Enhanced apoptotic index in hepatocytes, Kupffer cells, and inflammatory infiltrate showed positive correlation with hepatic lesion intensity, parasite load, and clinical status in naturally Leishmania-infected dogs.

It is unknown if Leishmania amastigote infections affect hepatocytes and Kupffer cell apoptosis, and the role played by apoptosis in liver lesions in leishmaniasis is still unclear. Clinically affected and subclinically infected dogs with leishmaniosis and uninfected controls were assessed. Parasite load, biochemical markers for evaluation of liver damage, morphometry (area, perimeter, number of inflammatory focus, major and minor diameters), apoptosis in hepatic tissue (hepatocytes, Kupffer cells, and inflammatory infiltrates) and cellularity in inflammatory foci were quantified. The parasite load in clinically affected dogs proved to be higher than in the other groups. All morphometric parameters (area, perimeter, number of inflammatory focus, major and minor diameters) from clinically affected were higher than the values found in the subclinically infected and uninfected control dogs. Only clinically affected dogs presented high levels of ALT, FA, GGT and cholesterol in serum. Strong positive correlation was observed between biochemical markers for evaluation of liver damage (ALT, FA, GGT and cholesterol) and hepatic apoptosis (hepatocytes, Kupffer cells, and inflammation). Clinically affected dogs showed a more intense hepatic lesion. Hepatocytes showed a higher rate of apoptosis in Leishmania-infected dogs than in uninfected control dogs. The Kupffer cell apoptotic index and apoptosis within the inflammatory infiltrates were higher in clinically affected dogs. The apoptotic index evaluated in hepatocytes, Kupffer cells, and inflammatory infiltrates showed a positive correlation with the intensity of the hepatic lesion, parasite load, and clinical status. Apoptotic cells also showed positive immunostaining for TUNEL, Bcl2, and Bax. Our data showed that hepatic apoptosis was related to the severity of liver damage, the progression of infection, and the parasite load in leishmaniasis. Apoptotic regulated cell recruitment modulated the inflammatory response and favored the survival and dissemination of parasites, depending on the clinical status of the Leishmania-infected dogs.

MCP-1/IL-12 ratio expressions correlated with adventitial collagen depositions in renal vessels and IL-4/IFN-γ expression correlated with interstitial collagen depositions in the kidneys of dogs with canine leishmaniasis.

Collagen deposition is a common event in chronic inflammation, and canine Leishmaniosis (CanL) is generally associated with a long and chronic evolution. Considering that the kidney shows fibrinogenic changes during CanL, and the balance of cytokines/chemokines regulates the profibrinogenic and antifibrinogenic immune responses differently, it can be hypothesized that the balance of cytokines/chemokines can be differentially expressed in the renal tissue in order to determine the expression of collagen depositions in the kidneys. This study aimed to measure collagen deposition and to evaluate cytokine/chemokine expressions in the kidney by means of qRT-PCR in sixteen Leishmania-infected dogs and six uninfected controls. Kidney fragments were stained with hematoxylin & eosin (H&E), Masson's Trichrome, Picrosirius Red, and Gomori's reticulin. Intertubular and adventitial collagen depositions were evaluated by the morphometric approach. Cytokine RNA expressions were measured by means of qRT-PCR to identify molecules involved in chronic collagen depositions in kidneys with CanL. Collagen depositions were related to the presence of clinical signs, and more intense intertubular collagen depositions occurred in infected dogs. Adventitial collagen deposition, as morphometrically measured by the average area of the collagen, was more intense in clinically affected dogs than in subclinically infected dogs. TNF-α/TGF-ß, MCP1/IL-12, CCL5/IL-12, IL-4/IFN-γ, and IL-12/TGF-ß expressions were associated with clinical manifestations in dogs with CanL. The IL-4/IFN-α ratio was more commonly expressed and upregulated in clinically affected dogs, and downregulated in subclinically infected dogs. Furthermore, MCP-1/IL-12 and CCL5/IL-12 were more commonly expressed in subclinically infected dogs. Strong positive correlations were detected between morphometric values of interstitial collagen depositions and MCP-1/IL-12, IL-12, and IL-4 mRNA expression levels in the renal tissues. Adventitial collagen deposition was correlated with TGF-ß, IL-4/IFN-γ, and TNF-α/TGF-ß. In conclusion, our results showed the association of MCP-1/IL-12 and CCL5/IL-12 ratios with an absence of clinical signs, as well as an IL-4/IFN-α ratio with adventitial and intertubular collagen depositions in dogs with visceral leishmaniosis.

Nucleolar organizer region proteins enhancement in nucleoplasm's of renal tubular cells is an indication of kidney impairment in Leishmania-infected dogs.

Hyperactivation of tubular cells contributes for the progression of kidney lesions. The exacerbated expression of immunological proteins and ribosomal DNA (rDNA) transcriptional activity are observed in tubular cells. This intensified expression results in more prominent hypertrophic changes and is often accompanied by increased expression of factors involved in different phases of ribosomal biosynthesis, such as the nucleolar organizer regions (NOR). The aim of this study was to evaluate whether there is an association between NOR proteins, renal impairment, and clinical status in Leishmania-infected dogs (CanL). Forty-five dogs with CanL and six uninfected controls were assessed in this study. PCR was performed to detect parasites' nucleic acids in kidney. Histopathological analyses were performed in kidney fragments, and NOR was detected by Ag stain (AgNOR). Leishmania-infected dogs showed more intense inflammation and collagen deposition compared with uninfected controls. Biochemical alterations were observed only in Leishmania-infected dogs. AgNORs per cell were significantly higher in clinically affected dogs and higher histopathological lesion score was observed in Leishmania-infected dogs. Positive correlations between number of NORs per cell in medullary region and histopathological lesion score were observed. Furthermore, AgNOR expression, intensity of renal lesions, and clinical sigs was associated in Leishmania-infected dogs. We propose that the detection of AgNOR proteins could be used to better estimate the kidney tubular damage at the time of examination in Leishmania-infected dogs as a marker to estimate renal impairment in dogs with CanL.

Enhanced apoptotic index, chemokines and inflammatory recruitment in renal tissues shows relationship with the clinical signs in Leishmania-infected dogs.

Apoptosis is associated with resolution of inflammation. However, apoptosis may also occur in active inflammation, balancing inflammatory recruitment instead of a resolution event. To test that hypothesis, we measured apoptosis and chemokines expression, involved in recruitment of inflammatory cells. Clinical affected and subclinically infected dogs with canine leishmaniosis (CanL) and uninfected controls were assessed. Apoptosis in renal tissue (glomeruli, tubules, and inflammatory infiltrate) and cellularity in inflammatory foci were quantified. Messenger RNA of CCL5, CCL4, MCP-1, MCP-2, Caspase (Casp) 3, Casp 8, Casp 9, Bax, Bcl2 and Fas were quantified by qRT PCR. Clinical affected dogs showed more intense inflammation and higher cellularity in the inflammatory infiltrates than subclinically infected ones, which were higher than controls. Glomerular and tubular cells showed higher apoptotic index in clinical affected dogs when compared to controls. Apoptosis within the inflammatory infiltrates was higher in clinical affected dogs. Bax/Bcl2 ratio and CCL4 showed higher expression in kidney from clinical affected when compared to subclinically infected dogs. Casp 3/CCL4 ratio expression were higher in subclinically infected dogs than in the clinical affected group. Additionally, results suggest that Casp 3/CCL4 ratio is balancing towards an inflammatory recruitment and CCL4 and Bax/Bcl2 ratio expression is associated with active inflammation in clinical affected CanL. Data demonstrate that apoptosis was not always correlated with resolution of inflammation, when a morphometric and a molecular evaluation were performed concomitantly. In kidneys of Leishmania infected dogs, apoptosis and chemokines may be balancing inflammatory recruitment. In conclusion, Bax/Bcl2 ratio, chemokines, Casp 8, Casp 3 and Fas were associated with renal apoptosis, active inflammation and increased inflammatory recruitment observed in clinical affected animals, influencing the clinical presentation of leishmaniosis.

Sodium Butyrate Downregulates Implant-Induced Inflammation in Mice.

Sodium butyrate (NaBu), a histone deacetylase inhibitor, has shown to exert beneficial actions attenuating inflammation in a number of intestinal and extra-intestinal diseases. However, the effects of NaBu on persistent inflammatory processes as in a response to implantation of foreign material have not been investigated. Synthetic matrix of polyether-polyurethane sponge was implanted in mice's subcutaneous layer of the dorsal region, and the animals were treated daily with oral administration of NaBu (100 mg/kg). After 7 days, the implants were removed and processed for assessment of inflammatory markers. Butyrate treatment caused a significant attenuation of neutrophil and macrophage infiltration in implants, which was reflected by the reduction of myeloperoxidase and N-acetyl-ß-D-glucosaminidase activities, respectively. Similar reduction was observed in intra-implants nitrite levels of NaBu-treated mice. NaBu treatment was also able to decrease mast cell recruitment/activation and the levels of CXCL1, CCL2, IL-6, TNF-ɑ, and TGF-ß1 in the implants but did not alter the levels of IL-10. In addition, NaBu administration decreased the concentration of proteins p65 and p50 in the nucleus as compared with the cytoplasm by western blot analysis. This result suggests that treatment with NaBu inhibited the NF-κB pathway. The circulating levels of TNF-ɑ and TGF-ß1 were also attenuated by NaBu. Persistent inflammation at sites of implanted devices very often impairs their functionality; therefore, our findings suggest that NaBu holds potential therapeutic value to control this adverse response to biomedical implants.

Kinetics of pancreatic tissue proliferation in a polymeric platform in mice.

BACKGROUND/OBJECTIVES: Pancreas regenerative capacity after injury is not always sufficient to comply with the body's requirement of digestive enzymes and hormones. We present an alternative system to induce pancreas parenchyma proliferation (exocrine and endocrine components), rather than regeneration or remodeling in normoglycemic mice. METHODS: Porous discs of polyether-polyurethane were surgically placed adjacent to the native pancreas and removed at days 15, 30 and 45 after implantation. No exogenous growth factors or extracellular matrix components were added to the platform. The synthetic matrix provided a platform that was filled with parenchymal and non-parenchymal pancreas tissue as detected by histological analysis. Immunohistochemistry analysis were performed to identify insulin positive cells in the newly formed tissue. In addition, angiogenic, inflammatory and metabolic parameters were carried out in those mice. RESULTS: At day 15, the pores of the platform were filled with inflammatory cells, spindled-shaped like fibroblasts, extracellular matrix components, blood vessels and clusters of pancreatic parenchyma (acini, ducts and islet-like structures). At days 30 and 45 the pancreas features remained well organized; its organization resembled that of a native pancreas. Interestingly, besides islet-like structures that showed positive cells to insulin, some ductal cells were also positive for insulin immunostaining. No significant differences in serum glucose and c-peptide concentrations during the experimental period were detected. CONCLUSIONS: The plain synthetic porous platform (without addition of exogenous molecules) placed adjacent to the native organ exhibits potential to restore and/or expand exocrine (acini, ducts) and endocrine (ß-cell mass) components in pancreatic injuries and in high metabolic demand.

Induction of liver proliferation using a polymeric platform in mice.

AIMS: Currently, animal models of liver regeneration are based on extensive lesions of the native organ and on cellular approaches using biomaterials to host growth factors and extracellular components to create artificial liver systems. We report a polymeric biological platform, minimally invasive, that induced sequential proliferation of liver parenchyma inside the scaffold in mice. MAIN METHODS: Porous discs of polyether-polyurethane were surgically placed under the left liver lobe and removed at days 4, 8, 12 and 25 after implantation. No exogenous growth factors or extracellular matrix components were added to the scaffold. Histological analysis of the implants was performed to identify hepatocytes, liver vascular structures and bile ducts in the newly formed tissue. In addition, systemic markers for hepatic function were determined. KEY FINDINGS: This biohybrid device provided a scaffold that was gradually filled with parenchymal and non-parenchymal liver tissue as detected by histological analysis. At day 4, the pores of the scaffold were filled with inflammatory cells and spindled-shaped like fibroblasts, and extracellular matrix components. At day 8, hepatocytes clusters, central lobular hepatic veins, portal space containing arteries, veins and biliary ducts were detected. By days 12 and 25 a liver-like structure filled 2/3 of the scaffold. Its organization resembled that of a mature liver. Serum concentration of ALT increased three-fold initially after implantation, returning gradually to control levels. SIGNIFICANCE: The plain synthetic scaffold (without addition of exogenous molecules) placed under the intact left liver lobe exhibits the potential to investigate physiological mechanisms that regulate liver parenchyma proliferation.

Synthetic matrix of polyether-polyurethane as a biological platform for pancreatic regeneration.

AIMS: Several alternative cellular approaches using biomaterials to host insulin-producing cells derived from stem cells have been developed to overcome the limitations of type 1 diabetes treatment (exogenous insulin injection). However, none seem to fulfill all requirements needed to induce pancreatic cells successful colonization of the scaffolds. Here, we report a polymeric platform adherent to the native mice pancreas filled with human adipose stem cells (hASCs) that was able to induce growth of pancreatic parenchyma. MAIN METHODS: Synthetic polyether-polyurethane discs were placed adjacent to pancreas of normoglycemic and streptozotocin-induced diabetic mice. At day 4 post implantation, 1×106 hASCs were injected intra-implant in groups of normoglycemic and diabetic mice. Immunohistochemistry analysis of the implants was performed to identify insulin positive cells in the newly formed tissue. In addition, metabolic, inflammatory and angiogenic parameters were carried out in those mice. KEY FINDINGS: This study provides evidence of the ability of a biohybrid device to induce the growth of differentiated pancreas parenchyma in both normoglycemic and streptozotocin-induced diabetic mice as detected by histological analysis. Glucose metabolism and body weight of hyperglycemic mice bearing hASCs implants improved. SIGNIFICANCE: The synthetic porous scaffold bearing hASC cells placed adjacent to the native animal pancreas exhibits the potential to be exploited in future cell-based type 1 diabetes therapies.

Platelet-activating factor receptor (PAFR) plays a crucial role in experimental global cerebral ischemia and reperfusion.

Stroke is one of the most frequent causes of death and disability worldwide leading to a significant clinical and socioeconomic burden. Although different mechanisms are involved in the pathogenesis of stroke, inflammatory response occurs after ischemia and contributes to the expansion of brain injury. Platelet-activating factor receptor (PAF) plays crucial roles in both physiological and pathological conditions in the brain. PAF receptor (PAFR) may be expressed on cellular and nuclear membranes of various cell types, especially leukocytes, platelets, endothelial cells, neuronal cells and microglia. Herein, using mice lacking the PAFR receptor (PAFR(-/-)), we investigate a potential role for this receptor during experimental transient global cerebral ischemia and reperfusion (BCCAo). In PAFR deficiency, we observed a significant improvement in the neurological deficits, which were associated with a reduction of brain infarcted area as evaluated by triphenyltetrazolium chloride (TTC). Moreover, a decrease in the percentage of necrotic cavities areas and in the frequency of ischemic neurons was also found by employing histometric analysis. In addition, in PAFR(-/-) mice there was prevention of caspase-3 activation and decreased vascular permeability and brain edema. Decreased brain levels of the cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and the chemokine (C-X-C motif) ligand 1 (CXCL1) by ELISA were also detected in PAFR(-/-) BCCAo animals. Taken together, our results suggest that PAFR activation might be crucial for the global brain ischemia and reperfusion injury.

Effect of triamcinolone in keloids morphological changes and cell apoptosis.

OBJECTIVE: to assess the effects of injectable triamcinolone on keloid scars length, height and thickness, and on the number of cells undergoing apoptosis. METHODS: This study consists in a prospective, controlled, randomized, single-blinded clinical trial, conducted with fifteen patients with ear keloids divided into two groups: group 1 - seven patients undergoing keloid excisions, and group 2 - eight patients undergoing keloid excisions after three sessions of infiltration with one ml of Triamcinolone hexacetonide (20mg/ml) with three week intervals between them and between the last session and surgery. The two groups were homogeneous regarding age, gender and evolution of the keloid scar. The keloid scars of patients in group 2 were measured for the length, height and thickness before triamcinolone injection and before surgery. A blinded observer performed morphological detailing and quantification of cells in hematoxylin-eosin-stained surgical specimens. An apoptotic index was created. RESULTS: The apoptotic index in group 1 was 56.82, and in group 2, 68.55, showing no significant difference as for apoptosis (p=0.0971). The reduction in keloid dimensions in Group 2 was 10.12% in length (p=0.6598), 11.94% in height (p=0.4981) and 15.62% in thickness (p=0.4027). CONCLUSION: This study concluded that the infiltration of triamcinolone in keloid scars did not increase the number of apoptosit and did not reduce keloids' size, length, height or thickness.

Effect of triamcinolone in keloids morphological changes and cell apoptosis / Efeito da triancinolona na apoptose celular e nas alterações morfológicas em queloides

OBJECTIVE: To assess the effects of injectable triamcinolone on keloid scars length, height and thickness, and on the number of cells undergoing apoptosis. METHODS: This study consists in a prospective, controlled, randomized, single-blinded clinical trial, conducted with fifteen patients with ear keloids divided into two groups: group 1 - seven patients undergoing keloid excisions, and group 2 - eight patients undergoing keloid excisions after three sessions of infiltration with one ml of Triamcinolone hexacetonide (20mg/ml) with three week intervals between them and between the last session and surgery. The two groups were homogeneous regarding age, gender and evolution of the keloid scar. The keloid scars of patients in group 2 were measured for the length, height and thickness before triamcinolone injection and before surgery. A blinded observer performed morphological detailing and quantification of cells in hematoxylin-eosin-stained surgical specimens. An apoptotic index was created. RESULTS:The apoptotic index in group 1 was 56.82, and in group 2, 68.55, showing no significant difference as for apoptosis (p=0.0971). The reduction in keloid dimensions in Group 2 was 10.12% in length (p=0.6598), 11.94% in height (p=0.4981) and 15.62% in thickness (p=0.4027). CONCLUSION: This study concluded that the infiltration of triamcinolone in keloid scars did not increase the number of apoptosit and did not reduce keloids' size, length, height or thickness.

OBJETIVO: Comparar o efeito da triancinolona injetável em cicatrizes queloidianas quanto ao número de células em apoptose e avaliar o efeito da triancinolona quanto às alterações no comprimento, altura e espessura dessas cicatrizes. MÉTODOS:Estudo clínico longitudinal, prospectivo, controlado, aleatorizado, unicego, com 15 pacientes portadores de queloides de orelha distribuídos em dois grupos: grupo 1, com sete pacientes submetidos apenas às exéreses dos queloides; e grupo 2, com oito pacientes submetidos às exéreses das lesões após três sessões de infiltração de 1ml de hexacetonida de triancinolona (20mg/mL), com intervalos de três semanas entre elas, assim como entre a última sessão e a operação. Os dois grupos foram homogêneos quanto à: idade (p=0,867), sexo (p=0,782) e tempo de evolução da cicatriz queloidiana (p=0,779). As cicatrizes queloidianas dos pacientes do grupo 2 foram medidas quanto ao comprimento, altura e espessura antes da injeção da triancinolona e antes do procedimento cirúrgico. Um observador mascarado realizou detalhamento morfológico e quantificação das células nas peças cirúrgicas, coradas com HE. Foi criado um índice apoptótico. RESULTADOS:Os dois grupos foram homogêneos quanto à: idade (p=0,867), sexo (p=0,782) e tempo de evolução da cicatriz queloidiana (p=0,779). o índice apoptótico no grupo 1 foi 56,82 e no grupo 2, 68,55, sem diferença (p=0,0971). As reduções nas dimensões dos queloides dos grupos 2 foram 10,12% para o comprimento (p=0,6598), 11,94% para a altura (p=0,4981) e 15,62% para a espessura (p=0,4027). CONCLUSÃO:A infiltração de triancinolona nas cicatrizes queloidianas não aumentou o número de apoptoses e não houve redução das dimensões, comprimento, altura e espessura dos queloides.

Apoptosis and expression of argyrophilic nucleolus organizer regions in epithelial neoplasms of the larynx / Apoptose e expressão de regiões argirófilas organizadoras de nucléolos em neoplasias epiteliais da laringe

INTRODUCTION: Occurrence of apoptosis and expression of proliferative markers are powerful tools to establish a prognosis in the follow-up of cancer. OBJECTIVE: To evaluate the growth fraction in papillomas and laryngeal squamous cell carcinomas with three degrees of differentiation through apoptosis and the expression of nucleolus organizer regions. METHODS: Retrospective study from which paraffin material was submitted to microtomy and hematoxylin-eosin and silver staining. Stained slides were used to quantify the apoptotic index and the number of nucleolus organizer regions by morphometry. RESULTS: Apoptosis was significantly more frequent in well differentiated carcinomas and in papillomas, and a higher growth fraction of expressed nucleolus organizer regions and cells that expressed a greater than average number of nucleolus organizer regions were more frequently noted in undifferentiated carcinomas. CONCLUSIONS: Thus, it was possible to verify that a high apoptotic index was associated with a lower chance of tumor differentiation in carcinomas, while a greater number of total nucleolus organizer regions, cells expressing nucleolus organizer regions above average and a higher growth fraction were associated with greater likelihood of abnormal cell proliferation and increased tumor differentiation. .

INTRODUÇÃO: A ocorrência de apoptose e a expressão de marcadores proliferativos são ferramentas poderosas no estabelecimento do prognóstico do câncer. OBJETIVO: Avaliar a fração de crescimento de papilomas e carcinomas laríngeos de células escamosas, com três graus de diferenciação, através da apoptose e expressão de regiões organizadoras de nucléolo. MÉTODO: Estudo retrospectivo, cujo material em blocado em parafina foi submetido à microtomia e coloração em hematoxilina-eosina, e pela prata. As lâminas coradas foram utilizadas para quantificar o índice apoptótico e o número de regiões organizadoras de nucléolo (NORs) através da morfometria. RESULTADOS: A apoptose foi significativamente mais frequente em carcinomas bem diferenciados e em papilomas; enquanto que uma maior fração de crescimento, de NORs expressos e de células que expressaram maior número de NORs, foram mais frequentes nos carcinomas indiferenciados. CONCLUSÕES: Foi possível verificar que o índice apoptótico elevado indica menores chances de diferenciação tumoral nos carcinomas, enquanto que um maior número de NORs totais e células expressando NORs acima da média, e uma maior fração de crescimento, determinam maiores chances de proliferação celular anormal e maior diferenciação tumoral. .

Apoptosis and expression of argyrophilic nucleolus organizer regions in epithelial neoplasms of the larynx.

INTRODUCTION: Occurrence of apoptosis and expression of proliferative markers are powerful tools to establish a prognosis in the follow-up of cancer. OBJECTIVE: To evaluate the growth fraction in papillomas and laryngeal squamous cell carcinomas with three degrees of differentiation through apoptosis and the expression of nucleolus organizer regions. METHODS: Retrospective study from which paraffin material was submitted to microtomy and hematoxylin-eosin and silver staining. Stained slides were used to quantify the apoptotic index and the number of nucleolus organizer regions by morphometry. RESULTS: Apoptosis was significantly more frequent in well differentiated carcinomas and in papillomas, and a higher growth fraction of expressed nucleolus organizer regions and cells that expressed a greater than average number of nucleolus organizer regions were more frequently noted in undifferentiated carcinomas. CONCLUSIONS: Thus, it was possible to verify that a high apoptotic index was associated with a lower chance of tumor differentiation in carcinomas, while a greater number of total nucleolus organizer regions, cells expressing nucleolus organizer regions above average and a higher growth fraction were associated with greater likelihood of abnormal cell proliferation and increased tumor differentiation.

Apoptose na infecção experimental de cães domésticos com Ehrlichia canis / Apoptosis in experimental infection with Ehrlichia canis in domestic dogs

A Erliquiose canina é uma zoonose causada pela Ehrlichia canis, bactéria Gram negativa de distribuição mundial. Alguns cães com erliquiose se tornam portadores assintomáticos enquanto outros desenvolvem uma doença aguda com morte rápida. A apoptose pode ser importante na eliminação de patógenos intracelulares, podendo, nas infecções por Ehrlichia sp. e Anaplasma sp., ocorrer modulação da apoptose celular para prolongar a sobrevivência desses organismos. Para avaliação do papel da apoptose na erliquiose canina, sete cães foram distribuídos em dois grupos. No Grupo inoculado, realizou-se a infecção por via intravenosa com sangue infectado com E. canis (isolado Jaboticabal), sendo realizada a inoculação com PBS estéril nos animais pertencentes ao Grupo Controle. Semanalmente e até 35 dias pós-inoculação, amostras de sangue foram coletadas e submetidas a n-PCR e reação de imunofluorescência (RIFI) para confirmação da infecção. No 36° dia pós-inoculação, os animais foram eutanasiados, sendo as amostras de baço, linfonodo, rim e fígado coletadas e processadas para as técnicas de TUNEL e imunohistoquímica (Bcl-2, Bax). Verificou-se pela n-PCR que os animais inoculados se tornaram positivos para E. canis a partir da segunda semana. Pela RIFI, verificou-se animais com sorologia positiva a partir da terceira semana pós-inoculação. No grupo controle, todos os testes realizados foram negativos para E. canis. Apesar da reação de TUNEL mostrar maior incidência de apoptose no Grupo Inoculado, sendo o baço e os linfonodos os órgãos que apresentaram maior marcação, os resultados da imunohistoquímica para Bcl-2 e Bax indicam que a via intrínseca de apoptose não é importante nos órgãos analisados.

Some dogs infected with Ehrlichia canis become asymptomatic while others develop an acute illness followed by quick death. Apoptosis may be an important mechanism for elimination of intracellular pathogens. Also, Ehrlichia sp. and Anaplasma sp. can modulate apoptosis to extend their survival. To evaluate the role of apoptosis in canine ehrlichiosis, 7 dogs were assigned into 2 groups, one with 4 animals inoculated intravenously with blood infected with Ehrlichia canis (Jaboticabal isolate) and a control with 3 dogs, inoculated with sterile PBS. Blood samples were collected weekly and 35 days post-inoculation to confirm the infection by nPCR and immunofluorescence. Thirty-six days after inoculation the animals were euthanized and samples from spleen, lymph nodes, kidney and liver were collected to carry out the TUNEL technique and immunohistochemistry (Bcl-2, Bax). Inoculated animals became positive for E. canis by nPCR already in the second week and by immunofluorescence in the third week after inoculation. The control group showed negative for E. canis in all tests. The TUNEL reaction showed a higher incidence of apoptosis in the inoculated group, with stronger labeling in the spleen and lymph nodes. The results of immunohistochemistry for Bcl-2 and Bax suggest that the intrinsic pathway of apoptosis is not important in the analyzed organs.

Apoptosis in eosinophilic nasal polyps treated in vitro with mitomycin C.

UNLABELLED: The etiopathogenesis of eosinophilic nasal polyps is yet to be explained. Eosinophils are key components in the inflammatory infiltrate and are related to the perpetuation of the inflammatory process in chronic rhinosinusitis with nasal polyps. OBJECTIVE: This paper aims to evaluate the in vitro action of mitomycin upon the apoptotic index of nasal polyps. MATERIALS AND METHODS: This is a self-paired prospective experimental study using biopsy fragments from 15 patients with eosinophilic nasal polyps. Biopsy fragments were divided into two groups. In the case group, the fragments were treated with 400 µg/ml of mitomycin for five minutes. The control group fragments were treated with culture medium. The pair of fragments contained in the two first compartments - control and case - were immediately sent to the histopathologist. The other pair of samples containing control and case fragments was incubated for 12 hours. The fragments were then taken to the histopathologist for testing. The apoptotic index was determined by the morphometry in hematoxylin and eosin staining and DNA fragmentation analysis (TUNEL reaction). RESULTS: The comparison between the two groups showed a statistically significant difference (p < 0,001) in the apoptotic index of the 12-hour incubated cultures. CONCLUSION: Mitomycin acts in vitro upon the eosinophilic nasal polyps inducing the rise of the eosinophilic apoptotic index.

Apoptosis, inflammatory response and parasite load in skin of Leishmania (Leishmania) chagasi naturally infected dogs: a histomorphometric analysis.

The skin has an important role in infection by Leishmania chagasi. Apoptosis modulates the inflammatory response acting distinctively either on the progression or regression of the lesions. The parasites interact with multiple regulatory systems inducing apoptosis in host cells, during cell invasion, stabilization and multiplication of pathogens. In this context, the aim of this study was to evaluate cell death within the inflammatory infiltrates, and to correlate these results with parasite load and clinical features of dogs naturally infected with L. chagasi. Fragments of skin pinnas (8 symptomatic+8 asymptomatic+6 negative controls) were used to characterize and measure the inflammatory response, parasite load and apoptosis. Diagnosis of canine leishmaniasis was confirmed by the detection of anti-Leishmania antibodies by IFA and ELISA in serum, direct visualization of the parasite and culture in spleen, liver, pinna, bone marrow and lymph nodes, and PCR (pinna). Histomorphometry was performed with images obtained from 20 representative histological fields in a light microscope. Ultra-thin sections were mounted over a 300 mesh grids, contrasted with 2% uranyl acetate and lead citrate and examined under a Transmission Electronic Microscopy. Amastigotes were only found in the skin of symptomatic animals (31.94 ± 18.81). The number of foci and cellularity of the inflammatory infiltrates in symptomatic dogs were higher than in other groups and in asymptomatics were higher than in controls (p<0.05; Tukey). The average area, perimeter and extreme diameters of the inflammatory infiltrates obtained in symptomatic dogs were higher than in controls (p<0.05; Tukey). The apoptotic index was higher in symptomatic than in other groups and there was no difference between asymptomatics and controls (p<0.05; Tukey). Ultrastructurally, apoptotic cells were shrunken, with condensed nuclear chromatin and cytoplasm. Condensed nuclei were frequently fragmented. Internucleosomal DNA fragmentation occurred only in symptomatic cases. Amastigotes were observed within neutrophils and macrophages. Apoptosis is directly related to parasite load, intensity of inflammatory response and clinical manifestations in L. chagasi naturally infected dogs.

Apoptose em pólipos nasais eosinofílicos submetidos in vitro ao tratamento com mitomicina C / Apoptosis in eosinophilic nasal polyps treated in vitro with mitomycin C

A etiopatogênese da polipose nasal eosinofílica ainda não foi esclarecida. Os eosinófilos constituem as principais células do infiltrado inflamatório e estão relacionados com a perpetuação do processo inflamatório na rinossinusite crônica com pólipos nasais. OBJETIVO: Avaliar a ação in vitro da mitomicina no índice apoptótico de pólipos nasais eosinofílicos. MATERIAL E MÉTODO: Estudo prospectivo experimental autopareado com amostra de biópsia de 15 pacientes com polipose nasal eosinofílica. Cada fragmento foi dividido em dois grupos. No grupo experimental aplicou-se mitomicina por cinco minutos, na dosagem de 400 µg/ml. O grupo controle foi submetido às mesmas manipulações, mas utilizando-se somente meio de cultura. Os fragmentos contidos nos dois primeiros compartimentos, controle e experimento, foram imediatamente submetidas ao preparo para histopatologia. O outro par de amostra, contendo controle e experimento, foi incubado por 12 horas. Após 12 horas, os fragmentos foram retirados para exame histopatológico. O índice apoptótico foi determinado pela morfometria na coloração hematoxilina-eosina e pela análise da fragmentação do DNA. RESULTADO: A comparação do dois grupos demonstrou diferença significativa (p < 0,001) no índice apoptótico das culturas incubadas por 12 horas. CONCLUSÃO: A mitomicina induz in vitro o aumento do índice apoptótico dos eosinófilos dos pólipos nasais eosinofílicos.

The etiopathogenesis of eosinophilic nasal polyps is yet to be explained. Eosinophils are key components in the inflammatory infiltrate and are related to the perpetuation of the inflammatory process in chronic rhinosinusitis with nasal polyps. OBJECTIVE: This paper aims to evaluate the in vitro action of mitomycin upon the apoptotic index of nasal polyps. MATERIALS AND METHODS: This is a self-paired prospective experimental study using biopsy fragments from 15 patients with eosinophilic nasal polyps. Biopsy fragments were divided into two groups. In the case group, the fragments were treated with 400 µg/ml of mitomycin for five minutes. The control group fragments were treated with culture medium. The pair of fragments contained in the two first compartments - control and case - were immediately sent to the histopathologist. The other pair of samples containing control and case fragments was incubated for 12 hours. The fragments were then taken to the histopathologist for testing. The apoptotic index was determined by the morphometry in hematoxylin and eosin staining and DNA fragmentation analysis (TUNEL reaction). RESULTS: The comparison between the two groups showed a statistically significant difference (p < 0,001) in the apoptotic index of the 12-hour incubated cultures. CONCLUSION: Mitomycin acts in vitro upon the eosinophilic nasal polyps inducing the rise of the eosinophilic apoptotic index.

Lesão de isquemia e reperfusão após clampagem contínua ou intermitente do pedículo hepático em coelhos / Ischemia/reperfusion injury after continuous or intermittent hepatic pedicle clamping in rabbits

RACIONAL: O controle do sangramento na hepatectomia é um desafio para os cirurgiões. A clampagem do pedículo hepático é manobra cirúrgica que pode promover redução do sangramento, mas provoca isquemia hepatocelular. Isso, junto com a reperfusão depois que a clampagem termina, leva à lesão de isquemia e reperfusão. OBJETIVO: Examinar os efeitos da lesão de isquemia e reperfusão no fígado após clampagem contínua e intermitente do pedículo hepático, usando a quantificação de apoptose como ferramenta. MÉTODO: Vinte coelhos New Zealand foram divididos em grupos 1 (controle), 2 (60 minutos de isquemia contínua) e 3 (60 minutos de isquemia intermitente alternando 12 minutos de isquemia e três minutos de reperfusão). Biópsias hepáticas foram colhidas antes e ao fim da isquemia e após seis horas de reperfusão, quando os animais eram sacrificados. Os fragmentos obtidos foram submetidos à análise histológica e histoquímica (reação de Tunel). Campos microscópicos foram analisados para caracterização e quantificação de apoptose. RESULTADOS: A isquemia levou à elevação do índice apoptótico em ambos os grupos experimentais em relação aos controles, mas similar entre eles. Depois da reperfusão os índices voltaram aos valores iniciais. CONCLUSÃO: A clampagem do pedículo hepático, tanto contínua quanto intermitente, induz a apoptose em células hepáticas de modo igual.

BACKGROUND : The control of bleeding in hepatectomy is a challenge for surgeons. The hepatic pedicle clamping is a surgical maneuver that can provide reduction in bleeding, but it provokes a hepatocellular suffering. This, along with reperfusion after the clamping finishes, leads to an injury known as ischemia/reperfusion injury. AIM: To examine the effects of the ischemia/reperfusion injury on the liver after continuous and intermittent hepatic pedicle clamping in an animal model, using the quantification of apoptosis for evaluation. METHOD: Twenty New Zealand rabbits were assigned to groups 1 (control), 2 (60 minutes of continuous ischemia) and 3 (60 minutes of intermittent ischemia alternating 12 minutes of ischemia and three minutes of reperfusion). Liver biopsies were collected before ischemia, at its end and after six hours of reperfusion, when the animals were killed. The liver fragments were subjected to histological analysis (paraffinization and hematoxilin-eosin staining) and histochemical (Tunel reaction). Microscope fields of view were scanned for characterization and quantification of apoptosis. RESULTS : Ischemia led to an increased apoptotic index in both experimental groups in comparison to controls, but similarly between them. After the reperfusion, the indexes returned to baseline values. CONCLUSION: Clamping of the hepatic pedicle, either continuous or intermittent, induces apoptosis in liver cells in a similar way.

Histopathology of Fasciola hepatica infection in Meriones unguiculatus

Três grupos de 60 Meriones unguiculatus foram inoculados, respectivamente, com 3, 5 e 8metacercárias de Fasciola hepatica e dez animais permaneceram como controle. Com intervalo de dez dias, seis animais do grupo infectado e um animal do grupo controle foram eutanasiados,sendo coletadas amostras para a histopatologia dos órgãos internos. Foram observadas áreas hemorrágicas, focos de necrose e fibrose no fígado, no rim direito e em órgãos internos contidosnas cavidades torácica e abdominal em 20,6por cento dos animais infectados, independentemente do número de metacercáreas. O fígado foi o órgão mais lesado, indicando o tropismo que o parasito apresenta durante seu desenvolvimento. Foram observadas alterações no espaço porta-hepático,dilatação do ducto biliar e de ramos da artéria hepática, rompimento de ramos da veia hepática e hemorragia periportal. Foram recuperados parasitos e ovos de F. hepatica aderidos à parede interna do ducto biliar. A análise histopatológica do fígado e do linfonodo mesentérico mostrou a presençade hemossiderose e de reação inflamatória macrofagocitária. No fígado, pulmões, diafragma, baço,linfonodos mesentéricos, rins, pâncreas e intestino delgado, havia infiltrado inflamatório misto formado por polimorfonucleares e mononucleares, áreas hemorrágicas, e focos de necrose e fibrose. Os resultados indicam que M. unguiculatus mantém a infecção por longos períodos, permitindo também o desenvolvimento do parasito em diferentes órgãos.

Ischemia/reperfusion injury after continuous or intermittent hepatic pedicle clamping in rabbits.

BACKGROUND: The control of bleeding in hepatectomy is a challenge for surgeons. The hepatic pedicle clamping is a surgical maneuver that can provide reduction in bleeding, but it provokes a hepatocellular suffering. This, along with reperfusion after the clamping finishes, leads to an injury known as ischemia/reperfusion injury. AIM: To examine the effects of the ischemia/reperfusion injury on the liver after continuous and intermittent hepatic pedicle clamping in an animal model, using the quantification of apoptosis for evaluation. METHOD: Twenty New Zealand rabbits were assigned to groups 1 (control), 2 (60 minutes of continuous ischemia) and 3 (60 minutes of intermittent ischemia alternating 12 minutes of ischemia and three minutes of reperfusion). Liver biopsies were collected before ischemia, at its end and after six hours of reperfusion, when the animals were killed. The liver fragments were subjected to histological analysis (paraffinization and hematoxilin-eosin staining) and histochemical (Tunel reaction). Microscope fields of view were scanned for characterization and quantification of apoptosis. RESULTS: Ischemia led to an increased apoptotic index in both experimental groups in comparison to controls, but similarly between them. After the reperfusion, the indexes returned to baseline values. CONCLUSION: Clamping of the hepatic pedicle, either continuous or intermittent, induces apoptosis in liver cells in a similar way.