the disadvantages of using a limit of quantification (LOQ) threshold for the analysis of microbial enumeration data. We show that, depending on the original distribution of concentrations and the LOQ value, it may be incorrect to treat artificial zeroes as censored below a quantification threshold. Next, a method is developed that estimates the true prevalence of contamination within a food lot and the parameters characterizing the within-lot distribution of concentrations, without assuming a LOQ, and using raw plate count data as an input. Counts resulting both from contaminated and uncontaminated sample units are analysed together. This procedure allows the estimation of the proportion of artificial zeroes among the total of zero counts, and therefore the estimation of true prevalence from enumeration results. We observe that this method yields best estimates of mean, standard deviation and prevalence at low true prevalence levels and low expected standard deviation. Furthermore, we conclude that the estimation of prevalence and the estimation of the distribution of concentrations are interrelated and therefore should be estimated simultaneously. We also conclude that one of the keys to an accurate characterization of the overall microbial contamination is the correct identification and separation of true and artificial zeroes. Our method for the analysis of quantitative microbial data shows a good performance in the estimation of true prevalence and the parameters of the distribution of concentrations, which indicates that it is a useful data analysis tool in the field of QMRA.

Umbilical cord blood CD34(+) stem cells and other mononuclear cell subtypes processed up to 96 h from collection and stored at room temperature maintain a satisfactory functionality for cell therapy.

BACKGROUND AND OBJECTIVES: Umbilical cord blood (UCB) is a good stem cell source for cell therapy. We recently demonstrated that cord blood mononuclear cell (MNCs) subtypes were viable and functional until 96 h after collection, even stored at room temperature. Now, we analyzed the viability and functionality of the cells before and after cryopreservation. MATERIALS AND METHODS: Twenty UCB units were analyzed at 24 and 96 h after collection, frozen for 6 months, thawed and re-evaluated. MNCs were analyzed by flow cytometry, viability by 7-AAD and clonogenic assays (CFU) were performed. RESULTS: After 96 h of storage, no substantial loss of MNC was found (median 7.320 × 10(6 ) × 6.05 × 10(6) ). Percentage and viability CD34(+) cells, B-cell precursors and mesenchymal stem cells were not affected. However, mature B and T lymphocytes as well as granulocytes had a substantial loss. CFU growth was observed in all samples. Prefreezing storage of 96 h was associated with a relative loss of colony formation (median 12%). Postthaw, this loss had a median of 49% (24 h samples) to 56% (96 h samples). CONCLUSION: The delay of 96 h before UCB processing is possible, without a prohibitive impairment of CD34(+) loss in number and functionality.

Chondrogenesis from umbilical cord blood cells stimulated with BMP-2 and BMP-6.

Umbilical cord blood contains undifferentiated mesenchymal stem cells (MSCs) with chondrogenic potential that may be used for the repair of joint damage. The role of growth factors during the process of chondrogenesis is still not entirely understood. The objective of this study was to evaluate the formation of chondrocytes, cartilaginous matrix and type II collagen from human umbilical cord blood stem cells exposed to two different growth factors, BMP-6 and BMP-2, while being cultured as a micromass or a monolayer. Umbilical cord blood was obtained from full-term deliveries, and then, mononuclear cells were separated and cultured for expansion. Afterward, these cells were evaluated by flow cytometry using antibodies specific for MSCs and induced to chondrogenic differentiation in micromass and monolayer cultures supplemented with BMP-2 and BMP-6. Cellular phenotype was evaluated after 7, 14 and 21 days by RT-PCR and Western blot analysis to identify the type II collagen and aggrecan. The expanded cells displayed surface antigens characteristic of mesenchymal progenitor cells and were negative for hematopoietic differentiation antigens. Type II collagen and aggrecan mRNAs were expressed from day 14 in cells stimulated with BMP-2 or BMP-6. Type II collagen was demonstrated by Western blotting in both groups, and the greatest expression was observed 21 days after the cells were stimulated with BMP-2 cultured in micromass. BMP-2 in micromass culture was more efficient to induce the chondrogenesis.

Produção de frutos para uso medicinal em Bromelia antiancatha (caraguatá): fundamentos para um extrastivismo sustentável / Fruit production for medicinal use in Bromelia antiacantha ("caraguatá"): foundations for sustainable extraction

Entre as várias espécies que têm sido utilizadas como fontes de subprodutos florestais estão a Bromelia antiacantha, espécie nativa da Mata Atlântica com grande potencial de uso com características alimentícias, ornamentais, industriais e farmacológicas. Os frutos da espécie são utilizados tradicionalmente no Planalto Norte Catarinense na confecção de xaropes para tratamento de males das vias respiratórias. Neste contexto, objetivou-se quantificar a produção de frutos e fundamentar estratégias para possível manejo de populações naturais de B. antiacantha. Foram acompanhadas 39 infrutescências de Janeiro/2008 a Agosto/2008 distribuídas em área de mata secundária na FLONA de Três Barras, SC. Nesta mesma área, indivíduos da espécie foram acompanhados através de estudos demográficos de 2001 a 2008. As infrutescências apresentaram em média 0,68m de comprimento, número médio de 187 frutos/infrutescência, o diâmetro médio dos frutos foi de 1,9 cm e o peso médio das infrutescências de 3,6 kg. A média de frutos aproveitáveis foi de 157 frutos/infrutescência totalizando 2,5 kg. A safra estimada para 2005 foi de 146 kg de frutos ha-1 e 80 kg de frutos ha-1 para 2008, e a renda líquida a partir da produção de xarope foi estimada em R$ 1168,00 por hectare, por ano. Os resultados mostraram que o manejo de B. antiacantha consiste em atividade economicamente interessante e que esta possibilidade, além de complementar a renda de comunidades locais onde a espécie se faz presente, também amplia o valor das áreas com cobertura florestal.

Among the large number of species that have been used as sources of forest byproducts is Bromelia antiacantha, a species native to the Atlantic Forest and that has great potential of use with nourishing, ornamental, industrial and pharmacological characteristics. The fruits of this species are traditionally used in the Northern Plateau of Santa Catarina State, Brazil, in the preparation of syrup for the treatment of respiratory disorders. In this context, the aim of this study was to quantify the production of fruits and to find strategies for the possible management of natural populations of B. antiacantha. A total of 39 inflorescences were observed from January/2008 to August/2008 distributed in a secondary forest area at FLONA (National Forest), Três Barras, Santa Catarina State. In this same area, B. antiacantha individuals were accompanied by demographic studies from 2001 to 2008. The inflorescences presented on average 0.68 m of length, mean number of 187 fruits/inflorescence, mean diameter of fruits of 1.9 cm and mean weight of inflorescences of 3.6 kg. The mean number of usable fruits was 157 fruits/inflorescence and the mean weight of these fruits was 2.5 Kg. The estimated harvest for 2005 was 146 Kg of fruits ha-1 and for 2008, 80 kg of fruits ha-1, and the net income from the syrup production could reach R$ 1168.00 per hectare per year. The results showed that the management of B. antiacantha consists in an economically interesting activity and that this possibility complements the income of local communities where the species occurs, besides increasing the value of forest areas.

Global gene expression reveals a set of new genes involved in the modification of cells during erythroid differentiation.

OBJECTIVES: Erythroid differentiation is a dynamic process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. In this study, gene expression profiles during differentiation of human erythroid cells of a normal blood donor were evaluated using SAGE. MATERIALS AND METHODS: Global gene expression was evaluated in cells collected immediately before addition of erythropoietin (0 h) and 192 and 336 h after addition of this hormone. Real-time PCR was used to evaluate activation of differentially expressed genes. RESULTS: The data indicate that global aspects of the transcriptome were similar during differentiation of the majority of the genes and that a relatively small set of genes is probably involved in modification of erythroid cells during differentiation. We have identified 93 differentially expressed genes during erythroid development, and expression of some of these was confirmed by qPCR. Various genes including EYA3, ERH, HES6, TIMELESS and TRIB3 were found to be homologous to those of Drosophila melanogaster and here are described for the first time during erythroid development. An important and unique carboxypeptidase inhibitor described in mammalians, LXN, was also identified. CONCLUSIONS: The results of this study amplify previously published data and may contribute to comprehension of erythroid differentiation and identification of new target genes involved in some erythroid concerning diseases.

Genetic characterization of a Juquitiba-like viral lineage in Oligoryzomys nigripes in Rio de Janeiro, Brazil.

Hantaviruses, family Bunyaviridae, are rodent-borne RNA viruses that have caused cases of hantavirus cardiopulmonary syndrome (HCPS) in various regions of the Americas. There are five hantaviral lineages associated with HCPS in Brazil: Juquitiba virus (JUQV), Araraquara virus (ARAV), Laguna Negra-like virus (LNV), Castelo dos Sonhos virus (CASV), and Anajatuba virus (ANAJV). Three additional hantaviruses have been described in rodents alone: Rio Mearim virus, Jaborá virus, and a hantavirus lineage related to Seoul virus. This study describes the genetic detection and characterization of a Juquitiba-like hantavirus in Oligoryzomys nigripes, or the black-footed pygmy rice rat, in the Serra dos Orgãos National Park, Rio de Janeiro State, where so far no cases of HCPS have been reported.

Early proliferation of umbilical cord blood cells from premature neonates.

BACKGROUND AND OBJECTIVE: Human umbilical cord blood (UCB) is an important source of haematopoietic stem cells; however, the behaviour of progenitor cells obtained from premature and full-term neonates is still a controversy subject. Thus, the aim of this study was to evaluate cell cycle parameters and the proliferative capacity of UCB progenitor cells from premature and full-term neonates. MATERIAL AND METHODS: Clonogenic assays were performed with methylcellulose, medium supplemented with recombinant stimulating growth factors and the colonies were scored on the seventh day and the 14th day of culture. A cell cycle study was carried out by DNA analysis using flow cytometry and 30 000 events were acquired; p107 and p130 expressions were analysed by Western blotting. RESULTS: Cultures obtained from UCB of premature neonates showed an early growth of colony-forming unit (CFU)-burst forming unit erythroid/CFU-granulocyte, erythrocyte, macrophage and megakaryocyte (BFU-E/GEMM), and CFU-granulocyte, macrophage (GM) by the seventh day of culture (P < 0.001). Therefore, the number and morphological characteristics of these colonies were comparable with those obtained from full-term neonates, on the 14th day of culture. At the 14th day, a large amount of CFU-GM was detected in the premature group (P < 0.0032). The premature culture on the 14th day showed fibroblasts and was comparable to those of full-term neonates on the 21st day in terms of number and morphology of the colonies. DNA analysis showed that the number of cells in S-phase was also higher in premature samples when compared to full-term neonates, P < 0.0021 (0 h = 12.8 vs. 2.5%; 16 h = 10.5 vs. 5.9%; 20 h = 13.5 vs. 10.3%; 24 h = 13.8 vs. 9.1%; 48 h = 14.0 vs. 5.4%; 72 h = 20.5 vs. 8.9%; 96 h = 13.8 vs. 7.7%). The Western blotting results demonstrated that p107 and p130 cell cycle protein expressions were higher in premature cells than in full-term cells. CONCLUSION: These results suggest that the higher capacity of proliferation and early differentiation of premature UCB might not be related only to the amount of stem/progenitor cells but also to a different timing of cell cycle entry.

Cardosins: a new and efficient plant enzymatic tool to dissociate neuronal cells for the establishment of cell cultures.

In the present work, we examined the feasibility of using cardosins, plant aspartic-proteinases from Cynara cardunculus L., to isolate cells from rat embryonic brain. Using morphological and functional assays, we compared cell cultures obtained with cardosins with those prepared with a well-established trypsin protocol. Cardosins and trypsin dissociation produced cells with similar yield, viability, and GABA release in response to a depolarizing stimulus. However, cardosins-dissociated cells appeared to recover faster in culture, as assessed by the MTT-test and by the number and length of neurtites, suggesting that cardosins are less aggressive to neurons than trypsin. This feature might be helpful for research and medical purposes requiring fast manipulations of cells.

beta-Spectrin S(ta) Bárbara: a novel frameshift mutation in hereditary spherocytosis associated with detectable levels of mRNA and a germ cell line mosaicism.

Hereditary spherocytosis (HS) is a common inherited anaemia characterized by the presence of spherocytic red cells and by a heterogeneous nature in terms of its clinical presentation, molecular basis and inheritance. Defects in several membrane protein genes have been involved in the pathogenesis of HS, including defects in the beta-spectrin gene. We detected a novel frameshift mutation in the beta-spectrin gene, a C deletion at codon 638, in a patient presenting with HS and spectrin deficiency. The mutant protein was not detected in the membrane or in other cellular compartments, but detectable levels of mutant mRNA were found in the patient. Interestingly, this mutation was not present in the patient's parents, suggesting a genetic mosaicism, especially as the patient has an affected brother with the same molecular defect. We analysed DNA from different tissues of the parents and the mutation was absent from all tissues analysed. This mutation seems to be confined to the germ cell lineage of the patient's mother and must present a mosaic pattern in these cells as the patient also has unaffected siblings.

Effect of residence times on River Mondego estuary eutrophication vulnerability.

The south arm of the Mondego estuary, located in the central western Atlantic coast of Portugal, is almost silted up in the upstream area. So, the water circulation is mostly driven by tides and the tributary river Pranto discharges. Eutrophication has been taking place in this ecosystem during last twelve years, where macroalgae reach a luxuriant development covering a significant area of the intertidal muddy flat. A sampling program was carried out from June 1993 to June 1994. Available data on salinity profiles and on nutrients loading into the south arm were used in order to get a better understanding of the ongoing changes. River Pranto flow discharges, controlled by a sluice, were also monitored. Integral formulations are typically based on assumptions of steady state and well-mixed systems and thus cannot take into account the space and time variability of estuarine residence times, due to river discharge flow, tidal coefficients, discharge(s) location and time of release during the tidal cycle. This work presents the hydrodynamics modelling (2D-H) of this system in order to estimate the residence times variability and to assess their effect on the estuarine eutrophication vulnerability, contributing to better environmental management strategies selection.

Diarrhea and malabsorption in primary humoral immunodeficiency.

Patients with Humoral immunodeficiency syndromes frequently present recurrent infections, mainly of the digestive and respiratory tracts. This study carried out a clinical and laboratorial evaluation in 15 humoral immunodeficiency patients presenting chronic gastrointestinal symptoms. Out results emphasize the relevance of immunodeficiency syndromes in the differential diagnosis of chronic diarrhea.