Latexin expression correlated with mineralization of articular cartilage during progression of post-traumatic osteoarthritis in a rat model.

As latexin has been linked with chondrocyte hypertrophic differentiation it is possible that this protein may also be involved in the mineralization of cartilage in OA. Therefore, we correlated latexin expression with the mineralization marker, alkaline phosphatase and determined the mineral deposition in the articular cartilage by analyzing the Ca/P ratio and the collagen fibrils pattern, during the progression of post-traumatic OA in a rat model. OA was induced by medial meniscectomy and post-surgery exercise for 5, 10, 20 and 45 days. Protein expression in articular cartilage was evaluated by immunofluorescence, histochemistry and Western blot. Minerals and structure of collagen fibrils in the superficial zone of cartilage were analyzed by energy dispersive X-ray spectroscopy (EDX) and atomic force microscopy (AFM) respectively. Protein expression analysis showed time-dependent up-regulation of latexin during OA progression. In the cartilage, latexin expression correlated with the expression and activity of alkaline phosphatase. EDX of the superficial zone of cartilage showed a Ca/P ratio closer to theoretical values for basic calcium phosphate minerals. The presence of minerals was also analyzed indirectly with AFM, as the collagen fibril pattern was less evident in the mineralized tissue. Latexin is expressed in articular cartilage from the early stages of post-traumatic OA; however, minerals were detected after latexin expression was up-regulated, indicating that its activity precedes and remains during the pathological mineralization of cartilage. Thus, our results contribute to the identification of molecules involved in the mineralization of articular chondrocytes.

Ultrastructural and biochemical basis for hepatitis C virus morphogenesis.

Chronic infection with HCV is a leading cause of cirrhosis, hepatocellular carcinoma and liver failure. One of the least understood steps in the HCV life cycle is the morphogenesis of new viral particles. HCV infection alters the lipid metabolism and generates a variety of microenvironments in the cell cytoplasm that protect viral proteins and RNA promoting viral replication and assembly. Lipid droplets (LDs) have been proposed to link viral RNA synthesis and virion assembly by physically associating these viral processes. HCV assembly, envelopment, and maturation have been shown to take place at specialized detergent-resistant membranes in the ER, rich in cholesterol and sphingolipids, supporting the synthesis of luminal LDs-containing ApoE. HCV assembly involves a regulated allocation of viral and host factors to viral assembly sites. Then, virus budding takes place through encapsidation of the HCV genome and viral envelopment in the ER. Interaction of ApoE with envelope proteins supports the viral particle acquisition of lipids and maturation. HCV secretion has been suggested to entail the ion channel activity of viral p7, several components of the classical trafficking and autophagy pathways, ESCRT, and exosome-mediated export of viral RNA. Here, we review the most recent advances in virus morphogenesis and the interplay between viral and host factors required for the formation of HCV virions.

Regulation of α5 and αV Integrin Expression by GDF-5 and BMP-7 in Chondrocyte Differentiation and Osteoarthritis.

The Integrin ß1 family is the major receptors of the Extracellular matrix (ECM), and the synthesis and degradation balance of ECM is seriously disrupted during Osteoarthritis (OA). In this scenario, integrins modify their pattern expression and regulate chondrocyte differentiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-ß) Superfamily, such as Growth differentiation factor 5 (Gdf-5) and Bone morphogenetic protein 7 (Bmp-7), play a key role in joint formation and could regulate the integrin expression during chondrocyte differentiation and osteoarthritis progression in an experimental OA rat model. Decrease of α5 integrin expression in articular cartilage was related with chondrocyte dedifferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressed αV integrin and was increased in the articular cartilage of rats with OA. Integrin expression during chondrocyte differentiation was also analyzed in a micromass culture system of mouse embryo mesenchymal cells, micromass cultures was treated with Gdf-5 or Bmp-7 for 4 and 6 days, respectively. Gdf-5 induced the expression of the α5 sub-unit, while Bmp-7 induced the expression of the αV sub-unit. This suggests a switch in signaling for prehypertrophic chondrocyte differentiation towards hypertrophy, where Gdf-5 could maintain the articular chondrocyte phenotype and Bmp-7 would induce hypertrophy. Decrease of Ihh expression during late stages of OA in rat model suggest that the ossification in OA rat knees and endochondral ossification could be activated by Bmp-7 and αV integrin in absence of Ihh. Thus, chondrocyte phenotype in articular cartilage is similar to prehypetrophic chondrocyte in growth plate, and is preserved due to the presence of Indian hedgehog (Ihh), Gdf-5 and α5 integrin to maintain articular cartilage and prevent hypertrophy.

Exercise modulates the expression of IL-1ß and IL-10 in the articular cartilage of normal and osteoarthritis-induced rats.

After a joint lesion, high-impact exercise is a risk factor for the development of osteoarthritis (OA). The degradation of articular cartilage in OA has been associated with the activation of inflammatory cytokine signaling pathways. However, differences in cytokine expression in healthy and injured cartilage after exercise have not yet been analyzed. We used immunofluorescence and Western blot to study the expression of IL-1ß and IL-10 in the articular cartilage of normal (N), sham-operated (S), and menisectomized (OA) rats subjected or not to high-impact exercise (E) for 3, 6, and 10 days (N, NE, S, SE, and OA groups). Cartilage integrity and proteoglycan content were only affected in the OA groups. Exercise increased the amount of IL-1ß and IL-10 positive chondrocytes in NE and SE groups compared with non-exercised groups (N and S). The expression of IL-1ß was up-regulated over time in the NE and OA groups, although in the late stages the increase was higher in the OA groups. In contrast, the expression of anti-inflammatory IL-10 was low in the OA group, whereas in the NE groups expression levels were higher at each time point analyzed. These results suggest that anti- and pro-inflammatory molecules in the cartilage might be tightly regulated to maintain the integrity of the tissue and that when this equilibrium is broken (when the meniscus is removed), the pro-inflammatory cytokines take over and OA develops.

Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus.

Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy.

Identification of exocytotic membrane proteins, syntaxin-1 and SNAP-25, in Entamoeba histolytica from hamster liver.

Entamoeba histolytica is a protozoan parasite causing dysentery and in some cases liver abscesses. These effects have been attributed to cytolytic substances released by exocytosis. In this study, the presence of the proteins syntaxin-1 and SNAP-25, which are assumed to be involved in exocytosis, were examined by immunohistochemistry, immunoelectron microscopy and western blot analysis. Syntaxin-1 and SNAP-25 were expressed in the vesicular, vacuolar and plasma membranes of E. histolytica trophozoites. It can be concluded that these proteins might be involved in exocytosis processes.

Ontogeny of rat chondrocyte proliferation: studies in embryo, adult and osteoarthritic (OA) cartilage.

The aim of this work was to study the ontogeny of chondrocyte cell division using embryo, adult and osteoarthritic (OA) cartilage. We searched for mitosis phases and performed a comparative evaluation of mitotic index, basic fibroblast growth factor b (FGFb), transforming growth factor beta1 (TGF-beta1) receptors, cyclin dependent kinase (CDK1) and Cyclin-B expression in fetal, neonate, 3, 5, 8 weeks old rats and experimental OA. Our results showed that mitosis phases were observed in all normal cartilage studied, although, we found a decrease in mitotic index in relation to tissue development. No mitosis was detected in OA cartilage. We also found a statistical significant reduction in cell number in OA cartilage, compared with the normal tissue. Furthermore, FGFb and TGF-beta1 receptors diminished in relation to tissue development, and were very scarce in experimental OA. Western blot assays showed CDK-1 expression in all cases, including human-OA cartilage. Similar results were observed for Cyclin-B, except for 8 weeks, when it was not expressed. Our results suggest that cell division seems to be scarce, if not absent within the OA cartilage studied. Nevertheless, the existence of factors essential for cell division leaves open the question concerning chondrocyte proliferation in OA cartilage, which is likely to be present in the early stages of the disease.

HCV core protein localizes in the nuclei of nonparenchymal liver cells from chronically HCV-infected patients.

Understanding the mechanism of hepatitis C virus (HCV) pathogenesis is an important part of HCV research. Recent experimental evidence suggests that the HCV core protein (HCcAg) has numerous functional activities. These properties suggest that HCcAg, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection. HCV is capable of infecting cells other than hepatocytes. Although the extrahepatic cellular tropism of HCV may play a role in the pathophysiology of this infection, the precise biological significance of the presence of HCV components in different liver cell types presently remains to be established. In this study, HCcAg was detected in nonparenchymal liver cells of six patients out of eight positive for serum HCV RNA. Immunostaining with anti-HCcAg mAbs revealed the presence of this protein in different liver cell types such as lymphocytes, Kupffer, polymorphonuclear, pit, endothelial, stellate, and fibroblast-like cells. Interestingly, HCcAg was immunolabeled not only in the cytoplasm but also in the nucleus of these cells. Remarkably, HCcAg co-localized with large lipid droplets present in stellate cells and with collagen fibers in the extracellular matrix. Moreover, HCcAg was immunolabeled in bile canaliculus suggesting the involvement of the biliary system in the pathobiology of HCV. Data suggest that nonparenchymal liver cells may constitute a reservoir for HCV replication. Besides, HCcAg may contribute to modulate immune function and fibrosis in the liver as well as steatosis.

A new role for chondrocytes as non-professional phagocytes. An in vitro study.

Chondrocytes are capable of engulfing latex particles, cell detritus, and necrotic and apoptotic remains in vitro. It is conceivable that chondrocytes might be involved in the clearance by phagocytosis of different materials within the cartilage. In fact, so far there is no evidence for the presence of "professional phagocytes" (macrophages and neutrophils) in this tissue. Chondrocyte suspensions obtained from rat knees and hips were cultured to assess phagocytosis of latex particles (1 microm), articular cartilage detritus, and necrotic and apoptotic chondrocyte remains (induced by VP-16 1 mM). We observed that chondrocytes phagocytosed latex particles as evaluated by confocal microscopy and flow cytometry. In addition, we observed that chondrocytes phagocytosed articular cartilage detritus and necrotic and apoptotic VP-16 induced-chondrocytes, as observed by bright field microscopy and transmission electron microscopy.

Lavado articular por punción versus artroscopia en el tratamiento de la osteoartritis de rodilla / Articular lavage by puncture versus arthroscopy in the treatment of knee osteoarthritis

Se sabe que el lavado articular y el debridamiento son opciones en el tratamiento de la osteoartritis de rodilla. Se realizó un estudio prospectivo, abierto y aleatorizado que incluyó 100 pacientes con OA de rodilla (criterio ACR) en estadios II y III de Kellgren y Lawrence para evaluar la utilidad y la eficacia del lavado articular por punción en comparación con el lavado y debridamiento artroscópico. Se distribuyeron en: grupo A (lavado por punción) y grupo B (lavado y debridamiento artroscópico). Se consideraron variables relacionadas con el dolor y la función articular que fueron analizadas al inicio del tratamiento y 90 d después; se realizó un análisis estadístico con el empleo del test de Chi cuadrado y el de t-Student, significación estadística p<0,05. Se observó que ambos grupos de pacientes experimentaron mejoría en todas las variables analizadas con las 2 variantes de tratamiento, sin diferencias estadísticamente significativas entre uno y otro grupo, los pacientes en un 90 por ciento aproximadamente de los casos experimentaron satisfacción con el proceder, sin ninguna complicación. Se concluyó que el lavado articular por punción y el lavado y debridamiento artroscópico mostraron ser útiles en el alivio de los síntomas en pacientes con OA ligera y moderada a los 3 meses de observación

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast.

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.

Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient.

Little is known about the life cycle of hepatitis C virus. Determination of the subcellular localization of HCV proteins may contribute to our understanding of the in vivo functions of the viral proteins. HCV core protein regulates multiple functions in host cells and it has been detected both in the cytoplasm and in the nucleus using different expression systems. In this study, nucleocapsid-like particles were observed in the nucleus of hepatocytes from a chronically HCV-infected patient. They were similar in size and shape to those of HCV core-like particles purified from recombinant Pichia pastoris cells. In addition the HCV core protein was detected not only in the cytoplasm but also in the nucleus and nucleolus of hepatocytes by immunoelectron microscopy. This is the first report showing nuclear localization of HCV core protein and nucleocapsid-like particles in hepatocytes during in vivo HCV infection.

Modifications of Golgi complex in chondrocytes from osteoarthrotic (OA) rat cartilage.

The status of the Golgi complex in normal vs osteoarthrotic (OA) cartilage has not yet been studied. A monoclonal antibody, MAb 58-K-9, allowed scoring of Golgi labeling intensity. In addition, ultrastructural assessment enabled us to focus on the distribution and relation between the endoplasmic reticulum (ER) and Golgi membranes. The study was performed in both normal and partially menisectomized OA-induced rat cartilage 20 and 45 days after surgery. Comparing Golgi immunolabeling intensities (mean +/- SEM) revealed a highly significant difference between normal (9.98 +/- 1.25), 20-day (2.49 +/- 0.34), and 45-day (0.82 +/- 0.22) cartilage. Moreover, chondrocytes from normal cartilage displayed 71.18% of labeling intensity in contrast to OA cartilage, in which chondrocyte labeling intensities were 24.95% (20 days) and 8.11% (45 days). OA chondrocytes appeared to display an overall reduction in Golgi labeling intensity, suggesting disruption of this organelle as the OA damage progressed. Interestingly, many 20-day OA-induced chondrocytes exhibited bubble-like Golgi immunolabeling compartmentalizing the cytoplasm, concomitant with putative apoptotic nuclear changes. At the same time, OA chondrocytes with a typical ultrastructural apoptotic pattern revealed a prominent ER gathered together with Golgi vesicles and saccules, also appearing to compartmentalize chondrocyte cytoplasm. We speculate about the role of Golgi modifications and apoptosis in OA pathogenesis.

Preservación de córneas para trasplante en medio de cultivo MCB / Corneal preservation for transplant in MCB culture

Se compara la viabilidad endotelial de córneas de conejo, conservadas en tres medios diferentes durante 48, 72 y 96 horas valoradas mediante la tinción con azul de tripano, microscopia de luz y microscopia electrónica. Material y métodos: Fueron analizadas 20 córneas divididas de la siguiente manera: ocho se conservaron en Optisol, 10 en MCB y dos en solución salina balanceada, mantenidas a 4o C. Resultados: En las córneas mantenidas en solución salina balanceada se observó destrucción de las células endoteliales a las 48 horas. En córneas preservadas en MCB y Optisol se apreció destrucción de un 5-10 por ciento de las células a las 72 horas. La destrucción fue del 20 por ciento después de 96 horas y de 30 por ciento luego de 105 horas en las células preservadas en el medio MCB. El estudio de microscopia electrónica de transmisión también confirmó que las células estructuralmente se encuentran preservadas hasta las 90 horas tanto en Optisol como en MCB. Conclusión: El medio de preservación para córneas producido en México (MCB) mantiene el tejido corneal hasta por 90 horas, de manera similar al Optisol, pero a un costo considerablemente menor.

Variability in the cell phenotype of aggregates or "clones" of human osteoarthritic cartilage. A case report

Human samples of articular cartilage from the knee of a clinically classified osteoarthritic patient, assessed by arthroscopy as part of the surgical treatment was studied by light and transmission electron microscopy. This particular case differed from others already reported in the variability of cell phenotype within the aggregates or "clones" frequently present in the osteoarthritic cartilage. The most common morphology of "clonal" cells forming the aggregates were large and rounded with an euchromatic nucleus. The cytoplasm was characterized by the presence of alternately clear and dense sites. At the ultrastructural level it was seen that the clear sites were formed by disrupted intermediates filaments and small particles, and that the dense sites were constituted by the segregation of different organelles of the chondrocytes. In addition, there were atypical aggregates composed only by secretory cells or by degenerating chondrocytes. Furthermore, a complex structure consisting of a very large cell inside a giant lacunae delimited by electron-dense material with small vesicles is described as a novel finding. The variability in the chondrocyte phenotype of the aggregates described here could be an indication of a better prognosis; nevertheless, the follow-up of the evolution of this patient is needed in order to know the final outcome.

Variability in the cell phenotype of aggregates or "clones" of human osteoarthritic cartilage. A case report

Human samples of articular cartilage from the knee of a clinically classified osteoarthritic patient, assessed by arthroscopy as part of the surgical treatment was studied by light and transmission electron microscopy. This particular case differed from others already reported in the variability of cell phenotype within the aggregates or "clones" frequently present in the osteoarthritic cartilage. The most common morphology of "clonal" cells forming the aggregates were large and rounded with an euchromatic nucleus. The cytoplasm was characterized by the presence of alternately clear and dense sites. At the ultrastructural level it was seen that the clear sites were formed by disrupted intermediates filaments and small particles, and that the dense sites were constituted by the segregation of different organelles of the chondrocytes. In addition, there were atypical aggregates composed only by secretory cells or by degenerating chondrocytes. Furthermore, a complex structure consisting of a very large cell inside a giant lacunae delimited by electron-dense material with small vesicles is described as a novel finding. The variability in the chondrocyte phenotype of the aggregates described here could be an indication of a better prognosis; nevertheless, the follow-up of the evolution of this patient is needed in order to know the final outcome.(AU)

Estimulación de los receptores de macrofagos en bajas concentraciones iónicas de Na+, K+, Ca++ y Mg++: potenciales de transmembrana / Stimulation of macrophages receptors in low ionic concentrations of Na+, K+, Ca++, Mg++: transmembrane potentials

Las células mononucleares fagocíticas obtenidas de la cavidad peritoneal de la rata, cambian las magnitudes de los potenciales de transmembranas de acuerdo con el estímulo fagocítico y en relación con el receptor estimulado. La incubación de estas células en medios iónicos, donde indistintamente se les bajó las concentraciones extracelulares de Na+, K+, Ca++ y Mg++ mostraron despolarizaciones de la membrana antes de inducir la fagocitosis que estimularon los receptores específicos Fc con latex cubierto con inmunoglobulinas IgG en los medios bajos Na+, Ca+, y Mg++, no se modificaron para los medios de K+. El estímulo fagocítico; provocó hiperpolarizaciones de la membrana; para todos los cambios iónicos pero fueron de menor magnitud para los decrementos de Na+, Ca+, y Mg++, respecto a lo registrado en las soluciones tyrode normales. Los tiempos en que ocurren estos cambios de PT también se modifican. Estos resultados muestran la importancia de las concentraciones iónicas extracelulares en la capacidad fagocítica de los macrófagos