Influência da qualidade microbiológica da água de dessedentação na morfologia intestinal de frangos de corte

Para avaliar se a qualidade microbiológica da água de dessedentação intervém na morfologia intestinal de frangos de corte, foram analisados o consumo semanal de água, a microbiologia de amostras de água, a microscopia eletrônica de varredura e a histologia do intestino delgado de frangos de corte tratados com água filtrada e não filtrada. Os frangos que ingeriram água filtrada tiveram acesso ao menor número de micro-organismos fecais (2,52±0,99 Número Mais Provável (NMP) de coliformes fecais e 1,17±1,25 NMP de Escherichia coli) em relação aos que ingeriram água não filtrada (3,62±0,67 NMP e 2,53±1,13 NMP). Aos 14, 21 e 45 dias de vida, foram colhidas amostras do duodeno, jejuno e íleo de 96 aves. Após rotina laboratorial, as amostras conservadas em glutaraldeído foram eletronmicrografadas e analisadas quanto à densidade de vilos e o material mantido em solução de Bouin foi destinado à confecção de lâminas histológicas que foram analisadas morfometricamente. O duodeno das aves que receberam água não filtrada apresentou maior densidade dos vilos em resposta à qualidade microbiológica da água. Na morfometria intestinal, observou-se que aves que receberam água não filtrada apresentaram aumento na profundidade das criptas intestinais e elevada altura das vilosidades em relação às aves que ingeriram água filtrada. Infere-se que a água filtrada, oferecida aos frangos de corte em um período de vida de 45 dias, favorece a manutenção da integridade intestinal.

Avaliação da resposta imunológica da mucosa intestinal de frangos de corte desafiados com diferentes sorovares de Salmonella

Objetivou-se com o presente estudo comparar o efeito de diferentes sorovares de Salmonella na resposta imune local da mucosa do intestino de frangos de corte. Aos sete dias de idade, as aves foram desafiadas com os sorovares S. Enteritidis, S. Typhimurium, S. Senftenberg, S. Mbandaka e S. Minnesota. Foi observado que todos os sorovares testados foram capazes de colonizar o intestino das aves sendo possível o isolamento de Salmonella em suabes de cloaca, 48 h após inoculação. De maneira geral, as aves do grupo controle negativo, que não foram desafiados apresentaram quantidade significativamente menor de células imunológicas na mucosa intestinal do que as aves desafiadas. Porém, verificou-se que os sorovares de Salmonella, utilizados neste estudo, apresentaram diferentes efeitos sobre a dinâmica celular da mucosa do íleo e ceco e afetaram de modo diferente o ganho de peso e ganho médio diário das aves demonstrando distintos graus de patogenicidade. Os sorovares Enteritidis e Typhimurium apresentaram um efeito mais intenso tanto no desempenho quanto na mobilização de células imunológicas na mucosa intestinal de frangos de corte

Desempenho e morfologia intestinal de frangos de corte na fase de crescimento, com e sem adição de nucleotídeos na dieta, em diferentes níveis proteicos

O experimento foi conduzido com o objetivo de avaliar o desempenho e a morfologia intestinal de frangos de corte na fase de crescimento, com e sem adição de nucleotídeos na dieta, em diferentes níveis proteicos. Foram utilizados 868 pintos de cortes machos de 21 dias de idade, da linhagem Cobb, submetidos a um delineamento inteiramente casualizado. As dietas foram compostas por dois controles, de alta e baixa proteína bruta, com 18,86% e 16,80% respectivamente, com a exigência de 1,062% de lisina digestível. Tendo como base a dieta controle de baixa proteína foram traçados mais cinco tratamentos com adição de 0,5 kg de nucleotídeos/ton de ração, e diferentes níveis de lisina digestível: 1,262%, 1,162%, 1,062%, 0962% e 0,862%, com quatro repetições cada. O consumo alimentar (g) diminuiu linearmente (P≤0,05) no período de 20 a 27, de 20 a 35 e de 20 a 42 dias de idade, em que aumentando os níveis de lisina digestível na dieta, observou-se diminuição no consumo de ração. A conversão alimentar teve efeito quadrático (P≤0,05) para as aves do período de 20 a 27, de 20 a 35 e de 20 a 42 dias de idade, diminuindo à medida que os níveis de lisina digestível aumentaram, atingindo o mínimo com 1,119, 1,187 e 1,132% de lisina digestível, respectivamente. A dieta com 1,062% de lisina digestível não diferiu (P>0.05) da dieta controle com alta proteína, para altura das vilosidades e profundidade de cripta, no duodeno, ilustrando então efeito benéfico do uso de nucleotídeos em dietas com baixa proteína bruta.

Avaliação dos efeitos de 5-hidroxitriptofano em-hidroxibenzilhidrazine associados a Lactobacillus spp. na morfometria intestinal e imunomarcação de serotonina em frangos de corte desafiados com Salmonella Enteridis

Resumo As células enterocromafins são um dos componentes da mucosa intestinal que liberam serotonina para o lúmen, promovendo atividades secretórias e crescimento celular de vários tecidos, incluindo vilosidades intestinais. O presente estudo avaliou as influências do 5-hidroxitriptofano (5HTP) e do m-hidroxibenzilhidrazine (NSD1015), associados a Lactobacillus spp., sobre o peso corporal e o desenvolvimento das vilosidades intestinais na porção proximal do duodeno de frangos de corte desafiados com Salmonella Enteritidis. Verificou-se também se a presença de Lactobacillus spp. e Salmonella Enteritidis influenciaram a imunomarcação de serotonina no duodeno e, para isso, o estudo foi dividido em dois experimentos, com e sem desafio por S. Enteritidis. No Experimento 1, em aves sem desafio, os pesos corporais não diferiram significantemente (p>0,05) e, no Experimento 2, aves com desafio, os tratamentos com o precursor isolado e associado a Lactobacillus spp. determinaram maior peso corporal das aves. Nos dois experimentos, as aves tratadas com 5HTP apresentaram aumento na densidade e altura das vilosidades no duodeno, sugerindo a atuação de 5HTP como um agente trófico. A administração de Lactobacillus spp. também determinou altura maior de vilosidades duodenais. Quanto a imunomarcação de serotonina, as aves tratadas com Lactobacillus spp. no Experimento 1 e as aves tratadas com Lactobacillus spp. e desafiadas com S. Enteritidis no Experimento 2, apresentaram valores superiores aos demais tratamentos, sugerindo que a presença destas bactérias promove maior liberação de serotonina para o duodeno, porém o mecanismo exato de como este processo ocorre necessita ser mais elucidado.

Homogeneous assay technologies in drug screening: Quenching Resonance Energy Transfer (QRET) technique

Information gained from the human genome project and improvements in compound synthesizing have increased the number of both therapeutic targets and potential lead compounds. This has evolved a need for better screening techniques to have a capacity to screen number of compound libraries against increasing amount of targets. Radioactivity based assays have been traditionally used in drug screening but the fluorescence based assays have become more popular in high throughput screening (HTS) as they avoid safety and waste problems confronted with radioactivity. In comparison to conventional fluorescence more sensitive detection is obtained with time-resolved luminescence which has increased the popularity of time-resolved fluorescence resonance energy transfer (TR-FRET) based assays. To simplify the current TR-FRET based assay concept the luminometric homogeneous single-label utilizing assay technique, Quenching Resonance Energy Transfer (QRET), was developed. The technique utilizes soluble quencher to quench non-specifically the signal of unbound fraction of lanthanide labeled ligand. One labeling procedure and fewer manipulation steps in the assay concept are saving resources. The QRET technique is suitable for both biochemical and cell-based assays as indicated in four studies:1) ligand screening study of β2 -adrenergic receptor (cell-based), 2) activation study of Gs-/Gi-protein coupled receptors by measuring intracellular concentration of cyclic adenosine monophosphate (cell-based), 3) activation study of G-protein coupled receptors by observing the binding of guanosine-5’-triphosphate (cell membranes), and 4) activation study of small GTP binding protein Ras (biochemical). Signal-to-background ratios were between 2.4 to 10 and coefficient of variation varied from 0.5 to 17% indicating their suitability to HTS use.

Effects of piperitenone oxide on the intestinal smooth muscle of the guinea pig

We investigated the effects of piperitenone oxide (PO), a major constituent of the essential oil of Mentha x villosa, on the guinea pig ileum. PO (30 to 740 µg/ml) relaxed basal tonus without significantly altering the resting membrane potential. In addition, PO relaxed preparations precontracted with either 60 mM K+ or 5 mM tetraethylammonium in a concentration-dependent manner. At concentrations from 0.1 to 10 µg/ml PO potentiated acetylcholine-induced contractions, while higher concentrations (>30 µg/ml) blocked this response. These higher PO concentrations also inhibited contractions induced by 60 mM K+. PO also blocked the components of acetylcholine contraction which are not sensitive to nifedipine or to solutions with nominal zero Ca2+ and EGTA. These results show that PO is a relaxant of intestinal smooth muscle and suggest that this activity may be mediated at least in part by an intracellular effect

Cytokines as determinants of resistance and pathology in human Schistosoma mansoni infection

The role of different cytokines in the peripheral blood mononuclear cell (PBMC) proliferative response and in in vitro granuloma formation was evaluated in a cross-sectional study with patients with the different clinical forms and phases of Schistosoma mansoni infection, as well as a group of individuals "naturally" resistant to infection named normal endemic (NE). The blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL-5 antibodies significantly reduced the PBMC proliferative response to soluble egg (SEA) and adult worm (SWAP) antigens in acute (ACT), chronic intestinal (INT) and hepatosplenic (HS) patients. Similar results were obtained in the in vitro granuloma formation. Blockage of IL-10 had no significant effect on either assay using PBMC from ACT or HS. In contrast, the addition of anti-IL-10 antibodies to PBMC cultures from INT patients significantly increased the proliferative response to SEA and SWAP as well as the in vitro granuloma formation. Interestingly, association of anti-IL-4 and anti-IL-10 antibodies did not increase the PBMC proliferative response of these patients, suggesting that IL-10 may act by modulating IL-4 and IL-5 secretion. Addition of recombinant IL-10 decreased the proliferative response to undetectable levels when PBMC from patients with the different clinical forms were used. Analysis of IFN-g in the supernatants showed that PBMC from INT patients secreted low levels of IFN-g upon antigenic stimulation. In contrast, PBMC from NE secreted high levels of IFN-g. These data suggest that IL-10 is an important cytokine in regulating the immune response and possibly controlling morbidity in human schistosomiasis mansoni, and that the production of IFN-g may be associated with resistance to infection.

Effect of Walker 256 tumor growth on intestinal absorption of leucine, methionine and glucose in newly weaned and mature rats

In tumor-bearing rats, most of the serum amino acids are used for synthesis and oxidation processes by the neoplastic tissue. In the present study, the effect of Walker 256 carcinoma growth on the intestinal absorption of leucine, methionine and glucose was investigated in newly weaned and mature rats. Food intake and carcass weight were decreased in newly weaned (NT) and mature (MT) rats bearing Walker 256 tumor in comparison with control animals (NC and MC). The tumor/carcass weight ratio was higher in NT than in MT rats, whereas nitrogen balance was significantly decreased in both as compared to control animals. Glucose absorption was significantly reduced in MT rats (MT = 47.3 ± 4.9 vs MC = 99.8 ± 5.3 nmol min-1 cm-1, Kruskal-Wallis test, P<0.05) but this fact did not hamper the evolution of cancer. There was a significant increase in methionine absorption in both groups (NT = 4.2 ± 0.3 and MT = 2.0 ± 0.1 vs NC = 3.7 ± 0.1 and MC = 1.2 ± 0.2 nmol min-1 cm-1, Kruskal-Wallis test, P<0.05), whereas leucine absorption was increased only in young tumor-bearing rats (NT = 8.6 ± 0.2 vs NC = 7.7 ± 0.4 nmol min-1 cm-1, Kruskal-Wallis test, P<0.05), suggesting that these metabolites are being used for synthesis and oxidation processes by the neoplastic cells, which might ensure their rapid proliferation especially in NT rats.

Association of hepatic nuclear factor-4 in the apolipoprotein B promoter: a preliminary report

Previous studies have examined the arrangement of regulatory elements along the apolipoprotein B (apoB) promoter region (-3067 to +940) and a promoter fragment extending from nucleotides -150 to +124 has been demonstrated to be essential for transcriptional activation of the apoB gene in hepatic and intestinal cells. It has also been shown that transcriptional activation of apoB requires a synergistic interaction between hepatic nuclear factor-4 (HNF-4) and CCAAT/enhancer-binding protein a (C/EBPa) transcription factors. Here, we have examined the hypothesis that HNF-4 factor binding to DNA may induce a DNA helix bend, thus facilitating the communication with a C/EBPa factor located one helix turn from this HNF-4 factor in the apoB promoter. A gel electrophoretic mobility shift assay using wild type double-stranded oligonucleotides or modified wild type duplex oligonucleotides with 10 nucleotides inserted between HNF-4 and C/EBPa factor motifs showed similar retarded complexes, indicating that HNF-4 and C/EBPa factors interact independently of the distance between binding sites. However, when only one base, a thymidine, was inserted at the -71 position of the apoB promoter, the complex shift was completely abolished. In conclusion, these results regarding the study of the mechanisms involving the interaction between HNF-4 and C/EBPa factors in the apoB promoter suggest that the perfect 5'-CCCTTTGGA-3' motif is needed in order to facilitate the interaction between the two factors.

Erythrocyte glucose-6-phosphate dehydrogenase activity assay and affinity for its substrate under "physiological" conditions

Glucose-6-phosphate dehydrogenase (G6PD) activity and the affinity for its substrate glucose-6-phosphate were investigated under conditions similar to the physiological environment in terms of ionic strength (I: 0.188), cation concentration, pH 7.34, and temperature (37oC). A 12.4, 10.4 and 21.4% decrease was observed in G6PD B, G6PD A+ and G6PD A- activities, respectively. A Km increase of 95.1, 94.4 and 95.4% was observed in G6PD B, G6PD A+ and G6PD A-, respectively, leading to a marked decrease in affinity. In conclusion, the observation of the reduced activity and affinity for its natural substrate reflects the actual pentose pathway rate. It also suggests a much lower NADPH generation, which is crucial mostly in G6PD-deficient individuals, whose NADPH availability is poor.

Decreased gastric emptying and gastrointestinal and intestinal transits of liquid after complete spinal cord transection in awake rats

We studied the effect of complete spinal cord transection (SCT) on gastric emptying (GE) and on gastrointestinal (GI) and intestinal transits of liquid in awake rats using the phenol red method. Male Wistar rats (N = 65) weighing 180-200 g were fasted for 24 h and complete SCT was performed between C7 and T1 vertebrae after a careful midline dorsal incision. GE and GI and intestinal transits were measured 15 min, 6 h or 24 h after recovery from anesthesia. A test meal (0.5 mg/ml phenol red in 5% glucose solution) was administered intragastrically (1.5 ml) and the animals were sacrificed by an iv thiopental overdose 10 min later to evaluate GE and GI transit. For intestinal transit measurements, 1 ml of the test meal was administered into the proximal duodenum through a cannula inserted into a gastric fistula. GE was inhibited (P<0.05) by 34.3, 23.4 and 22.7%, respectively, at 15 min, 6 h and 24 h after SCT. GI transit was inhibited (P<0.05) by 42.5, 19.8 and 18.4%, respectively, at 15 min, 6 h and 24 h after SCT. Intestinal transit was also inhibited (P<0.05) by 48.8, 47.2 and 40.1%, respectively, at 15 min, 6 h and 24 h after SCT. Mean arterial pressure was significantly decreased (P<0.05) by 48.5, 46.8 and 41.5%, respectively, at 15 min, 6 h and 24 h after SCT. In summary, our report describes a decreased GE and GI and intestinal transits in awake rats within the first 24 h after high SCT.

A polymerase chain reaction-based assay to identify genotype F of hepatitis B virus

We have developed a polymerase chain reaction (PCR) assay which distinguishes genotype F from the other genotypes of hepatitis B virus (HBV). The method was used to characterize HBV strains isolated in urban areas of the Brazilian Amazon. DNA was amplified in 54 of a total of 78 HBsAg-positive serum samples, using universal, non-genotype-specific primers. Only 4 (7.4%) were identified as genotype F by our genotype-specific PCR assay. This proportion is notably lower than that previously reported in Argentina, Venezuela, Peru, and Central America.

Measurement of intestinal permeability using mannitol and lactulose in children with diarrheal diseases

The excretion ratio of lactulose/mannitol in urine has been used to assess the extension of malabsorption and impairment of intestinal permeability. The recovery of lactulose and mannitol in urine was employed to evaluate intestinal permeability in children with and without diarrhea. Lactulose and mannitol probes were measured using high-performance liquid chromatography with pulsed amperometric detection (HPLC-PAD). Two groups of solutions containing 60 µM sugars were prepared. Group I consisted of glucosamine, mannitol, melibiose and lactulose, and group II of inositol, sorbitol, glucose and lactose. In the study of intra-experiment variation, a sample of 50 µl from each group was submitted to 4 successive determinations. The recovered amounts and retention times of each sugar showed a variation <2 and 1%, respectively. The estimated recovery was >97%. In the study of inter-experiment variation, we prepared 4 independent samples from groups I and II at the following concentrations: 1.0, 0.3, 0.1, 0.03 and 0.01 mM. The amounts of the sugars recovered varied by <10%, whereas the retention times showed an average variation <1%. The linear correlation coefficients were >99%. Retention (k'), selectivity (a) and efficiency (N) were used to assess the chromatographic conditions. All three parameters were in the normal range. Children with diarrhea presented a greater lactulose/mannitol ratio compared to children without diarrhea.

Monitoring human cytomegalovirus viral load in peripheral blood leukocytes of renal transplant recipients by a simple limiting dilution-PCR assay

To assess the clinical relevance of a semi-quantitative measurement of human cytomegalovirus (HCMV) DNA in renal transplant recipients within the typical clinical context of a developing country where virtually 100% of both receptors and donors are seropositive for this virus, we have undertaken HCMV DNA quantification using a simple, semi-quantitative, limiting dilution polymerase chain reaction (PCR). We evaluated this assay prospectively in 52 renal transplant patients from whom a total of 495 serial blood samples were collected. The samples scored HCMV positive by qualitative PCR had the levels of HCMV DNA determined by end-point dilution-PCR. All patients were HCMV DNA positive during the monitoring period and a diagnosis of symptomatic infection was made for 4 of 52 patients. In symptomatic patients the geometric mean of the highest level of HCMV DNAemia was 152,000 copies per 106 leukocytes, while for the asymptomatic group this value was 12,050. Symptomatic patients showed high, protracted HCMV DNA levels, whereas asymptomatic patients demonstrated intermittent low or moderate levels. Using a cut-off value of 100,000 copies per 106 leukocytes, the limiting dilution assay had sensitivity of 100%, specificity of 92%, a positive predictive value of 43% and a negative predictive value of 100% for HCMV disease. In this patient group, there was universal HCMV infection but relatively infrequent symptomatic HCMV disease. The two patient groups were readily distinguished by monitoring with the limiting dilution assay, an extremely simple technology immediately applicable in any clinical laboratory with PCR capability.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.