Different cellular and immunohistochemical abdominal wall cicatrization parameters evaluation in comparison with sublay, onlay, and ipom technique in an experimental rat model.

PURPOSE: Incisional hernia (IH) occurs when there is a partial or complete solution of continuity of a fascia previously incised. Systematic reviews demonstrate that surgical treatment of IHs with the use of meshes are approximately 16%. Meta-analyses have demonstrated the superiority of mesh placement using sublay technique, but without a pathophysiological explanation. Thus, we aim to evaluate the different techniques of mesh positioning in an experimental model. METHODS: Fifty rats were distributed into five groups; control; simulation (SM)-submitted to laparotomy only; onlay-the mesh was positioned in onlay fashion; retromuscular (SL)-the mesh was positioned in a sublay fashion; intraperitoneal (IPOM)-positioning of the mesh adjacent to the transversalis fascia, inside the cavity. After 60 days, adhesions, tensiometry, histology, and immunohistochemistry were addressed. RESULTS: The IPOM group had the most adhesions, together with the SL group, with significantly relevant results. The SL group had higher values of tensiometric evaluation, while the IPOM group had the lowest mean in the tensiometry evaluation, being even lower than the SM group. Regarding histological and immunohistochemical findings, the SL group had a higher pixel number count compared to the groups, with statistical significance, in addition to higher expression of polymorphonuclear infiltrate and CD68 markers. CONCLUSION: The mesh positioning in sublay compartment is associated with the development of more pronounce minimum tensile force required for detaching the surrounding abdominal wall tissues it was incorporated. The intensity of these findings correlates to the different histological and immunohistochemical profiles observed following each repair, since SL group was characterized by a higher proportion of collagen, inflammatory, and reparative elements. Characterizing these pro-healing elements and its counterparts will allow the development of new therapeutic tools which could be added to the still far-from-ideal current therapeutic options for IH treatment.

The follicular thyroid cell line PCCL3 responds differently to laminin and to polylaminin, a polymer of laminin assembled in acidic pH.

The extracellular-matrix protein laminin forms polymers both in vivo and in vitro. Acidification of pH leads to the formation of an artificial polymer with biomimetic properties, named polylaminin (polyLM). Follicle cells in the thyroid are in close contact with laminin, but their response to this important extracellular signal is still poorly understood. PCCL3 thyroid follicular cells cultured on glass, on regular laminin (LM) or on laminin previously polymerized in acidic pH (polyLM) showed different cell morphologies and propensities to proliferate, as well as differences in the organization of their actin cytoskeleton. On polyLM, cells displayed a typical epithelial morphology and radially organized actin fibers; whereas on LM, they spread irregularly on the substrate, lost cell contacts, and developed thick actin fibers extending through the entire cytoplasm. Iodide uptake decreased similarly in response to both laminin substrates, in comparison to glass. On both the LM and polyLM substrates, the expression of the sodium iodide symporter (NIS) decreased slightly but not significantly. NIS showed dotted immunostaining at the plasma membrane in the cells cultured on glass; on polyLM, NIS was observed mainly in the perinuclear region, and more diffusely throughout the cytoplasm on the LM substrate. Additionally, polyLM specifically favored the maintenance of cell polarity in culture. These findings indicate that PCCL3 cells can discriminate between LM and polyLM and that they respond to the latter by better preserving the phenotype observed in the thyroid tissue.

Low somatostatin receptor subtype 2, but not dopamine receptor subtype 2 expression predicts the lack of biochemical response of somatotropinomas to treatment with somatostatin analogs.

OBJECTIVES: To evaluate somatostatin receptor 2A (SSTR2A) and dopamine receptor 2 (DR2) protein expression in somatotropinomas and to relate it to response to somatostatin analogues (SA). DESIGN AND PATIENTS: SSTR2A and DR2 expression was analyzed by immunohistochemistry in 88 somatotropinomas from patients submitted to either pre-surgical or adjuvant SA treatment. Tumors were scored according to percentage of immunostained cells: 0 (< 25%), 1 (25-50%), and 2 (> 50%). Relation between protein expression and response to SA was performed in 66 patients. Response to SA was assessed by percent IGF-I reduction, being considered as an IGF-I per cent reduction higher than 50%. Disease control was also assessed (GH < 1.0 ng/ml and normal IGF-I). RESULTS: SSTR2A and DR2 were expressed in 100% and 98% of tumors, respectively. Biochemical response and disease control rates were 48% and 32%, respectively. Median IGF-I percent reduction after 3 months of SA treatment was lower in the SSTR2A score 0 than in the scores 1 and 2 (p < 0.001, both), and after 6 months in the score 0 than in the score 1 (p = 0.001) and 2 (p < 0.001). Biochemical response and disease control were associated with SSTR2 expression (p < 0.001 and p = 0.004, respectively). A negative predictive value for biochemical response of 100% was found when a SSTR2A expression < 25%of immunostained cells cut-off point was considered. No relation was found between DR2 expression and biochemical response and disease control. CONCLUSION: SSTR2A and DR2 are highly expressed in somatotropinomas. Low SSTR2A, but not DR2, expression is a negative predictive factor to response to SA.

Validation of immunohistochemistry for somatostatin receptor subtype 2A in human somatotropinomas: comparison between quantitative real time RT-PCR and immunohistochemistry.

Somatostatin receptors subtype 2 (SSTR2) expression in somatotropinomas is recognized as a predictor of response to the currently available somatostatin analogs and may be analyzed, mainly, by quantitative RT-PCR or immunohistochemistry (IHC). The former has the advantages of a higher sensitivity and of being quantitative, while the latter, although semi-quantitative, evaluates protein expression and is routinely used in the evaluation of pituitary adenomas. We aimed to evaluate the SSTR2A protein expression in somatotropinomas and to compare it to our previous data regarding mRNA expression, assessed by quantitative real time RTPCR. Thirteen somatotropinomas were analyzed by IHC and the tumors were scored according to percent of immunostained cells: 0 (<25%), 1 (25-50%) and 2 (>50%). SSTR2A immunostaining was present in all but one somatotropinoma, 4 (31%) tumors were classified as score 0, 4 (31%) as score 1, and 5 (38%) as score 2. Median SSTR2 mRNA content was significantly different among the three IHC scores (p=0.036) and was lower in the score 0 than in the score 2 (p=0.016). The finding that there is a positive correlation between RT-PCR and IHC indicates that IHC can be applied in order to assess the SSTR2A content in somatotropinomas.

The role of P2X7 purinergic receptors in inflammatory and nociceptive changes accompanying cyclophosphamide-induced haemorrhagic cystitis in mice.

BACKGROUND AND PURPOSE: ATP is released in response to cellular damage, and P2X7 receptors have an essential role in the onset and maintenance of pathological changes. Haemorrhagic cystitis (HC) is a well-known adverse effect of therapy with cyclophosphamide used for the treatment of many solid tumours and autoimmune conditions. Here we have evaluated the role of P2X7 receptors in a model of HC induced by cyclophosphamide. EXPERIMENTAL APPROACH: Effects of pharmacological antagonism or genetic deletion of P2X7 receptor on cyclophosphamide-induced HC in mice was assessed by nociceptive and inflammatory measures. In addition, the presence of immunoreactive P2X7 receptors was assessed by immunohistochemistry. KEY RESULTS: Pretreatment with the selective P2X7 receptor antagonist A-438079 or genetic ablation of P2X7 receptors reduced nociceptive behaviour scores in the HC model. The same strategies decreased both oedema and haemorrhage indices, on macroscopic or histological evaluation. Treatment with A-438079 decreased the staining for c-Fos in the lumbar spinal cord and brain cortical areas. Treatment with A-438079 also prevented the increase of urinary bladder myeloperoxidase activity and macrophage migration induced by cyclophosphamide and reduced the tissue levels of IL-1ß and TNF-α. Finally, P2X7 receptors were markedly up-regulated in the bladders of mice with cyclophosphamide-induced HC. CONCLUSIONS AND IMPLICATIONS: P2X7 receptors were significantly involved in a model of HC induced by cyclophosphamide. Pharmacological inhibition of these receptors might represent a new therapeutic option for this pathological condition.

Patient's cells colonize the biofilm of Tenckhoff catheters used in peritoneal dialysis.

Peritoneal dialysis (PD) is a renal substitutive therapy based on the infusion of a dialysate in the peritoneum, which induces through an osmotic gradient the ultrafiltration of water and the clearance of blood stream impurities by the peritoneal membrane. The colonization of Tenckhoff catheters (TCs) used in PD by pathogenic microorganisms can lead to peritonitis, and probably catheter removal. Here, optical microscopy and scanning electron microscopy were applied to study biofilm formation in 11 TCs. Biofilms varied in their morphology and thickness. Short-term catheters (6 months) presented thinner deposits (3 µm) with granular or flat morphologies, either on the intraluminal or external surfaces. Bacterial colonies were found on catheters from infected patients. A tendency was observed for long-term catheters (6-8 years) to present thicker biofilms (30-35 µm). Surprisingly, patients' cells colonized the deep layers of the thicker biofilms, forming a complex multicelullar community. It was concluded that the presence of a biofilm is not necessarily related with peritonitis, and biofilm features may correlate to the therapy time.

Bone marrow-derived mononuclear cell therapy attenuates silica-induced lung fibrosis.

This study tests the hypothesis that bone marrow-derived mononuclear cell (BMDMC) therapy may reduce lung inflammation and fibrosis leading to an improvement in respiratory mechanics in a murine model of silicosis. 52 female C57BL/6 mice were randomly assigned into four groups. In the silica group (SIL), silica suspension (20 mg/50 µL in saline) was intratracheally instilled. In the control animals, 50 µL saline was administered intratracheally. At 1 h, the control and SIL groups were further randomised, receiving BMDMC (2×106 i.v. control-cell and SIL-cell) or saline (50 µL i.v. control and SIL). BMDMC were obtained from male donor mice. At day 15, lung mechanics, histology, and the presence of Y chromosome, interleukin (IL)-1ß, IL-1α, IL-1 receptor antagonist (IL-1RN), IL-1 receptor type 1, transforming growth factor (TGF)-ß and caspase-3 mRNA expressions in lung tissue were analysed. In the SIL-cell group, the fraction area of granuloma, the number of macrophages and the collagen fibre content were reduced, yielding improved lung mechanics. The presence of male donor cells in lung tissue was not confirmed using detection of Y chromosome DNA. Nevertheless, caspase-3, IL-1ß, IL-1α, IL-1RN and TGF-ß mRNA expression diminished after cell therapy. In conclusion, BMDMC acted on inflammatory and fibrogenic processes improving lung function through paracrine effects.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats.

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5%, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 +/- 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 +/- 0.02 cm, P < 0.01) vs control (0.16 +/- 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 +/- 2.64%, P < 0.001; EtOH + CCl4/8wR: 10.4 +/- 1.36%, P < 0.05) vs control (2.2 +/- 1.21%). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 +/- 1.3%, P < 0.01; collagen III: 1.3 +/- 0.2%, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 +/- 0.06%, P < 0.05; collagen III: 1.5 +/- 0.8%, P < 0.01) vs control (collagen I: 0.38 +/- 0.11%; collagen III: 0.25 +/- 0.06%). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 +/- 8%, P < 0.01; EtOH + CCl4/8wR: 58.8 +/- 21%, P < 0.01) vs control (7.9 +/- 0.8%). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.

An ultrasound and histomorphological analysis of experimental liver cirrhosis in rats

We investigated whether liver injury by dual exposure to ethanol and carbon tetrachloride (EtOH + CCl4) for 15 weeks would persist after hepatotoxic agents were removed (EtOH + CCl4/8wR). After 15 weeks of hepatic injury with ethanol (5.5 percent, m/v) and carbon tetrachloride (0.05, mL/kg, ip), 5 of 11 female Wistar rats were sacrificed. The other 6 rats were maintained for an additional 8 weeks without hepatotoxic agents. Ultrasonography showed increased liver echogenicity and dilation of portal vein caliber in both groups (EtOH + CCl4: 0.22 ± 0.01 cm, P < 0.001; EtOH + CCl4/8wR: 0.21 ± 0.02 cm, P < 0.01) vs control (0.16 ± 0.02 cm). Histopathology showed regenerative nodules in both experimental groups. Histomorphometry revealed increased fibrosis content in both groups (EtOH + CCl4: 12.6 ± 2.64 percent, P < 0.001; EtOH + CCl4/8wR: 10.4 ± 1.36 percent, P < 0.05) vs control (2.2 ± 1.21 percent). Collagen types I and III were increased in groups EtOH + CCl4 (collagen I: 2.5 ± 1.3 percent, P < 0.01; collagen III: 1.3 ± 0.2 percent, P < 0.05) and EtOH + CCl4/8wR (collagen I: 1.8 ± 0.06 percent, P < 0.05; collagen III: 1.5 ± 0.8 percent, P < 0.01) vs control (collagen I: 0.38 ± 0.11 percent; collagen III: 0.25 ± 0.06 percent). Tissue transglutaminase increased in both groups (EtOH + CCl4: 66.4 ± 8 percent, P < 0.01; EtOH + CCl4/8wR: 58.8 ± 21 percent, P < 0.01) vs control (7.9 ± 0.8 percent). Cirrhosis caused by the association of CCl4-EtOH remained for at least 8 weeks after removal of these hepatotoxic agents. Ultrasound images can be a useful tool to evaluate advanced hepatic alterations.

Expression of metalloproteinases and their tissue inhibitors in inflamed gingival biopsies.

OBJECTIVES: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are known to be involved in the periodontal disease process. Results of in vivo MMPs and TIMPs gene expressions in the gingiva, though, are still controversial. In the present study, we compared the gene expression of MMP-1, -2, -9, -13 and TIMP-1, -2 in healthy and inflamed gingiva. METHODS: 38 gingival samples were collected from gingivitis (n = 10), advanced chronic periodontitis (n = 10), generalized aggressive periodontitis (n = 8) and periodontally healthy individuals (n = 10). Total RNA isolated from those samples was subjected to reverse transcription followed by amplification by polymerase chain reaction (RT-PCR). Products were visualized in agarose gels and quantified by optical densitometry. Samples were also processed for gelatin zymography and Western blotting for MMP-2 and MMP-9 in order to assess for post-transcriptional MMP regulation at the protein level. RESULTS: The frequencies and levels of transcripts encoding MMPs and TIMPs were found to be not significantly different among groups (p > 0.05, Fisher's Exact and Kruskall-Wallis tests). There is a trend towards higher MMP-2 and -9 gelatinase activities in the inflamed samples, although not statistically significant. In contrast, zymography and Western blotting studies show that MMP-2 is virtually absent in the chronic periodontitis group. CONCLUSION: These results could reflect a complex regulation of MMPs and TIMPs' gene expression in the course of gingival inflammation. They also reveal a great biological diversity even among individuals with similar periodontal status.

Kinetics of mobilization and differentiation of lymphohematopoietic cells during experimental murine schistosomiasis in galectin-3 -/- mice.

Galectin-3 (gal-3), a beta-galactoside-binding animal lectin, plays a role in cell-cell and cell-extracellular matrix interactions. Extracellular gal-3 modulates cell migration and adhesion in several physiological and pathological processes. Gal-3 is highly expressed in activated macrophages. Schistosoma mansoni eggs display a large amount of gal-3 ligands on their surface and elicit a well-characterized, macrophage-dependent, granulomatous, inflammatory reaction. Here, we have investigated the acute and chronic phases of S. mansoni infection in wild-type and gal-3(-/-) mice. In the absence of gal-3, chronic-phase granulomas were smaller in diameter, displaying thinner collagen fibers with a loose orientation. Schistosoma-infected gal-3(-/-) mice had remarkable changes in the monocyte/macrophage, eosinophil, and B lymphocyte subpopulations as compared with the infected wild-type mice. We observed a reduction of macrophage number, an increase in eosinophil absolute number, and a decrease in B lymphocyte subpopulation (B220(+/high) cells) in the periphery during the evolution of the disease in gal-3(-/-) mice. B lymphopenia was followed by an increase of plasma cell number in bone marrow, spleen, and mesenteric lymph nodes of the infected gal-3(-/-) mice. The plasma IgG and IgE levels also increased in these mice. Gal-3 plays a role in the organization, collagen distribution, and mobilization of inflammatory cells to chronic-phase granulomas, niches for extramedullary myelopoiesis, besides interfering with monocyte-to-macrophage and B cell-to-plasma cell differentiation.

The role of purinergic P2X7 receptors in the inflammation and fibrosis of unilateral ureteral obstruction in mice.

Receptors of the P2X7 type have been demonstrated in granulocytes, monocytes/macrophages, B and T lymphocytes, and have been involved in several cellular mechanisms including those related to inflammation and immunological response. This study attempted to investigate the role of these receptors on the inflammatory and fibrogenic response in the kidneys of unilateral ureteral obstruction (UUO), by using P2X7 knockout mice (-/-). C57Bl6 mice were submitted to left UUO and killed after 7 and 14 days. Histopathology using hematoxylin-eosin, periodic-acid Schiff and Sirius-red staining, immunohistochemistry for macrophages, myofibroblasts, transforming growth factor-beta (TGF-beta)1 and P2X7, and immunofluorescence for apoptotic cells (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling) were performed. Protocols were as follows: (1) control; (2) sham; (3) control P2X7 (-/-); (4) sham P2X7 (-/-); (5) UUO wild type (WT); (6) UUO P2X7 (-/-). Myofibroblasts and Sirius-red staining were significantly lower in UUO P2X7 (-/-) mice at days 7 and 14, compared to UUO WT. Kidneys from UUO P2X7 (-/-) mice showed reduced number of inflammatory cells at day 14 but not at day 7, compared to UUO WT. TGF-beta1 was less in UUO P2X7 (-/-) mice at days 7 and 14 when compared to UUO WT. Macrophage infiltration and tubular apoptosis were lower in UUO P2X7 (-/-) at day 14 but not at day 7, compared to UUO WT. P2X7 was expressed only in tubular epithelial cells at day 7 of UUO WT mice. These findings constitute the first evidence that P2X7 receptors are implicated in macrophage infiltration, collagen deposition and apoptosis in response to ureteral obstruction in mice.

Fructose-1,6 diphosphate as a protective agent for experimental ischemic acute renal failure.

Cold ischemia time is a risk factor for the development of acute renal failure in the immediate post-transplant period. In this study, we aimed to determine if intravenous fructose-1,6-diphosphate (FDP), given before nephrectomy, attenuates renal cell injury in a cold ischemia model. Male adult Wistar rats were subjected to infusion of either FDP 350 mg/kg (group F, n=6), an equal volume of 0.9% NaCl (group S, n=6), an equal volume/osmolality of mannitol (group M, n=6) or no infusion (group C, n=7). Kidneys were then perfused in situ with Collins solution and nephrectomy was performed. Other kidney slices were stored in Collins solution at 4 degrees C. Adenosine triphosphate (ATP) levels and lactate dehydrogenase (LDH) release were examined at 0, 24, 48 and 72 h. Other slices, obtained after 50 min immersion in Collins solution at 37 degrees C, were frozen for characterization of cytoskeletal preservation using phalloidin-FITC staining. Apical fluorescence intensity of proximal tubule cells, indicative of the F-actin concentration, was measured in a fluorescence microscope interfaced with computer image analysis system. Adenosine triphosphate levels, after up to 72 h of tissue incubation, were higher (P<0.05) in the FDP group when compared to other groups. In addition, LDH release was smaller (P<0.0001) in the FDP group. The F-actin concentration of proximal tubule cells cells was greater in the FDP group (P<0.0001). Results indicate that FDP is a useful tool to increase tissue viability in a rat kidney subjected to cold ischemia, by maintaining ATP cell content, decreasing LDH release and preventing microfilament disruption of proximal tubule cells.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration.

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies.

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4%), followed by genotype 3 (21.4%), and genotype 2 (7.2%). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4%), mild (57.2%), and moderate (21.4%). Viral RNA was detected in liver cells from nine patients (64.3%). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Liver injury and viremia in mice infected with dengue-2 virus.

The goal of this study was to test the feasibility of BALB/c mice as an experimental model in the study of dengue disease. BALB/c mice were intraperitoneal infected with DENV-2 obtained from a human patient. Histopathological analysis of infected animals revealed liver injury with viral antigens detection. In initial stages, the most prominent lesions were vacuolization and diffuse steatosis in hepatocytes. Serum levels of ALT and AST increased progressively, reaching the highest values 7 days p.i. and decreasing at the 14th day. Since levels of circulating virus were very low, viremia was analyzed in C6/36 cells. Virus presence was detected by ultrastructural analysis, confirmed by RT-PCR assays. Period of viremia was analyzed by flow cytometry with cells incubated with mouse-infected sera collected in different days, revealing peak virus levels at the 7th day p.i. All such data correlate to the development of the disease described in humans.

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4 percent), followed by genotype 3 (21.4 percent), and genotype 2 (7.2 percent). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4 percent), mild (57.2 percent), and moderate (21.4 percent). Viral RNA was detected in liver cells from nine patients (64.3 percent). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.

Histopathological aspects of Dengue-2 virus infected mice tissues and complementary virus isolation.

The difficulty in studying dengue virus (DENV) infection in humans and in developing a virus vaccine is the absence of a suitable animal model which develops the full spectra of the Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS). Despite the fact that viruses have been found in various animal tissues, we isolated DENV from tissues of adult BALB/c mice, inoculated with DENV serotype 2 (DENV-2) obtained from human serum. Viruses were ultrastructurally identified and immunolocalized by immunofluorescence techniques in C6/36 mosquito cell cultures, inoculated with tissues (liver, lung, kidney and cerebellum) macerate supernatant from mice, 48 h post-infection (p.i.). These organs, collected at the same stage of infection, were examined histologically. The histopathological analysis revealed focal alterations in all tissues examined. Liver contained focal ballooned hepatocytes, but without modifying the average diameter of the majority of hepatocytes. Sinusoidal lumen was significantly diminished at this stage but portal and centrolobular veins became congested. Lungs exhibited hemorrhagic foci in the alveolar space, vascular congestion and focal alveolitis. Cerebellar tissue showed rare foci of neuronal compactation (Purkinje cells) and perivascular oedema. In kidneys it was observed an increase in glomerular volume with augmented endocapillary and mesangial cellularity, with reactivity to anti-IgM in all glomeruli of infected mice. In conclusion, DENV-2 was found in all tissues examined early in the evolution of infection. Presence of viruses in tissues has mainly led to hemodynamic alterations with generalized vascular congestion and increased permeability, and mast cell recruitment in lungs. The latter could participate in the vascular modifications in tissues.