Proteolysis of the collagen fibril in osteoarthritis

The development of cartilage pathology in osteoarthritis involves excessive damage to the collagen fibrillar network, which appears to be mediated primarily by the chondrocyte-generated cytokines interleukin-1 and tumour necrosis factor alpha and the collagenases matrix metalloproteinase-1 (MMP-1) and MMP-13. The damage to matrix caused by these and other MMPs can result in the production of sufficient degradation products that can themselves elicit further degradation, leading to chondrocyte differentiation and eventually matrix mineralization and cell death. Knowledge of these MMPs, cellular receptors and cytokine pathways, and the ability to selectively antagonize them by selective blockade of function, may provide valuable therapeutic opportunities in the treatment of osteoarthritis and other joint diseases involving cartilage resorption, such as rheumatoid arthritis. The ability to detect the products of these degradative events released into body fluids of patients may enable us to monitor disease activity, predict disease progression and determine more rapidly the efficacy of new therapeutic agents.     

Cytologic findings of epithelioid angiosarcoma of the thyroid. A case report

BACKGROUND: Angiosarcoma of the thyroid is a rare and aggressive tumor and occurs mainly in patients from central Europe, especially the alpine region. The fine needle aspiration findings of a keratin-positive epithelioid angiosarcoma of the thyroid occurring in a nonmountainous area in South America is described. CASE: A 65-year-old male from S?o Paulo, Brazil, presented with a mass in the anterior part of the neck with progressive enlargement for three months. The cytologic findings on the fine needle aspirate were a cellular smear composed of single cells and small clusters of neoplastic cells, oval and round. Cell borders were indistinct, and the cytoplasm was vacuolated. The nuclei were eccentrically located, with irregular nuclear membranes; single, prominent nucleoli; and a coarse chromatin pattern. Features suggestive of intracytoplasmic lumens were identified. Open surgical biopsy demonstrated a tumor infiltrating the thyroid gland and composed of large, round, atypical epithelioid cells lining vascular spaces. These neoplastic cells were immunoreactive for AE1:AE3, CK7, vimentin, CD31 and factor VIII. CONCLUSION: Epithelioid angiosarcoma should be considered in the differential diagnosis of epithelioid neoplasms of the thyroid. An immunohistochemical panel should include vascular markers even in the presence of immunoreactivity for epithelial markers.     

Connection between elastin haploinsufficiency and increased cell proliferation in patients with supravalvular aortic stenosis and Williams-Beuren syndrome

To elucidate the pathomechanism leading to obstructive vascular disease in patients with elastin deficiency, we compared both elastogenesis and proliferation rate of cultured aortic smooth-muscle cells (SMCs) and skin fibroblasts from five healthy control subjects, four patients with isolated supravalvular aortic stenosis (SVAS), and five patients with Williams-Beuren syndrome (WBS). Mutations were determined in each patient with SVAS and in each patient with WBS. Three mutations found in patients with SVAS were shown to result in null alleles. RNA blot hybridization, immunostaining, and metabolic labeling experiments demonstrated that SVAS cells and WBS cells have reduced elastin mRNA levels and that they consequently deposit low amounts of insoluble elastin. Although SVAS cells laid down approximately 50% of the elastin made by normal cells, WBS cells deposited only 15% of the elastin made by normal cells. The observed difference in elastin-gene expression was not caused by a difference in the stability of elastin mRNA in SVAS cells compared with WBS cells, but it did indicate that gene-interaction effects may contribute to the complex phenotype observed in patients with WBS. Abnormally low levels of elastin deposition in SVAS cells and in WBS cells were found to coincide with an increase in proliferation rate, which could be reversed by addition of exogenous insoluble elastin. We conclude that insoluble elastin is an important regulator of cellular proliferation. Thus, the reduced net deposition of insoluble elastin in arterial walls of patients with either SVAS or WBS leads to the increased proliferation of arterial SMCs. This results in the formation of multilayer thickening of the tunica media of large arteries and, consequently, in the development of hyperplastic intimal lesions leading to segmental arterial occlusion.     

Development of a biosensor-based method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors

A biosensor-based assay, using a surface plasmon resonance detection system, was developed to detect and isotype anti-adenoviral antibodies in patients dosed with an adenoviral-based gene therapy vector. In the assay, whole, intact virus was immobilized onto the sensor chip surface. Electron microscopy and monoclonal antibody studies provide evidence that the virus remains intact after immobilization. The patients tested had preexisting serum levels of anti-adenoviral antibodies. A classic anamnestic response was observed in patients dosed with the gene-therapy agent. Isotyping experiments indicated that IgG antibodies predominated in serum even at the predose time point. Analysis of ascites fluid samples from some patients indicated detectable levels of IgA in addition to IgG. Results obtained using the biosensor-based assay corresponded to an existing enzyme-linked immunosorbent assay. The assay was easy to perform and the automated instrument reduced the required "hands on" time. In addition to studying the development of anti-adenoviral antibodies, the techniques described may be applied to virus:receptor interaction studies or antiviral drug:virus interaction studies.     

Effect of GP120 on glutathione peroxidase activity in cortical cultures and the interaction with steroid hormones

GP120 (the protein component of the HIV viral coat) is neurotoxic and may contribute to the cell loss associated with AIDS-related dementia. Previously, it has been shown in rat cortical mixed cultures that gp120 increased the accumulation of hydrogen peroxide and superoxide, two reactive oxygen species (ROS). We now demonstrate that gp120 increased activity of the key antioxidant glutathione peroxidase (GSPx), presumably as a defensive mechanism against the increased ROS load. Both estrogen and glucocorticoids (GCs), the adrenal steroid released during stress, blunted this gp120 effect on GSPx activity. The similar effects of estrogen and of GCs are superficially surprising, given prior demonstrations that GCs exacerbated and estrogens protected against gp120 neurotoxicity. We find that these similar effects of estrogen and GCs on GSPx regulation arose, in fact, from very different routes, which are commensurate with these prior reports. Specifically, estrogen has demonstrated antioxidant properties that may prevent the ROS increase (therefore acting as a neuroprotective agent) and rendered unnecessary the compensatory GSPx increased activity. To verify this we have added H2O2 to estrogen + gp120-treated cells, and GSPx activity was increased. However, with addition of H2O2 to GCs + gp120-treated cells there was no increase in activity. GCs appeared to decrease enzyme production and or activity and therefore under insult conditions ROS levels rose in the cell resulting in increased neurotoxicity. Overexpression of GSPx enzyme via herpes vector system reversed the GCs-induced loss of enzyme and eliminated the GCs exacerbation of gp120 neurotoxicity.     

Epstein-Barr virus BALF1 is a BCL-2-like antagonist of the herpesvirus antiapoptotic BCL-2 proteins

Cellular BCL-2 family proteins can inhibit or induce programmed cell death in part by counteracting the activity of other BCL-2 family members. All sequenced gammaherpesviruses encode a BCL-2 homologue that potently inhibits apoptosis and apparently escapes some of the regulatory mechanisms that govern the functions of their cellular counterparts. Examples of these protective proteins include BHRF1 of Epstein-Barr virus (EBV) and KSBcl-2 of Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8. The gamma-1 subgroup of these viruses, such as EBV, encodes a second BCL-2 homologue. We have now found that this second BCL-2 homologue encoded by EBV, BALF1, inhibits the antiapoptotic activity of EBV BHRF1 and of KSBcl-2 in several transfected cell lines. However, BALF1 failed to inhibit the cellular BCL-2 family member, BCL-x(L). Thus, BALF1 acts as a negative regulator of the survival function of BHRF1, similar to the counterbalance observed between cellular BCL-2 family members. Unlike the cellular BCL-2 family antagonists, BALF1 lacked proapoptotic activity and could not be converted into a proapoptotic factor in a manner similar to cellular BCL-2 proteins by caspase cleavage or truncation of the N terminus. Coimmunoprecipitation experiments and immunofluorescence assays suggest that a minimal amount, if any, of the BHRF1 and BALF1 proteins colocalizes inside cells, suggesting that mechanisms other than direct interaction explain the suppressive function of BALF1.     

Fluid shear stress-induced alignment of cultured vascular smooth muscle cells

The study objectives were to quantify the time- and magnitude-dependence of flow-induced alignment in vascular smooth muscle cells (SMC) and to identify pathways related to the orientation process. Using an intensity gradient method, we demonstrated that SMC aligned in the direction perpendicular to applied shear stress, which contrasts with parallel alignment of endothelial cells under flow SMC alignment varied with the magnitude of and exposure time to shear stress and is a continuous process that is dependent on calcium and cycloskeleton based mechanisms. A clear understanding and control of flow-induced SMC alignment will have implications for vascular tissue engineering.     

Population of Lytechinus variegatus (Echinoidea: Toxopneustidae) and structural characteristics of seagrass of Thalassia testudinum in Mochima Bay,Venezuela)

To compare the general features of Thalassia testudinum seagrass at Mochima Bay with sea urchin (Lxtechinus variegatus) abundance and distribution, three T. testudinum seagrass beds were selected, from the mouth (strong wave exposure) to the inner bay (calm waters). Each site was surveyed by using 5 line transects (20 m long) parallel to the coast and 1 m2 quadrats. In situ measurements of T. testudinum cover, shoot and leaf density were taken. Estimation of dry biomass for each seagrass fraction (leaves, rhizomes and roots) and leaf length were obtained from 25 vegetation samples extracted per site using cores (15 cm diameter). A multivariate analysis of variance (Manova) and a less significative difference test (LSD) were performed to examine differences between sites and within sites at different depths. A stepwise multiple regression analysis was done, dependent variable was sea urchin density; independent variables: vegetation values at each site. The only seagrass species found in the three sites was T. testudinum, and cover was 56-100%, leaf density 100-1000 leaf/m2, lengths 6-18.8 cm and shoot density 20-475 shoots/m2. The highest sea urchin densities were found at Isla Redonda and Ensenada Toporo (1-3.6 ind/m2), the lowest at Playa Colorada (0.6-0.8 ind/m2). Significant differences in seagrass features between sites were obtained (Manova p < 0.001), but not between depths (Manova p < 0.320). The regression coefficient between sea urchin density and seagrass parameters was statistically significant (r2 = 0.154, p < 0.007), however, total biomass was the only variable with a significant effect on sea urchin distribution (beta = 0.308, p < 0.032). The other variables did not explain satisfactorily L. variegatus abundance and distribution.