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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.

Temporomandibular joint (TMJ) is sensitive to loading and mechanical stress that provokes morphological changes produced by the impact in the occlusal plane. Here, this impact is evaluated in TMJ articular disc and articular cartilage using an in vivo model of unilateral occlusal plane impact and by analysis of serial tissue sections stained with Hematoxylin-Eosin (H-E) or with Masson trichrome technique. Thus, six groups of 5 Wistar rats (200 - 250 g) are subjected to biomechanical dental stimulation by placing unilateral resin occlusal interference, or unilateral tooth wear made by upper left molars artificial mechanical devastation (1 control and 2 experimental groups for each treatment). Each treatment is evaluated two times at 1 and 15 days post-treatment. By H-E staining, control groups show chondrocytes arrangement as several cord cell groups in comparison with the experimental groups, which show an arrangement in one cord cell along of articular disc. However, this yields no significant difference (p < 0.05) in cell number between control and experimental groups. In contrast, in articular cartilage chondrocytes are random distributed along the superficial zone in control groups, while in experimental groups cell-free regions are observed in superficial zone. An image Blue hue analysis for trichrome stain is performed to quantify collagen; this shows a significant collagen decrease (p < 0.05) in almost all experimental groups compared with the controls. A degenerative process biomechanically induced by unilateral occlusal plane modification, causes cell and tissue changes on the TMJ structures that remain the degenerative changes observed in early osteoarthritis.

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-1β 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-1β and IL-10 positive chondrocytes in NE and SE groups compared with non-exercised groups (N and S). The expression of IL-1β 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.

The balance between synthesis and degradation of the cartilage extracellular matrix is severely altered in osteoarthritis, where degradation predominates. One reason for this imbalance is believed to be due to the ligation of the α5β1 integrin, the classic fibronectin (FN) receptor, with soluble FN fragments instead of insoluble FN fibrils, which induces matrix metalloproteinase (MMP) expression. Our objective was to determine whether the lack of α5β1-FN binding influences cartilage morphogenesis in vivo and whether non-ligated α5β1 protects or aggravates the course of osteoarthritis in mice. We engineered mice (Col2a-Cre;Fn1RGE/fl), whose chondrocytes express an α5β1 binding-deficient FN, by substituting the aspartic acid of the RGD cell-binding motif with a glutamic acid (FN-RGE). At an age of 5 months the knee joints were stressed either by forced exercise (moderate mechanical load) or by partially resecting the meniscus followed by forced exercise (high mechanical load). Sections of femoral articular knees were analysed by Safranin-O staining and by immunofluorescence to determine tissue morphology, extracellular matrix proteins and matrix metalloproteinase expression. The articular cartilage from untrained control and Col2a-Cre;Fn1RGE/fl mice was normal, while the exposure to high mechanical load induced osteoarthritis characterized by proteoglycan and collagen type II loss. In the Col2a-Cre;Fn1RGE/fl articular cartilage osteoarthritis progressed significantly faster than in wild type mice. Mechanistically, we observed increased expression of MMP-13 and MMP-3 metalloproteinases in FN-RGE expressing articular cartilage, which severely affected matrix remodelling. Our results underscore the critical role of FN-α5β1 adhesion as ECM sensor in circumstances of articular cartilage regeneration.

We identify changes in the expression and localization of α5, α4, and α2 integrins during osteoarthritis (OA) pathogenesis in a rat experimental model. The changes were concomitant with variations in the extracellular matrix (ECM) content and the increase of metalloproteinases (MMPs) activity during OA pathogenesis, which were analyzed by immunofluorescence and Western blot assays. Our results showed an increased expression of α5 and α2 integrins at OA late stages, which was co-related with changes in the ECM content, as a consequence of the MMPs activity. In addition, this is the first report that has shown the presence of α4 integrin since OA early stages, which was co-related with the loss of proteoglycans and clusters formation. However, at late OA stages, the increased expression of α4 integrin in the middle and deep zones of the cartilage was also co-related with the abnormal endochondral ossification of the cartilage through its interaction with osteopontin. Finally, we conclude that ECM-chondrocytes interaction through specific cell receptors is essential to maintain the cartilage homeostasis. However, due to integrins cell signaling is ligand-dependent; changes in the ECM contents could induce activation of either anabolic or catabolic processes, which limits the reparative capacity of chondrocytes, favoring OA severity. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1161–1166, 2014.

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.

The death of chondrocytes and the loss of extracellular matrix are the central features in cartilage degeneration during Osteoarthritis (OA) pathogenesis. The mechanism by which chondrocytes are removed in OA cartilage are still not totally defined, although previous reports support the presence of apoptotic as well as non apoptotic signals. In addition, in 2004 Roach and co-workers suggested the term "Chondroptosis" to design the type of cell death present in articular cartilage, which include the presence of some apoptotic and autophagic processes. To identify the mechanisms, as well as the chronology by which chondrocytes are eliminated during OA pathogenesis, we decided to evaluate apoptosis (by active caspase 3 and TUNEL signal) and autophagy (by LC3II molecule and cytoplasmic vacuolization) using Immunohistochemistry and Western blot techniques in an animal OA model. During OA pathogenesis, chondrocytes exhibit modifications in their death process in each zone of the cartilage. At early stages of OA, the death of chondrocytes starts with apoptosis in the superficial and part of the middle zones of the cartilage, probably as a consequence of a constant mechanical damage in the joint. As the degenerative process progresses, high incidence of active caspase 3 as well as LC3II expression are observed in the same cell, which indicate a combination of both death processes. In contrast, in the deep zone, due the abnormal subchondral bone ossification during the OA pathogenesis, apoptosis is the only mechanism observed.

Despite of recent advances on acknowledge of hepatitis B virus (HBV) structure and biology, little is known about the morphogenesis and release of virions. In this study, the ultrastructural analysis of HBV components in liver biopsies from chronically HBV-infected patients disclosed complex assembly and morphogenesis pathways for HBV. HBV core (HBcAg) and surface (HBsAg) antigens were specifically identified in all liver biopsies from HVB-infected patients. HBcAg containing core-like particles 24-28 nm in diameter were observed both in nucleus and cytoplasm of hepatocytes. Dane’s-like particles were detected either budding to or into the lumen of ER close to HBsAg containing tubular structures. Besides, Dane’s-like particles were detected in different vesicular compartments resembling multivesicular endosomes. Other kind of enveloped HBV-like particles similar to the previously described cobra-shaped and horn-shaped particles were also observed in hepatocytes. Some of these particles were connected to the vesicle membrane through a stalk 20-22 nm in diameter. On the other hand, spherical subviral particles (SVP) were frequently observed in cytoplasmic vesicles. Moreover, a minor proportion of enveloped HBV-like particles budding through the plasma membrane to the extracellular space and bile canaliculi were detected. Interestingly, Dane’s-like particles in the bile canaliculi and entering into ductular-like cells were shown. Strikingly, Dane’s-like particles close to tubular structures and specific immunolabeling for HBcAg both in cytoplasm and nuclei of stellate-like cells were detected. Remarkably, HVB-like particles appeared entering hepatocytes from large cytoplasmic processes of fibrocytes raising the interesting possibility of a cell to cell passage of HBV through direct transcellular migration.

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.

The morphology of the normal human and rat articular cartilage was assessed using transmission electron microscopy (TEM), atomic force microscopy (AFM), and two-photon exci- tation (2PE) microscopy. Spurr-embedded sections from fixed human cartilage were simultaneously evaluated using TEM and AFM. The presences of tracks among the chondrocytes from the superficial zone of the cartilage were observed. In order to ratify the presence of interconnecting tracks among superficial zone chondrocytes, whole fixed human and rat cartilage, as well as fresh whole rat carti- lage, were examined under the 2PE. In all cases, these tracks were observed. In addition, porous ma- trix, well-defined lacunae, and cytoplasmic projections anchored to the extracellular matrix (ECM) were also detected. We conclude that normal human and rat flattened superficial chondrocytes might be interconnected by tracks running through the ECM. In addition, cytoplasmic projections were observed anchored to the ECM. All these structures may possibly be related to cell/cell and ECM/ chondrocytes signaling. Our findings provide new information that possibly will be of relevant impor- tance for a more profound study of normal cartilage physiology and eventually, the pathogenesis of osteoarthritis. Microsc. Res. Tech. 70:1072-1078, 2007. V C 2007 Wiley-Liss, Inc.

Recently, it has been shown that a truncated HCV core (HCcAg) variant, covering the first 120 aa (HCcAg.120), interacts with plasmid DNA vaccine (pIDKE2), encoding the HCV structural proteins (HCcAg, E1 and E2). In the present work, HCcAg.120-pIDKE2 complexes, forming heterogeneous packaged structures, were visualized using a negative stain/rotary shadow technique. Interestingly, 72 hours after intramuscular injection of HCcAg.120-pIDKE2 complexes in Balb/c mice, E2 protein was immunolabeled in muscle cells. In fact, HCcAg.120-pIDKE2 complexes induced anti-HCV humoral and cellular immune responses in mice when inoculated by both, parenteral or mucosal routes, although intranasal administration generally rendered weaker results. On the other hand, data demonstrated that Alum enhanced the HCV-specific IgG antibody production. However, the analysis of the HCV-specific cellular immune response showed that HCcAg.120-pIDKE2 delivered in PBS by the intramuscular route induced the strongest HCV-specific lymphoproliferative response, especially against E1 and induced viremia control in a vaccinia virus surrogate challenge model. These results support the use of HCcAg.120-pIDKE2 complexes in the rational design of therapeutic or preventive vaccine strategies against HCV infection.

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.

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.

Understanding the mechanism of Hepatitis C Virus (HCV) pathogenesis is an important part of HCV research. In this study, the presence of apoptosis in HCV-infected liver and Peripheral Blood Mononuclear Cells (PBMC) from patients positive for anti-HCV antibodies and negative for serum HCV-RNA was investigated. The samples obtained from 21 patients were studied by in situ Hybridization (ISH), Immunofluorescence, TUNEL reaction and caspase 3 activation assays. The findings show that both DNA fragmentation by TUNEL assay and activation of caspase 3 were detected in hepatocytes from patients histologically confirmed as bearing chronic hepatitis or with abnormal ALAT or GGTP as well as in patients with no histological evidences of chronic hepatitis and normalization of transaminases. Apoptotic cells were also detected in PBMC samples by the TUNEL assay. ISH analysis of liver biopsies and PBMC samples showed both positive and negative strands of the HCV genome localized in some cells showing nuclear characteristics of apoptosis such as chromatin margination, condensation and fragmentation. These typical morphological changes of apoptotic cell death were also observed in some hepatocytes showing reaction products suggestive of HCcAg. Data suggest that under certain conditions HCV induces apoptosis in the absence of liver injury. Induction of apoptosis in HCV-infected cells may interfere with viral replication, which may lead to undetectable levels of HCV-RNA in serum.

Analysis of Hepatitis C Virus (HCV)-infected hepatocytes at the cellular level may contribute to elucidate the mechanisms of HCV pathogenesis. In this work, the presence of HCV components and pathological reactions in apoptotic hepatocytes from chronic HCV-infected patients were studied by electron microscopy and confocal microscopy. Eight samples of liver biopsies from patients with chronic hepatitis C were studied by laser scanning confocal microscopy, Transmission Electron Microscopy (TEM) and Immunoelectron Microscopy (IEM). Data provide evidence for apoptosis of hepatocytes from HCV-infected liver biopsies during chronic HCV infection. Confirmation of this process was based on the morphological data by TEM including cell shrinkage; chromatin condensation; formation of apoptotic bodies; phagocytosis by neighbouring cells; and the presence of DNA fragmentation by TUNEL assay and caspase 3 activation. Interestingly, Hepatitis C core protein (HCcAg) was specifically immunolabeled in the rough endoplasmic reticulum, mitochondria as well as in the nucleus of apoptotic hepatocytes. In addition, E1 was specifically immunostained in the cytoplasm and in the mitochondria of some hepatocytes. The presence of Crystalloid Bodies (CB) similar to those observed in recombinant P. pastoris expressing HCcAg was observed in the cytoplasm of some hepatocytes. Immunogold labelling showed that HCcAg co-localized with these CB. In addition, structures forming a paracristalline array and particles with a diameter of 50 nm appeared in the mitochondria of some apoptotic hepatocytes. Moreover, unstructured large aggregates containing HCcAg similar to those detected at late stages of HCcAg expression in recombinant P. pastoris cells were frequently observed in damaged hepatocytes. Of note, these aggregates were specifically immunostained with anti-HCcAg. Data suggest the possibility for a direct role of these HCV-related structures as well as HCcAg and E1 in apoptosis and pathogenicity.

Recently, it has been shown that HCV core proteins (HCcAg) with C-terminal deletions assemble in vitro into virus-like particles (VLPs) in the presence of structured RNA molecules. Results presented in this work showed that a truncated HCcAg variant covering the first 120 aa (HCcAg.120) with a 32 aa N-terminal fusion peptide (6xHistag-XpressTMepitope) interacts with plasmid DNA vaccine. Interestingly, the buoyant density of VLPs containing HCcAg.120 in CsCl gradients changed from 1.15-1,17 g mLˉ1 to 1.30-1.34 g mLˉ1 after addition of plasmid DNA to assembly reactions. In addition, a delay in electrophoretic mobility of HCcAg.120-plasmid samples on agarose gels was observed indicating a direct interaction between VLPs and nucleic acids. Remarkably, addition of either plasmid DNA or tRNA to assembly reactions leaded to heterogeneous and larger VLPs formation than those observed in HCcAg.120 assembly reactions. VLPs containing HCcAg.120 induced a specific IgG antibodies in mice that reacted with hepatocytes from HCV-infected patients. VLPs obtained in this work would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure. Besides, the capacity of particles containing HCcAg.120 to interact with nucleic acids could be used in the development of DNA vaccines and viral vectors based on these particles.

In this study, we examined the expression of hepatitis C virus (HCV) components in hepatocytes and peripheral blood mononuclear cells (PBMC) from patients positive for anti-HCV antibodies and negative for serum HCV-RNA. The samples obtained from 25 randomly selected patients (13 of 25 patients showed no histological evidences of chronic hepatitis and had normal serum ALAT and GGTP levels) were studied by in situ Hybridization and Immunofluorescence assays. The findings show that HCV-RNA of both positive and negative polarity was carried in the hepatocytes of more than 80% of cases. The proportion of cells expressing the negative strand (mean ± SD, 10.25% ± 5.56%) was lower than those expressing positive strand (mean ± SD, 19.88% ± 9.19%) (p=0.0076; Student’s t test). In addition, reaction products suggestive of HCV core, E1 and E2 antigens within hepatocytes were also observed. Both hybridization and immunofluorescence signals were localized in the cytoplasm suggesting that this is the place of active HCV replication. On the other hand, the HCV-RNA of positive polarity was detected in PBMC from 16 out of 17 samples analyzed (94%) while the HCV-RNA of negative polarity was detected in 82% of samples investigated. Positive hybridization signals were localized in the cytoplasm of PBMC. Interestingly, 12 out of 13 patients with clinical and histological resolution of hepatitis, contain HCV-RNA in either liver or PBMC. These results provide further evidences that the intermediate replicative form of the HCV genome can persist in hepatocytes and PBMC after apparently complete resolution of chronic hepatitis C.

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 μm), 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. Microsc. Res. Tech. 64:269–278, 2004. © 2004 Wiley-Liss, Inc.

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.

The Integrin β1 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 differen-tiation in the articular cartilage. Members of the Transforming growth factor beta (Tgf-β) Su-perfamily, 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 dedif-ferentiation during OA progression, while increase of α1, α2, and α3 integrin expression was related with fibrous areas in articular cartilage during OA. Hypertrophic chondrocytes expressedαV 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 theα5 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 hy-pertrophy.

Several microscopical techniques, such as high resolution light microscopy, Normaski microscopy, laser confocal and transmission electron microscopy, were used in a correlative morphological study of human osteoarthritic (OA) cartilage. Emphasis was made on the characterization of chondrocytes heterogeneity observed in this tissue. Novel findings were assessed in the morphological and immunocytological study of the chondrocytes organized in aggregates or "clones" typical of this degenerative disease, consisting of the modification of certain elements of the cytoskeleton that influence changes in the cell shape. Also, the presence of cilia and centrioles found in certain cell raised the question if chondrocytes are able to move and regroup as an alternative mechanism to mitosis in the formation of cell clusters or "clones." The presence of two types of secretory chondrocytes was observed and discussed. The use of a correlative approach of several microscopical techniques in a systematic morphological and immunocytological characterization of chondrocyte population within the fibrillated and nonfibrillated human osteoarthritic cartilage gave complementary information that could be important for a better understanding of the histopathogenesis of OA.

Calcium deposits have been related to articular cartilage (AC) degeneration and have been observed in late stages of osteoarthritis (OA). However, the role of those deposits, whether they induce the OA pathogenesis or they appear as a consequence of such process, is still unknown. In this work, we present the kinetics of expression and tissue localisation of osteopontin (OPN), a mineralisation biomarker, and calcium deposits in samples from (normal, sham) and osteoarthritic cartilage (in a rat model). Immunohistochemical and Western blot assays for OPN, as well as Alizarin red staining for calcium deposits were performed; superficial, middle, and deep zones of AC were analysed. An increased expression of OPN and calcium deposits was found in the osteoarthritic cartilage compared with that of control groups, particularly in the superficial zone of AC in early stages of OA. In addition, the expression and localisation of OPN and calcium deposits during the OA pathogenesis suggest that the pathological AC mineralisation starts in the superficial zone during OA pathogenesis.

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.

SummaryKnee articular cartilage samples obtained by arthroscopy from ten patients with well defined knee osteoarthritis (OA) were studied by light and transmission electron microscopy. The morphological phenotype of cells from fibrillated and non-fibrillated regions of OA cartilage was characterized. Three different cell sub-populations were identified. Type 1 cells were found in the superficial and upper middle zones and comprised single chondrocytes and cells organized in aggregates or ‘clones’ that showed a typical chondrocyte phenotype. Type 2 cells displayed a secretory phenotype. Type 3 cells comprised chondrocytes undergoing a degenerative process and were distributed throughout all zones of the cartilage. Changes in the cytoskeletal arrangement, presence of abundant filopodia, peripheral localization of centrioles, and presence of primary cilia were found in many chondrocytes suggesting that they are active motile cells. No mitotic figures were found in this study. Morphometrical analysis was performed to determine the total number of cells and the number of chondrocytes per lacuna in the superficial and upper middle zones of fibrillated and non-fibrillated OA cartilage. There were no statistically significant differences in the total number of cells. In contrast, fibrillated OA cartilage contained a statistically significantly higher percentage of lacunae containing four of more chondrocytes than non-fibrillated OA cartilage samples. The absence of mitotic figures and the presence of motile elements in many chondrocytes raise the possibility that cell aggregates or ‘clones’ in damaged OA cartilage originate by an active process of cell migration rather than by cellular division.

Osteoarthritis (OA) is the most common form of arthritis and represents a global health problem. OA is estimated to affect 40% of the population over 70 years of age and is a major cause of pain and physical disability (Lawrence, 2008). This is a condition that predominantly involves hip, knee, spine, foot and hands. Several risk factors have been associated with the initiation and progression of OA, increasing age, sex, obesity, occupational loading, malalignment, articular trauma and crystal deposition. OA is a chronic degenerative joint disorder characterized primarily by destruction of articular cartilage, formation of reparative fibrocartilage and subchondral bone remodelling. However, not only the cartilage and bone are affected, also the synovium and the jointstabilizing structures such as ligaments and meniscus (Baker‐LePain, 2010). Apparently, all the tissues of the joint respond to mechanical stress with the consequent loss of function and clinical deterioration. The application of mechanical forces under physiological conditions is a preponderant factor in cartilage homeostasis. It is now well know, that environmental factors, such as compressive and tensile forces, load and shear stress have a significant influence on the chondrocyte metabolism. Also, conditions that alter the load distribution on the articular surface can induce the development of OA (Roos, 2005). This degradative process of the cartilage in OA is a consequence of an imbalance between anabolism and catabolism of chondrocytes. Generally, chondrocytes respond with increased expression of inflammatory mediators and matrix-degrading proteinases (Kurz, 2005). Other effect observed in chondrocytes under mechanical stimulation is the change in membrane and osmotic potential (Wright, 1992, Bush, 2001). In OA, chondrocytes undergo depolarization instead of hyperpolarization (Millward-Sadler, 2000), and the decrease of the osmotic potential has been associated with loss of volume control in early stages of OA (Stockwell, 1991, Bush, 2003). However, the sequence of mechanobiological events necessary for the maintenance of extracellular matrix (ECM) homeostasis and its involvement in the pathogenesis of OA is still poorly understood.

Epidemiological studies correlate low levels of vitamin D with the osteoarthritis (OA) progression. Cytokines and metalloproteases play a major role in OA promoting the inflammation and degradation of the cartilage and can be induced through the Toll-like receptor (TLR) pathway. The aim of this study was to evaluate the protective effect of vitamin D supplementation on the development of osteoarthritis (OA) through examining the genetic regulation of TLRs, cytokines, and metalloproteases in chondrocytes as well as the wideness of cartilage in rats with OA. Our results demonstrate that the signaling through TLR-4 is a proinflammatory mechanism in osteoarthritis that drives the upregulation of MMP-3, IL-1β, and TNF-αgene expression, leading to cartilage degradation and inflammation. Vitamin D supplementation had a protective effect during the onset but not during the chronic stage of OA in the rat model.

Nanotechnology is expected to open new avenues to fight and prevent disease using atomic scale tailoring of materials. Among the most promising nanomaterials with antibacterial properties are metallic nanoparticles, which exhibit increased chemical activity due to their large surface to volume ratios and crystallographic surface structure. The study of bactericidal nanomaterials is particularly timely considering the recent increase of new resistant strains of bacteria to the most potent antibiotics. This has promoted research in the well known activity of silver ions and silver-based compounds, including silver nanoparticles. The present work studies the effect of silver nanoparticles in the range of 1-100 nm on Gram-negative bacteria using high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM). Our results indicate that the bactericidal properties of the nanoparticles are size dependent, since the only nanoparticles that present a direct interaction with the bacteria preferentially have a diameter of approximately 1-10 nm.

Objective: This study has aimed to study different culture systems that might stimulate an increase in cell proliferation of normal and osteoarthritis chondrocytes from articular cartilage in rat model. Material and Methods: Three culture systems using chondrocytes embedded in alginate beads were tested: chondrocytes cultured in Dulbecco’s modified Eagle’s medium (DMEM) as control, a co-culture system consisting of a monolayer of de-differentiated chondrocytes as a source of mitotic factors, and an enriched medium containing culture medium obtained from a monolayer of chondrocytes and DMEM. Normal and osteoarthritis chondrocytes were stained with 5-carboxyfluorescein diacetate succinimidyl ester and were cultured in each of the three systems. After 5 days of culture cell, proliferation was detected by flow cytometry. Chondrocyte phenotype was confirmed by collagen type II and MMP-3 expression. To determine possible molecules released into the medium by the cultured chondrocyte monolayer and which would probably be involved in cell proliferation, a study of mRNA and expression of transforming growth factor- β 1 (TGF- β 1), fibroblastic growth factor-2 (FGF-2), epidermal growth factor (EGF), platelet derived growth factor-A (PDGF-A) and insulin-like growth factor-1 (IGF-1) proteins was conducted. Results and Conclusions: Chondrocytes in the co-culture system or in enriched medium showed an increase in proliferation; only when osteoarthritis chondrocytes were cultured in enriched medium would they display a statistically significant increase in their proliferation rate and in their viability. When chondrocytes from the monolayer were analysed, differential mRNA expression of TGF- β 1 and IGF-1 was found during all passages, which suggests that these two growth factors might be involved in chondrocyte proliferation.