Diagnosis of acute graft-versus-host disease in the gastrointestinal tract of patients undergoing allogeneic hematopoietic stem cell transplantation. A descriptive and critical study of diagnostic tests

ABSTRACT Introduction: Acute graft-versus-host disease (GVHD) is one of the major causes of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (AHSCT) and has become the subject of several studies to understand and treat it. Objective: This study does a descriptive analysis of the apoptotic index (AI) evaluation and intestinal permeability (IP) alterations in association with the clinical, endoscopic and histopathological data on patients undergoing AHSCT, with emphasis on acute intestinal graft-versus-host disease (GVHD) diagnosis. Methods: Thirty-one patients were divided into two groups—one of patients with a clinical GVHD diagnosis and one of those without GVHD diagnosis. Results: Thirteen deaths (41.9%) occurred during the study period, thereby reaffirming the severity of the alterations found in the patients. Fifteen patients subjected to 21 esophagogastroduodenoscopy procedures prior to D + 90 post-transplant had visible endoscopic alterations and 19 biopsies revealed histological alterations to the stomach and duodenum. Higher apoptotic indices, not reaching statistical significance, were observed in patients who died of graft versus host disease (GVHD), in the more acute forms of GVHD and where clinical GVHD was present. The intestinal permeability evaluation was performed on nine patients able to undergo it in the three proposed study periods, which showed alterations, some of which were pronounced even during pre-transplant and, therefore, the pre-conditioning phase. Conclusion: Clinical judgment remains a fundamental tool in the diagnosis of GVHD. This study points to the known limitations of traditional diagnostic aids (endoscopy and histology) and points to new methods not usually employed in clinical practice.

Diagnosis of acute graft-versus-host disease in the gastrointestinal tract of patients undergoing allogeneic hematopoietic stem cell transplantation. A descriptive and critical study of diagnostic tests.

INTRODUCTION: Acute graft-versus-host disease (GVHD) is one of the major causes of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (AHSCT) and has become the subject of several studies to understand and treat it. OBJECTIVE: This study does a descriptive analysis of the apoptotic index (AI) evaluation and intestinal permeability (IP) alterations in association with the clinical, endoscopic and histopathological data on patients undergoing AHSCT, with emphasis on acute intestinal graft-versus-host disease (GVHD) diagnosis. METHODS: Thirty-one patients were divided into two groups-one of patients with a clinical GVHD diagnosis and one of those without GVHD diagnosis. RESULTS: Thirteen deaths (41.9%) occurred during the study period, thereby reaffirming the severity of the alterations found in the patients. Fifteen patients subjected to 21 esophagogastroduodenoscopy procedures prior to D + 90 post-transplant had visible endoscopic alterations and 19 biopsies revealed histological alterations to the stomach and duodenum. Higher apoptotic indices, not reaching statistical significance, were observed in patients who died of graft versus host disease (GVHD), in the more acute forms of GVHD and where clinical GVHD was present. The intestinal permeability evaluation was performed on nine patients able to undergo it in the three proposed study periods, which showed alterations, some of which were pronounced even during pre-transplant and, therefore, the pre-conditioning phase. CONCLUSION: Clinical judgment remains a fundamental tool in the diagnosis of GVHD. This study points to the known limitations of traditional diagnostic aids (endoscopy and histology) and points to new methods not usually employed in clinical practice.

Testing a new surgical instrument to obtain bovine hoof biopsy samples / Teste de viabilidade de um instrumento cirúrgico para obtenção de biópsias do tecido laminar do casco de bovinos

The histological and molecular analysis of biopsy samples are fundamental steps for the understanding of physiopathology, diagnosis and prognosis of the diseases. However, harvest of tissue biopsies from hoof lamellar tissue is a procedure with limitations due to lack of effective surgical instruments and techniques. The aim of the current study is to develop and test in vivo a surgical instrument with the specific purpose of harvesting lamellar tissue in cattle. A prototype called Falcão-Faleiros' lamellotome (INPIBR102013018765-8) was designed, produced and tested. After sedation, five adult cattle were restrained in lateral recumbency and locally anesthetized in two digits. The stratum corneum was worn down using a rotary tool coupled to a 3/8" high-speed cutter until the soft tissue proximity was reached. Next, the inner edge of the worn area was bounded with a scalpel. The lamellotome was introduced to obtain and hold the sample. The histological specimens of 16mm length by 6mm depth were stained with HE, PAS, Masson's thricome and Shorr. The structures of interest were differentiated in the histological analysis without technical artifacts and a mean number of 85 epidermal laminae per sample were counted. No relevant lameness or wound complication were seen following the procedure. In conclusion the technique using the lamellotme was effective in obtaining lamellar tissue biopsy samples without causing clinical harm in cattle. The procedure showed potential to be used in clinical research or even as a supplementary diagnosis method for routine bovine podiatry.(AU)

A avaliação das propriedades histológicas e da expressão de genes e proteínas em biópsias tem sido determinante para o entendimento da fisiopatologia, o diagnóstico e o prognóstico das enfermidades. Entretanto, a obtenção de biópsias do casco é um procedimento com limitações devido à ausência de técnicas e instrumentos específicos. O objetivo foi desenvolver e testar, na espécie bovina, um instrumento cirúrgico especificamente desenvolvido para realização de biópsias de casco nominado lamelótomo de Falcão-Faleiros (INPI, BR102013018765-8). Utilizaram-se cinco bovinos adultos que foram sedados, contidos em decúbito lateral e tiveram dois dígitos anestesiados. Em seguida, uma serra circular acoplada a uma microretífica foi usada para o desgaste do estrato córneo na parede dorsal até próximo do estrato lamelar. Após incisões retilíneas delimitando a borda interna da área desgastada, utilizou-se o lamelótomo para obtenção da amostra. Os fragmentos de 16mm de comprimento e 6mm de profundidade foram fixados em formalina e processados para histologia com colorações HE, PAS, Shorr e tricrômico de Masson. Nenhum dos animais apresentou claudicação ou complicação relevantes no período pós-opertório. As amostras foram consideradas adequadas quanto à integridade das lâminas e à preservação de sua arquitetura. Obtiveram-se média de 85 lâminas epidérmicas viáveis por biópsia. Conclui-se que o lamelótomo de Falcão-Faleiros é apropriado e seguro para a obtenção de biópsias de casco em bovinos, se mostrando promissor para uso em estudos clínicos e na rotina de diagnóstico de problemas podais em bovinos.(AU)

Testing a new surgical instrument to obtain bovine hoof biopsy samples / Teste de viabilidade de um instrumento cirúrgico para obtenção de biópsias do tecido laminar do casco de bovinos

The histological and molecular analysis of biopsy samples are fundamental steps for the understanding of physiopathology, diagnosis and prognosis of the diseases. However, harvest of tissue biopsies from hoof lamellar tissue is a procedure with limitations due to lack of effective surgical instruments and techniques. The aim of the current study is to develop and test in vivo a surgical instrument with the specific purpose of harvesting lamellar tissue in cattle. A prototype called Falcão-Faleiros' lamellotome (INPIBR102013018765-8) was designed, produced and tested. After sedation, five adult cattle were restrained in lateral recumbency and locally anesthetized in two digits. The stratum corneum was worn down using a rotary tool coupled to a 3/8" high-speed cutter until the soft tissue proximity was reached. Next, the inner edge of the worn area was bounded with a scalpel. The lamellotome was introduced to obtain and hold the sample. The histological specimens of 16mm length by 6mm depth were stained with HE, PAS, Masson's thricome and Shorr. The structures of interest were differentiated in the histological analysis without technical artifacts and a mean number of 85 epidermal laminae per sample were counted. No relevant lameness or wound complication were seen following the procedure. In conclusion the technique using the lamellotme was effective in obtaining lamellar tissue biopsy samples without causing clinical harm in cattle. The procedure showed potential to be used in clinical research or even as a supplementary diagnosis method for routine bovine podiatry.(AU)

A avaliação das propriedades histológicas e da expressão de genes e proteínas em biópsias tem sido determinante para o entendimento da fisiopatologia, o diagnóstico e o prognóstico das enfermidades. Entretanto, a obtenção de biópsias do casco é um procedimento com limitações devido à ausência de técnicas e instrumentos específicos. O objetivo foi desenvolver e testar, na espécie bovina, um instrumento cirúrgico especificamente desenvolvido para realização de biópsias de casco nominado lamelótomo de Falcão-Faleiros (INPI, BR102013018765-8). Utilizaram-se cinco bovinos adultos que foram sedados, contidos em decúbito lateral e tiveram dois dígitos anestesiados. Em seguida, uma serra circular acoplada a uma microretífica foi usada para o desgaste do estrato córneo na parede dorsal até próximo do estrato lamelar. Após incisões retilíneas delimitando a borda interna da área desgastada, utilizou-se o lamelótomo para obtenção da amostra. Os fragmentos de 16mm de comprimento e 6mm de profundidade foram fixados em formalina e processados para histologia com colorações HE, PAS, Shorr e tricrômico de Masson. Nenhum dos animais apresentou claudicação ou complicação relevantes no período pós-opertório. As amostras foram consideradas adequadas quanto à integridade das lâminas e à preservação de sua arquitetura. Obtiveram-se média de 85 lâminas epidérmicas viáveis por biópsia. Conclui-se que o lamelótomo de Falcão-Faleiros é apropriado e seguro para a obtenção de biópsias de casco em bovinos, se mostrando promissor para uso em estudos clínicos e na rotina de diagnóstico de problemas podais em bovinos.(AU)

Testing a new surgical instrument to obtain bovine hoof biopsy samples

ABSTRACT: The histological and molecular analysis of biopsy samples are fundamental steps for the understanding of physiopathology, diagnosis and prognosis of the diseases. However, harvest of tissue biopsies from hoof lamellar tissue is a procedure with limitations due to lack of effective surgical instruments and techniques. The aim of the current study is to develop and test in vivo a surgical instrument with the specific purpose of harvesting lamellar tissue in cattle. A prototype called Falcão-Faleiros lamellotome (INPIBR102013018765-8) was designed, produced and tested. After sedation, five adult cattle were restrained in lateral recumbency and locally anesthetized in two digits. The stratum corneum was worn down using a rotary tool coupled to a 3/8 high-speed cutter until the soft tissue proximity was reached. Next, the inner edge of the worn area was bounded with a scalpel. The lamellotome was introduced to obtain and hold the sample. The histological specimens of 16mm length by 6mm depth were stained with HE, PAS, Massons thricome and Shorr. The structures of interest were differentiated in the histological analysis without technical artifacts and a mean number of 85 epidermal laminae per sample were counted. No relevant lameness or wound complication were seen following the procedure. In conclusion the technique using the lamellotme was effective in obtaining lamellar tissue biopsy samples without causing clinical harm in cattle. The procedure showed potential to be used in clinical research or even as a supplementary diagnosis method for routine bovine podiatry.

RESUMO: A avaliação das propriedades histológicas e da expressão de genes e proteínas em biópsias tem sido determinante para o entendimento da fisiopatologia, o diagnóstico e o prognóstico das enfermidades. Entretanto, a obtenção de biópsias do casco é um procedimento com limitações devido à ausência de técnicas e instrumentos específicos. O objetivo foi desenvolver e testar, na espécie bovina, um instrumento cirúrgico especificamente desenvolvido para realização de biópsias de casco nominado lamelótomo de Falcão-Faleiros (INPI, BR102013018765-8). Utilizaram-se cinco bovinos adultos que foram sedados, contidos em decúbito lateral e tiveram dois dígitos anestesiados. Em seguida, uma serra circular acoplada a uma microretífica foi usada para o desgaste do estrato córneo na parede dorsal até próximo do estrato lamelar. Após incisões retilíneas delimitando a borda interna da área desgastada, utilizou-se o lamelótomo para obtenção da amostra. Os fragmentos de 16mm de comprimento e 6mm de profundidade foram fixados em formalina e processados para histologia com colorações HE, PAS, Shorr e tricrômico de Masson. Nenhum dos animais apresentou claudicação ou complicação relevantes no período pós-opertório. As amostras foram consideradas adequadas quanto à integridade das lâminas e à preservação de sua arquitetura. Obtiveram-se média de 85 lâminas epidérmicas viáveis por biópsia. Conclui-se que o lamelótomo de Falcão-Faleiros é apropriado e seguro para a obtenção de biópsias de casco em bovinos, se mostrando promissor para uso em estudos clínicos e na rotina de diagnóstico de problemas podais em bovinos.

Apoptose e maturação placentária bovina: um estudo imunohistoquímico e morfométrico / Apoptosis and maturation of the bovine placenta: an immunohistochemical and morphometric study

A liberação da placenta após o parto envolve a perda da adesão materno-fetal e ocorre somente após a maturação completa do placentoma, que está relacionada com a diminuição da celularidade dos tecidos fetal e materno. A apoptose é requerida tanto para a maturação quanto para a liberação normal da placenta após o parto. O objetivo do presente estudo foi avaliar a ocorrência de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomas de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imunoistoquímica e histoquímica. As lâminas obtidas foram coradas em HE, Picrosirius Red e submetidas à análise imunoistoquímica das proteínas Caspase 3, Caspase 8, Bax e Bid. O aumento no número de vasos não necessariamente se associou ao aumento do calibre destes durante a evolução da gestação. Os resultados de histomorfometria revelaram aumento da marcação para Bax e Caspases 3 e 8 em células trofoblásticas binucleadas no final da gestação, enquanto o Bid se manteve sem alteração significativa. A histomorfometria das células trofoblásticas mononucleadas revelou expressão alta para Bax no início de gestação, com diminuição aos 6 meses de gestação e aumento das imunomarcações para Caspases 3 e 8, e Bid com o avanço gestacional. Os colágenos tipo I e III não aumentaram do terço médio ao final da gestação, o que é importante para a diminuição da adesão materno-fetal. Esses resultados confirmam que as Caspases 3 e 8, e o Bax estão envolvidos nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial e/ou extrínseca ao longo da gestação em células trofoblásticas binucleadas, e que nas células trofoblásticas mononucleadas o Bax deixa de ser importante, enquanto o Bid e as Caspases 3 e 8 se tornam os mais significativos.(AU)

Placental release after birth involves loss of maternal-fetal adhesion and occurs only after complete maturation of the placentoma related to the decrease in cellularity of fetal and maternal tissues. Apoptosis is required for both the normal maturation and release of the placenta after birth. The aim of this study was to evaluate the occurrence of apoptosis in samples of the placenta of cows in different stages of gestation. Samples of 15 healthy cow placentomas at 4 (n=5), 6 (n=5) and 9 (n=5) months of gestation were harvested and processed for routine histology, immunohistochemistry and histochemistry. The slides were stained with HE, PicroSirius Red and subjected to immunohistochemical analysis of proteins Caspase 3, Caspase 8, Bax and Bid. Increase in number of vessels was not associated with increase in vascular area during progression of gestation. The results of histomorphometry revealed increased labeling for Bax and Caspases 3 and 8 in trophoblastic binucleated cells in late pregnancy, where the Bid remained without significant change. Histomorphometry analyzing the mononuclear trophoblast cells showed a high expression for Bax in early pregnancy, but decreased at 6 months of gestation. Immunolabeling revealed increased Caspases 3/8 and Bid with advancing of gestation. Further evaluation of type I and III collagen showed a decrease of both types of collagens at the end of gestation, what is very important for the reduction of maternal-fetal adhesion. These results confirm that Caspases 3 and 8 and Bax are involved in the mechanisms of activation of apoptosis through mitochondrial intrinsic and/or extrinsic pathway during pregnancy in trophoblastic binucleated cells. In mononuclear trophoblast cells Bax looses importance in the apoptosis process, awhile Bid and caspases 3 and 8 become the most significant.(AU)

Apoptose e maturação placentária bovina: um estudo imunohistoquímico e morfométrico / Apoptosis and maturation of the bovine placenta: an immunohistochemical and morphometric study

A liberação da placenta após o parto envolve a perda da adesão materno-fetal e ocorre somente após a maturação completa do placentoma, que está relacionada com a diminuição da celularidade dos tecidos fetal e materno. A apoptose é requerida tanto para a maturação quanto para a liberação normal da placenta após o parto. O objetivo do presente estudo foi avaliar a ocorrência de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomas de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imunoistoquímica e histoquímica. As lâminas obtidas foram coradas em HE, Picrosirius Red e submetidas à análise imunoistoquímica das proteínas Caspase 3, Caspase 8, Bax e Bid. O aumento no número de vasos não necessariamente se associou ao aumento do calibre destes durante a evolução da gestação. Os resultados de histomorfometria revelaram aumento da marcação para Bax e Caspases 3 e 8 em células trofoblásticas binucleadas no final da gestação, enquanto o Bid se manteve sem alteração significativa. A histomorfometria das células trofoblásticas mononucleadas revelou expressão alta para Bax no início de gestação, com diminuição aos 6 meses de gestação e aumento das imunomarcações para Caspases 3 e 8, e Bid com o avanço gestacional. Os colágenos tipo I e III não aumentaram do terço médio ao final da gestação, o que é importante para a diminuição da adesão materno-fetal. Esses resultados confirmam que as Caspases 3 e 8, e o Bax estão envolvidos nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial e/ou extrínseca ao longo da gestação em células trofoblásticas binucleadas, e que nas células trofoblásticas mononucleadas o Bax deixa de ser importante, enquanto o Bid e as Caspases 3 e 8 se tornam os mais significativos.

Placental release after birth involves loss of maternal-fetal adhesion and occurs only after complete maturation of the placentoma related to the decrease in cellularity of fetal and maternal tissues. Apoptosis is required for both the normal maturation and release of the placenta after birth. The aim of this study was to evaluate the occurrence of apoptosis in samples of the placenta of cows in different stages of gestation. Samples of 15 healthy cow placentomas at 4 (n=5), 6 (n=5) and 9 (n=5) months of gestation were harvested and processed for routine histology, immunohistochemistry and histochemistry. The slides were stained with HE, PicroSirius Red and subjected to immunohistochemical analysis of proteins Caspase 3, Caspase 8, Bax and Bid. Increase in number of vessels was not associated with increase in vascular area during progression of gestation. The results of histomorphometry revealed increased labeling for Bax and Caspases 3 and 8 in trophoblastic binucleated cells in late pregnancy, where the Bid remained without significant change. Histomorphometry analyzing the mononuclear trophoblast cells showed a high expression for Bax in early pregnancy, but decreased at 6 months of gestation. Immunolabeling revealed increased Caspases 3/8 and Bid with advancing of gestation. Further evaluation of type I and III collagen showed a decrease of both types of collagens at the end of gestation, what is very important for the reduction of maternal-fetal adhesion. These results confirm that Caspases 3 and 8 and Bax are involved in the mechanisms of activation of apoptosis through mitochondrial intrinsic and/or extrinsic pathway during pregnancy in trophoblastic binucleated cells. In mononuclear trophoblast cells Bax looses importance in the apoptosis process, awhile Bid and caspases 3 and 8 become the most significant.

Foreign body response to subcutaneous implants in diabetic rats.

Implantation of synthetic matrices and biomedical devices in diabetic individuals has become a common procedure to repair and/or replace biological tissues. However, an adverse foreign body reaction that invariably occurs adjacent to implant devices impairing their function is poorly characterized in the diabetic environment. We investigated the influence of this condition on the abnormal tissue healing response in implants placed subcutaneously in normoglycemic and streptozotocin-induced diabetes in rats. In polyether-polyurethane sponge discs removed 10 days after implantation, the components of the fibrovascular tissue (angiogenesis, inflammation, fibrogenesis, and apoptosis) were assessed. Intra-implant levels of hemoglobin and vascular endothelial growth factor were not different after diabetes when compared with normoglycemic counterparts. However, there were a lower number of vessels in the fibrovascular tissue from diabetic rats when compared with vessel numbers in implants from non-diabetic animals. Overall, the inflammatory parameters (neutrophil accumulation--myeloperoxidase activity, tumor necrosis factor alpha, and monocyte chemotactic protein-1 levels and mast cell counting) increased in subcutaneous implants after diabetes induction. However, macrophage activation (N-acetyl-ß-D-glucosaminidase activity) was lower in implants from diabetic rats when compared with those from normoglycemic animals. All fibrogenic markers (transforming growth factor beta 1 levels, collagen deposition, fibrous capsule thickness, and foreign body giant cells) decreased after diabetes, whereas apoptosis (TUNEL) increased. Our results showing that hyperglycemia down regulates the main features of the foreign body reaction induced by subcutaneous implants in rats may be relevant in understanding biomaterial integration and performance in diabetes.

Diabetes alters inflammation, angiogenesis, and fibrogenesis in intraperitoneal implants in rats.

The increased prevalence of diabetes worldwide is associated with increasing numbers of diabetic individuals receiving synthetic matrices and biomedical implants to repair and/or replace biological tissues. This therapeutic procedure invariably leads to adverse tissue healing (foreign body reaction), thus impairing the biomedical device function of subcutaneous implants. However, the influence of diabetes on abnormal tissue healing in intraperitoneal implants is unclear. We investigated key components of foreign body reactions in diabetic rats. Polyether-polyurethane sponge discs were placed intraperitoneally in rats previously injected with streptozotocin for induction of diabetes and in non-diabetic rats. Implants removed 10 days after implantation were assessed by determining the components of the fibrovascular tissue (angiogenesis, inflammation, and fibrogenesis). In implants from diabetic rats, fibrous capsule thickness and fibrovascular tissue infiltration (hematoxylin & eosin and picrosirius staining) were reduced in comparison with implants from non-diabetic rats. Hemoglobin (Hb) content (vascular index) and VEGF levels (pro-angiogenic cytokine) were increased after diabetes. However, the number of vessels (H&E and CD31-immunostaining) in the fibrovascular tissue from diabetic rats was decreased when compared with vessel numbers in implants from non-diabetic animals. Overall, all inflammatory parameters (macrophage accumulation-NAG activity; TNF-α and MCP-1 levels) increased in intraperitoneal implants after diabetes induction. The pro-fibrogenic cytokine (TGFß-1) increased after diabetes, but collagen deposition remained unaltered in the implants from diabetic rats. These important diabetes-related changes (increased levels of pro-inflammatory and angiogenic and fibrogenic cytokines) in peritoneal implant healing provide an insight into the mechanisms of the foreign body response in the diabetic environment in rats.

Histopathology of dairy cows' hooves with signs of naturally acquired laminitis / Histopatologia do casco de vacas de aptidão leiteira com e sem sinais de laminite naturalmente adquirida

The purpose of this study was to investigate histological changes in dairy cows' hooves with or without injuries from naturally acquired laminitis. Cull cows with no clinical signs of hoof abnormalities (G1, n=9) and those with macroscopic lesions associated with laminitis without (G2, n=23) or with lameness (G3, n=7) were used in the study. After slaughter, samples of dermo-epidermal junctions of sole, axial and dorsal regions of the hoof were obtained and histologically processed using HE and PAS staining. Congestion, hemorrhage and inflammatory infiltrate in the dermis of sole, axial and dorsal regions were blindly and semiquantitatively evaluated by the same researcher. Inflammatory infiltrate was evaluated in the dermal laminae of axial and dorsal regions. The morphology of epidermal cells and the presence of irregularities in three regions of the basement membrane (BM) length were examined using PAS staining. Scores of lesions in different regions of the hoof in the same group and in different groups for each region of the hoof were compared using non-parametric analyses. Inflammatory infiltrate in the dermis of all regions of the hoof was detected in all groups with no significant statistical difference. Cows with no clinical signs of hoof abnormalities secondary to laminitis (G1) have inflammation scores and epidermal cell changes similar to those of groups with laminitis injuries, suggesting the existence of a prodromal phase for this disease in bovines. BM had irregularities with a variable intensity along its length, however, with no difference among groups. The pattern of BM irregularities found has not been reported so far and does not resemble the BM collapse described in horses and cattle with induced acute laminitis. Is it concluded that even in the absence of macroscopic hoof signs associated to laminitis, dairy cows have histological injuries compatible with inflammation of the dermo-epidermal junction as in affected animals. Basement membrane of cows with or without laminitis associated lesions had irregularities with an irregular distribution along its length which need to be further studied.(AU)

O objetivo deste trabalho foi investigar as alterações histológicas no casco de vacas leiteiras sem e com lesões de laminite, naturalmente, adquirida. Utilizaram-se animais de descarte sem lesões macroscópicas no casco (G1 n=9) e com lesões macroscópicas associadas à laminite sem (G2 n=23), ou com claudicação (G3 n=7). Após o abate, amostras da junção derme-epiderme das regiões solear, axial e dorsal do casco foram obtidas e processadas, histologicamente, nas colorações de HE e PAS. Avaliou-se, semiquantitativamente, às cegas e por um mesmo pesquisador, congestão, hemorragia e infiltrado inflamatório na derme das regiões solear, axial e dorsal. Avaliou-se infiltrado inflamatório na lâmina dermal das regiões axial e dorsal. A morfologia das células epidermais e a presença de irregularidades em três regiões do comprimento da membrana basal (MB) foram examinadas na coloração de PAS. Os escores das lesões nas diferentes regiões do casco no mesmo grupo e nos diferentes grupos para cada região do casco foram comparados através de análises não paramétricas (P<0,05). Infiltrado inflamatório na derme de todas as regiões do casco foi detectado em todos os grupos sem diferença estatística (P>0,05). Vacas sem lesões macroscópicas secundárias à laminite (G1) apresentaram escores de inflamação e alteração de células epidermais semelhantes aos dos grupos com lesões de laminite, sugerindo a existência de fase prodrômica para a doença em bovinos. A MB apresentou irregularidades com intensidade variável ao longo de seu comprimento (P<0,05), porém, sem diferença entre grupos (P>0,05). O padrão de irregularidades na MB encontrado não foi relatado até o momento e não se assemelha ao colapso de MB descrito em equinos e bovinos com laminite induzida. Concluiu-se que, mesmo na ausência de lesões macroscópicas no casco causadas por laminite, vacas leiteiras apresentam lesões histológicas compatíveis com inflamação da junção derme-epiderme como em animais afetados. A membrana basal de bovinos sem e com lesões de laminite apresenta irregularidades com distribuição irregular ao longo de seu comprimento que precisam ser melhor estudadas.(AU)

Histopathology of dairy cows' hooves with signs of naturally acquired laminitis / Histopatologia do casco de vacas de aptidão leiteira com e sem sinais de laminite naturalmente adquirida

The purpose of this study was to investigate histological changes in dairy cows' hooves with or without injuries from naturally acquired laminitis. Cull cows with no clinical signs of hoof abnormalities (G1, n=9) and those with macroscopic lesions associated with laminitis without (G2, n=23) or with lameness (G3, n=7) were used in the study. After slaughter, samples of dermo-epidermal junctions of sole, axial and dorsal regions of the hoof were obtained and histologically processed using HE and PAS staining. Congestion, hemorrhage and inflammatory infiltrate in the dermis of sole, axial and dorsal regions were blindly and semiquantitatively evaluated by the same researcher. Inflammatory infiltrate was evaluated in the dermal laminae of axial and dorsal regions. The morphology of epidermal cells and the presence of irregularities in three regions of the basement membrane (BM) length were examined using PAS staining. Scores of lesions in different regions of the hoof in the same group and in different groups for each region of the hoof were compared using non-parametric analyses. Inflammatory infiltrate in the dermis of all regions of the hoof was detected in all groups with no significant statistical difference. Cows with no clinical signs of hoof abnormalities secondary to laminitis (G1) have inflammation scores and epidermal cell changes similar to those of groups with laminitis injuries, suggesting the existence of a prodromal phase for this disease in bovines. BM had irregularities with a variable intensity along its length, however, with no difference among groups. The pattern of BM irregularities found has not been reported so far and does not resemble the BM collapse described in horses and cattle with induced acute laminitis. Is it concluded that even in the absence of macroscopic hoof signs associated to laminitis, dairy cows have histological injuries compatible with inflammation of the dermo-epidermal junction as in affected animals. Basement membrane of cows with or without laminitis associated lesions had irregularities with an irregular distribution along its length which need to be further studied.

O objetivo deste trabalho foi investigar as alterações histológicas no casco de vacas leiteiras sem e com lesões de laminite, naturalmente, adquirida. Utilizaram-se animais de descarte sem lesões macroscópicas no casco (G1 n=9) e com lesões macroscópicas associadas à laminite sem (G2 n=23), ou com claudicação (G3 n=7). Após o abate, amostras da junção derme-epiderme das regiões solear, axial e dorsal do casco foram obtidas e processadas, histologicamente, nas colorações de HE e PAS. Avaliou-se, semiquantitativamente, às cegas e por um mesmo pesquisador, congestão, hemorragia e infiltrado inflamatório na derme das regiões solear, axial e dorsal. Avaliou-se infiltrado inflamatório na lâmina dermal das regiões axial e dorsal. A morfologia das células epidermais e a presença de irregularidades em três regiões do comprimento da membrana basal (MB) foram examinadas na coloração de PAS. Os escores das lesões nas diferentes regiões do casco no mesmo grupo e nos diferentes grupos para cada região do casco foram comparados através de análises não paramétricas (P<0,05). Infiltrado inflamatório na derme de todas as regiões do casco foi detectado em todos os grupos sem diferença estatística (P>0,05). Vacas sem lesões macroscópicas secundárias à laminite (G1) apresentaram escores de inflamação e alteração de células epidermais semelhantes aos dos grupos com lesões de laminite, sugerindo a existência de fase prodrômica para a doença em bovinos. A MB apresentou irregularidades com intensidade variável ao longo de seu comprimento (P<0,05), porém, sem diferença entre grupos (P>0,05). O padrão de irregularidades na MB encontrado não foi relatado até o momento e não se assemelha ao colapso de MB descrito em equinos e bovinos com laminite induzida. Concluiu-se que, mesmo na ausência de lesões macroscópicas no casco causadas por laminite, vacas leiteiras apresentam lesões histológicas compatíveis com inflamação da junção derme-epiderme como em animais afetados. A membrana basal de bovinos sem e com lesões de laminite apresenta irregularidades com distribuição irregular ao longo de seu comprimento que precisam ser melhor estudadas.

A comparative study of apoptosis in reticular and erosive oral lichen planus

The oral lichen planus (OLP) is a chronic inflammatory disease, probably autoimmune, with different clinical forms. The most common types are the reticular and the erosive ones. Apoptosis participates in the destruction of basal keratinocytes, but its role in the perpetuation of the subepithelial lymphocytic infiltrates was not yet investigated. To evaluate the involvement of apoptosis in the epithelium and in subepithelial lymphocytic infiltrates, 15 samples of reticular and erosive OLP and 10 samples of healthy oral mucosa were collected and processed histologically. Apoptosis was quantified in the epithelium and in inflammatory cell infiltrates. TUNEL reaction was used to measure apoptosis in the infiltrates. Erosive OLP showed more intense epithelial apoptosis than reticular OLP and controls. In contrast, apoptosis in the inflammatory cell infiltrates was more frequent in reticular than in erosive OLP. Lymphocytes were the predominant cells within the inflammatory cell infiltrates and were more frequent in erosive OLP than in reticular type. These results suggest that different apoptotic levels are involved in the erosive/reticular switch in OLP, determining different clinical presentations. In conclusion, decreased apoptosis in inflammatory infiltrates may contribute to the persistence of T lymphocytes, worsening the attack to the epithelium in erosive OLP.

O líquen plano oral (LPO) é uma doença crônica inflamatória, provavelmente auto-imune, com diferentes formas clínicas. Os tipos mais comuns são o reticular e o erosivo. A apoptose participa da destruição dos ceratinócitos basais, no entanto o seu papel na perpetuação do infiltrado linfocitário subepitelial ainda não foi investigado. Para avaliar o envolvimento da apoptose no epitélio e no infiltrado linfocitário subepitelial, quinze amostras de LPO reticular, quinze de LPO erosivo e dez amostras de mucosa oral saudável foram coletadas e processadas histologicamente. A apoptose foi quantificada no epitélio e nas células do infiltrado inflamatório. A reação de TUNEL foi usada para mensurar a apoptose no infiltrado. A intensidade da apoptose no epitélio mostrou ser maior no LPO erosivo que no LPO reticular e estes foram maiores que no controle. Em contraste, a apoptose nas células do infiltrado inflamatório foi mais freqüente no LPO reticular que no LPO erosivo. Os linfócitos foram as células predominantes dentro do infiltrado inflamatório e foram mais freqüentes no tipo erosivo de LPO que no tipo reticular. Estes resultados sugerem que diferentes níveis de apoptose estão envolvidos no tipo erosivo e reticular de LPO, determinando as diferenças nas apresentações clínicas. Em conclusão, a diminuição da apoptose no infiltrado inflamatório pode contribuir para a persistência dos linfócitos T, piorando o ataque ao epitélio no LPO erosivo.

A comparative study of apoptosis in reticular and erosive oral lichen planus.

The oral lichen planus (OLP) is a chronic inflammatory disease, probably autoimmune, with different clinical forms. The most common types are the reticular and the erosive ones. Apoptosis participates in the destruction of basal keratinocytes, but its role in the perpetuation of the subepithelial lymphocytic infiltrates was not yet investigated. To evaluate the involvement of apoptosis in the epithelium and in subepithelial lymphocytic infiltrates, 15 samples of reticular and erosive OLP and 10 samples of healthy oral mucosa were collected and processed histologically. Apoptosis was quantified in the epithelium and in inflammatory cell infiltrates. TUNEL reaction was used to measure apoptosis in the infiltrates. Erosive OLP showed more intense epithelial apoptosis than reticular OLP and controls. In contrast, apoptosis in the inflammatory cell infiltrates was more frequent in reticular than in erosive OLP. Lymphocytes were the predominant cells within the inflammatory cell infiltrates and were more frequent in erosive OLP than in reticular type. These results suggest that different apoptotic levels are involved in the erosive/reticular switch in OLP, determining different clinical presentations. In conclusion, decreased apoptosis in inflammatory infiltrates may contribute to the persistence of T lymphocytes, worsening the attack to the epithelium in erosive OLP.

Caspase-3/caspase-8, bax and bcl2 in pulps of human primary teeth with physiological root resorption.

OBJECTIVE. Physiological root resorption is a programmed event that takes place in primary teeth leading to elimination of all root structures. The mechanism behind pulp elimination indicates apoptosis, but its pathway has not been well characterised yet. To better understand this event, we evaluated the gene expression of bax, bcl-2, caspase-3 and caspase-8 through real-time polymerase chain reaction (PCR) and immunohistochemistry expression of Caspase-8 and Bax in pulps. METHODS. Samples were split into two groups: pulps from primary teeth with physiological root resorption (n = 40) and control (n =40), pulps from permanent teeth. Samples of each group were split into PCR (n = 20) and immunohistochemistry (n = 20). RESULTS. Pulps from primary teeth showed a higher caspase-3 mRNA level than pulps from permanent teeth. The expression of bax gene was more intense than caspase-8 but both did not show difference between groups. The bcl-2 mRNA level was incipient and similar between groups. Histopath slides did not show any evidence of inflammatory infiltration, which implies that extrinsic via is not likely to be involved. Immunohistochemistry reaction to Bax and Caspase-8 supported PCR results. CONCLUSIONS. Pulp apoptosis is likely to occur via caspase-3 activation through the mitochondrial pathway.

MyD88 and STING signaling pathways are required for IRF3-mediated IFN-ß induction in response to Brucella abortus infection.

Type I interferons (IFNs) are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-ß in macrophages and splenocytes. Further, IFN-ß induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-ß expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-ß and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-αßR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-γ and NO production by IFN-αßR KO spleen cells and reduced apoptosis.

Apoptose na maturação placentária de vacas em diferentes estágios de gestação: evidenciação imuno-histoquímica e bioquímica / Apoptosis in placental maturation of cows in different stages of pregnancy: Immunohistochemical and biochemical evidences

Apoptose tem um papel importante na manutenção da homeostase placentária, e o desequilíbrio desse processo pode comprometer a gestação. O objetivo do presente estudo foi avaliar a ocorrencia de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomos de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imuno-histoquímica e isolamento de DNA. As lâminas obtidas foram coradas em HE, ou submetidas à análise imuno-histoquímica das proteínas pró-apoptóticas caspase-3 e Bax, e da proteína anti-apoptótica Bcl-2. O DNA isolado foi submetido à eletroforese em gel de agarose para detecção da fragmentação internucleossômica do genoma. Os resultados de histomorfometria revelaram que as células apoptóticas aumentaram progressivamente com o avanço da gestação. Confirmou-se a apoptose pela fragmentação característica do DNA genômico, visualizada pelo clássico "padrão em escada" na eletroforese em gel de agarose. Adcionalmente, a imunoexpressão de caspase-3, Bax e Bcl-2 foram observadas em todas as amostras. Entretanto, a proteína caspase-3 apresentou marcação mais intensa em todos os tempos gestacionais, quando comparada com a marcação das proteínas Bcl-2 e Bax. Esses resultados confirmam e reforçam a importância da apoptose na maturação placentária. Além disto, indica que caspase-3, Bax e Bcl-2 estão envolvidas nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial ao longo da gestação, contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.(AU)

Apoptosis is important for placental homeostasis maintenance, and a misbalance in this process may compromise the success of pregnancy. The aim of this study is to evaluate apoptosis in bovine placental samples at different stages of pregnancy. Placentome samples from 15 healthy cows, at gestational ages of 4 (n=5), 6 (n=5) and 9 (n=5) months were collected and routinely processed for histological and immunohistochemical analysis, and for DNA isolation. Histopathology sections were stained with HE. Others were submitted to immunohistochemistry, using pro-apoptotic caspase-3 and Bax protein, and anti-apoptotic Bcl-2 specific antibodies. Isolated DNAs were processed for electrophoresis in agarose gel, for detection of internucleosomal fragmentation. Histomorphometry results demonstrated that apoptotic cells gradually increase with the advance of the gestation. Also, the DNA ladder pattern was observed in all groups. In addition, caspase-3, Bax and Bcl-2 were all expressed in the three different gestation periods. However, caspase-3 presented a higher expression in all groups, in comparison to Bcl-2 and Bax. These results confirm the importance of the apoptosis in the placental maturation. Also, these results indicated that caspase-3, Bcl-2 and Bax are involved in apoptotic activation mechanisms by mitochondrial intrinsic pathway during placental maturation, contributing for physiological cellularity and cellular turn over balance in bovine placenta.(AU)

Apoptose na maturação placentária de vacas em diferentes estágios de gestação: evidenciação imuno-histoquímica e bioquímica / Apoptosis in placental maturation of cows in different stages of pregnancy: Immunohistochemical and biochemical evidences

Apoptose tem um papel importante na manutenção da homeostase placentária, e o desequilíbrio desse processo pode comprometer a gestação. O objetivo do presente estudo foi avaliar a ocorrencia de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomos de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imuno-histoquímica e isolamento de DNA. As lâminas obtidas foram coradas em HE, ou submetidas à análise imuno-histoquímica das proteínas pró-apoptóticas caspase-3 e Bax, e da proteína anti-apoptótica Bcl-2. O DNA isolado foi submetido à eletroforese em gel de agarose para detecção da fragmentação internucleossômica do genoma. Os resultados de histomorfometria revelaram que as células apoptóticas aumentaram progressivamente com o avanço da gestação. Confirmou-se a apoptose pela fragmentação característica do DNA genômico, visualizada pelo clássico "padrão em escada" na eletroforese em gel de agarose. Adcionalmente, a imunoexpressão de caspase-3, Bax e Bcl-2 foram observadas em todas as amostras. Entretanto, a proteína caspase-3 apresentou marcação mais intensa em todos os tempos gestacionais, quando comparada com a marcação das proteínas Bcl-2 e Bax. Esses resultados confirmam e reforçam a importância da apoptose na maturação placentária. Além disto, indica que caspase-3, Bax e Bcl-2 estão envolvidas nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial ao longo da gestação, contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.

Apoptosis is important for placental homeostasis maintenance, and a misbalance in this process may compromise the success of pregnancy. The aim of this study is to evaluate apoptosis in bovine placental samples at different stages of pregnancy. Placentome samples from 15 healthy cows, at gestational ages of 4 (n=5), 6 (n=5) and 9 (n=5) months were collected and routinely processed for histological and immunohistochemical analysis, and for DNA isolation. Histopathology sections were stained with HE. Others were submitted to immunohistochemistry, using pro-apoptotic caspase-3 and Bax protein, and anti-apoptotic Bcl-2 specific antibodies. Isolated DNAs were processed for electrophoresis in agarose gel, for detection of internucleosomal fragmentation. Histomorphometry results demonstrated that apoptotic cells gradually increase with the advance of the gestation. Also, the DNA ladder pattern was observed in all groups. In addition, caspase-3, Bax and Bcl-2 were all expressed in the three different gestation periods. However, caspase-3 presented a higher expression in all groups, in comparison to Bcl-2 and Bax. These results confirm the importance of the apoptosis in the placental maturation. Also, these results indicated that caspase-3, Bcl-2 and Bax are involved in apoptotic activation mechanisms by mitochondrial intrinsic pathway during placental maturation, contributing for physiological cellularity and cellular turn over balance in bovine placenta.

Canine distemper virus induces apoptosis in cervical tumor derived cell lines.

Apoptosis can be induced or inhibited by viral proteins, it can form part of the host defense against virus infection, or it can be a mechanism for viral spread to neighboring cells. Canine distemper virus (CDV) induces apoptotic cells in lymphoid tissues and in the cerebellum of dogs naturally infected. CDV also produces a cytopathologic effect, leading to apoptosis in Vero cells in tissue culture. We tested canine distemper virus, a member of the Paramyxoviridae family, for the ability to trigger apoptosis in HeLa cells, derived from cervical cancer cells resistant to apoptosis. To study the effect of CDV infection in HeLa cells, we examined apoptotic markers 24 h post infection (pi), by flow cytometry assay for DNA fragmentation, real-time PCR assay for caspase-3 and caspase-8 mRNA expression, and by caspase-3 and -8 immunocytochemistry. Flow cytometry showed that DNA fragmentation was induced in HeLa cells infected by CDV, and immunocytochemistry revealed a significant increase in the levels of the cleaved active form of caspase-3 protein, but did not show any difference in expression of caspase-8, indicating an intrinsic apoptotic pathway. Confirming this observation, expression of caspase-3 mRNA was higher in CDV infected HeLa cells than control cells; however, there was no statistically significant change in caspase-8 mRNA expression profile. Our data suggest that canine distemper virus induced apoptosis in HeLa cells, triggering apoptosis by the intrinsic pathway, with no participation of the initiator caspase -8 from the extrinsic pathway. In conclusion, the cellular stress caused by CDV infection of HeLa cells, leading to apoptosis, can be used as a tool in future research for cervical cancer treatment and control.

Hydrocortisone decreases apoptosis in jejunum of horses subjected to experimental ischemia and reperfusion / Hidrocortisona diminui a apoptose no jejuno de equinos sujeitos a isquemia e reperfusão experimentais

In order to evaluate the effect of hydrocortisone on apoptosis in the jejunum of horses subjected to ischemia and reperfusion, ten horses were paired and grouped into two groups - treated (n=5) and non treated (n=5). Segments of the jejunum were used as controls (C), or as venous ischemia (VIsc), which were subjected to 2h of ischemia followed by 2 or 12h of reperfusion. C samples were collected at time zero (prior to ischemia) and VIsc samples were collected at 2h of ischemia and at 2 and 12h of reperfusion. TUNEL positive apoptotic cells were counted in 10 microscopical fields in deep mucosa from each horse throughout the time course. After 12h of reperfusion, the number of apoptotic cells in treated group were significantly lower than in untreated animals, indicating that hydrocortisone inhibits apoptosis. These results indicate that hydrocortisone has a beneficial effects favoring the maintenance of jejunal integrity in horses with ischemia and reperfusion injuries by preventing apoptotic cell death.(AU)

Com o objetivo de avaliar o efeito da hidrocortisona sobre a apoptose no jejuno de equinos submetidos à is-quemia e reperfusão, dez cavalos foram agrupados em dois grupos: tratado (n=5) e não-tratado (n=5). Foi utilizado um segmento do jejuno como controle (C) e outro foi submetido a isquemia venosa (VIsc) por 2h seguida de 2 ou 12 h de re-perfusão. Amostras de C foram coletadas no tempo zero (antes da isquemia) e amostras de VIsc foram coletadas após 2h de isquemia e a 2 e 12h de reperfusão. Células apoptóticas TUNEL positivas foram contadas em 10 campos microscópicos da mucosa na região das criptas de cada animal em cada tempo. Após 12h de reperfusão, o número de células apoptóticas no grupo tratado foram significativamente menores do que no grupo não-tratado, indicando que a hidrocortiso-na inibe a apoptose. Esses resultados mostram que a hidro-cortisona tem efeito benéfico favorecendo a manutenção da integridade do jejuno em cavalos com lesão de isquemia e reperfusão por prevenir a morte celular por apoptose.(AU)

Hydrocortisone decreases apoptosis in jejunum of horses subjected to experimental ischemia and reperfusion / Hidrocortisona diminui a apoptose no jejuno de equinos sujeitos a isquemia e reperfusão experimentais

In order to evaluate the effect of hydrocortisone on apoptosis in the jejunum of horses subjected to ischemia and reperfusion, ten horses were paired and grouped into two groups - treated (n=5) and non treated (n=5). Segments of the jejunum were used as controls (C), or as venous ischemia (VIsc), which were subjected to 2h of ischemia followed by 2 or 12h of reperfusion. C samples were collected at time zero (prior to ischemia) and VIsc samples were collected at 2h of ischemia and at 2 and 12h of reperfusion. TUNEL positive apoptotic cells were counted in 10 microscopical fields in deep mucosa from each horse throughout the time course. After 12h of reperfusion, the number of apoptotic cells in treated group were significantly lower than in untreated animals, indicating that hydrocortisone inhibits apoptosis. These results indicate that hydrocortisone has a beneficial effects favoring the maintenance of jejunal integrity in horses with ischemia and reperfusion injuries by preventing apoptotic cell death.

Com o objetivo de avaliar o efeito da hidrocortisona sobre a apoptose no jejuno de equinos submetidos à is-quemia e reperfusão, dez cavalos foram agrupados em dois grupos: tratado (n=5) e não-tratado (n=5). Foi utilizado um segmento do jejuno como controle (C) e outro foi submetido a isquemia venosa (VIsc) por 2h seguida de 2 ou 12 h de re-perfusão. Amostras de C foram coletadas no tempo zero (antes da isquemia) e amostras de VIsc foram coletadas após 2h de isquemia e a 2 e 12h de reperfusão. Células apoptóticas TUNEL positivas foram contadas em 10 campos microscópicos da mucosa na região das criptas de cada animal em cada tempo. Após 12h de reperfusão, o número de células apoptóticas no grupo tratado foram significativamente menores do que no grupo não-tratado, indicando que a hidrocortiso-na inibe a apoptose. Esses resultados mostram que a hidro-cortisona tem efeito benéfico favorecendo a manutenção da integridade do jejuno em cavalos com lesão de isquemia e reperfusão por prevenir a morte celular por apoptose.