Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.     

Common Antigenic Determinant in a Rumen Organism and in Salmonellae Containing the Antigen O4

Nineteen of 28 strains of rumen organisms isolated from a cow on a high roughage diet and identified morphologically as butyrivibrios, reacted to a low agglutinin titer with salmonella antisera, forming five groups. However only one strain reacted with polyvalent O salmonella antiserum. This strain reacted with O4 factor serum and with antisera to Salmonella strains containing the antigen O4, and agglutinin absorption tests showed the presence of an antigen identical to O4. When 16 further strains of butyrivibrio-like rumen organisms isolated from three cows and one steer were examined, one possessed an antigen similar to but not identical with the antigen O9, and two strains reacted with specific O6,7 factor serum but were not examined further. These four strains were presumptively identified by physiological tests as butyrivibrios. The possible site of antigenic stimulation by such organisms is discussed.     

Lymphoproliferative disorder in CTLA-4 knockout mice is characterized by CD28-regulated activation of Th2 responses

Mice lacking CTLA-4 die at an age of 2-3 wk due to massive lymphoproliferation, leading to lymphocytic infiltration and destruction of major organs. The onset of the lymphoproliferative disease can be delayed by treatment with murine CTLA4Ig (mCTLA4Ig), starting day 12 after birth. In this study, we have characterized the T cells present in CTLA-4-deficient mice before and after mCTLA4Ig treatment. The T cells present in CTLA-4-deficient mice express the activation markers, CD69 and IL-2R; down-regulate the lymphoid homing receptor, CD62L; proliferate spontaneously in vitro and cannot be costimulated with anti-CD28 mAb consistent with a hyperactivated state. The T cells from CTLA-4-deficient mice survive longer in culture correlating with higher expression of the survival factor, Bcl-xL, in these cells. Most significantly, the CD4+ T cell subset present in CTLA-4-deficient mice secretes high levels of IL-4 and IL-5 upon TCR activation. Treatment of CTLA-4-deficient mice treated with mCTLA4Ig reverses the activation and hyperproliferative phenotype of the CTLA-4-deficient T cells and restores the costimulatory activity of anti-CD28 mAb. Furthermore, T cells from mCTLA4Ig-treated mice are not skewed toward a Th2 cytokine phenotype. Thus, CTLA-4 regulates CD28-dependent peripheral activation of CD4+ T cells. This process results in apoptosis-resistant, CD4+ T cells with a predominantly Th2 phenotype that may be involved in the lethal phenotype in these animals.     

Neural Stem Cells as Engraftable Packaging Lines Can Mediate Gene Delivery to Microglia: Evidence from Studying Retroviral env-Related Neurodegeneration

The induction of spongiform myeloencephalopathy by murine leukemia viruses is mediated primarily by infection of central nervous system (CNS) microglia. In this regard, we have previously shown that CasBrE-induced disease requires late, rather than early, virus replication events in microglial cells (W. P. Lynch et al., J. Virol. 70:8896-8907, 1996). Furthermore, neurodegeneration requires the presence of unique sequences within the viral env gene. Thus, the neurodegeneration-inducing events could result from microglial expression of retroviral envelope protein alone or from the interaction of envelope protein with other viral structural proteins in the virus assembly and maturation process. To distinguish between these possible mechanisms of disease induction, we engineered the engraftable neural stem cell line C17-2 into packaging/producer cells in order to deliver the neurovirulent CasBrE env gene to endogenous CNS cells. This strategy resulted in significant CasBrE env expression within CNS microglia without the appearance of replication competent virus. CasBrE envelope expression within microglia was accompanied by increased expression of activation markers F4/80 and Mac-1 (CD11b) but failed to induce spongiform neurodegenerative changes. These results suggest that envelope expression alone within microglia is not sufficient to induce neurodegeneration. Rather, microglia-mediated disease appears to require neurovirulent Env protein interaction with other viral proteins during assembly or maturation. More broadly, the results presented here prove the efficacy of a novel method by which neural stem cell biology may be harnessed for genetically manipulating the CNS, not only for studying neurodegeneration but also as a paradigm for the disseminated distribution of retroviral vector-transduced genes.     

Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype

Escherichia coli DEAD-box protein A (DbpA) is an ATP-dependent RNA helicase with specificity for 23S ribosomal RNA. Although DbpA has been extensively characterized biochemically, its biological function remains unknown. Previous work has shown that a DbpA deletion strain is viable with little or no effect on growth rate. In attempt to elucidate a phenotype for DbpA, point mutations were made at eleven conserved residues in the ATPase active site, which have exhibited dominant-negative phenotypes in other DExD/H proteins. Biochemical analysis of these DbpA mutants shows the expected decrease in RNA-dependent ATPase activity and helix unwinding activity. Only the least biochemically active mutation, R331A, produces small colony phenotype and a reduced growth rate. This dominant slow growth mutant will be valuable to determine the cellular function of DbpA.     

Morphometric analysis of collagen in gestational and retained bovine placentomes

Bovine placentome collagen was quantified (P<0.01) at four gestational stages (90, 150, 210 and 270 d, n = 8 d ), at 2 h post partum without (n = 4) and at 2 and 12 h post partum with (n = 8) experimentally-induced placental retention. Placentome sections were fixed and stained for collagen. Fetal cotyledonary (FC) collagen volume fraction (V(V)) increased over days of gestation studied (V(V)=0.03+/-0.01, 0.06+/-0.01, 0.13+/-0.01 and 0.19+/-0.01). Fetal cotyledonary hydroxyproline (3.15+/-0.41, 4.55+/-0.41 and 7.04+/-0.41 mg/g) and FC protein (432.0+/-17.1, 479.9+/-17.1, 585.4+/-17.1 mg/g) increased over Days 90, 150 and 210 and were similar on Days 210 and 270. Fetal cotyledonary collagen V(V) and hydroxyproline did not differ between Day 270, retained and nonretained cotyledons. Protein concentration was higher in 2 h (578.1+/-18.5 mg/g) and 12 h (526.0+/-18.5 mg/g) retained versus nonretained (400.4+/-36.2 mg/g) cotyledons. Maternal caruncular (MC) collagen V(V) and protein concentration were higher on Days 90 and 150 than on Days 210 and 270. Maternal caruncular hydroxyproline was similar from Day 90 to 210 and increased from Day 210 to 270. Maternal caruncular collagen V(V), hydroxyproline and protein concentrations were similar on Day 270 and in 2 h and 12 h retained membrane caruncles. Gestational increases in placentome collagen occurred from FC sources. No difference in FC or MC collagen V(V) existed between Day 270, retained and nonretained placentomes.     

Non-linear regression of biological temperature-dependent rate models based on absolute reaction-rate theory

Biological temperature-dependent rate models based on Arrhenius' and Eyring's equations have been formulated by Johnson & Lewin (1946), Hultin (1955), and Sharpe & DeMichele (1977). The original formulation of Sharpe and DeMichele is poorly suited for non-linear regression. Very high correlations of parameter estimators occassionally make regression with their equation impossible using Marquardt's algorithm (1963).This analysis describes a new formulation of Sharpe and DeMichele's model that greatly alleviates the non-linear regression problem. It is partly based on Hultin's formulation (1955). Biological and graphical interpretation of the model parameters is discussed. Regression suitability is illustrated with a typical data set. Similar modifications to the equations of Hultin (1955) and Johnson & Lewin (1946) are described.     

Mutations in the WRN gene in mice accelerate mortality in a p53-null background

Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.     

Identification and partial characterization of two inducible gelatin-cleavage activities localized to the sea urchin extraembryonic matrix,the hyaline layer

We have identified two inducible, gelatin-cleaving activities in the sea urchin extraembryonic matrix, the hyaline layer. Isolated hyaline layers, incubated in the presence of benzamidine, were devoid of gelatin-cleavage activities with apparent molecular mass less then 80k. However, when layers were incubated for 9-11 h in the absence of benzamidine, gelatin-cleavage activities, with apparent molecular mass 40- and 50k, were detected. Induction required the presence of NaCl and CaCl(2) at concentrations similar to those found in seawater and readdition of the reversible serine protease inhibitor benzamidine prevented induction. Both gelatin-cleaving activities were activated by calcium at a concentration similar to the calcium concentration found in seawater. Magnesium, also a major cationic species present in seawater, could not replace calcium as the activating ion. In addition, magnesium could not compete with calcium for binding to the gelatinases. Both cleavage activities showed substrate specificity and each failed to cleave bovine serum albumin, bovine hemoglobin or casein. Cleavage activity towards gelatin was inhibited by benzamidine and aminoethyl benzenesulfonyl fluoride, indicating that both activities belonged to the serine class of proteases. The induced 40-kDa activity displayed similar properties to those of a comigrating, gelatin-cleaving activity present in 69-h-old embryos.