José Luiz Guerra

Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants.

The ability of Lys49 and Asp49 phospholipases A(2) (PLA(2)), from Bothrops asper snake venom, to cause hyperalgesia was investigated in rats, using the paw pressure test. Intraplantar injection of both toxins (5-20 micro g/paw) caused hyperalgesia, which peaked 1h after injections. Incubation of both proteins with heparin, prior to their injection, partially reduced this response. Chemical modification of Asp49 PLA(2) with p-bromophenacyl bromide (p-BPB), which abrogates its PLA(2) activity, also abolished hyperalgesia. Intraplantar injection of a synthetic peptide corresponding to the C-terminal sequence 115-129 of Lys49 PLA(2), caused hyperalgesia of similar time course, but varying magnitude, than that induced by the native protein. In contrast, a homologous peptide derived from the Asp49 PLA(2) did not show any nociceptive effect. Hyperalgesia induced by both PLA(2)s was blocked by the histamine and serotonin receptor antagonists promethazine and methysergide, respectively, by the bradykinin B(2) receptor antagonist HOE 140 and by antibodies to tumor necrosis factor alfa (TNFalpha) and interleukin 1 (IL-1). Pretreatment with guanethidine, atenolol, prazosin and yohimbine, inhibitors of sympathomimetic amines, or with indomethacin, inhibitor of the cyclo-oxygenase pathway, reduced Lys49 PLA(2)-induced hyperalgesia without interfering with the nociceptive activity of Asp49 PLA(2). The hyperalgesic response to both myotoxins was not modified by pretreatment with celecoxib, an inhibitor of the cyclo-oxygenase type II, by zileuton, an inhibitor of the lipoxygenase pathway or by N(g)-methyl-L-arginine (LNMMA), an inhibitor of nitric oxide synthase. These results suggest that Asp49 and Lys49 PLA(2)s are important hyperalgesic components of B. asper venom, and that Lys49 and Asp49 PLA(2)s exert their algogenic actions through different molecular mechanisms.

Does prolonged oral exposure to cyanide promote hepatotoxicity and nephrotoxicity

Long-term exposure to cyanide and/or its main metabolite, thiocyanate, has been associated with goiter, pancreatic diabetes and several neurological disorders. However, very little is found in the literature relating the nephrotoxic and hepatotoxic effects of these substances. Thus, the objective of the present study was to verify the effects of prolonged exposure to potassium cyanide (KCN) in these organs. Forty-six male adults rats, weighing approximately 200 g at the beginning of the experiment, were distributed into five groups-four experimental and one control. Experimental groups were dosed with target doses of 0.3, 0.9, 3.0 or 9.0 mg KCN/kg per day, in the drinking water, during 15 days and the control groups received only tap water. At the end of this experiment, all rats were subjected to euthanasia and plasma samples were obtained in order to determine thiocyanate and thyroidal hormones levels and fragments of thyroid, kidney and liver were collected. Rats treated with the highest cyanide dose (9.0 mg KCN/kg per day) showed lower body weight gain. An increase in the thiocyanate levels was verified in all experimental groups. The histopathologic study revealed hydropic degeneration of the renal tubular epithelial cells in those animals, which received KCN at the dose of 3.0-9.0 mg/kg per day. This study also showed hydropic degeneration of the hepatocytes of those animals, which received KCN at a dose of 9.0 mg/kg per day, and in the thyroid gland an increase was observed in the number of reabsorption vacuoles on follicular colloid, in a dose-dependent manner, in all animals of the experimental groups.

CD4 + T cells participate in the nephropathy of canine visceral leishmaniasis

Renal involvement in visceral leishmaniasis (VL) is very frequent. The renal lesions of humans and dogs are similar but their pathogenesis has not been clearly elucidated. There is growing evidence that the cellular immune response is involved in the pathogenesis of immunologically mediated glomerulonephritis. Since T cells could participate in the pathogenesis of nephropathy, in the present study we investigated the possible involvement of CD4(+) and CD8(+) T cells in the nephropathy of canine VL. Six dogs naturally infected with Leishmania (Leishmania) chagasi from the endemic area in the Northeast of Brazil, the town of Teresina in the State of Piauí, were studied. An expressive inflammatory infiltrate of CD4(+) T cells both in glomeruli and in interstitium was present in 4 animals and absent in 2. CD8(+) T cells were detected only in one animal. CD4(+) T cells alone were observed in 3 animals; when CD8+ T cells were present CD4(+) T cells were also present. CD4(+) T cells were observed in cases of focal segmental glomerulosclerosis, diffuse membranoproliferative glomerulonephritis, diffuse mesangial proliferative glomerulonephritis and crescentic glomerulonephritis. CD8(+) T cells were present only in a case of crescentic glomerulonephritis. Leishmania antigen was detected in glomeruli and in interstitial inflammatory infiltrate in 4 animals and immunoglobulins were observed in 4 dogs. In this study we observed that T cells, in addition to immunoglobulins, are present in the renal lesion of canine VL. Further studies are in progress addressing the immunopathogenic mechanisms involving the participation of immunoglobulins and T cells in canine VL nephropathy.

Effect of β-carotene on the development of the solid Ehrlich tumor in mice

The effect of beta-Carotene on the development of the solid Ehrlich tumor in BALB/c mice was investigated. Male mice received orally, on alternate days, three different doses of beta-Carotene (1, 3.5 or 7 mg/100 g) or corn oil as the control. This protocol started 14 days before tumor inoculation (1.75 x 10(5) cells) into mouse footpad and lasted until 10 days after. The tumor growth was evaluated by daily measurement of the footpad thickness, and the tumor mass was evaluated morphometrically. The proliferation rate of tumor was investigated by counting PCNA (proliferating cell nuclear antigen) positive nuclei in the 10th day of the tumor development. Histopathological examination of the lymphoid tissue: thymus, spleen and popliteal lymph node were also performed. beta-Carotene treatment, at dose 3.5 mg/100 g, increased the tumor growth, proliferative rate and the relative weight of popliteal lymph nodes, showing up an adverse effect only when this intermediate dose was used. No effects were obtained when the smaller (1,0 mg/100 g) or the higher (7.0 mg/100 g) doses were used. These results suggest that depending on the dose, beta-Carotene may determine an undesirable effect upon the tumor growth. This should be taken into account in chemopreventive experiments and human applications.

Tuberculosis lymphadenitis in slaughtered swine from S. Paulo State (Brazil): gross lesions, histopathology and demonstration of mycobacteria

INTRODUCTION: As the occurrence of tuberculosis lymphadenitis in swine constitutes a public health risk, especially in immunosuppressed individuals, the distribution of tuberculoid lesions and the presence of micobacteria in lymphonodes and hepatic and muscular tissue in swine for slaughter, in the State of S. Paulo, SP (Brazil), in the period from 1993 to 1994, were studied. MATERIAL E METODO: Tuberculous lesions in 60 carcasses of swine, slaughtered at abattoirs during the period 1993-1994, were studied. When macroscopic lesions were observed, a representative sampling of lymphnodes, hepatic and muscular tissues (masseter and diaphragm) were examined using histophatological and micobacterial isolation techniques. RESULTS: The macroscopic lesions were found predominantly in mesenteric lymphnodes. The MAI Complex (M.avium-intracellulare) was isolated mainly in the carcass group that showed lesions and was not found in the control group. Microorganisms of the MAI Complex were not isolated from hepatic and muscular tissues. CONCLUSION: No clear relationship between the type of mycobacteria isolated and the macroscopic lesions observed during the carcass inspection was found.INTRODUCAO: Como a ocorrencia de linfadenites tuberculoides nos suinos oferece riscos a saude publica, sobretudo em individuos imunocomprometidos, foi estudada a distribuicao das lesoes tuberculoides e a presenca de micobacterias em linfonodos, tecido hepatico e muscular de suinos de abate, no Estado de Sao Paulo, SP (Brasil), no periodo de 1993-1994. MATERIAL E METODO: Foram estudadas 60 carcacas de suinos abatidos, sendo que, trinta apresentavam lesoes tuberculoides macroscopicas (grupo A) e trinta estavam livres de tais lesoes (grupo B ou controle). Foram analisadas seis localizacoes: linfonodos (retrofaringeos, jejunais e mediastinicos), tecido hepatico e tecido muscular (masseter e diafragma). Os exames executados foram: histopatologia e o cultivo para micobacterias. RESULTADOS: No grupo A, 14 em 30 carcacas apresentaram granuloma na histopatologia e, em 14, houve o isolamento de representantes do Complexo MAC (Mycobacterium avium-intracellulare). No grupo B nao foram observadas lesoes na histopatologia, e de quatro carcacas houve o isolamento de micobacterias de rapido crescimento. As lesoes macroscopicas foram encontradas predominantemente nos linfonodos mesentericos. O Complexo MAC foi isolado apenas em linfonodos do grupo com lesao, nao sendo isolado em tecido hepatico e muscular. CONCLUSAO: Nao foi possivel o estabelecimento de relacao entre o tipo de micobacteria isolada e as caracteristicas macroscopicas das lesoes.

T cells, adhesion molecules and modulation of apoptosis in visceral leishmaniasis glomerulonephritis

BackgroundImmune complex deposition is the accepted mechanism of pathogenesis of VL glomerulopathy however other immune elements may participate. Further in the present study, no difference was seen between immunoglobulin and C3b deposit intensity in glomeruli between infected and non-infected dogs thus T cells, adhesion molecules and parameters of proliferation and apoptosis were analysed in dogs with naturally acquired VL from an endemic area. The dog is the most important domestic reservoir of the protozoa Leishmania (L.) chagasi that causes visceral leishmaniasis (VL). The similarity of VL manifestation in humans and dogs renders the study of canine VL nephropathy of interest with regard to human pathology.MethodsFrom 55 dogs with VL and 8 control non-infected dogs from an endemic area, kidney samples were analyzed by immunohistochemistry for immunoglobulin and C3b deposits, staining for CD4+ and CD8+ T cells, ICAM-1, P-selectin and quantified using morphometry. Besides proliferation marker Ki-67, apoptosis markers M30 and TUNEL staining, and related cytokines TNF-α, IL-1α were searched and quantified.ResultsWe observed similar IgG, IgM and IgA and C3b deposit intensity in dogs with VL and non-infected control dogs. However we detected the Leishmania antigen in cells in glomeruli in 54, CD4+ T cells in the glomeruli of 44, and CD8+ T cells in 17 of a total of 55 dogs with VL. Leishmania antigen was absent and T cells were absent/scarse in eight non-infected control dogs. CD 4+ T cells predominate in proliferative patterns of glomerulonephritis, however the presence of CD4+ and CD8+ T cells were not different in intensity in different patterns of glomerulonephritis. The expression of ICAM-1 and P-selectin was significantly greater in the glomeruli of infected dogs than in control dogs. In all patterns of glomerulonephritis the expression of ICAM-1 ranged from minimum to moderately severe and P-selectin from absent to severe. In the control animals the expression of these molecules ranged from absent to medium intensity. It was not observed any correlation between severity of the disease and these markers. There was a correlation between the number of Leishmania antigen positive cells and CD4+ T cells, and between the number of CD4+ T cells and CD8+ T cells. In dogs presenting different histopathological patterns of glomerulonephritis, parameters of proliferation and apoptosis were studied. Ki-67, a proliferative marker, was not detected locally, but fewer apoptotic cells and lower TNF-α expression were seen in infected animals than in non-infected controls.ConclusionImmunopathogenic mechanisms of VL glomerulonephritis are complex and data in the present study suggest no clear participation of immunoglobulin and C3b deposits in these dogs but the possible migration of CD4+ T cells into the glomeruli, participation of adhesion molecules, and diminished apoptosis of cells contributing to determine the proliferative pattern of glomerulonephritis in VL.

Effects of Long-term Cyanide Ingestion by Pigs

Animal performance and health status are adversely affected by long-term cyanide ingestion; however, the effects of cyanide ingestion by pigs have not been fully determined. The aim of the present study was to determine the effects of prolonged exposure to different doses of potassium cyanide (KCN) in growing-finishing swine. Twenty-four pigs, 45 days of age, were divided into four equal groups and treated with different doses of KCN: 0, 2.0, 4.0 or 6.0 mg per kg body weight per day for 70 consecutive days. The results showed a significant alteration in thiocyanate, creatinine and urea levels and in alanine aminotransferase activity of swine dosed with 4.0 and 6.0 mg/kg/KCN. Thyroid weight was significantly increased in those pigs from 4.0 mg/kg KCN group, but no change in cholesterol, triiodothyronine or thyroline levels were observed. Body and carcase weights, body weight gain, and bacon thickness were not affected by KCN treatment. The histopathological study revealed increased numbers of vacuoles in the colloid of thyroid follicles, degeneration of cerebellar white matter and Purkinje cells, degeneration of renal tubular epithelial cells, caryolysis and pyknosis in hepatocytes, and disturbance of the normal lobular architecture of the liver in all treated pigs. Thus, long-term administration of KCN to swine affects several tissues and could adversely affect animal production.

Retrospective review and systematic study of mammary tumors in dogs and characteristics of the extracellular matrix

A finalidade do presente trabalho foi efetuar um estudo retrospectivo, de 1932 a 1999 , afim de se estabelecer a casuistica desses tumores nos arquivos do Departamento de Patologia da Faculdade de Medicina Veterinaria - USP , bem como a frequencia de desmoplasia, metaplasia cartilaginosa e ossea em 578 desses tumores. Entre os 537 tumores malignos, 13.05% foram adenocarcinomas tubulares simples, 3.91% foram adenocarcinomas tubulares compostos, 7.26% adenocarcinomas papiliferos simples, 4.28% adenocarcinomas papiliferos compostos, 23.27% cistoadenocarcinomas papiliferos simples, 8.37% cistoadenocarcinomas papiliferos compostos, 16.38% adenocarcinomas solidos simples, 6.70% adenocarcinomas solidos compostos, 2.04% carcinomas de celulas espinhosas simples, 1.11% carcinomas de celulas espinhosas compostos, 2.79% carcinomas mucinosos ,8.19% carcinomas anaplasicos ,0.93% carcinomas escamosos, 1.30% fibrossarcomas,.0.18% condrossarcoma, 0.18% osteossarcoma e entre os 41 tumores benignos ,51.21% foram adenomas, 12.19% cistadenomas pailiferos,7.31% papilomas, 4.87% fibroadenomas e 24.39% foram fibroadenomas. Tanto a desmoplasia como a metaplasia foi um achado frequente nas neoplasias benignas e malignas, mas foram mais frequentes entre os adenocarcinomas tubulares compostos: 38% apresentaram desmoplasia, 57% metaplasia cartilaginosa e 28% metaplasia ossea. Entre as neoplasias benignas, adenoma apresentou a maior frequencia: 14 % com desmoplasia, 29% com metaplasia cartilaginosa e 24% com metaplasia ossea. Os resultados deste estudo enfatizam a complexidade da inter-relacao entre as macromoleculas da matriz extracelular e as celulas tumorais.

Osteosarcoma in dogs: clinical-morphological study and prognostic correlation

Osteosarcoma is defined as a bone matrix-producing malignant mesenchymal tumor. It is relatively rare among domestic animals, but corresponds to 85% of all malignant bone tumors in dogs. To study canine osteosarcoma in animals living in Brazil and correlate the clinical- morphological findings with prognosis, forty-six cases of dogs with osteosarcoma were studied retrospectively out of a total of 56 malignant primary bone tumors on file. The animals were assessed in terms of age, sex, breed, topographic tumor distribution, and presence of metastases at diagnosis. The tumors were classified histologically by the method of Pool 1 . Osteosarcomas corresponded to 82% of primary malignant bone dysplasias in the sample studied. Age ranged from 6 months to 14 years (C = 8 y), sex: 32 F/18 M; breed: large dogs (86%), topographic location: appendicular skeleton (64%), axial skeleton (28.5%) and extraskeletal (7.5%); presence of metastases in 17.4%. Histopathology: osteoblastic (32.6%), chondroblastic (9.6%) and telangiectatic (5.8%) patterns; combined: 52%. Osteosarcoma mainly affects adult females. Breed is a predisposing factor, the appendicular skeleton is most often involved, and an osteoblastic histological pattern predominates. The lungs are a common site of metastasis, with pure- bred male dogs being most predisposed regardless of the morphological pattern of osteosarcoma.

Beta-carotene reduces the ductular (oval) cell reaction in the liver of Wistar rats submitted to the resistant hepatocyte model of carcinogenesis

Summary The morphology of livers of Wistar rats treated with beta‐carotene (BC), vitamin A (VA, retinol acetate) or corn oil (CO, controls) and submitted to the resistant hepatocyte model of carcinogenesis was studied. Preneoplastic lesions (PNL) were smaller and less numerous in the BC group. The latter group also presented fewer placental glutathione‐S‐transferase (GST‐P) positive and hematoxylin and eosin (H&E) distinguishable PNL, with smaller mean areas and smaller mean areas of the liver occupied by PNL. Clear cell foci predominated in BC livers. In picrosirius‐stained liver sections, fibrosis, whether or not accompanying the bile ductular cells, surrounded only 16.67% of PNL in the BC group, as compared to 35.71% in the VA group and 87.72% in the CO group. Moreover, the ductular cell reaction was smaller in the BC group. Smooth muscle actin‐positive cells surrounded some PNL, mostly in CO rats, and less frequently in the VA and BC groups. Examination by transmission electron microscopy (TEM) showed that cells with nuclei similar to those of perisinusoidal cells, devoid of cytoplasmic fat globules, probably represented myofibroblasts derived from Ito cells and accompanied the ductular cell reaction. On the basis of these results, we suggest that BC reduced not only the PNL but also the ductular (oval) cell reaction in this experimental model.Abbreviations: ASMA, anti‐smooth muscle actin antibody; BC, beta‐carotene; CO, corn oil; GST‐P, placental glutathione‐S‐transferase; PNL, preneoplastic lesion(s); TEM, transmission electron microscopy; VA, vitamin A.