Gastrointestinal dysfunction in mice with a targeted mutation in the gene encoding vasoactive intestinal polypeptide: a model for the study of intestinal ileus and Hirschsprung's disease.

In 1970, Drs. Said and Mutt isolated a novel peptide from porcine intestinal extracts with powerful vasoactive properties, and named it vasoactive intestinal peptide (VIP). Since then, the biological actions of VIP in the gut as well as its signal transduction pathways have been extensively studied. A variety of in vitro and in vivo studies have indicated that VIP, expressed in intrinsic non-adrenergic non-cholinergic (NANC) neurons, is a potent regulator of gastrointestinal (GI) motility, water absorption and ion flux, mucus secretion and immune homeostasis. These VIP actions are believed to be mediated mainly by interactions with highly expressed VPAC(1) receptors and the production of nitric oxide (NO). Furthermore, VIP has been implicated in numerous physiopathological conditions affecting the human gut, including pancreatic endocrine tumors secreting VIP (VIPomas), insulin-dependent diabetes, Hirschsprung's disease, and inflammatory bowel syndromes such as Crohn's disease and ulcerative colitis. To further understand the physiological roles of VIP on the GI tract, we have begun to analyze the anatomical and physiological phenotype of C57BL/6 mice lacking the VIP gene. Herein, we demonstrate that the overall intestinal morphology and light microscopic structure is significantly altered in VIP(-/-) mice. Macroscopically there is an overall increase in weight, and decrease in length of the bowel compared to wild type (WT) controls. Microscopically, the phenotype was characterized by thickening of smooth muscle layers, increased villi length, and higher abundance of goblet cells. Alcian blue staining indicated that the latter cells were deficient in mucus secretion in VIP(-/-) mice. The differences became more pronounced from the duodenum to the distal jejunum or ileum of the small bowel but, became much less apparent or absent in the colon with the exception of mucus secretion defects. Further examination of the small intestine revealed larger axonal trunks and unusual unstained patches in myenteric plexus. Physiologically, the VIP(-/-) mice showed an impairment in intestinal transit. Moreover, unlike WT C57BL/6 mice, a significant percentage of VIP(-/-) mice died in the first postnatal year with overt stenosis of the gut.

Characterization of the pharmacology, signal transduction and internalization of the fluorescent PACAP ligand, fluor-PACAP, on NIH/3T3 cells expressing PAC1.

Fluor-PACAP, a fluorescent derivative of PACAP-27, has been confirmed to share a high affinity for PAC1 receptors transfected into NIH/3T3 cells and to have comparable pharmacological characteristics to the unconjugated, native form. Through competitive binding with 125I-PACAP-27, the two ligands exhibited similar dose- dependent inhibition. Additional examination of the efficacy of activating adenylyl cyclase revealed that both ligands analogously stimulated the production of cyclic AMP. Furthermore, PAC1 internalization visualized by our Fluor-PACAP, is compareable to that performed with the radioligand, 125I-PACAP-27, with maximal internalization achieved within thirty minutes. Thus, Fluor-PACAP exhibits intracellular signaling abilities homologous to the native ligand.

Inactivated suckling mouse brain rabies vaccine provides short-term immunity in capuchin monkeys (Cebus apella).

Eight capuchin monkeys (Cebus apella) were vaccinated against rabies with an inactivated suckling mouse brain vaccine (SMBV). Three 1-ml doses of 2% brain tissue suspension were given by i.m. injection at 0, 30, and 60 days. Blood samples were collected at 0, 30, 60, 90, 150, 210, 240, 300, and 365 days and were tested by simplified fluorescence inhibition to titer-neutralizing antibodies. All of the animals developed neutralizing antibodies with titers >0.5 IU/ml after vaccination, but the immune response persisted for only 122.3 +/- 32.6 days. The SMBV was able to induce immune response in the capuchin monkeys, but protection was short-lived.

Identification of an essential amino acid motif within the C terminus of the pituitary adenylate cyclase-activating polypeptide type I receptor that is critical for signal transduction but not for receptor internalization.

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) receptor is a G protein-coupled receptor and class II receptor member. The receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues (Tr406), 53 residues (Tr416), 49 residues (Tr420), 44 residues (Tr424), and 37 residues (Tr433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radioligand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. (125)I-PACAP-27 binding (K(d) = 0.6-1.5 nm) for the Tr406 and Tr433 were similar to wild type Hop and Null splice variants (K(d) = approximately 1.1 nm). Although internalization of ligand for both the Tr406 and Tr433 mutants was reduced to 50-60% at 60 min compared with 76-87% for WT, loss of G protein coupling did not account for differences in internalization. Despite similar binding properties Tr406 and Tr416 mutants showed no AC or PLC response. Addition of 14 amino acids distal to HopTr406 resulted in normal AC and PLC responses. Site-directed mutagenesis indicated that Arg(416) and Ser(417) are essential for G protein activation. The proximal C terminus mediates signal transduction, and the distal is involved with internalization. Two residues within the C terminus, Arg(416) and Ser(417) conserved among class II receptors are the likely sites for G protein coupling.

ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci.

In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra) and crude larvae extract (T-Tcra), and from Taenia solium extracts of scolex (S-Ts) and of larvae (T-Ts). A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0% and 80.0% sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5% and 100.0%. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.

Performance of the ELISA test for swine cysticercosis using antigens of Taenia solium and Taenia crassiceps cysticerci.

Studies were conducted to evaluate antigens of Taenia solium (Tso) and Taenia crassiceps (Tcra) cysticerci in the ELISA test for the diagnosis of swine cysticercosis. The samples analyzed were cysticercosis positive and negative control sera and heterologous sera. Four antigens were assayed: vesicular fluid (VF) and crude (T) Tcra and scolex (S) and crude (T) Tso. All antigens showed good performance, but VF-Tcra was the best followed by T-Tcra. Sensitivity rates of ELISA were respectively, in 2nd and 3rd standard deviation cut-offs, 96.0 and 80.0% for the VF antigen and specificity of 97.5 and 100.0%. Cross-reactivity was verified only for hidatidosis and ascaridiosis. Due to the high performance observed, the ELISA test using Tcra antigens should be recommended for the diagnosis of swine cysticercosis.

Essential structural motif in the C-terminus of the PACAP type I receptor for signal transduction and internalization.

The objectives of the study reported here were to identify amino acid residues of the C-terminus that are critical for intracellular signaling. A total of nine amino acid substitution and truncation mutants were constructed by PCR and confirmed by sequencing. Mutant and wildtype receptors were stably transfected into NIH/3T3 fibroblasts and studied for their ability to bind PACAP-27 and activate phospholipase C (PLC) and adenylyl cyclase (AC). Receptor affinity of 125I-PACAP-27 for the wildtype and mutants were similar (Kd = 0.6-1.5 nM). However, truncation of the entire 63 amino acids of the hPAC1 resulted in no signaling to either AC or IP. Addition of the proximal 10 amino acids of the C-terminus failed to restore AC or IP signaling, whereas addition of the proximal 27 amino acids of the C-terminus resulted in reconstitution of complete AC and IP responses, identical to the WT. Point mutations within this 17 amino acid region identified specific amino acids involved in PAC1 signaling. These results indicate that a structural motif within the proximal region of the carboxyl terminus is critical for G protein coupling.

Ocorrência de anticorpos antitoxoplasma em gatos infectados naturalmente pelo vírus da imunodeficiência dos felinos / Ocurrence of antibodies to Toxoplasma in cats naturally infected with feline immunodeficiency virus

Foram avaliadas, pela técnica de imunofluorescência indireta, a freqüência e a magnitude dos títulos de anticorpos antitoxoplasma (Toxoplasma gondii, Nicolle; Manceaux, 1909), em gatos infectados naturalmente pelo vírus da imunodeficiência dos felinos (VIF). Utilizaram-se 115 amostras de soro sangüíneo de gatos negativos ao vírus da leucemia felina que foram divididas em 3 grupos. Os 22 animais do grupo I eram positivos ao VIF. Os 58 animais que compuseram o grupo II eram doentes porém negativos na pesquisa de anticorpos anti-VIF e os 35 felinos do grupo III em hígidos e negativos ao VIF. Os resultados obtidos permitiram concluir que a freqüência de anticorpos antitoxoplasma foi maior no grupo I do que nos grupos II e III. A análise estatística mostrou forte associaçäo entre a infecçäo pelo VIF e a presença de anticorpos antitoxoplasma. Näo se observou diferença entre a magnitude dos títulos de anticorpos antitoxoplasma nos animais positivos e negativos ao VIF. Embora gatos que desenvolvam imunidade raramente eliminem oocistos, näo se sabe exatamente como esta imunidade pode influenciar a eliminaçäo de oocistos naqueles gatos infectados pelo VIF. Em face da alta freqüência de anticorpos antitoxoplasma observada nos animais positivos ao VIF, acredita-se que todos os gatos positivos a esse vírus devam ser avaliados quanto à presença de anticorpos antitoxoplasma

[Aflatoxins: current concepts on mechanisms of toxicity and their involvement in the etiology of hepatocellular carcinoma]. / Aflatoxinas: conceitos sobre mecanismos de toxicidade e seu envolvimento na etiologia do câncer hepático celular.

Current concepts derived from intensive research over the last decade, on biotransformation, mechanisms of toxicity and evidences for the involvement of aflatoxins in the etiology of human liver cancer are summarily presented. Aflatoxin B1(AFB1), the main metabolite produced by moulds of genus Aspergillus, exerts its effects after conversion to the reactive compound AFB1-epoxide, by the action of cytochrome P450-dependent enzymes. This epoxide can form derivatives with cellular macromolecules, including proteins, RNA and DNA. The reaction with DNA occurs with guanines in the códon 249 of tumor suppressor gene p53. Primary biotransformation of AFB1 also produces hydroxylated and less toxic derivatives, such as aflatoxins Q1 and P1. Differences intra and interspecies in the pathways of activation/detoxification are directly related to the susceptibility of animals to aflatoxin effects. In humans, studies of individual biomonitoring of AFB1 metabolites such as AFB1-N7-guanine have demonstrated that aflatoxins constitute an important risk factor for hepatocellular carcinoma (HCC) in exposed populations. Some of these studies also show a synergistic action between aflatoxins and the hepatitis B virus in the development of human HCC. In view of these concepts, and taking into account the frequent detection of aflatoxins in Brazilian foodstuffs, the need for investigation into the level of exposure to these toxins and its impact on human health is stressed.

Immunochemical assessment of aflatoxin M1 in milk powder consumed by infants in São Paulo, Brazil.

Aflatoxin M1 was surveyed in 300 samples of whole milk powder consumed by infants at municipal schools and nurseries in São Paulo, Brazil. The analyses were performed by using commercially available test systems of a direct competitive enzyme-linked immunosorbent assay (ELISA). Samples were reconstituted in water (1:8), centrifuged at 1630 x gav for 15 min, and submitted directly to the assay without clean-up procedures. Results showed 33 (11%) positive samples for aflatoxin M1 at levels of 0.10-1.00 ng/ml (mean: 0.27 +/- 0.20 ng/ml). By using data on milk consumption patterns for 4-month-old children (highest intake), a mean daily intake of 3.7 ng/kg body weight/day was estimated. The implications of these data on human health are discussed.

[Evaluation of enzyme-linked immunosorbent assay (ELISA) in milk powder contaminated with known concentrations of aflatoxin M1]. / Avaliação do desempenho do método do ensaio por enzimas imuno-adsorvidas (ELISA) em leite em pó reconstituído contaminado experimentalmente com aflatoxina M1.

The evaluation of commercially available test systems of competitive enzyme-linked immunosorbent assay (ELISA), for Aflatoxin M1 (AFM1) was performed in experimental conditions, through repeated analysis, in samples of milk powder contaminated with known concentrations of the toxin. Recoveries of AFM1 added to milk at levels of 0.10, 0.20, 0.50 and 1.00 ng/ml were 83.0%, 87.5%, 103.0% and 111.8% respectively. Relative standard deviations for the above mentioned concentrations were 65.5%, 31.8%, 10.9% and 13.6%, respectively (n = 10, per spiking level). According to these results the ELISA is appropriate for AFM1 research and/or surveying, mainly for concentrations between 0.20-1.00 ng/ml.

[Health risk due to the consumption of raw milk commercialized without due authorization]. / Risco à saúde representado pelo consumo de leite cru comercializado clandestinamente.

Sixty raw milk samples commercialized without due authorization in the counties of Botucatu and S. Manuel, State of S. Paulo (Brazil), were submitted to mesophilic microorganism and coagulase-positive Staphylococcus and most probable number of total coliform and fecal coliform counts. Forty-one (68.3%) and 50 (83.3%) of the samples were found, respectively to contain mesophilic microorganisms and total coliforms above the maximum limits established by the Health Ministry for type C pasteurized milk. Thirty (50.0%) and 11 (18.3%) of the samples were found, respectively, to the contaminated by coagulase-positive Staphylococcus and fecal coliforms. Only 5 (8.3%) samples were found to comply with the required legal standards. The results showed the unsatisfactory hygienic and sanitary conditions of the raw milk and suggest the existence of great risk to the health of the consumers, especially when the product is taken without being boiled.

[Seroepidemiologic survey of leptospirosis among environmental sanitation workers in an urban locality in the southern region of Brazil]. / Levantamento soroepidemiológico de leptospirose em trabalhadores do serviço de saneamento ambiental em localidade urbana da região sul do Brasil.

Sera from 386 environmental sanitation workers, concerned with water supply, drains and drainage galleries, sewers, garbage collection and road sweepers, were examined for leptospiral agglutinins by the microscopic agglutination test. Altogether 40 of the 386 workers (10.4%) were positive to one or more serovars; however, the difference in seropositivity between the professional categories was not significant (p < 0.05). Twelve serovars were recorded among the seropositive workers with predominance of L. castelonis and L. australis; but the difference between the serovars was not statistically significant (p > 0.05). Of the seropositive workers, 86.9% had agglutination titres > or = 100 and < or = 400; the rates for titres 100 and 400 were higher than 800, 1,600 and 3,200 (p < 0.05).

[Advances in rabies research]. / Avanços na pesquisa da raiva.

Recent advances in research into rabies, based on the technological process of the biological sciences mainly molecular biology and genetic engineering, are mentioned. The current knowledge of the characteristics, properties pathogeny and immunology of the rabies views is presented, as well as the development of new diagnostic and vaccine evaluation techniques. The epidemiological importance of the identification of different immunogenic rabies virus strains and the role in immunoprophylaxis of the production of highly immunogenic vaccines, are set out.

Comparison of virus neutralisation and enzyme-linked immunosorbent assay for the identification of antibodies against vesicular stomatitis (Indiana 3) virus.

Bovine, equine and swine sera from areas free from vesicular stomatitis virus (VSV) Indiana 3 (IND3)--namely Argentina, Chile, Italy and Uruguay--and endemic areas (in Brazil) were examined for anti-VSV IND3 virus antibodies in order to compare results obtained using the virus neutralisation (VN) test and liquid-phase blocking enzyme-linked immunosorbent assay (ELISA). Statistical analysis of the data showed close agreement between the two techniques (K = 0.92). The ELISA gave a higher specificity, positive predictive value and efficiency than the VN test. The ELISA offers the additional advantage of using a non-infectious antigen and can therefore be used for antibody quantification in diagnostic laboratories in VSV-free areas to support epidemiological surveillance programmes.

[Presence of intestinal parasites in vegetables sold in the metropolitan area of São Paulo-SP, Brazil. II--Research on intestinal protozoans]. / Estudo da ocorrência de enteroparasitas em hortaliças comercializadas na região metropolitana de São Paulo-SP, Brasil. II-Pesquisa de protozoários intestinais.

Vegetables in nature, commercially traded in the metropolitan area of S. Paulo, Brazil, were analyzed by means of the appropriate methodology with a view to discovering and identifying protozoan cysts of medical interest. The vegetables under study consisted of 50 samples of each of the varieties listed below: lettuce (Lactuca sativa)-oily leaves and crisp-head varieties, endive (Chicorium sp) and watercress (Nasturtium officinale). Results showed high rates of contamination in all the varieties of vegetable analysed. However, the watercress was the one which presented the highest frequencies of enteroparasites. Both the oily leaves and crisp-head varieties of lettuce presented the lowest rates of contamination, whereas endive presented values ranking, in general, between those of the lettuce and those of the watercress. A great variety of those protozoans which occur frequently in the population resident in the metropolitan area of S. Paulo were observed in the samples, the most frequent being Entamoeba sp (with 4 and 8 nuclei) and Giardia sp. Cysts of Iodamoeba sp, Endolimax sp and Chilomastix sp were also recovered from the samples, thus corroborating the occurrence of high rates of fecal contamination. The significance of these kinds of food in the transmission of protozoans is discussed in the light of the results obtained.

[Presence of intestinal parasites in vegetables sold in the metropolitan region of São Paulo, SP, Brazil. I--Search of helminths]. / Estudo da ocorrência de enteroparasitas em hortaliças comercializadas na região metropolitana de São Paulo, SP, Brasil. I--Pesquisa de helmintos.

Vegetables in natura, commercially traded in the metropolitan area of S. Paulo, were analysed by the appropriate methodology with a view to discovering an identifying the paths of transmission of enteroparasites of medical interest. The vegetables under study consisted of 50 samples of each variety listed below: lettuce (Lactuca sativa)-oily leaves and crisphead varieties, endive (Chicorium sp) and water-cress (Nasturtium Officinale). Results showed high rates of contamination in all the vegetable varieties analysed. However, the water-cress was that which presented the highest frequencies of enteroparasites. The endive presented middle values ranking, in general, between the lettuces and the water-cress. Though high, the average number of helminth eggs and larvae obtained per 100 gr. of sample did not present statistically significant differences as between the four vegetable varieties studied. A great variety of helminths and protozoans such as occur frequently in the resident population of the metropolitan area of S. Paulo, were observed in the samples. However, the most frequent were: hookworms and Ascaris sp. Eggs of Toxocara sp, Fasciola sp and Trichostrongylidae were also recovered from the samples thus corroborating the occurrence of vegetables contamination with faeces of domestic animals. In view of the results obtained, the importance of these kinds of food in the transmission of enteroparasites is stressed, as well as the need for actions which improve the sanitary conditions of these products.

[Prevalence of bovine cysticercosis in the state of São Paulo (Brazil)]. / Prevalência da cisticercose bovina no Estado de São Paulo (Brasil).

A retrospective study of Cysticercus bovis in cattle raised and slaughtered in the State of S. Paulo, Brazil, during the year of 1986, was undertaken on the basis of official records of the Brazilian Meat Inspection Service. The analysis of its special distribution was carried out in accordance with the political-administrative division adopted by the State Government, composed of 11 Administrative Regions (ARs) and one Metropolitan Region (MR), subdivided into 42 Governmental Regions (GRs), totaling 572 municipalities. The prevalence rates of each political-administrative unit were statistically compared using the "Z" test of two proportions. From records relating to 896,654 head, 48,957 cases of cysticercosis were detected, equivalent to a prevalence of 5.5% in the State. The prevalence results were obtained for 385 municipalities, in 42 GRs, 11 ARs and in the MR. Statistically significant prevalence values were observed in 97 municipalities, in 14 GRs and 4 ARs.