Thiago Pinheiro Arrais Aloia

Identification of hepatic stem/progenitor cells in canine hepatocellular and cholangiocellular carcinoma

Hepatic progenitor cells (HPCs) are bipotential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic or cholangiocytic lineages. HPCs are present in both hepatocellular (HCC) and cholangiocellular carcinoma (CC) in humans; and a small percentage of HCC can originate from cancer stem cells. However, its distribution in canine liver tumour has not been studied. Herein, we searched for stem/progenitor cells in 13 HCC and 7 CC archived samples by immunohistochemical analysis. We found that both liver tumours presented a higher amount of K19-positive HPCs. Besides, 61.6% of HCC cases presented immature CD44-positive hepatocytes. Nevertheless, only two cases presented CD133-positive cells. As observed in humans, hepatic canine tumours presented activated HPCs, with important differentiation onto hepatocytes-like cells and minimal role of cancer stem cells on HCC. These findings reiterate the applicability of canine model in the search for new therapies before application in humans.

Maternal immune activation in late gestation increases neuroinflammation and aggravates experimental autoimmune encephalomyelitis in the offspring.

Multiple sclerosis (MS) is characterized by an autoimmune response against myelin antigens driven by autoreactive T cells. Several lines of evidence indicate that environmental factors, such as previous infection, can influence and trigger autoimmune responses. However, the importance of the gestational period, particularly under inflammatory conditions, on the modulation of MS and related neuroinflammation by the offspring is unknown. This study aimed to evaluate the impact of prenatal exposure to lipopolysaccharide (LPS) during late gestation on the neuroinflammatory response in primary mixed glial cultures and on the progression of experimental autoimmune encephalomyelitis (EAE, an animal model of MS) in the offspring. LPS (Escherichia coli 0127:B8, 120μg/kg) was administered intraperitoneally to pregnant C57BL/6J mice on gestational day 17, and the offspring were assigned to two experiments: (1) mixed glial cultures generated using the brain of neonates, stimulated in vitro with LPS, and (2) adult offspring immunized with MOG35-55. The EAE clinical symptoms were followed for 30days. Different sets of animals were sacrificed either during the onset (7days post-immunization [p.i.]), when spleen and lymph nodes were collected, or the peak of disease (20days p.i.), when CNS were collected for flow cytometry, cytokine production, and protein/mRNA-expression analysis. The primary CNS cultures from the LPS-treated group produced exaggerated amounts of IL-6, IL-1β and nitrites after in vitro stimulus, while IL-10 production was lowered compared to the data of the control group. Prenatal exposure to LPS worsened EAE disease severity in adult offspring, and this worsening was linked to increased CNS-infiltrating macrophages, Th1 cells and Th17 cells at the peak of EAE severity; additionally, exacerbated gliosis was evidenced in microglia (MHC II) and astrocytes (GFAP protein level and immunoreactivity). The IL-2, IL-6 and IL-17 levels in the spleen and lymph nodes were increased in the offspring of the LPS-exposed dams. Our results indicate that maternal immune activation during late gestation predispose the offspring to increased neuroinflammation and potentiate the autoimmune response and clinical manifestation of EAE.

Connexin 43 deficiency accelerates skin wound healing and extracellular matrix remodeling in mice

BACKGROUND Cellular channels composed of connexin 43 are known to act as key players in the life cycle of the skin and consequently to underlie skin repair. OBJECTIVE This study was specifically set up to investigate the suite of molecular mechanisms driven by connexin 43-based channels on wound healing. METHODS To this end, a battery of parameters, including re-epithelialization, neovascularization, collagen deposition and extracellular matrix remodeling, was monitored over time during experimentally induced skin repair in heterozygous connexin 43 knockout mice. RESULTS It was found that connexin 43 deficiency accelerates re-epithelialization and wound closure, increases proliferation and activation of dermal fibroblasts, and enhances the expression of extracellular matrix remodeling mediators. CONCLUSION These data substantiate the notion that connexin 43 may represent an interesting therapeutic target in dermal wound healing.

Monoacylglycerol lipase (MAGL) inhibition attenuates acute lung injury in mice.

Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for 2-Arachidonoylglycerol (2-AG), is mediated by monoacylglycerol lipase (MAGL). The piperidine carbamate, 4-nitrophenyl- 4-(dibenzo[d] [1,3]dioxol-5-yl (hydroxy) methyl) piperidine- 1-carboxylate (JZL184), is a drug that inhibits MAGL and presents high potency and selectivity. Thus, JZL184 increases the levels of 2-AG, an endocannabinoid that acts on the CB1 and CB2 cannabinoid receptors. Here, we investigated the effects of MAGL inhibition, with a single dose (16 mg/kg, intraperitoneally (i.p.)) of JZL184, in a murine model of lipopolysaccharide (LPS) -induced acute lung injury (ALI) 6, 24 and 48 hours after the inflammatory insult. Treatment with JZL184 decreased the leukocyte migration into the lungs as well as the vascular permeability measured through the bronchoalveolar lavage fluid (BAL) and histological analysis. JZL184 also reduced the cytokine and chemokine levels in the BAL and adhesion molecule expression in the blood and BAL. The CB1 and CB2 receptors were considered involved in the anti-inflammatory effects of JZL184 because the AM281 selective CB1 receptor antagonist (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and the AM630 selective CB2 receptor antagonist ([6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone) blocked the anti-inflammatory effects previously described for JZL184. It was concluded that MAGL inhibition, and consequently the increase in 2-AG levels, produced anti-inflammatory effects in a murine model of LPS-induced ALI, a finding that was considered a consequence of the activation of the CB1 and CB2 receptors.

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 (CCl4)‐induced hepatic fibrosis in mice. We administered CCl4 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 CCl4‐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 CCl4‐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. Microsc. Res. Tech., 2011.

Modulation of extracellular matrix by nutritional hepatotrophic factors in thioacetamide-induced liver cirrhosis in the rat.

Nutritional substances associated to some hormones enhance liver regeneration when injected intraperitoneally, being denominated hepatotrophic factors (HF). Here we verified if a solution of HF (glucose, vitamins, salts, amino acids, glucagon, insulin, and triiodothyronine) can revert liver cirrhosis and how some extracellular matrices are affected. Cirrhosis was induced for 14 weeks in 45 female Wistar rats (200 mg) by intraperitoneal injections of thioacetamide (200 mg/kg). Twenty-five rats received intraperitoneal HF twice a day for 10 days (40 mL.kg-1.day-1) and 20 rats received physiological saline. Fifteen rats were used as control. The HF applied to cirrhotic rats significantly: a) reduced the relative mRNA expression of the genes: Col-alpha1 (-53%), TIMP-1 (-31.7%), TGF-beta1 (-57.7%), and MMP-2 (-41.6%), whereas Plau mRNA remained unchanged; b) reduced GGT (-43.1%), ALT (-17.6%), and AST (-12.2%) serum levels; c) increased liver weight (11.3%), and reduced liver collagen (-37.1%), regenerative nodules size (-22.1%), and fibrous septum thickness. Progranulin protein (immunohistochemistry) and mRNA (in situ hybridization) were found in fibrous septa and areas of bile duct proliferation in cirrhotic livers. Concluding, HF improved the histology and serum biochemistry of liver cirrhosis, with an important reduction of interstitial collagen and increased extracelullar matrix degradation by reducing profibrotic gene expression.

Effects of long-term heat stress in an experimental model of avian necrotic enteritis

Stressful conditions are predisposing factors for disease development. Heat stress is one of the most important stressors in poultry production. The reemergence of some previously controlled diseases [e.g., avian necrotic enteritis (NE)] has been extensively reported. The combination of bacterial infection and certain environmental factors have been reported to trigger the disease. The aim of this study was to analyze the effects of long-term heat stress (35 ± 1°C) on the development of NE in broiler chickens. For this purpose, 60 male broiler chickens were divided into the following 6 groups: control group (C), heat stressed control group (C/HS35), thioglycolate group (T), thioglycolate heat-stressed group (T/HS35), infected group (I), and infected heat-stressed group (I/HS35). The poultry of groups I and I/HS35 were experimentally infected with Clostridium perfringens via their feed from 15 to 21 d of life. Heat stress (35 ± 1°C) was constantly applied to the birds of the stressed groups from 14 to 21 d of life. The infected and heat-stressed broiler chickens presented a trend toward a decrease in gross lesion scores and significantly lower microscopic scores of necrosis in the duodenum and jejunum (P < 0.05), lower fusion of villi in the duodenum (P < 0.05), and lower congestion scores in the jejunum and ileum (P < 0.05) in relation to infected and non-heat-stressed chickens. Broilers of I/HS35 group also exhibited small number of heterophils in the duodenum and jejunum compared with those of the I group (P < 0.05). Furthermore, the duodenum and jejunum of infected and heat-stressed broilers showed lower number of clostridia on the intestinal mucosa (P < 0.05). Data were discussed in light of a heat stress induced reduction on intestinal inflammation via a decrease in heterophil migration to the intestinal mucosa, which in turn might have reduced tissue damage during inflammation, hence preventing the development of a more severe form of NE.

Hypervitaminosis A-induced hepatic fibrosis in a cat

Rationale: The excessive intake of vitamin A in the form of vitamin concentrate, supplement or vitamin-rich liver can result in hypervitaminosis A in man and animals. Although osteopathologies resulting from chronic vitamin A intoxication in cats are well characterized, no information is available concerning feline hypervitaminosis A-induced liver disease. Clinical summary: We report the first case of hepatic stellate cell lipidosis and hepatic fibrosis in a domestic cat that had been fed a diet based on raw beef liver. Radiographic examination revealed exostoses and ankylosis between vertebrae C1 and T7, compatible with deforming cervical spondylosis. Necropsy showed a slightly enlarged and light yellow to bronze liver. Microscopic and ultrastructural analyses of liver tissues revealed diffuse and severe liver fibrosis associated with hepatic stellate cell hyperplasia and hypertrophy. These cells showed immunopositive staining for α-smooth muscle actin and desmin markers. The necropsy findings of chronic liver disease coupled with osteopathology supported the diagnosis of hypervitaminosis A. Practical relevance: As in human hepatology, if there is dietary evidence to support increased intake of vitamin A, then hypervitaminosis A should be considered in the differential diagnosis of chronic liver disease in cats.

Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice

Abstract Objective: Liver fibrosis results from the perpetuation of the normal wound healing response to several types of injury. Despite the wealth of knowledge regarding the involvement of intracellular and extracellular signaling pathways in liver fibrogenesis, information about the role of intercellular communication mediated by gap junctions is scarce. Methods: In this study, liver fibrosis was chemically induced by carbon tetrachloride in mice lacking connexin32, the major liver gap junction constituent. The manifestation of liver fibrosis was evaluated based on a series of read-outs, including collagen morphometric and mRNA analysis, oxidative stress, apoptotic, proliferative and inflammatory markers. Results: More pronounced liver damage and enhanced collagen deposition were observed in connexin32 knockout mice compared to wild-type animals in experimentally triggered induced liver fibrosis. No differences between both groups were noticed in apoptotic signaling nor in inflammation markers. However, connexin32 deficient mice displayed decreased catalase activity and increased malondialdehyde levels. Conclusion: These findings could suggest that connexin32-based signaling mediates tissue resistance against liver damage by the modulation of the antioxidant capacity. In turn, this could point to a role for connexin32 signaling as a therapeutic target in the treatment of liver fibrosis.

Modelo de suplementação nutricional com fatores hepatotróficos aumenta proliferação celular em fígado de ratos sadios

ABSTRACT Two protocols of hepatotrophic factors (HF) administration, in solution composed by aminoacids, vitamins, mineral salts, glucose, insulin, glucagon, and triiodothyronine were evaluated in healthy rats. This solution was administered for 10 days, (40mg/kg/day) i.p., in two (group 2xFH; n=15) or three daily doses (group 3xFH n=15). The effects on hepatocytes cell proliferation, angiogenesis, and hepatic extracellular matrix, and also possible adverse reactions were analyzed. Animals of groups 2xFH and 3xFH presented an increase in hepatic mass of 30.1% and 22.5%, respectively, when compared rats of control group (CT; n=15). Hepatocellular proliferation index was higher in rats of groups 2xFH (1.4%) and 3xFH (1.2%) when compared to CT group animals (0.53%), and the relative densitometry of the vascular endothelial growth factor analyzed with immunoblot did not show a significant difference among the three groups. Rats of groups 2xFH and 3xFH showed a reduction of interstitial collagen when compared to CT rats. HF solution stimulated hepatic growth and reduced the volume of perisinusoidal collagen. Administration in three daily doses resulted in 26.7% mortality, possibly due to excessive stress from manipulation and lower physiological adaptation of rats, which did not occur in rats of groups 2xFH and CT. The more appropriate and safer experimental procedure for this approach in rats with higher chance of animal adaptation and significant results is the application of HF in two daily doses. Keywords: rat, collagen, hepatotrophic factors, experimental model, parenteral nutrition