Research of Salmonella spp. and evaluation of pathogenicity, cytotoxicity of Escherichia coli isolates proceeding from sparrows (Passer domesticus)

The aim of this study was to research the occurrence of Salmonella spp. and Escherichia coli in feces samples of sparrows, as well as to identify the pathogenicity, cytotoxicity and sensitivity profile of the isolates to antimicrobial use. Two hundred and twenty eight sparrows were captured in eight farms. The in vitro pathogenicity test was performed by the isolates culture on congo red-magnesium oxalate Agar, whilst the in vivo pathogenicity test was performed in one day-old chicks. In order to study the cytotoxic effects of indicators, samples were inoculated into Vero cells. The results obtained for Escherichia coli isolation confirmed the presence of this microorganism in 30 (13.2%) of the evaluated samples. Out of those isolates, 10 (33.3%) presented the capacity of absorbing ongo red. As for in vivo pathogenicity a 68.0% of mortality rate of the evaluated samples was observed. Out of 20 isolates tested for cytotoxin production, none of them presented cytotoxic effect in the Vero cells. The Salmonella spp was isolated only in one sample (0.04%), and it was identified as Salmonella enterica subspecies houtenae. Results obtained through this research indicate the need for new studies to identify other virulence factors of E. coli samples and to delineate the phylogenetic profile of the isolates in order to establish a relation with colibacillosis outbreaks in chickens and broilers in the studied region, as well as to analyze the critical points in the aviculture productive chain to identify the source of Salmonella enterica subspecies houtenae.

Chemoreceptors and cardiovascular control in acute and chronic systemic hypoxia

This review describes the ways in which the primary bradycardia and peripheral vasoconstriction evoked by selective stimulation of peripheral chemoreceptors can be modified by the secondary effects of a chemoreceptor-induced increase in ventilation. The evidence that strong stimulation of peripheral chemoreceptors can evoke the behavioural and cardiovascular components of the alerting or defence response which is characteristically evoked by novel or noxious stimuli is considered. The functional significance of all these influences in systemic hypoxia is then discussed with emphasis on the fact that these reflex changes can be overcome by the local effects of hypoxia: central neural hypoxia depresses ventilation, hypoxia acting on the heart causes bradycardia and local hypoxia of skeletal muscle and brain induces vasodilatation. Further, it is proposed that these local influences can become interdependent, so generating a positive feedback loop that may explain sudden infant death syndrome (SIDS). It is also argued that a major contributor to these local influences is adenosine. The role of adenosine in determining the distribution of O2 in skeletal muscle microcirculation in hypoxia is discussed, together with its possible cellular mechanisms of action. Finally, evidence is presented that in chronic systemic hypoxia, the reflex vasoconstrictor influences of the sympathetic nervous system are reduced and/or the local dilator influences of hypoxia are enhanced. In vitro and in vivo findings suggest this is partly explained by upregulation of nitric oxide (NO) synthesis by the vascular endothelium which facilitates vasodilatation induced by adenosine and other NO-dependent dilators and attenuates noradrenaline-evoked vasoconstriction.

The insulin receptor substrate 1 associates with phosphotyrosine phosphatase SHPTP2 in liver and muscle of rats

Insulin stimulates the tyrosine kinase activity of its receptor resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate-1 (IRS-1) which, in turn, associates with proteins containing SH2 domains. It has been shown that IRS-1 associates with the tyrosine phosphatase SHPTP2 in cell cultures. While the effect of the IRS-1/SHPTP2 association on insulin signal transduction is not completely known, this association may dephosphorylate IRS-1 and may play a critical role in the mitogenic actions of insulin. However, there is no physiological demonstration of this pathway of insulin action in animal tissues. In the present study we investigated the ability of insulin to induce association between IRS-1 and SHPTP2 in liver and muscle of intact rats, by co-immunoprecipitation with anti-IRS-1 antibody and anti-SHPTP2 antibody. In both tissues there was an increase in IRS-1 association with SHPTP2 after insulin stimulation. This association occurred when IRS-1 had the highest level of tyrosine phosphorylation and the decrease in this association was more rapid than the decrease in IRS-1 phosphorylation levels. The data provide evidence against the participation of SHPTP2 in IRS-1 dephosphorylation in rat tissues, and suggest that the insulin signal transduction pathway in rat tissues is related mainly to the mitogenic effects of the hormone.

Serum cytokine levels in autoimmune and non-autoimmune hyperthyroid states

Although the role of interleukin-2 (IL-2) and interferon gamma (gIFN) is still poorly understood in hyperthyroid diseases, it is reasonable to assume that these cytokines may be present at higher levels in Graves' disease (GD) than in other primarily non-autoimmune thyroid diseases. In order to look for an easy method to distinguish GD from primarily non-autoimmune causes of hyperthyroidism, we compared 13 healthy individuals with 21 treated and untreated hyperthyroid GD patients and with 19 patients with hyperthyroidism due to other etiologies: 7 cases of multinodular goiter, 5 cases of excessive hormone replacement and 7 cases of amiodarone-associated hyperthyroidism. All patients presented low TSH levels and a dubious clinical thyroid state. We found a good correlation between TSH and serum IL-2 levels (r = 0.56; P<0.01). Serum IL-2 (P<0.01) and gIFN (P<0.01) levels were lower in the hyperthyroid group of patients than in control subjects, suggesting a depressed TH1 pattern in the T-cell subset of hyperthyroid patients. GD had normal IL-2 levels, while patients with other forms of thyrotoxicosis presented decreased IL-2 levels (P<0.05). There was no difference between treated and untreated GD patients. We suggest that the direct measurement of serum IL-2 level may help to confirm hyperthyroidism caused by GD.

Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ß-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ß-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ß-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ß-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ß-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

Effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices

It has been suggested that glucocorticoids released during stress might impair neuronal function by decreasing glucose uptake by hippocampal neurons. Previous work has demonstrated that glucose uptake is reduced in hippocampal and cerebral cortex slices 24 h after exposure to acute stress, while no effect was observed after repeated stress. Here, we report the effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices and on plasma glucose and corticosterone levels. Male adult Wistar rats were exposed to restraint 1 h/day for 50 days in the chronic model. In the acute model there was a single exposure. Immediately or 24 h after stress, the animals were sacrificed and the hippocampus and cerebral cortex were dissected, sliced, and incubated with Krebs buffer, pH 7.4, containing 5 mM glucose and 0.2 µCi D-[U-14C] glucose. CO2 production from glucose was estimated. Trunk blood was also collected, and both corticosterone and glucose were measured. The results showed that corticosterone levels after exposure to acute restraint were increased, but the increase was smaller when the animals were submitted to repeated stress. Blood glucose levels increased after both acute and repeated stress. However, glucose utilization, measured as CO2 production in hippocampal and cerebral cortex slices, was the same in stressed and control groups under conditions of both acute and chronic stress. We conclude that, although stress may induce a decrease in glucose uptake, this effect is not sufficient to affect the energy metabolism of these cells.

GLUT4 protein expression in obese and lean 12-month-old rats: insights from different types of data analysis

GLUT4 protein expression in white adipose tissue (WAT) and skeletal muscle (SM) was investigated in 2-month-old, 12-month-old spontaneously obese or 12-month-old calorie-restricted lean Wistar rats, by considering different parameters of analysis, such as tissue and body weight, and total protein yield of the tissue. In WAT, a ~70% decrease was observed in plasma membrane and microsomal GLUT4 protein, expressed as µg protein or g tissue, in both 12-month-old obese and 12-month-old lean rats compared to 2-month-old rats. However, when plasma membrane and microsomal GLUT4 tissue contents were expressed as g body weight, they were the same. In SM, GLUT4 protein content, expressed as µg protein, was similar in 2-month-old and 12-month-old obese rats, whereas it was reduced in 12-month-old obese rats, when expressed as g tissue or g body weight, which may play an important role in insulin resistance. Weight loss did not change the SM GLUT4 content. These results show that altered insulin sensitivity is accompanied by modulation of GLUT4 protein expression. However, the true role of WAT and SM GLUT4 contents in whole-body or tissue insulin sensitivity should be determined considering not only GLUT4 protein expression, but also the strong morphostructural changes in these tissues, which require different types of data analysis.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5' sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Building bone tissue: matrices and scaffolds in physiology and biotechnology

Deposition of bone in physiology involves timed secretion, deposition and removal of a complex array of extracellular matrix proteins which appear in a defined temporal and spatial sequence. Mineralization itself plays a role in dictating and spatially orienting the deposition of matrix. Many aspects of the physiological process are recapitulated in systems of autologous or xenogeneic transplantation of osteogenic precursor cells developed for tissue engineering or modeling. For example, deposition of bone sialoprotein, a member of the small integrin-binding ligand, N-linked glycoprotein family, represents the first step of bone formation in ectopic transplantation systems in vivo. The use of mineralized scaffolds for guiding bone tissue engineering has revealed unexpected manners in which the scaffold and cells interact with each other, so that a complex interplay of integration and disintegration of the scaffold ultimately results in efficient and desirable, although unpredictable, effects. Likewise, the manner in which biomaterial scaffolds are "resorbed" by osteoclasts in vitro and in vivo highlights more complex scenarios than predicted from knowledge of physiological bone resorption per se. Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue engineering strategies.

Frequency of Fanconi anemia in Brazil and efficacy of screening for the FANCA 3788-3790del mutation

Fanconi anemia (FA) is an autosomal recessive genetic disease characterized by progressive bone marrow failure, susceptibility to cancer and multiple congenital anomalies. There is important clinical variability among patients and the knowledge of factors which might predict outcome would greatly help the decision making regarding the choices of treatment and the appropriate time to start it. Future studies of the possible correlation between specific mutations with specific clinical presentations will provide the answer to one of these factors. At our Center we standardized a rapid and precise screening test using a mismatch PCR assay for a specific mutation (3788-3790del in exon 38 of gene FANCA) in Brazilian FA patients. We present the results obtained after screening 80 non-consanguineous FA patients referred from all regions of Brazil with a clinical diagnosis of FA supported by cellular hypersensitivity to diepoxybutane. We were able to detect the 3788-3790del allele in 24 of the 80 (30%) FA patients studied. Thirteen of the 80 (16.25%) were homozygotes and 11 of the 80 (13.75%) were compound heterozygotes, thus confirming the high frequency of the FANCA 3788-3790del mutation in Brazilian FA patients. The identification of patients with specific mutations in the FA genes may lead to a better clinical description of this condition, also providing data for genotype-phenotype correlations, to a better understanding of the interaction of this specific mutation with other mutations in compound heterozygote patients, and ultimately to the right choices of treatment for each patient with improvement of the prognosis on future studies.

Description of Lyme disease-like syndrome in Brazil: is it a new tick borne disease or Lyme disease variation?

An emerging clinical entity that reproduces clinical manifestations similar to those observed in Lyme disease (LD) has been recently under discussion in Brazil. Due to etiological and laboratory particularities it is named LD-like syndrome or LD imitator syndrome. The condition is considered to be a zoonosis transmitted by ticks of the genus Amblyomma, possibly caused by interaction of multiple fastidious microorganisms originating a protean clinical picture, including neurological, osteoarticular and erythema migrans-like lesions. When peripheral blood of patients with LD-like syndrome is viewed under a dark-field microscope, mobile uncultivable spirochete-like bacteria are observed. PCR carried out with specific or conservative primers to recognize Borrelia burgdorferi sensu stricto or the genus Borrelia has been negative in ticks and in biological samples. Two different procedures, respectively involving hematoxylin and eosin staining of cerebrospinal fluid and electron microscopy analysis of blood, have revealed spirochetes not belonging to the genera Borrelia, Leptospira or Treponema. Surprisingly, co-infection with microorganisms resembling Mycoplasma and Chlamydia was observed on one occasion by electron microscopy analysis. We discuss here the possible existence of a new tick-borne disease in Brazil imitating LD, except for a higher frequency of recurrence episodes observed along prolonged clinical follow-up.

Trypanosoma cruzi infection induces up-regulation of cardiac muscarinic acetylcholine receptors in vivo and in vitro

The pathogenesis of chagasic cardiomyopathy is not completely understood, but it has been correlated with parasympathetic denervation (neurogenic theory) and inflammatory activity (immunogenic theory) that could affect heart muscarinic acetylcholine receptor (mAChR) expression. In order to further understand whether neurogenic and/or immunogenic alterations are related to changes in mAChR expression, we studied two models of Trypanosoma cruzi infection: 1) in 3-week-old male Sprague Dawley rats chronically infected with T. cruzi and 2) isolated primary cardiomyocytes co-cultured with T. cruzi and peripheral blood mononuclear cells (PBMC). Using [³H]-quinuclidinylbenzilate ([³H]-QNB) binding assays, we evaluated mAChR expression in homogenates from selected cardiac regions, PBMC, and cultured cardiomyocytes. We also determined in vitro protein expression and pro-inflammatory cytokine expression in serum and cell culture medium by ELISA. Our results showed that: 1) mAChR were significantly (P < 0.05) up-regulated in right ventricular myocardium (means ± SEM; control: 58.69 ± 5.54, N = 29; Chagas: 72.29 ± 5.79 fmol/mg, N = 34) and PBMC (control: 12.88 ± 2.45, N = 18; Chagas: 20.22 ± 1.82 fmol/mg, N = 19), as well as in cardiomyocyte transmembranes cultured with either PBMC/T. cruzi co-cultures (control: 24.33 ± 3.83; Chagas: 43.62 ± 5.08 fmol/mg, N = 7 for both) or their conditioned medium (control: 37.84 ± 3.84, N = 4; Chagas: 54.38 ± 6.28 fmol/mg, N = 20); 2) [³H]-leucine uptake was increased in cardiomyocytes co-cultured with PBMC/T. cruzi-conditioned medium (Chagas: 21,030 ± 2321; control 10,940 ± 2385 dpm, N = 7 for both; P < 0.05); 3) plasma IL-6 was increased in chagasic rats, IL-1β, was increased in both plasma of chagasic rats and in the culture medium, and TNF-α level was decreased in the culture medium. In conclusion, our results suggest that cytokines are involved in the up-regulation of mAChR in chronic Chagas disease.

Lactobacillus delbrueckii UFV-H2b20 induces type 1 cytokine production by mouse cells in vitro and in vivo

Lactobacillus delbrueckii UFV-H2b20 has been shown to increase clearance of bacteria injected into the blood of germ-free mice. Moreover, it induces the production of type 1 cytokines by human peripheral mononuclear cells. The objective of the present study was to investigate the production of inflammatory cytokines [interleukin-12 (IL-12 p40), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ)] triggered in vitro by live, heat-killed or lysozyme-treated L. delbrueckii UFV-H2b20 and in vivo by a live preparation. Germ-free, L. delbrueckii-monoassociated and lipopolysaccharide (LPS)-resistant C3H/HeJ mice were used as experimental models. UFV-H2b20 induced the production of IL-12 p40 and TNF-α by peritoneal cells and IFN-γ by spleen cells from germ-free or monoassociated Swiss/NIH mice and LPS-hyporesponsive mice (around 40 ng/mL for IL-12 p40, 200 pg/mL for TNF-α and 10 ng/mL for IFN-γ). Heat treatment of L. delbrueckii did not affect the production of these cytokines. Lysozyme treatment decreased IL-12 p40 production by peritoneal cells from C3H/HeJ mice, but did not affect TNF-α production by these cells or IFN-γ production by spleen cells from the same mouse strain. TNF-α production by peritoneal cells from Swiss/NIH L. delbrueckii-monoassociated mice was inhibited by lysozyme treatment. When testing IL-12 p40 and IFN-γ levels in sera from germ-free or monoassociated Swiss/NIH mice systemically challenged with Escherichia coli we observed that IL-12 p40 was produced at marginally higher levels by monoassociated mice than by germ-free mice (40 vs 60 ng/mL), but IFN-γ was produced earlier and at higher levels by monoassociated mice (monoassociated 4 and 14 ng/mL 4 and 8 h after infection, germfree 0 and 7.5 ng/mL at the same times). These results show that L. delbrueckii UFV-H2b20 stimulates the production of type 1 cytokines in vitro and in vivo, therefore suggesting that L. delbrueckii might have adjuvant properties in infection in which these cytokines play a major role.

Cardiac and pulmonary alterations in symptomatic and asymptomatic dogs infectednaturally with Leishmania (Leishmania) chagasi

Fifteen symptomatic and seven asymptomatic dogs infected naturally with Leishmania chagasi were examined in order to identify the presence of parasites and changes in heart and lung. Histopathological, cytological, and immunohistochemical analyses were performed on samples of heart and lung tissues. An inflammatory reaction characterized by inflammatory mononuclear, perivascular and intermuscular infiltrates was observed in both symptomatic and asymptomatic animals on histopathological analysis of the heart. In the lung, there was thickening of the alveolar septa due to congestion, edema, inflammatory infiltrate, and fibroblast proliferation. A focal reaction was observed although a diffuse reaction was present in both groups. On cytological examination, heart and lung imprints revealed amastigotes in two symptomatic animals and heart imprints were found in 1 asymptomatic dog. Immunoperoxidase staining showed amastigotes in the lung and heart of only 1 of 6 symptomatic animals examined. Within the ethical principles and limits of this research, it can be inferred that the study of heart and lung alterations in canine visceral leishmaniasis is increasingly important for understanding the problem related to humans. Dogs with visceral leishmaniasis were a good experimental model, since infection was caused by the same agent and the animals developed clinical, pathological and immunological alterations similar to those observed in humans.

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines

Endothelins (ETs) and sarafotoxins (SRTXs) belong to a family of vasoconstrictor peptides, which regulate pigment migration and/or production in vertebrate pigment cells. The teleost Carassius auratus erythrophoroma cell line, GEM-81, and Mus musculus B16 melanocytes express rhodopsin, as well as the ET receptors, ETB and ETA, respectively. Both cell lines are photoresponsive, and respond to light with a decreased proliferation rate. For B16, the doubling time of cells kept in 14-h light (14L):10-h darkness (10D) was higher compared to 10L:14D, or to DD. The doubling time of cells kept in 10L:14D was also higher compared to DD. Using real-time PCR, we demonstrated that SRTX S6c (12-h treatment, 100 pM and 1 nM; 24-h treatment, 1 nM) and ET-1 (12-h treatment, 10 and 100 pM; 24- and 48-h treatments, 100 pM) increased rhodopsin mRNA levels in GEM-81 and B16 cells, respectively. This modulation involves protein kinase C (PKC) and the mitogen-activated protein kinase cascade in GEM-81 cells, and phospholipase C, Ca2+, calmodulin, a Ca2+/calmodulin-dependent kinase, and PKC in B16 cells. Cells were kept under constant darkness throughout the gene expression experiments. These results show that rhodopsin mRNA levels can be modulated by SRTXs/ETs in vertebrate pigment cells. It is possible that SRTX S6c binding to the ETB receptors in GEM-81 cells, and ET-1 binding to ETA receptors in B16 melanocytes, although activating diverse intracellular signaling mechanisms, mobilize transcription factors such as c-Fos, c-Jun, c-Myc, and neural retina leucine zipper protein. These activated transcription factors may be involved in the positive regulation of rhodopsin mRNA levels in these cell lines.