Electroporation transiently decreases GJB2 (connexin 26) expression in B16/BL6 melanoma cell line.

Connexins are proteins that form gap junctions. Perturbations in the cell membrane reportedly promote changes in the expression profile of connexins. Electroporation promotes destabilization by applying electrical pulses, and this procedure is used in electrochemotherapy and gene therapy, among others. This in vitro work aimed to study the interference of electroporation on the expression profile of GJB2 (Cx26 gene) and Connexin 26 in melanoma cell line B16/BL6. The techniques of immunocytochemistry, Western blot, and real-time PCR were used. After electroporation, cells showed a transient decrease in GJB2 mRNA. The immunostaining of Cx26 showed no noticeable change after electroporation at different time points. However, Western blot showed a significant reduction in Cx26 30 min after electroporation. Our results showed that electroporation interferes transiently in the expression of Connexin 26 in melanoma and are consistent with the idea that electroporation is a process of intense stress that promotes cell homeostatic imbalance and results in disruption of cell physiological processes such as transcription and translation.

Walker 256 tumor-bearing rats as a model to study cancer pain.

UNLABELLED: An animal model of cancer pain induced by injection of Walker 256 carcinoma cells into the plantar surface of rat hind paw is described. Tumor growth and the occurrence of metastasis were investigated by histopathological analysis. Tumor cell growth was also analyzed plethysmographically by the increase in paw volume. For characterization of pain symptoms, hyperalgesia, allodynia, and spontaneous pain were evaluated 5 to 8 days after cell injection. The volume of the inoculated paw started to increase on day 2 after inoculation, being 40% higher on day 5 after injection. At this time, there was a marked proliferation of tumor cells, with the presence of anaplastic and pleomorphic cells, nucleoli, and atypical mitotic features. On days 7 and 8 after injection, histopathological analysis of popliteal lymph nodes showed the presence of tumor cells. The intraplantar injection of Walker 256 cells caused hyperalgesia at day 5 after cell inoculation. Low-threshold mechanical allodynia was significant 2 days after cell injection, being increased on day 5. In addition, inoculation of tumor cells induced gross behavior, characterized by a significant increase in licking and lifting of the injected paw 5 days after injection. The pain-enhancing effect caused by cell inoculation was partially inhibited by indomethacin on day 2 after cell injection, whereas morphine blocked allodynia on days 2 and 5. These results indicate that intraplantar injection of Walker 256 cells cause pain symptoms characteristic of cancer pain. This experimental model can then be used to investigate new analgesic or anti-tumor drugs. PERSPECTIVE: This article presents a new animal model for studying cancer pain and metastasis. This model could help in understanding the mechanisms involved in cancer pain symptoms and may be used for the investigation of new analgesic or anti-tumor drugs.

Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content.

Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.

[Immunohistochemical staining of the macrophagic and astrocytic response in the brainstem of Wistar rats submitted to the ethidium bromide gliotoxic model and treated with cyclophosphamide]. / Marcação imuno-histoquímica da resposta macrofágica e astrocitária no tronco encefálico de ratos Wistar submetidos ao modelo gliotóxico do brometo de etídio e tratados com ciclofosfamida.

The gliotoxic ethidium bromide (EB) was used to study morphologically the macrophagic and astrocytic response under immunosuppression by cyclophosphamide (CY). Astrocyte immunoreactivity to glial fibrillary acidic protein (GFAP) and vimentin (VIM) and macrophagic immunoreactivity to ED1 were investigated after EB injection. Male Wistar rats were injected with 0.9% saline solution (group I), 0.1% BE (group II) and 0.1% EB associated with CY treatment (group III). Brainstem samples were collected from the 1st to the 21st day post-injection for GFAP, VIM and ED1 immunostaining. In groups II and III, it was observed increased immunoreactivity to GFAP and reexpression of VIM. In group II, ED1-positive cells were noted after the 2nd day and in group III, after the 3rd day. On the 14th day post-injection, it was observed a greater quantity of ED1- positive cells in group III than in group II. Apparently, CY did not change the astrocytic response pattern.

Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii.

A vaccine containing crude Toxoplasma gondii rhoptry proteins incorporated in the immunostimulating complexes (ISCOM) adjuvant was tested in pigs for protecting against tissue cyst formation. For this, 38 mixed breed pigs were divided into four groups, G1 (vaccinated challenged, n=10) received two doses (100 microg/dose) of the rhoptry vaccine at days 0 and 21, G2 (vaccinated challenged, n=10) received viable tachyzoites (7 x 10(7)) of the RH strain at day 0, G3 (unvaccinated challenged, n=10) and G4 (unvaccinated unchallenged, n=8). Pigs were challenged with 4 x 10(4) VEG strain oocysts 57 days later. The G1 pigs produced high IgG antibody levels in the indirect enzyme-linked immunosorbent assay (ELISA) after the second dose of rhoptry vaccine, but were not clinically protected against a high dose oocyst challenge. Partial protection was observed in G1 at the chronic phase of infection, when compared with G3. Pigs in group 2 developed high antibody levels and were protected against clinic signs. T. gondii was not detected in two (G1) and three (G2) pigs by mouse bioassay. The results indicate partial protection in pigs vaccinated with a rhoptry vaccine.

Chemical carcinogenesis by DMBA (7, 12-dimethylbenzanthracene) in female BALB/c mice: new facts / Carcinogenese quimica por DMBA (7, 12-dimethylbenzanthracene) em camundongos femeas BALB/c: novos fatos

Polycyclic aromatic hydrocarbons are known carcinogens used in rodent experimental models. In this study, the carcinogen DMBA (7,12-dimethylbenzanthracene) was administered by gavage, diluted in corn oil, to female BALB / c mice at hebdomadary doses of 1 mg per animal for 1, 3, 6 or 9 weeks. Animals were weighed and monitored weekly until death. Remaining animals were euthanized at the age of 53 weeks. At necropsy, representative fragments of neoplasms were collected and routinely processed for histopathological analysis. Of all mice that received DMBA, 68.57% developed some type of tumor. Of the 70 mice treated with various doses of DMBA, 22 (31.43%) developed mammary tumors. The adenoacanthoma was the most commonly (18.75%) diagnosed histological type of breast cancer. Lung (15.71%), lymphoid tissue (11.43%), stomach (7.14%) and skin (2.86%) were also primary sites of tumor development. One third (33.33%) of the mice receiving 1 mg of DMBA developed lung cancer. Therefore, the administration of DMBA was shown to be an efficient model of carcinogenesis in mice, especially for the study of breast cancer, when using the highest dose, and lung, when using the lowest dose. Carcinogenesis models have been used for several purposes in cancer research. These results represent new facts for a classic carcinogenesis model.

Hidrocarbonetos policíclicos e aromáticos são carcinógenos usados em modelos experimentais em roedores. Neste estudo, o carcinógeno DMBA (7,12-dimetilbenzantraceno) foi administrado por gavagem, diluído em óleo de milho, para camundongos BALB/c em doses hebdomadárias de 1 mg por animal por 1, 3, 6 ou 9 semanas. Os animais foram pesados e monitorados semanalmente até a morte. Os animais remanescentes foram eutanasiados com a idade de 53 semanas. Na necroscopia, fragmentos representativos das neoplasias foram colhidos e rotineiramente processados para exame histopatológico. De todos os animais que receberam DMBA, 68,57% desenvolveram algum tipo de tumor. Entre os 70 camundongos tratados com diferentes doses de DMBA, 22 (31,43%) desenvolveram neoplasias mamárias. O adenoacantoma foi o tumor mamário mais comumente diagnosticado (18,75%). Pulmões (15,71%), tecido linfoide (11,43%), estômago (7,14%) e pele (2,86%) foram também locais primários de desenvolvimento de neoplasias. Um terço (33,33%) dos camundongos que receberam 1 mg de DMBA desenvolveram neoplasias pulmonares. Portanto, a administração de DMBA foi considerada um modelo eficiente de carcinogênese em camundongos, especialmente para o estudo de neoplasias mamárias, quando a maior dose é utilizada, e de neoplasias pulmonares, quando utilizada a menor dose. Os modelos de carcinogênese química têm sido usados para diversos estudos na pesquisa em câncer, os resultados aqui apresentados mostram novos fatos para um modelo clássico de carcinogênese.

Chemical carcinogenesis by DMBA (7, 12-dimethylbenzanthracene) in female BALB/c mice: new facts / Carcinogenese quimica por DMBA (7, 12-dimethylbenzanthracene) em camundongos femeas BALB/c: novos fatos

Polycyclic aromatic hydrocarbons are known carcinogens used in rodent experimental models. In this study, the carcinogen DMBA (7,12-dimethylbenzanthracene) was administered by gavage, diluted in corn oil, to female BALB / c mice at hebdomadary doses of 1 mg per animal for 1, 3, 6 or 9 weeks. Animals were weighed and monitored weekly until death. Remaining animals were euthanized at the age of 53 weeks. At necropsy, representative fragments of neoplasms were collected and routinely processed for histopathological analysis. Of all mice that received DMBA, 68.57% developed some type of tumor. Of the 70 mice treated with various doses of DMBA, 22 (31.43%) developed mammary tumors. The adenoacanthoma was the most commonly (18.75%) diagnosed histological type of breast cancer. Lung (15.71%), lymphoid tissue (11.43%), stomach (7.14%) and skin (2.86%) were also primary sites of tumor development. One third (33.33%) of the mice receiving 1 mg of DMBA developed lung cancer. Therefore, the administration of DMBA was shown to be an efficient model of carcinogenesis in mice, especially for the study of breast cancer, when using the highest dose, and lung, when using the lowest dose. Carcinogenesis models have been used for several purposes in cancer research. These results represent new facts for a classic carcinogenesis model. (AU)

Hidrocarbonetos policíclicos e aromáticos são carcinógenos usados em modelos experimentais em roedores. Neste estudo, o carcinógeno DMBA (7,12-dimetilbenzantraceno) foi administrado por gavagem, diluído em óleo de milho, para camundongos BALB/c em doses hebdomadárias de 1 mg por animal por 1, 3, 6 ou 9 semanas. Os animais foram pesados e monitorados semanalmente até a morte. Os animais remanescentes foram eutanasiados com a idade de 53 semanas. Na necroscopia, fragmentos representativos das neoplasias foram colhidos e rotineiramente processados para exame histopatológico. De todos os animais que receberam DMBA, 68,57% desenvolveram algum tipo de tumor. Entre os 70 camundongos tratados com diferentes doses de DMBA, 22 (31,43%) desenvolveram neoplasias mamárias. O adenoacantoma foi o tumor mamário mais comumente diagnosticado (18,75%). Pulmões (15,71%), tecido linfoide (11,43%), estômago (7,14%) e pele (2,86%) foram também locais primários de desenvolvimento de neoplasias. Um terço (33,33%) dos camundongos que receberam 1 mg de DMBA desenvolveram neoplasias pulmonares. Portanto, a administração de DMBA foi considerada um modelo eficiente de carcinogênese em camundongos, especialmente para o estudo de neoplasias mamárias, quando a maior dose é utilizada, e de neoplasias pulmonares, quando utilizada a menor dose. Os modelos de carcinogênese química têm sido usados para diversos estudos na pesquisa em câncer, os resultados aqui apresentados mostram novos fatos para um modelo clássico de carcinogênese. (AU)

Cytotoxic effects of butanolic extract from Pfaffia paniculata (Brazilian ginseng) on cultured human breast cancer cell line MCF-7.

Roots of Pfaffia paniculata have been well documented for multifarious therapeutic values and have also been used for cancer therapy in folk medicine. This study has been performed in a human breast tumor cell line, the MCF-7 cells. These are the most commonly used model of estrogen-positive breast cancer, and it has been originally established in 1973 at the Michigan Cancer Foundation from a pleural effusion taken from a woman with metastatic breast cancer. Butanolic extract of the roots of P. paniculata showed cytotoxic effect MCF-7 cell line, as determined with crystal violet assay, cellular death with acridine orange/ethidium bromide staining, and cell proliferation with immunocytochemistry of bromodeoxyuridine (BrdU). Subcellular alterations were evaluated by electron microscopy. Cells treated with butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. The results show that this butanolic extract indeed presents cytotoxic substances, and its fractions merit further investigations.

Toxoplasma gondii: comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs.

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7x10(7) viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10=2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as "gold standard" and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISAxIFAT (kappa = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 +/- 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs.

Microscopical study of experimental wound healing in Notothenia coriiceps (Cabeçuda) at 0 degrees C.

Notothenia coriiceps (Cabeçuda) is an Antarctic benthic fish frequently found with lesions in the tegument caused by seal predation. We have investigated epidermal repair in these animals by means of a microscopic study of experimental wound healing at 0 degrees C. At 24--48 h after wound induction, mucous exudate and necrotic lining cells covered the wound. At 7--14 days, an epidermal "tongue" could be discerned, folded at the tip, with intercellular oedema between the tip and the wound border. After 23--30 days, the wound was completely closed and the migrating epidermis, with intercellular oedema, was reduced. By 45--90 days, melanocytes progressively increased in the epidermis but no scales were formed. The inflammatory infiltrate was mainly composed of neutrophils after 7 days, at which time they were mostly replaced by macrophages; lymphocytes and plasma cells were also present. The border epidermis slid towards the centre, folding at the tip and finally fusing to form a diaphragm. The cells of the epidermis began to multiply only after complete closure of the wound. The lack of scale formation on induced and naturally found wounds, even after 90 days, suggests that different mechanisms in wound repair occur at 0 degrees C from those in fish from temperate and tropical environment. This is the first report of successful wound repair at polar temperatures, indicating the adaptation of N. coriiceps to the Antarctic environment.

Toxoplasma gondii: isolation of tachyzoites rhoptries and incorporation into Iscom.

Rhoptries have been isolated from Toxoplasma gondii tachyzoites by subcellular fractionation in isopynic density sucrose gradient. Five bands were observed, and transmission electron microscopy of these indicated that rhoptries were in band 3. This band had a density of 1.17 g/cm(3). Fraction 1 had membrane structures of the parasite. Fraction 2 contained membranes and mitochondria. Fraction 4 had mostly conoid structure and fraction 5 showed ghosts. The electrophoretic and Western blotting analysis of the fractions indicated the presence of a number of proteins. Iscoms were constructed from band 3, which contained the rhoptry structures. Iscom showed a only protein incorporated of 55 kDa. Isolation of the parasite organelles has got in this work is necessary to identification, characterization, and function elucidation of the organelle proteins.

Descrição histológica da via genital masculina e hermafrodita de escargots das espécies Achatina fulica E Achatina monochromatica / Histology description of the male and hermafrodite genital vias of the escargots Achatina fulica AND Achatina monochromatica

O objetivo deste trabalho foi descrever a histologia da via genital masculina e hermafrodita dos escargots das espécies Achatina fulica e Achatina monochromatica. Foram usados 10 exemplares adultos de cada espécie para análise histológica. Após o processo seletivo, pesagem e tomada das medidas da concha, os espécimes foram sacrificados por congelamento em freezer, a -2ºC por aproximadamente 10min. Para a exposição dos órgãos reprodutores foi efetuada uma incisão longitudinal iniciada na extremidade cranial e, acompanhando a torção do corpo, até a extremidade caudal. Cada animal foi dissecado, sobre uma placa de Petri, sob imersão em formol a 10% para melhor preservação dos tecidos. Foram coletados fragmentos representativos de cada segmento do sistema reprodutor e observou-se que a estrutura histológica dos mesmos não apresentou diferenças notórias, com exceção do ovotestis do Achatina fulica, no qual se constatou um número maior de células pigmentadas no epitélio germinativo do que, nesse mesmo epitélio, no Achatina monochromatica.

The aim of this work was to make histological description of the male and hermaphrodite genital system of escargots of the species Achatina fulica and Achatina monochromatica. Ten adult copies of each species were used for such analysis. After a selective process, weighting and measuring the shell, the specimens were sacrificed by freezing in freezer to -2ºC for approximately 10min. Reproductive organs were exposed by longitudinal incision made in the cranial extremity and by accompanying the sprain of the body, until the tail extremity. Each animal was dissected over a Petri plate, under immersion in 10% phormol for better preservation of the tissues. Representative fragments of each segment of the reproductive system were collected and it was observed that their histological structure did not present evident differences, except for the Achatina fulica ovotestis in which a larger number of pigmented cells was observed in the germinative epithelium than in the same epithelium in the Achatina monochromatica.

Descrição histológica da via genital masculina e hermafrodita de escargots das espécies Achatina fulica e Achatina monochromatica / Histology description of the male and hermafrodite genital vias of the escargots Achatina fulica and Achatina monochromatica

O objetivo deste trabalho foi descrever a histologia da via genital masculina e hermafrodita dos escargots das espécies Achatina fulica e Achatina monochromatica. Foram usados 10 exemplares adultos de cada espécie para análise histológica. Após o processo seletivo, pesagem e tomada das medidas da concha, os espécimes foram sacrificados por congelamento em freezer, a -2ºC por aproximadamente 10min. Para a exposição dos órgãos reprodutores foi efetuada uma incisão longitudinal iniciada na extremidade cranial e, acompanhando a torção do corpo, até a extremidade caudal. Cada animal foi dissecado, sobre uma placa de Petri, sob imersão em formol a 10% para melhor preservação dos tecidos. Foram coletados fragmentos representativos de cada segmento do sistema reprodutor e observou-se que a estrutura histológica dos mesmos não apresentou diferenças notórias, com exceção do ovotestis do Achatina fulica, no qual se constatou um número maior de células pigmentadas no epitélio germinativo do que, nesse mesmo epitélio, no Achatina monochromatica.(AU)

The aim of this work was to make histological description of the male and hermaphrodite genital system of escargots of the species Achatina fulica and Achatina monochromatica. Ten adult copies of each species were used for such analysis. After a selective process, weighting and measuring the shell, the specimens were sacrificed by freezing in freezer to -2ºC for approximately 10min. Reproductive organs were exposed by longitudinal incision made in the cranial extremity and by accompanying the sprain of the body, until the tail extremity. Each animal was dissected over a Petri plate, under immersion in 10% phormol for better preservation of the tissues. Representative fragments of each segment of the reproductive system were collected and it was observed that their histological structure did not present evident differences, except for the Achatina fulica ovotestis in which a larger number of pigmented cells was observed in the germinative epithelium than in the same epithelium in the Achatina monochromatica.(AU)

Effects of endosulfan sublethal concentrations oncarp (Cyprinus carpio, Linnaeus, 1758): Morphometric, hystologic, ultrastructural analysesand cholinesterase activity evaluation / Efeitos do endosulfano em concentrações subletais em carpas (Cyprinuscarpio, Linnaeus, 1758): Análises morfométricas, histológicas eultraestrutural e avaliação da atividade da colinesterase

Endosulfan, an organochlorine pesticide, has been banned by most developed countries, although it is still produced, sold and used in developing countries. Used for control in crops, as well as for insect control in public health programs in some countries, its effects on the environment and its toxicity are still in discussion. For some researchers, its bioaccumulation in terrestrial organisms is considered irrelevant but for aquatic life it should be considered carefully. The present research work was to carry out an study on the effects of sublethal concentrations of endosulfan on the fresh water fish carp (Cyprinus carpio, Linnaeus, 1758). The fishes were exposed during 15 days to 0.001 mg/L of endosulfan using dimethylsulfoxide 0.1% (DMSO) as solvent. The acetylcholinesterase activity on the brain and axial muscle, as well as liver morphometric, histopathologic and ultrastructural analysis were studied. The hepatic somatic index and the livers weight showed smaller values when compared with the control groups, besides being also observed histopathological and ultrastructural alterations. It has not been observed significant alterations in the cholinesterase activity of both brain and striated muscle. These results suggest that the organochloride endosulfan caused toxic effects in the hepatic metabolism of the fish exposed to it in sub lethal doses

Endosulfano, um pesticida organoclorado, tem sido banido pela maioria dos países desenvolvidos, embora seja ainda produzido e utilizado deliberadamente em países em desenvolvimento. Utilizado no controle de pragas, assim como no controle de insetos em Programas de Saúde Pública em alguns países, seus efeitos no meio ambiente e sua toxicidade continuam em discussão. Para alguns pesquisadores a bioacumulação nos organismos terrestres é considerada irrelevante, mas não para a vida aquática. O objetivo da presente pesquisa foi estudar os efeitos das concentrações subletais do endosulfano em peixes de água doce Cyprinus carpio, (Linnaeus, 1758). Os peixes foram expostos durante 15 dias, a uma concentração de 0,001mg/L de endosulfano utilizando o dimetilsulfóxido (DMSO) a 0,1% como solvente. A atividade da acetilcolinesterase do músculo axial e cerebral assim como a morfometria, histopatologia e a ultraestrutura do fígado desses peixes foram avaliadas. O índice somático hepático e o peso dos fígados mostraram valores menores quando comparados ao grupo controle, observando-se também, alterações histopatológicas e ultraestruturais. Nenhuma alteração significante na atividade da acetilcolinesterase muscular e cerebral foram observadas. Os resultados sugerem que o organoclorado endosulfano causou efeitos tóxicos no metabolismo hepático dos peixes expostos a doses subletais

Effects of endosulfan sublethal concentrations on carp (Cyprinus carpio, Linnaeus, 1758): Morphometric, hystologic, ultrastructural analyses and cholinesterase activity evaluation / Efeitos do endosulfano em concentrações subletais em carpas (Cyprinuscarpio, Linnaeus, 1758): Análises morfométricas, histológicas e ultraestrutural e avaliação da atividade da colinesterase

Endosulfan, an organochlorine pesticide, has been banned by most developed countries, although it is still produced, sold and used in developing countries. Used for control in crops, as well as for insect control in public health programs in some countries, its effects on the environment and its toxicity are still in discussion. For some researchers, its bioaccumulation in terrestrial organisms is considered irrelevant but for aquatic life it should be considered carefully. The present research work was to carry out an study on the effects of sublethal concentrations of endosulfan on the fresh water fish carp (Cyprinus carpio, Linnaeus, 1758). The fishes were exposed during 15 days to 0.001 mg/L of endosulfan using dimethylsulfoxide 0.1% (DMSO) as solvent. The acetylcholinesterase activity on the brain and axial muscle, as well as liver morphometric, histopathologic and ultrastructural analysis were studied. The hepatic somatic index and the livers weight showed smaller values when compared with the control groups, besides being also observed histopathological and ultrastructural alterations. It has not been observed significant alterations in the cholinesterase activity of both brain and striated muscle. These results suggest that the organochloride endosulfan caused toxic effects in the hepatic metabolism of the fish exposed to it in sub lethal doses.(AU)

Endosulfano, um pesticida organoclorado, tem sido banido pela maioria dos países desenvolvidos, embora seja ainda produzido e utilizado deliberadamente em países em desenvolvimento. Utilizado no controle de pragas, assim como no controle de insetos em Programas de Saúde Pública em alguns países, seus efeitos no meio ambiente e sua toxicidade continuam em discussão. Para alguns pesquisadores a bioacumulação nos organismos terrestres é considerada irrelevante, mas não para a vida aquática. O objetivo da presente pesquisa foi estudar os efeitos das concentrações subletais do endosulfano em peixes de água doce Cyprinus carpio, (Linnaeus, 1758). Os peixes foram expostos durante 15 dias, a uma concentração de 0,001mg/L de endosulfano utilizando o dimetilsulfóxido (DMSO) a 0,1% como solvente. A atividade da acetilcolinesterase do músculo axial e cerebral assim como a morfometria, histopatologia e a ultraestrutura do fígado desses peixes foram avaliadas. O índice somático hepático e o peso dos fígados mostraram valores menores quando comparados ao grupo controle, observando-se também, alterações histopatológicas e ultraestruturais. Nenhuma alteração significante na atividade da acetilcolinesterase muscular e cerebral foram observadas. Os resultados sugerem que o organoclorado endosulfano causou efeitos tóxicos no metabolismo hepático dos peixes expostos a doses subletais.(AU)

Experimental chronic granulomatous inflammatory process in fish: a morphological, ultrastructural anel immunocytochemical study / Processo inflamatório crônico granulomatoso experimental em peixes: um estudo morfológico, ultraestrutural e imunohistoquímico

The purpose of the present srudy was to evaluate the chronic granulomatous inflammatory process experimentally induced in Oreochromis niloticus by the inoculation ofBCG and to clarify aspects of the inflammatory reaction in fish for a better understanding of the phylogeny of the process. Results obtained by light microscopy and ultrastructural analysis showed the participation of macrophages, thrombocytes, lymphocytes, eosinophils, plasma cells and foreign type giant cells in the inflammatory process. Besides these cell types, a granulomatous reaction mainly consisting of epithelioid cells was also observed ultrastrucrurally. These epithelioid cells developed desmosomes throughout the experiment, and also began to express receptors for cytokeratin. Both findings are immunocytochemicaliy characteristic of epithelial cells. Pigment cells (melanomacrophages) could also be seen surrounding ali the granulomatoid formation in a crescent fashion and participating in the chronic granulomatous inflammatory reaction.(AU)

O objetivo do presente estudo foi avaliar o processo inflamatório crônico granulomatoso induzido experimentalmente em Oreochromis niloticus através da inoculação de BCG e elucidar aspectos da reação inflamatória em peixes para uma melhor compreensão da filogenia do processo. Os resultados obtidos por microscopia de luz comum e ultra-estrutural demonstraram a participação de macrófagos, trombócitos, linfócitos, eosinófilos, células plasmáticas e células gigante tipo corpo estranho no processo inflamatório. Além desses tipos celulares, uma reação granulomatosa constituída predominantemente de células epitelióides também foram observadas ultraestruturalmente. Essas células epitelióides desenvolveram desmossomos ao longo do experimento, e também passaram a expressar receptores para citoqueratina, características estas de células epiteliais. Células pigmentares (melanomacrófagos), envolvendo de maneira crescente toda a formação granulomatóide e, participando ativamente da reação inflamatória crônica granulomatosa.(AU)

Marcação imuno-histoquímica da resposta macrofágica e astrocitária no tronco encefálico de ratos Wistar submetidos ao modelo gliotóxico do brometo de etídio e tratados com ciclofosfamida / Immunohistochemical staining of the macrophagic and astrocytic response in the brainstem of Wistar rats submitted to the ethidium bromide gliotoxic model and treated with cyclophosphamide

O gliotóxico brometo de etídio (BE) foi utilizado para o estudo da resposta macrofágica e astrocitária sob imunossupressão com ciclofosfamida (CY). Investigou-se a imunorreatividade astrocitária à proteína glial fibrilar ácida (GFAP) e à vimentina (VIM), e a imunorreatividade macrofágica ao ED1 após injeção do BE. Foram utilizados ratos Wistar adultos injetados na cisterna basal com salina a 0,9 por cento (grupo I), BE a 0,1 por cento (grupo II) e BE a 0,1 por cento, imunossuprimidos com CY (grupo III). Fragmentos do tronco encefálico foram colhidos do 1° ao 21° dia pós-injeção para estudo imuno-histoquímico da GFAP, VIM e ED1. Nos grupos II e III, observou-se imunorreatividade aumentada para GFAP e re-expressão de VIM. No grupo II, células ED1-positivas foram observadas a partir do 2° dia e no grupo III, a partir do 3° dia. Aos 14 dias pós-injeção, havia mais células ED1-positivas no grupo III. A CY aparentemente não alterou a resposta astrocitária.

The gliotoxic ethidium bromide (EB) was used to study morphologically the macrophagic and astrocytic response under immunosuppression by cyclophosphamide (CY). Astrocyte immunoreactivity to glial fibrillary acidic protein (GFAP) and vimentin (VIM) and macrophagic immunoreactivity to ED1 were investigated after EB injection. Male Wistar rats were injected with 0.9 percent saline solution (group I), 0.1 percent BE (group II) and 0.1 percent EB associated with CY treatment (group III). Brainstem samples were collected from the 1st to the 21st day post-injection for GFAP, VIM and ED1 immunostaining. In groups II and III, it was observed increased immunoreactivity to GFAP and reexpression of VIM. In group II, ED1-positive cells were noted after the 2nd day and in group III, after the 3rd day. On the 14th day post-injection, it was observed a greater quantity of ED1- positive cells in group III than in group II. Apparently, CY did not change the astrocytic response pattern.

Teste de tuberculinização em caprinos (Capra hircus) experimentalmente sensibilizados / Tuberculin test in experimentally sensitized goats (Capra hircus)

Foram estabelecidos critérios de interpretação do teste de tuberculinização aplicado ao diagnóstico da tuberculose em grupos de caprinos experimentalmente sensibilizados. Dos 30 animais utilizados, dez foram sensibilizados com Mycobacterium avium - amostra D4 (grupo A) e dez com Mycobacterium bovis - amostra AN5 (grupo B). Dez caprinos não foram sensibilizados e constituíram o grupo controle (grupo C). No teste cervical simples, realizado com tuberculina bovina (M. bovis) e lido às 72 horas pós-tuberculinização (p.t.), as reações positivas foram aquelas em que houve aumento da espessura da dobra de pele igual ou superior a 3,9mm; reações inconclusivas, quando situadas entre 1,8 e 3,8mm, e negativas quando iguais ou menores que 1,7mm. A análise dos resultados do teste cervical comparativo, realizado com o M. avium e M. bovis e lido às 72 horas p.t., indicou reação positiva quando o aumento da espessura da dobra da pele induzida pela tuberculina bovina superou a reação à aviária em pelo menos 2,5mm; reação inconclusiva quando a diferença entre a reação à tuberculina bovina e à tuberculina aviária ficou situada entre 1,9 e 2,4mm; e negativa quando a reação bovina ultrapassou a aviária em até 1,8mm. As 96 horas após a injeção da tuberculina, foi efetuada a avaliação histológica do local das reações tuberculínicas colhendo-se amostras de pele de cinco caprinos dos grupos A e B e de quatro animais do grupo controle; os resultados confirmaram a presença, nos grupos sensibilizados, de infiltrado inflamatório, constituído, preferencialmente, por células mononucleares.

The tuberculin skin test was established with the aim to be applied in the diagnosis of tuberculosis in experimentally sensitized goats. Thirty goats were alocated into three groups with ten animals each. The animals in group A were sensitized with Mycobacterium avium sample D4; group B with Mycobacterium bovis sample AN5; and group C (control) was inoculated with saline solution. The results of the simple cervical test after 72h of bovine tuberculin inoculation was interpreted as follow: positive reaction, when there was an increase in the skin fold thickness greater than 3.9mm; suspicious, when from 1.8 to 3.8mm; and negative when less than 1.7mm. The analysis of the results of the comparative cervical test between M. avium and M. bovis and analysed, 72h after tuberculin, indicated positive reactions, when the increase in skin fold thickness induced by M. bovis was greater than that one induced by the avian tuberculin at least 2.5mm; it was considered suspicious when the difference between the bovine and avian tuberculin reactions was from 1.9 to 2.4mm; and negative when this difference was smaller than 1.8mm. The histological evaluation of the local specific tuberculin response, were performed in skin samples collected from five goats in group A, five in group B, and four in group C, the results were the presence of mononuclear inflammatory infiltrate at 96h after tuberculin inoculation.

Cytokine detention at the site of Leishmania (Leishmania)Amazonensis subcutaneous inoculation in mice depleted of Natural Killer cells / Detecção de citocinas no sítio de inoculação subcutânea de Leishmania (Leishmania) amazonensis em camundongos depletados de células Natural Killer

BALBI c mice depleted and non-depleted of Natural Killer (NK) cells were infected subcutaneously with 10 7 stationary phase promastigotes of Leishmania (Leishmania) amazonensis and samples were taken at 24 hours and 7 days after infection. In NK cell-depleted mice, the NK cytotoxic activityof spleen cellsdecreased at 7 days after infection and more parasites were found in the lesion. The NK cell populations were analyzed by immunohistochemistry in spleen cryosections. An increase of NK1.1 + expression and a decrease of NK5E6+ antigen expression was observed in NK cell-depleted mice compared to non-depleted mice, When the presence ofIFN -g, IL-12 and IL-4 at the site of parasite inoculation was analyzed by immunohistochemistry, a large amount of cytokines was detected in K cell-depleted mice at 24 hours and 7 days afrer infection. In nondepleted mice, there was a small amount ofIL-12 at 24 hours and of IL-4 at 7 days after infection. These data cells suggest that K cell depletion by 90Srresults in increased parasitism in the lesion. The increase of NK1.1 + expression, which mainly produces IL-4, may take part in the progression of the infection. (AU)

Camundongos BALB/ c depletados e não depletados em células Natural IVller (NK) foram infectados subcutaneamente com 107 promastigotas de Leishmania (Leishmania) amazonensis em fase estacionária de crescimento e amostras foram colhidas às 24 horas e 7 dias de infecção. Nos camundongos depletados em células NK, a atividade citotóxica NK de células esplênicas estava diminuída aos 7 dias de infecção e mais parasitos foram encontrados na lesão. Populações de células NK foram analisadas por imuno-histoquimica em cortes congelados de baço. Foi observado aumento na expressão de células NK1.1+ e diminuição na expressão do antígeno NK5E6+ nos animais depletados em células NK comparados aos camundongos não depletados. A presença de IFN-g, IL-12 e IL-4, analisada por imunohistoquimica no sítio de inoculação dos parasitos, mostrou que maior quantidade de citocinas foram detectadas nos camundongos depletados em células NK às 24 horas e 7 dias depois da infecção. Nos camundongos não depletados, havia pequenas quantidades de IL-12 às 24 horas e de IL-4 aos 7 dias de infecção. Estes dados sugerem que a depleção de células NK por 90Sr resulta em aumento de parasitismo na lesão. O aumento na expressão de células NK1.1+, as quais produzem principalmente IL-4, pode representar um dos mecanismos que colaborariam para a progressão da infecção. (AU)

Granulosa cell tumour with metastasis in a cat

The clinical, gross and microscopical features of a granulosa cell tumour in the ovary of a 2-year-old cat are described. This is a rare tumour in cats, moreover because of the animal's age. It was diagnosed clinically by ultrasound examination. The tumour, a cystic mass in the region of the left ovary, metastasized to the epiplon and lungs. Tumour cells expressed vimentin and were arranged in a diffuse sarcomatous pattern, supported by a fine fibrovascular stroma. The presence of hair loss and repeated oestrus as clinical signs are indicative of orthoendocrine paraneoplastic syndrome, due to excessive oestrogen stimulation. This tumour should be considered in the differential diagnosis of behavioural disturbances in female cats