Carlos Thadeu Schmidt Cerski

Acute and Subchronic Toxicity Evaluation of Poly(ɛ-Caprolactone) Lipid-Core Nanocapsules in Rats

Owing to concerns over the effects of the physicochemical properties of nanoparticles and their interaction with biological systems, further investigation is required. We investigated, for the first time, the toxicity of lipid-core nanocapsules (LNCs) containing a polymeric wall of poly(ε-caprolactone) and a coating of polysorbate 80 used as drug delivery devices (~245nm) in Wistar rats after single- and repeated-dose treatments. The suspensions were prepared by interfacial deposition of the polymer and were physicochemically characterized. Toxicological effects were determined after single doses of 18.03, 36.06, and 72.12 × 10(12) LNC/kg and repeated doses of 6.01, 12.02, and 18.03 × 10(12) LNC/kg for 28 days by ip administration. The results for both the treatments showed no mortality or permanent body weight changes during the experiments. A granulomatous foreign body reaction was observed in the liver and spleen of higher dose groups in acute and subchronic treatments. Most of the hepatotoxicity and nephrotoxicity markers were within the reference values and/or were similar to the control group. However, a slight alteration in the hematologic parameters was observed in both the studies. Thus, to verify a possible methodological influence, we performed an in vitro test to confirm such influence. These findings are in agreement with earlier reports regarding no appreciable toxicity of biodegradable polymeric nanoparticles, indicating that LNC might be a safe candidate for drug delivery system. Furthermore, the results presented in this study are important for health risk assessment and to implement strategies for testing biodegradable polymeric nanoparticles.

Melatonin Activates Endoplasmic Reticulum Stress and Apoptosis in Rats with Diethylnitrosamine-Induced Hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence, its metastatic potential and the low efficacy of conventional treatment. Inactivation of apoptosis is implicated in tumour progression and chemotherapy resistance, and has been linked to the presence of endoplasmic reticulum stress. Melatonin, the main product of the pineal gland, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects in HCC cells, but these effects still need to be confirmed in animal models. Male Wistar rats in treatment groups received diethylnitrosamine (DEN) 50 mg/kg intraperitoneally twice/once a week for 18 weeks. Melatonin was given in drinking water at 1 mg/kg/d, beginning 5 or 12 weeks after the start of DEN administration. Melatonin improved survival rates and successfully attenuated liver injury, as shown by histopathology, decreased levels of serum transaminases and reduced expression of placental glutathione S-transferase. Furthermore, melatonin treatment resulted in a significant increase of caspase 3, 8 and 9 activities, polyadenosine diphosphate (ADP) ribose polymerase (PARP) cleavage, and Bcl-associated X protein (Bax)/Bcl-2 ratio. Cytochrome c, p53 and Fas-L protein concentration were also significantly enhanced by melatonin. Melatonin induced an increased expression of activating transcription factor 6 (ATF6), C/EBP-homologous protein (CHOP) and immunoglobulin heavy chain-binding protein (BiP), while cyclooxygenase (COX)-2 expression decreased. Data obtained suggest that induction of apoptosis and ER stress contribute to the beneficial effects of melatonin in rats with DEN-induced HCC.

In vivo toxicological evaluation of polymeric nanocapsules after intradermal administration

Polymeric nanocarriers have shown great promise as delivery systems. An alternative strategy has been to explore new delivery routes, such as intradermal (i.d.), that can be used for vaccines and patch-based drug delivery. Despite their many advantages, there are few toxicity studies, especially in vivo. We report a safety assessment of biodegradable poly(ɛ-caprolactone) lipid-core nanocapsules (LNC) with a mean size of 245±10nm following single and repeated intradermal injections to Wistar rats. Suspensions were prepared by interfacial deposition of polymer. The animals (n=6/group) received a single-dose of saline solution (1.2ml/kg) or LNC (7.2×10(12)LNC/kg), or repeated-doses of two controls, saline solution or Tween 80 (0.9ml/kg), or three different concentrations of LNC (1.8, 3.6, and 5.4×10(12)LNC/kg) for 28 consecutive days. Clinical and physiological signs and mortality were observed. Samples of urine, blood, and tissue were used to perform toxicological evaluation. There were no clinical signs of toxicity or mortality, but there was a slight decrease in the relative body weights in the Tween 80-treated group (p<0.01) after repeated administration. No histopathological alterations were observed in tissues or significant changes in blood and urinary biomarkers for tissue damage. Mild alterations in white blood cells count with increases in granulocytes in the Tween-80 group (p<0.05) were found. Genotoxicity was evaluated through the comet assay, and no statistical difference was observed among the groups. Therefore, we conclude that, under the conditions of these experiments, biodegradable LNC did not present appreciable toxicity after 28 consecutive days of intradermal administration and is promising for its future application in vaccines and patch-based devices for enhancing the delivery of drugs.

Histopathological diagnosis of intra- and extrahepatic neonatal cholestasis

The histopathology of the liver is fundamental for the differential diagnosis between intra- and extrahepatic causes of neonatal cholestasis. However, histopathological findings may overlap and there is disagreement among authors concerning those which could discriminate between intra- and extrahepatic cholestasis. Forty-six liver biopsies (35 wedge biopsies and 11 percutaneous biopsies) and one specimen from a postmortem examination, all from patients hospitalized for neonatal cholestasis in the Pediatrics Service of Hospital de Clínicas de Porto Alegre, were prospectively studied using a specially designed histopathological protocol. At least 4 of 5 different stains were used, and 46 hepatic histopathological variables related to the differential diagnosis of neonatal cholestasis were studied. The findings were scored for severity on a scale from 0 to 4. Sections which showed less than 3 portal spaces were excluded from the study. Sections were examined by a pathologist who was unaware of the final diagnosis of each case. Bile tract permeability was defined by scintigraphy of the bile ducts and operative cholangiography. The F test and discriminant analysis were used as statistical methods for the study of the hepatic histopathological variables. The chi-square method with Yates correction was used to relate the age of the patients on the date of the histopathological study to the discriminatory variables between intra- and extrahepatic cholestasis selected by the discriminant function test. The most valuable hepatic histopathological variables for the discrimination between intra- and extrahepatic cholestasis, in decreasing order of importance, were periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, foci of myeloid metaplasia, and portal-portal bridges. The only variable which pointed to the diagnosis of intrahepatic cholestasis was myeloid metaplasia. Due to the small number of patients who were younger than 60 days on the date of the histopathological study (N = 6), no variable discriminated between intra- and extrahepatic cholestasis before the age of 2 months and all of them, except for the portal expansion, were discriminatory after this age. In infants with cholestasis, foci of myeloid metaplasia, whenever present in the liver biopsy, suggested intrahepatic cholestasis. Periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, portal cholestasis and portal-portal bridges suggested extrahepatic obstructive cholestasis.

O colágeno em fáscia transversal de pacientes com hérnia inguinal direta submetidos à videolaparoscopia

PURPOSE: To analyse in respect to thickness, constitutive elements and total collagen quantification, samples of transversalis fascia from direct inguinal hernia patients, between twenty and sixty years of age and colected at the moment of laparoscopic repair, compairing with samples of the same tissue, obtained from corpse. METHODS: We collected 23 samples from patients and 22 samples from corpse. The samples were stained for Hematoxiline-eosine and Sirius-Red. The video captured images were analysed by computer-assisted videomorphometric techenic. RESULTS: We noticed the thickness of normal transversalis fascia to be 4.5 milimetres. Otherwise, the transversalis fascia from patients were 58 % thinner (p< 0.001). There was no evidence of any senile degenerative process involving the collagen fibres. The Dense connective tissue was the principal constitutive element in both groups being responsable for 75 % of controls fascia and 49 % for the hernia one (p< 0.001). The collagens mean percentual area in the sample field from patients were half the controls area (p< 0.001), stablishing that there are less collagen in the hernias wall. CONCLUSION: The direct inguinal hernia patients have less collagen in transversalis fascia either absolutely or relatively to other constitutional elements.

Pharmacological Preconditioning Using Intraportal Infusion of L-Arginine Protects Against Hepatic Ischemia Reperfusion Injury

OBJECTIVE The present study examined the effects of intraportal infusion of L-arginine on ischemia/reperfusion injury (I/RI) in pig livers, by observing changes in the liver function, liver cell morphology, and changes in the mitochondrial ultrastructure. BACKGROUND The involvement of the nitric oxide (NO) pathway in the reperfusion-ischemic phenomenon is complex and not fully understood. Likewise, little is known about the possible benefit of intraportal infusion of L-arginine (substrate for the NO synthesis) on liver I/RI. METHODS A pig model consisting of 90 min of hepatic ischemia and 180 min of reperfusion was employed. Eighteen female hybrid pigs were randomly divided into three groups: sham-operated, non-preconditioned, and pharmacologically preconditioned group (intraportal infusion of L-arginine 400 mg/kg) 10 min before being subjected to ischemia and reperfusion. Serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), thiobarbituric acid reactive substances (TBARS), and the bile flow were measured. Liver biopsies were taken 180 min after reperfusion for histology, caspase-3 immunohistochemistry, and ultrastructural examination of mitochondria. RESULTS In the pharmacologically preconditioned group, we observed increased bile flow (P < 0.01) and improved serum AST levels (P < 0.01) relative to the non-preconditioned group. Serum concentrations of TBARS did not differ between the groups. Sinusoidal congestion (P = 0.02) was more evident in the non-preconditioned group than in the sham operated group. Infiltrating PMNs (P = 0.01) were more evident in the non-preconditioned group than in the sham and pharmacologically preconditioned group. The pharmacologically preconditioned group showed an approximately 2.5-fold decrease in caspase-3 activity relative to the non-preconditioned group (P < 0.01). Notably, damage to the mitochondrial ultrastructure in the pharmacologically preconditioned group was reduced relative to the other groups (P < 0.01). CONCLUSIONS Pharmacological preconditioning with intraportal L-arginine provided protection against hepatic I/RI in early phases of the reperfusion period. The mechanisms underlying the protective effect may include preservation of the mitochondrial structure and inhibition of caspase-3 activity.

Pancreas β-cells morphology, liver antioxidant enzymes and liver oxidative parameters in alloxan-resistant and alloxan-susceptible Wistar rats : a viable model system for the study of concepts into reactive oxygen species

The aim of this study was to investigate biochemical and antioxidant parameters in alloxan‐resistant (ALR) and alloxan‐susceptible (ALS) rats. Diabetes was induced in 60‐day‐old male Wistar rats by a single intraperitonial injection of alloxan (AL, 150 mg/kg). Ten days after induction, a group of rats showed a significant decrease in glycemia. This group was named alloxan‐resistant group. Susceptible rats showed a remarkable increase in the plasma lipid content, blood glucose and HbA1. Glycogen content in the liver decreased significantly in the ALS group (2.08 ± 0.41 mg%) compared with ALR group (4.22 ± 0.18). Aspartate aminotransferase and alanine aminotransferase activities were quantified in the plasma. Interestingly, ALR rats showed a decrease in both activities (42.1 ± 6.11 and 21.7 ± 5.54 U/mL) when compared with ALS rats (59.1 ± 6.55 and 58.1 ± 7.28 U/mL). The TBARS index was significantly increased in the ALS liver (0.38 ± 0.08 nm/mg protein) when compared with the ALR liver (0.18 ± 0.04). Superoxide dismutase and catalase activities in the ALR (230 ± 13 and 131 ± 15 U/mg protein) liver showed a marked increase when compared with the ALS liver (148 ± 13 and 68 ± 5 U/mg protein). The immunohistochemical and hematoxilin–eosin analysis also revealed that pancreatic islets of ALR rats display a different morphology amongst the groups. These results suggest an increased regenerative or recovery process in the ALR rat pancreatic islets and an increased hepatic antioxidant defenses in these group of alloxan‐resistant rats.

Spatial distribution of cancer stem cells in head and neck squamous cell carcinomas

BACKGROUND CD44 and aldehyde dehydrogenase 1 (ALDH1) are considered putative markers of highly tumorigenic cells (i.e., cancer stem-like cells) in head and neck squamous cell carcinomas. This small subset of cells is believed to be the primary responsible for tumor initiation and progression. The objectives of this study were (i) to evaluate the patterns of CD44 and ALDH1 expression in the tumor center and in the invasive front, as well as in adjacent non-tumor epithelium, and (ii) to correlate these findings with clinical parameters. MATERIALS AND METHODS The sample comprised 44 patients with primary head and neck squamous cell carcinomas. Hematoxylin and eosin (HE) staining was used for histopathological tumor grading and for morphological analysis of adjacent non-tumor epithelium. Semiquantitative analysis was performed in histological sections immunostained for CD44 and ALDH1. RESULTS ALDH1 immunostaining in the invasive front showed positive association with tumor size, regional metastasis, tumor histopathological grading, and disease progression. Moreover, expression of this marker in both tumor invasive front and adjacent non-tumor epithelium was related with more aggressive tumors. CD44 immunostaining was heterogeneous in all areas evaluated and did not show association with clinical data. CONCLUSION Collectively, these data suggest that ALDH1 immunostaining in the invasive front and in adjacent non-tumor epithelium may help identify tumors with a more aggressive behavior, potentially contributing to improving treatment customization and the monitoring of patients with head and neck cancer.

Relationship between serum concetrations of type III procollagen, hyluronic acid and histopathological findings in the liver of HCV-positive blood donors

RACIONAL: Marcadores sorologicos tem sido propostos para monitorar fibrose hepatica em doenca cronica do figado. Dentre os marcadores de fibrose, acido hialuronico e procolageno tipo III tem sido estudados nestes pacientes. OBJETIVO: Avaliar a associacao de marcadores sericos de fibrose com achados histologicos. METODOS: Foi realizado estudo transversal prospectivo em doadores de sangue anti-HCV positivos. A populacao estudada incluiu homens e mulheres com idade entre 18-60 anos com provas de funcao hepatica alteradas (niveis de alanina aminotransferase >1.5 vezes do normal e alteracoes de dois ou mais dos seguintes: qualquer alteracao nos niveis de alanina aminotransferase, aspartato aminotransferase, bilirrubina conjugada, gamaglobulina, gamaglutamiltranspeptidase, albumina, plaquetas, niveis de fosfatase alcalina >1,5 vezes o valor normal, tempo de protrombina abaixo de 70% e acima de 60%). Quarenta e nove pacientes foram submetidos a biopsia hepatica e coleta de sangue para analise de procolageno tipo III, acido hialuronico e provas funcionais hepaticas. RESULTADOS: Nao houve relacao entre elevacao de provas de funcao hepatica e a presenca de fibrose - ALT (>1,5 vezes acima do normal, risco de fibrose = 18,8%; <1,5 vezes, 11,8%). Procolageno tipo III elevado foi correlacionado com 66,7% chances de fibrose, enquanto nivel normal, 9,3%. Acido hialuronico, quando elevado, demonstrou chance de 33,3% de fibrose; quando normal, 12.5%. CONCLUSOES: Nao houve associacao entre provas de funcao hepatica, acido hialuronico e fibrose, mas houve entre esta ultima e procolageno tipo III. Talvez este marcador possa ser util para avaliar fibrose em pacientes com hepatite cronica pelo virus C.BACKGROUND Serologic markers have been proposed for monitoring hepatic fibrosis in chronic liver disease. Among fibrosis markers, type III procollagen (PIIIP) and hyaluronic acid have been studied in these patients. AIM To evaluate the association between these serum markers with histological findings. METHODS A prospective cross-sectional study was carried out with HCV-positive blood donors. The studied population included men and women whose age ranged from 18 to 60 years, with elevated liver function tests [ALT levels > 1.5 times the normal value and alterations of two or more of the following: any changes in the levels of ALT, aspartate aminotransferase, conjugated bilirrubin, gammaglobulin, gammaglutamyltranspeptidase, albumin, platelet count; alkaline phosphatase levels >1.5 times the normal value, or prothrombin time below 70% and above 60%]. Fourty-nine patients were submitted to liver biopsy, blood analysis of PIIIP, hyaluronic acid, besides liver function tests. RESULTS Liver function tests were not associated with tissular fibrosis, as assessed by ALT (>1.5 times above normal, fibrosis risk=18.8%; <1.5 times, 11.8%). Elevated PIIIP was correlated with 66.7% chance of fibrosis, whereas normal levels, 9.3%. Hyaluronic acid, when elevated, gave a chance of 33.3% of fibrosis; when normal, 12.5%. CONCLUSION There was no association between liver function tests, hyaluronic acid and fibrosis. However, PIIIP was related with liver fibrosis. Maybe, this marker should be useful to assess fibrosis in patients with chronic hepatitis C.

Oxidative stress and cell damage in a model of precancerous lesions and advanced hepatocellular carcinoma in rats

Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer deaths throughout the world. This study was aimed to analyze oxidative stress and cell damage in a multistage model of liver carcinogenesis induced by diethylnitrosamine (DEN) in rats. Male Wistar rats weighing 145–150 g were divided into three groups: control, precancerous lesions (PL) (which received 100 mg DEN once a week every 6 weeks up to 28 weeks), and advanced HCC (50 mg DEN once/twice per week up to 19 weeks). Lipid peroxidation (TBARS), superoxide dismutase (SOD) activity, and expression of transforming growth factor-1 beta (TGF)-1β, endothelial and inducible nitric oxide syntahese (eNOS, iNOS), NADPH quinone oxireductase (NQO)-1, nuclear factor erythroid 2-related factor (NrF)2, kelch-like ECH-associated protein (Keap)1 and heat shock protein (HSP)70 were measured. TBARS concentration was augmented in the PL and advanced HCC groups. SOD activity, TGF-1β and Nrf2 expression were higher in animals with precancerous lesions. In advanced HCC, expression of NQO1 and iNOS increased while there was a decrease in HPS70 expression. Data obtained provide evidence for the differential activation of proteins involved in oxidative stress and cell damage during progression of carcinogenesis in an animal model of HCC.