Quantification of Global DNA Methylation in Canine Melanotic and Amelanotic Oral Mucosal Melanomas and Peripheral Blood Leukocytes From the Same Patients With OMM: First Study.

Oral mucosal melanomas (OMMs) are aggressive and resistant cancers of high importance in veterinary oncology. Amelanotic OMM produces comparatively less melanin and is considered to be more aggressive than melanotic OMM. Global DNA methylation profiles with hypomethylated or hypermethylated patterns have both been associated with aggressive neoplasms; however, global DNA hypomethylation seems to correlate to higher aggressiveness. Accordingly, global DNA methylation in peripheral blood leukocytes has been investigated to understand the role of systemic or environmental factors in cancer development. This study aimed to quantify global DNA methylation in canine melanotic and amelanotic OMM samples and in the peripheral blood leukocytes of the same dogs. Tumor tissue samples were collected from 38 dogs, of which 19 were melanotic and 19 were amelanotic OMM. These were submitted to immunohistochemistry (IHC) with anti-5-methylcytosine (5mC) and anti-Ki67 primary antibodies. Ki67- and 5mC-positive nuclei were manually scored with the help of an image analysis system. Peripheral blood samples were collected from 18 among the 38 OMM-bearing dogs and from 7 additional healthy control dogs. Peripheral blood leukocytes were isolated from the 25 dogs, and DNA was extracted and analyzed by high-performance liquid chromatography (HPLC) for global DNA methylation. The pattern of global DNA methylation in both canine melanotic and amelanotic OMM indicated higher percentages of weakly or negatively stained nuclei in most of the OMM cells, presuming predominant global DNA hypomethylation. In addition, Ki67 counts in amelanotic OMM were significantly higher than those in melanotic OMM (p < 0.001). Global DNA methylation different immunostaining patterns (strong, weak or negative) correlated with Ki67 scores. Global DNA methylation in circulating leukocytes did not differ between the 9 melanotic and 9 amelanotic OMM or between the 18 OMM-bearing dogs and the 7 healthy dogs. This study provides new information on canine melanotic and amelanotic OMM based on global DNA methylation and cell proliferation.

Effects of Alpha-Connexin Carboxyl-Terminal Peptide (aCT1) and Bowman-Birk Protease Inhibitor (BBI) on Canine Oral Mucosal Melanoma (OMM) Cells.

Oral mucosal melanomas (OMM) are aggressive cancers in dogs, and are good models for human OMM. Gap junctions are composed of connexin units, which may have altered expression patterns and/or subcellular localization in cancer cells. Cell-to-cell communication by gap junctions is often impaired in cancer cells, including in melanomas. Meanwhile, the upregulated expression of the gap junction protein connexin 43 (Cx43) inhibits melanoma progression. The α-connexin carboxyl-terminal (aCT1) peptide reportedly maintains Cx43 expression and cell-cell communication in human mammary cells and increases the communication activity through gap junctions in functional assays, therefore causing decreased cell proliferation. The Bowman-Birk protease inhibitor (BBI), a component of soybeans, induces Cx43 expression in several tumor cells as a trypsin-chymotrypsin inhibition function, with antineoplastic effects. This study investigated the effect of aCT1 peptide and BBI treatment, alone or in combination, on TLM1 canine melanoma cell viability. Cell viability after treatment with aCT1, the reverse sequence peptide (R-pep), and/or BBI for 5 days was analyzed by PrestoBlue assay. Immunofluorescence was used to observe Cx43 localization and expression. aCT1 (200 µM) alone did not significantly decrease cell viability in TLM1 cells, whereas BBI (400 µg/ml) alone significantly decreased the TLM1 viability. Combined treatment with both aCT1 (200 µM) and BBI (400 µg/ml) significantly decreased cell viability in TLM1 cells. Cx43 expression, as identified by immunostainings in TLM1 cells, was increased in the cell membrane after the combination treatment with BBI and aCT1. This dual treatment can be combined to achieve the anticancer activity, possibly by increasing Cx 43 expression and affecting Cx43 migration to the cell membrane. In conclusion, a treatment strategy targeting Cx43 with BBI and aCT1 may possibly lead to new effective therapies for canine OMM.

Testicular subcutaneous allografting followed by immunosuppressive treatment promotes maintenance of spermatogonial cells in rainbow trout (Oncorhynchus mykiss).

Germ cell transplantation and testis graft represent promising biotechnologies that can be applied for the reproduction of commercial or endangered species. However, mechanisms of rejection from the host immune system might remove the transplanted donor cells/tissues and limit the surrogate production of gametes. In this work, we administered emulsion containing-immunosuppressants to verify whether they are capable to prevent immune rejection and promote survival of testis allografts in rainbow trout. In the first part of this study, we demonstrated in vitro that tacrolimus and cyclosporine were able to affect viability, inhibit leucocyte proliferation, and suppress il2 expression in vitro. In in vivo experiments, both doses of tacrolimus (0.5 and 1.5 mg/kg) and the lower dose of cyclosporine (20 mg/kg) significantly inhibited the expression of il2 in head kidney, three days post-injection. A higher dose of cyclosporine (40 mg/kg) was able to inhibit il2 expression for up to seven days post-injection. In the second part, testis allografts were conducted in fish treated weekly with emulsion containing-tacrolimus. Immunohistochemical, conventional histology, and qRT-PCR (vasa) analysis demonstrated the presence of spermatogonial cells by the fifth week, in animals treated with 0.5 mg/kg of tacrolimus similar as found in autografted group. In the group treated with the highest tacrolimus dose (1.5 mg/kg) and in the non-treated group (without immunosuppressant), no germ cells or their respective markers were detected. il2 expression in head kidney was also suppressed in grafted animals treated with tacrolimus compared to non-treated group. These results suggest that tacrolimus may be a promising immunosuppressant for testis allografts or germ cell transplantation in rainbow trout. Co-administration combining tacrolimus (at lower dose) with other immunosuppressive drugs for inhibiting other activation pathways of the immune system, as performed in human organ transplantation, could be an alternative approach to optimize the immunosuppressive effects in host organisms.

Inhibitory effects of Euphorbia tirucalli latex on murine B16/F10 melanoma cells and lung metastasis.

Euphorbia tirucalli (E. tirucalli) is a tropical and subtropical plant that produces a latex which is used for several purposes. The components of E. tirucalli latex include triterpenes, diterpenes and steroids. The aim of the present study was to evaluate the effects of diluted E. tirucalli latex on murine B16/F10 melanoma cells and lung metastasis. For this purpose, an in vitro study was first performed, in which B16/F10 cells were treated with diluted (1/2 to 1/11,192) E. tirucalli latex. In a second study, B16/F10 melanoma cells were inoculated into the tail vein of mice to generate lung metastases; the mice then received 0.467 µg of latex diluted in 200 ml saline by gavage for 14 days. A significant decrease in B16/F10 cell viability was observed using the MTT assay at 24 and 48 h after treatment with E. tirucalli latex. In addition, a significant decrease in the volume fraction occupied by B16/F10 metastatic colonies in the lungs was observed in mice treated with E. tirucalli latex. These results confirm the antineoplastic effects of diluted E. tirucalli latex.

Biochemical and Utralmicroscopic Evaluation of Myofibril Proteins and Collagen during Ageing in Broiler Chicken PSE (Pale, Sof, Exudative) Meat

Abstract The aim of this work was to study the myofibril proteins and collagen fraction changes in broiler chickens PSE (pale, soft, exudative) meat during ageing and their relationship to meat quality. The results presented an increase of myofibril proteins and collagen solubility promoted by the enhanced proteases activities during storage. Ultramicroscopically, the PSE meat samples revealed intracellularly a sarcomere super contraction and lacunas within the A and I bands while Z-lines appeared very dense and fragmented in comparison to normal samples. This observation was noticed already at 4h storage while extracellularly collagen fibrils decreased visually within the endomysium only after 24h of conditioning. These results influenced the quality as the PSE meat presented better functional properties at the first hours of conditioning before further proteins degradation by proteases. Thereafter, at the later ageing stage a further disintegration of the abnormal meat structure would affect the meat functional properties.

Stereology shows that damaged liver recovers after protein refeeding.

OBJECTIVE: The aim of the present study was to investigate the putative effects of a low-protein diet on the three-dimensional structure of hepatocytes and determine whether this scenario could be reversed by restoring the adequate levels of protein to the diet. METHODS: Using design-based stereology, the total number and volume of hepatocytes were estimated in the liver of mice in healthy and altered (by protein malnutrition) conditions and after protein renutrition. RESULTS: This study demonstrated a 65% decrease in the liver volume (3302 mm3 for the control for undernourished versus 1141 mm3 for the undernourished group) accompanied by a 46% reduction in the hepatocyte volume (8223 µm3 for the control for undernourished versus 4475 µm3 for the undernourished group) and a 90% increase in the total number of binucleate hepatocytes (1 549 393 for the control for undernourished versus 2 941 353 for the undernourished group). Reinstating a normoproteinic diet (12% casein) proved to be effective in restoring the size of hepatocytes, leading to an 85% increase in the total number of uninucleate hepatocytes (15 988 560 for the undernourished versus 29 600 520 for the renourished group), and partially reversed the liver atrophy. CONCLUSIONS: Awareness of these data will add to a better morphologic understanding of malnutrition-induced hepatopathies and will help clinicians improve the diagnosis and treatment of this condition in humans and in veterinary practice.

Histology of the gastrointestinal tract from Bothrops jararaca and Crotalus durissus / Aspectos histológicos do trato gastrointestinal de Bothrops jararaca e Crotalus durissus

Recent studies have discussed the importance of snakes, going beyond the context of Public Health (snakebites). Promising research demonstrates the utility of snake venom in several fields, including oncology, diagnosis, anticoagulant therapy, and pain treatment. Nevertheless, there are still gaps in the basic knowledge on these animals, specifically regarding the histological characterization of the gastrointestinal tract, which ultimately hinder comparative histology and pathology studies. In this context the present study intends to contribute with the advance of current knowledge on snake histology by describing and analyzing histological samples of the gastrointestinal tracts of Bothrops jararaca and Crotalus durissus. Samples were collected from 12 individuals (six from each species), up to 6 months of age and equally distributed between males and females. Histological slides were prepared from the organs collected and were stained with hematoxylin and eosin, periodic acid-Schiff and alcian blue. The slides were photographed with a high-resolution camera to create a portfolio representative of the histology of the gastrointestinal systems of these animals.(AU)

Na atualidade, a importância das serpentes extravasa a conotação em saúde pública (acidentes ofídicos), e pesquisas promissoras têm demonstrado a utilidade dos venenos das serpentes em diversas áreas como oncologia, diagnóstico, terapia anticoagulante e tratamento da dor. Ainda assim, existem lacunas no conhecimento básico sobre esses animais, tais como a caracterização histológica do seu sistema gastrointestinal, que se tornam obstáculos para estudos nos campos da histologia e patologia comparadas. Nesse contexto, o presente trabalho pretende contribuir com o avanço do conhecimento sobre histologia de serpentes a partir da descrição e análise histológica de amostras do trato gastrointestinal de exemplares das espécies Bothrops jararaca e Crotalus durissus. Para tanto, foram coletadas amostras de 12 indivíduos com até seis meses de idade, seis por espécie (distribuídos igualmente entre machos e fêmeas). A partir dos órgãos colhidos, lâminas histológicas foram preparadas e coradas pela técnica de hematoxilina e eosina, bem como PAS com Alcian Blue. As lâminas foram fotografadas por câmera de alta resolução, resultando em um portfólio representativo da histologia do sistema gastrointestinal desses animais.(AU)

Connexins, Pannexins, and Their Channels in Fibroproliferative Diseases.

Cellular and molecular mechanisms of wound healing, tissue repair, and fibrogenesis are established in different organs and are essential for the maintenance of function and tissue integrity after cell injury. These mechanisms are also involved in a plethora of fibroproliferative diseases or organ-specific fibrotic disorders, all of which are associated with the excessive deposition of extracellular matrix components. Fibroblasts, which are key cells in tissue repair and fibrogenesis, rely on communicative cellular networks to ensure efficient control of these processes and to prevent abnormal accumulation of extracellular matrix into the tissue. Despite the significant impact on human health, and thus the epidemiologic relevance, there is still no effective treatment for most fibrosis-related diseases. This paper provides an overview of current concepts and mechanisms involved in the participation of cellular communication via connexin-based pores as well as pannexin-based channels in the processes of tissue repair and fibrogenesis in chronic diseases. Understanding these mechanisms may contribute to the development of new therapeutic strategies to clinically manage fibroproliferative diseases and organ-specific fibrotic disorders.

Parenteral solution of nutritional hepatotrophic factors improves regeneration in thioacetamide-induced cirrhotic livers after partial hepatectomy.

Liver resection is a suitable option for the treatment of certain hepatic conditions, particularly hepatocarcinomas, in patients with cirrhosis. However, this disease impairs liver regeneration, which increases the risk of liver failure and postoperative death. Supportive treatments for regeneration of the remaining liver may be useful for the recovery of these patients. We demonstrated that nutritional hepatotrophic factors (NHF) is an effective regenerative stimulus for cirrhotic livers in rats subjected to partial hepatectomy (PH). The rats with thioacetamide-induced cirrhosis were subjected to PH, and they were divided into 2 groups. One group received intraperitoneal administration of NHF, and the other group received saline solution. After 12 days, biometric data, collagen content, hepatocyte regeneration (proliferation cell nuclear antigen immunochemistry), and profibrotic gene expression (Collagen-α1, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1, and transforming growth factor beta 1) were assessed. The results indicated that the rats treated with NHF after PH had an increased liver size, a reduced amount of collagen, and a higher hepatocyte proliferation index compared with the rats that underwent PH alone. In addition, collagen-α1 gene expression was decreased in the NHF-treated rats. Thus, postoperative improvement in the liver morphology following NHF treatment may cause a significant decrease in the risk of liver failure and mortality after hepatic resection.

Morfometria das fibras colágeno cardíaco em ratos sadios e diabéticos suplementados com vitamina C / Morphometry of the collagen fibers in healthy and diabetic rats treated with vitamin C

The increase of heart collagen fibers in diabetics is a well known fact, but the consequences are not defined. The aim was to quantify the cardiac collagen fibers in normal and diabetic rats treated with vitamin C. We selected 32 Wistar rats, 16 diabetic animals induced endovenously with streptozootocin, and 16 healthy animals, half of them, diabetics and normals, were treated with vitamin C for 90 days. After the experimental proceeding, the hearts were removed and processed accordingly to conventional protocol for optical microscopy and specific staining for collagen. The results showed that the diabetic rats presented increase in the number of cardiac collagen fibers, but the ones treated with vitamin C showed little accumulation of fibers. It could be concluded that treatment with vitamin C is important for the prevention of heart failure in diabetic animals.

O aumento do conteúdo de fibras colágenas no coração de diabéticos é um fato bastante conhecido, suas conseqüências ainda são objeto de estudo e causam certa controvérsia, portanto este trabalho objetivou estudar a variação na quantidade das fibras de colágeno cardíacas em animais normais e diabéticos tratados pela vitamina C. Para isso foram selecionados 32 ratos Wistar, 16 diabéticos induzidos pela injeção endovenosa de estreptozootocina e 16 normais, sendo metade deles tratados com Vitamina C (diabéticos e normais) por um período de 90 dias. Após período experimental, os corações foram retirados e processados segundo protocolo convencional para microscopia óptica e coloração específica para colágeno. Os resultados mostram que animais diabéticos apresentam maior quantidade de fibras de colágeno cardíacas e que o tratamento com a vitamina C determinou um menor acúmulo na quantidade dessas fibras.

Clareamento da melanina na epiderme do lobo-marinho-sul-americano e sua aplicação na imunohistoquímica enzimática / Bleaching of melanin in the epidermis of south American fur seal and its application on enzyme immunohistochemistry

O Lobo-marinho-sul-americano (Arctocephalus australis) é um mamífero marinho anfíbio distribuído ao longo da Costa do Atlântico e do Pacífico da América do Sul. Esta espécie está bem adaptada a diferentes habitats devido à morfologia dos membros em forma de nadadeira e de seu sistema tegumentar. Estudos imuno-histoquímicos são importantes para avaliar os mecanismos de adaptação da pele devido a diferencial expressão dos antígenos presentes no tecido dependendo da região da superfície corporal. Entretanto, sua epiderme altamente pigmentada (melanina) impede a visualização dos marcadores cromógenos utilizados na imunohistoquímica. Neste trabalho foi desenvolvido um método de clarear a melanina para permitir a visualização dos cromógenos sem alterar a afinidade antígeno-anticorpo para a imuno-histoquímica. A análise do índice do PCNA (proliferating cell nuclear antigen) na epiderme de A. australis, com diaminobenzidina (DAB) como cromógeno foi usada para testar o método. O clareamento da melanina permitiu obter o índice de proliferação celular na epiderme e evitar resultados falso-positivos sem afetar os resultados imuno-histoquímicos.

The South American fur seal (Arctocephalus australis) is an amphibious marine mammal distributed along the Atlantic and Pacific coasts of South America. The species is well adjusted to different habitats due to the morphology of its fin-like members and due to some adaptations in their integumentary system. Immunohistochemical studies are very important to evaluate the mechanisms of skin adaptation due the differential expression of the antigens present in the tissue depending of the region of the body surface. However, its strongly pigmented (melanin) epidermis prevents the visualization of the immunohistochemical chromogens markers. In this study a melanin bleaching method was developed aimed to allow the visualization of the chromogens without interfering in the antigen-antibody affinity for immunohistochemistry. The analysis of PCNA (proliferating cell nuclear antigen) index in the epidermis of A. australis by immunohistochemistry with diaminobenzidine (DAB) as chromogen was used to test the method. The bleaching of the melanin allowed to obtain the cell proliferation index in epidermis and to avoid false positive results without affecting the immunohistochemical results.

Morphological and molecular pathology of CCL4-induced hepatic fibrosis in connexin43-deficient mice.

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4) -induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis.

Adaptation of the gill epithelium of an euryhaline fish, the guppy (Poecilia vivipara), to freshwater / Adaptação do epitélio branquial de peixes eurialinos, guaru (Poecilia vivipara), para água doce

The south-american euryhaline fish Poecilia vivipara (BLOCH; SNEIDER, 1801), the guppy, is found both in estuary and river waters, which suggests high adaptability to environments of different salinity. In this work we studied the adaptation of the interlamellar, bars and rakers epithelia of the gills of estuary fish to freshwater conditions. The results reveal that the gill epithelia of Poecilia vivipara can adjust itself to freshwater by decreasing the VP of mucous cells of the interlamellar epithelium and increase the volumetric proportion (VP) of chloride cells. However, there was no evidence of similar morphological alteration in the rakers region. The epithelia of the rakers appears to be part of a different compartment that is less sensitive to variation of salinity.

O peixe eurihalino sul-americano Poecilia vivipara (BLOCH; SNEIDER, 1801), o guppy, é encontrado tanto em estuários quanto em águas de rios, o que sugere uma alta adaptabilidade aos diferentes ambientes de salinidade. Neste trabalho, estudamos a adaptação do epitélio interlamelar, do arco e do rastelo das brânquias dos peixes de estuário de água doce. Os resultados revelam que o epitélio branquial de Poecilia vivipara pode ajustar-se à água doce, diminuindo a proporção volumétrica (PV) de células mucosas do epitélio interlamelar e aumentando a PV de células clorídricas. No entanto, não houve nenhuma evidência de alteração morfológica semelhante na região do rastelo branquial. O epitélio do rastelo branquial parece ser parte de um compartimento diferente que é menos sensível a variações de salinidade.

Hepatotrophic factors reduce hepatic fibrosis in rats.

CONTEXT: Hepatic fibrosis occurs in response to several aggressive agents and is a predisposing factor in cirrhosis. Hepatotrophic factors were shown to stimulate liver growth and to restore the histological architecture of the liver. They also cause an improvement in liver function and accelerate the reversion of fibrosis before it progresses to cirrhosis. OBJECTIVE: To test the effects of hepatic fibrosis solution composed by amino acids, vitamins, glucose, insulin, glucagon and triiodothyronine on hepatic fibrosis in rats. METHODS: Fibrosis was induced in rats by gastric administration of dimethylnitrosamine (10 mg/kg) for 5 weeks. After liver biopsy, the rats received either hepatotrophic factors solution (40 mg/kg/day) or saline solution for 10 days by intraperitoneal injection. Blood samples and liver fragments were collected for hepatic function analysis, standard histopathology evaluation, and morphometric collagen quantification. RESULTS: Rats in the hepatotrophic factors group showed a decrease of the histopathological components of fibrosis and an increase of their hepatic mass (12.2%). There was no development of neoplasic lesions in both groups. Compared with the saline group, the hepatotrophic factors group also had a decrease of blood levels of hepatic-lesion markers (AST, ALT) and a decrease of collagen content in the portal spaces (31.6%) and perisinusoidal spaces (42.3%), as well as around the hepatic terminal vein (57.7%). Thus, hepatotrophic factors administration in the portal blood promoted a regenerative hepatic response, with an overall reduction of the volumetric density of collagen, improved hepatic function, and a general improvement in the histopathological aspects of fibrosis. CONCLUSION: Taken together, these results suggest the potential therapeutic use of this hepatotrophic factors solution to treat chronic liver diseases.

Hepatotrophic factors reduce hepatic fibrosis in rats / Fatores hepatotróficos reduzem a fibrose hepática em ratos

CONTEXT: Hepatic fibrosis occurs in response to several aggressive agents and is a predisposing factor in cirrhosis. Hepatotrophic factors were shown to stimulate liver growth and to restore the histological architecture of the liver. They also cause an improvement in liver function and accelerate the reversion of fibrosis before it progresses to cirrhosis. OBJECTIVE: To test the effects of hepatic fibrosis solution composed by amino acids, vitamins, glucose, insulin, glucagon and triiodothyronine on hepatic fibrosis in rats. METHODS: Fibrosis was induced in rats by gastric administration of dimethylnitrosamine (10 mg/kg) for 5 weeks. After liver biopsy, the rats received either hepatotrophic factors solution (40 mg/kg/day) or saline solution for 10 days by intraperitoneal injection. Blood samples and liver fragments were collected for hepatic function analysis, standard histopathology evaluation, and morphometric collagen quantification. RESULTS: Rats in the hepatotrophic factors group showed a decrease of the histopathological components of fibrosis and an increase of their hepatic mass (12.2 percent). There was no development of neoplasic lesions in both groups. Compared with the saline group, the hepatotrophic factors group also had a decrease of blood levels of hepatic-lesion markers (AST, ALT) and a decrease of collagen content in the portal spaces (31.6 percent) and perisinusoidal spaces (42.3 percent), as well as around the hepatic terminal vein (57.7 percent). Thus, hepatotrophic factors administration in the portal blood promoted a regenerative hepatic response, with an overall reduction of the volumetric density of collagen, improved hepatic function, and a general improvement in the histopathological aspects of fibrosis. CONCLUSION: Taken together, these results suggest the potential therapeutic use of this hepatotrophic factors solution to treat chronic liver diseases.

CONTEXTO: A fibrose hepática ocorre em resposta a diversos agentes agressores e é um fator predisponente da cirrose. Fatores hepatotróficos são conhecidos por estimular o crescimento hepático e restaurar a arquitetura histológica do fígado. Promovem, também, melhora na função hepática e aceleram a reversão da fibrose antes de sua progressão para cirrose. OBJETIVO: Testar os efeitos de uma solução de fatores hepatotróficos, composta por aminoácidos, vitaminas, glicose, insulina, glugacon e triiodotironina na fibrose hepática em ratos. MéTODOS: No presente estudo, a fibrose foi induzida em ratos pela administração de dimetilnitrosamina (10 mg/kg) durante 5 semanas. Após a biopsia do fígado, os ratos receberam a solução de fatores hepatotróficos (40 mg/kg/dia) ou solução salina por injeção intraperitonial, durante 10 dias. Amostras sanguíneas e fragmentos do fígado foram coletados para análise da função hepática, avaliação do critério histopatológico e quantificação morfométrica do colágeno. RESULTADOS: Os ratos do grupo fatores hepatotróficos demonstraram diminuição dos componentes histopatológicos da fibrose e aumento de massa hepática (12,2 por cento). Não houve o desenvolvimento de lesões neoplásicas em ambos os grupos. Comparado com o grupo de salina, no grupo fatores hepatotróficos também houve diminuição nos níveis dos marcadores sanguíneos de lesão hepática (AST e ALT), e diminuição da quantidade de colágeno nos espaços porta (31,6 por cento) e espaços perissinusoidais (42,3 por cento), assim como ao redor das veias terminais hepáticas (57,7 por cento). Assim, a administração de fatores hepatotróficos no sangue portal promoveu resposta regenerativa hepática, com redução da densidade volumétrica de colágeno, melhora na função hepática e melhora geral nos aspectos histopatológicos da fibrose. CONCLUSÃO: Juntos, estes resultados sugerem o potencial uso terapêutico desta solução de fatores hepatotróficos para tratar doenças hepáticas crônicas.

Roles of gap junctions and connexins in non-neoplastic pathological processes in which cell proliferation is involved.

Cell proliferation is an important process for reproduction, growth and renewal of living cells and occurs in several situations during life. Cell proliferation is present in all the steps of carcinogenesis, initiation, promotion and progression. Gap junctions are the only specialization of cell membranes that allows communication between adjacent cells. They are known to contribute to tissue homeostasis and are composed of transmembrane proteins called "connexins." These junctions are also known to be involved in cell proliferation control. The roles of gap junctions and connexins in cell proliferation are complex and still under investigation. Since pioneer studies by Loewenstein, it is known that neoplastic cells lack communicating junctions. They do not communicate with their neighbors or with non-neoplastic cells from the surrounding area. There are many studies and review articles dedicated to neoplastic tissues. The aim of this review is to present evidence on the roles of gap junctions and connexins in non-neoplastic processes in which cell proliferation is involved.

Altered expression of connexins in urethane-induced mouse lung adenomas.

Lung carcinogenesis is a multistep process whose molecular alterations can be studied in mouse models. Urethane, a specific lung tumor carcinogen, can induce adenomas in mice. Mouse lung alveolar cells reportedly generate lung neoplasms, and express connexins 26, 32, 43 and 46. The aim of the present study was to evaluate the expression of connexins in urethane-induced lung adenomas. Fifteen-day-old CD1 male mice received 2 i.p. injections of urethane (1.5 g/kg bw). The mice were euthanized 25 weeks after urethane injection, and lung adenomas were quantified. Lung tissue and lung adenomas were harvested and the RNA was extracted. The expression of connexins 26, 32, 43 and 46 was evaluated by Real-Time PCR, and these proteins were identified by Western blot. Immunohistochemistry revealed the distribution pattern of these connexins in lung tissue and adenomas. The treatment with urethane was associated with the downregulation of Cx26, 32 and 46 expressions, and with the upregulation of Cx43 expression in lung tissue. Surprisingly, in lung adenomas Cx32 and Cx43 expressions were not detected, although the expression of connexins 26 and 46 was present. Western blot and immunohistochemistry corroborated the RT-PCR data. These results may indicate a role of Cx32 and Cx43 in urethane-induced lung carcinogenesis, since their absence may contribute to the development of urethane induced lung tumors. The role of Cx26 and Cx46 is yet to be determined.

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

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

Increased susceptibility to urethane-induced lung tumors in mice with decreased expression of connexin43.

Gap junction intercellular communication capacity and connexin expression are reportedly decreased in human lung cancer. The mechanisms by which connexins, the gap junction proteins, act as tumor suppressors are unclear. In order to understand the involvement of connexins in tumorigenesis, we analyzed the effect of the heterologous deletion of Gja1 [the connexin43 (Cx43) gene] on the development of lung adenomas in mice. Heterozygous (Cx43(+/-)) and wild-type mice (Cx43(+/+)) were treated or not with single doses of urethane at 15 and 17 days after birth. Twenty-five weeks later, both the number and size of nodules were increased in Cx43(+/-) mice as compared with Cx43(+/+) mice. Moreover, the lesions were histologically more aggressive in the heterozygous mice. However, no increase in spontaneous lesions was observed in the lungs of untreated Cx43(+/-) mice. Heterozygous mice effectively presented lower expression of Cx43 genes and decreased amounts of Cx43. In conclusion, our results indicate that deletion of one allele of the Cx43 gene clearly favors the carcinogenic effect of urethane administration and results in a higher susceptibility to lung adenoma formation in mice.

Stimulation of intact rat liver by exogenous hepatotrophic factors with additional growth of its mass

It was previously demonstrated that it is possible to increase the size of the intact liver of rats, contray to the biological determination that ties liver size to animal size bay intraperitoneal (portal) administration of exogenous hepatotrophic factors (a solution containing glucose, amino acids, insulin, glucagon, vitamins, and electrolytes) which qualitatively mimie substances and hormones normally present in splanchnic blood (PARRA et al.). In the present investigation, we studied the possible action of fat (LipofundinR), human milk growth factors, co-factors (folic acid, vitamin B12 and zinc), and thyroid hormone (T3 on the regenerative stimulus when added to the above solution. T3 was found to be an important factor which increased liver mass when compared to the basic solution (67.54 por cento) versus 36.07 por cento). Although these values were not compared statistically due to the small number of surviving animals, they are important as guidelines for future research about new compositions of hepatotrophic solutions that will maintain or improve the level of stimulation obtained thus far, with a possible reduction in animal mortality