Cytokine profiles in the joint depend on pathology,but are different between synovial fluid,cartilage tissue and cultured chondrocytes

Introduction

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

Methods

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).

Results

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

Conclusions

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.     

Two distinct endogenous type C viruses isolated from the asian rodent Mus cervicolor: conservation of virogene sequences in related rodent species.

The cocultivation of a lung cell line from the Southeast Asian mouse Mus cervicolor with cells from heterologous species has resulted in the isolation of two new distinct type C viruses. Both viruses are endogenous to M. cervicolor and are present in multiple copies in the cellular DNA of these mice. One of the viruses, designated M. cervicolor type CI, replicates readily in the SIRC rabbit cell line and is antigenically related to the infectious primate type C viruses isolated from a woolly monkey (simian sarcoma-associated virus) and gibbon apes (gibbon ape leukemia virus). This virus is also closely related by both immunological and nucleic acid hybridization criteria to a type C virus previously isolated from a second Asian murine species, Mus caroli. The isolation of the M. cervicolor type C I virus thus provides further evidence that the infectious primate type C viruses originated by trans-species infection of primates by an endogenous virus of mice. The second virus, designated M. cervicolor type C II, replicates well in various cell lines derived from the laboratory mouse Mus musculus. While antigenically related to type C viruses derived from M. musculus, the M. cervicolor type C II virus isolate can be readily distinguished from standard murine leukemia viruses. Both new type C viruses from M. cervicolor are unrelated to the previously described retrovirus (M432) isolated from the same Mus species. The DNA of M. cervicolor therefore contains multiple copies of at least three distinct classes of endogenous viral genes. An examination of the cellular DNA of other rodent species for nucleic acid sequences related to the genomes of both M. cervicolor type C I and II reveals that both viruses have been highly conserved evolutionarily, and that other species of rodents, such as laboratory mice and rats, contain endogenous virogenes related to those in the DNA of M. cervicolor.     

A new genetic locus, Bevi, on human chromosome 6 which controls the replication of baboon type C virus in human cells.

Somatic cell hybrids derived from seven independent fusions between mouse X human and hamster X human parental cells were examined for their ability to support the replication of the baboon endogenous type C virus. These hybrids preferentially segregated human chromosomes while retaining rodent chromosomes, as demonstrated by karyotypic and isozyme analysis. A total of 41 primary colonies and 33 secondary subclones were analyzed for viral replication, as well as for the presence of enzyme structural gene markers for 19 of 23 human chromosomes. A syntenic association was seen between the ability of the baboon type C virus to infect and replicate in hybrid cultures and the expression of human malic enzyme-1 (assigned to human chromosome 6). Analysis of 86 highly segregated subclones derived from cells preinfected with baboon type C virus showed that the continued production of baboon type C virus segregated concordantly with the expression of three enzyme genes assigned to human chromosome 6 (malic enzyme-1, phosphoglucomutase-3 and superoxide dismutase-2). Subclones of infected hybrids which lost chromosome 6 and failed to release virus also failed to synthesize the virus-coded major structural protein p30. No syntenic association between baboon virus expression and any of 18 other human chromosomes was observed. These studies define a new gene (designated Bevi) on human chromosome 6 which dominantly controls the replication of baboon type C virus. The data suggest that Bevi may be a preferred integration site for the baboon type C DNA provirus in the human genome.     

How Effective Is Help on the Doorstep? A Longitudinal Evaluation of Community-Based Organisation Support

Community-based responses have a lengthy history. The ravages of HIV on family functioning has included a widespread community response. Although much funding has been invested in front line community-based organisations (CBO), there was no equal investment in evaluations. This study was set up to compare children aged 9–13 years old, randomly sampled from two South African provinces, who had not received CBO support over time (YC) with a group of similarly aged children who were CBO attenders (CCC). YC baseline refusal rate was 2.5% and retention rate was 97%. CCC baseline refusal rate was 0.7% and retention rate was 86.5%. 1848 children were included—446 CBO attenders compared to 1402 9–13 year olds drawn from a random sample of high-HIV prevalence areas. Data were gathered at baseline and 12–15 months follow-up. Standardised measures recorded demographics, violence and abuse, mental health, social and educational factors. Multivariate regression analyses revealed that children attending CBOs had lower odds of experiencing weekly domestic conflict between adults in their home (OR 0.17; 95% CI 0.09, 0.32), domestic violence (OR 0.22; 95% CI 0.08, 0.62), or abuse (OR 0.11; 95% CI 0.05, 0.25) at follow-up compared to participants without CBO contact. CBO attenders had lower odds of suicidal ideation (OR 0.41; 95% CI 0.18, 0.91), fewer depressive symptoms (B = -0.40; 95% CI -0.62, -0.17), less perceived stigma (B = -0.37; 95% CI -0.57, -0.18), fewer peer problems (B = -1.08; 95% CI -1.29, -0.86) and fewer conduct problems (B = -0.77; 95% CI -0.95, -0.60) at follow-up. In addition, CBO contact was associated with more prosocial behaviours at follow-up (B = 1.40; 95% CI 1.13, 1.67). No associations were observed between CBO contact and parental praise or post-traumatic symptoms. These results suggest that CBO exposure is associated with behavioural and mental health benefits for children over time. More severe psychopathology was not affected by attendance and may need more specialised input.     

Photoresponse in the heterotrophic marine dinoflagellate Oxyrrhis marina

Expressed rhodopsins were detected by proteomic analysis in an investigation of potential signal receptors in the cell membrane of the marine heterotrophic dinoflagellate Oxyrrhis marina (CCMP604). We inferred these to be sensory rhodopsins, a type of G-protein-coupled receptor trans-membrane signaling molecule. Because phototactic behavior based on sensory rhodopsins has been reported in other protists, we investigated the photosensory response of O. marina. This dinoflagellate exhibited strongest positive phototaxis at low levels (2-3 μE/m(2)/s) of white light when the cells were previously light adapted and well fed. Positive phototaxis was also found for blue (450 nm), green (525 nm), and red (680 nm) wavelengths. In a further test, O. marina showed significantly greater phototaxis toward concentrated algal food illuminated by blue light to stimulate red chlorophyll-a autofluorescence in the prey, compared with using bleached algae as prey. Concentration of a cytoplasmic downstream messenger molecule, cyclic adenosine monophosphate, a component of the signaling pathway of G-protein-coupled receptor molecules, rapidly increased in O. marina cells after exposure to white light. In addition, treatment with hydroxylamine, a rhodopsin signaling inhibitor, significantly decreased their phototactic response. Our results demonstrate that a heterotrophic marine dinoflagellate can orient to light based on rhodopsins present in the outer cell membrane and may be able to use photosensory response to detect algal prey based on chlorophyll autofluorescence.     

Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin-3 gallate (EGCG), which possess anti-oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen-induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12-dimethylbenz(a)anthracene (DMBA) Sprague-Dawley (S-D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor-bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA-MB-231 estrogen receptor-negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA-transformed D3-1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27(Kip1) cyclin-dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27(Kip1) protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen-induced mammary tumorigenesis in female S-D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27(Kip1) CKI.     

Genomic organization, chromosomal localization, and independent expression of human cyclin D genes.

Murine cDNA clones for three cyclin D genes that are normally expressed during the G1 phase of the cell cycle were used to clone the cognate human genes. Bacteriophage and cosmid clones encompassing five independent genomic loci were partially sequenced and chromosomally assigned by an analysis of somatic cell hybrids containing different human chromosomes and by fluorescence in situ hybridization to metaphase spreads from normal peripheral blood lymphocytes. The human cyclin D1 gene (approved gene symbol, CCND1) was assigned to chromosome band 11q13, cyclin D2 (CCND2) to chromosome band 12p13, and cyclin D3 (CCND3) to chromosome band 6p21. Pseudogenes containing sequences related to cyclin D2 and cyclin D3 mapped to chromosome bands 11q13 and 6p21, respectively. Partial nucleotide sequence analysis of exons within each gene revealed that the authentic human cyclin D genes are more related to their mouse counterparts than to each other. These genes are ubiquitously transcribed in human tumor cell lines derived from different cell lineages, but are independently and, in many cases, redundantly expressed. The complex patterns of expression of individual cyclin D genes and their evolutionary conservation across species suggest that each family member may play a distinct role in cell cycle progression.     

Nucleotide sequence of the gag gene and gag-pol junction of feline leukemia virus.

The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.     

Characterization of anti-idiotypic suppressor T cells (Tsid) induced after antigen priming

The induction and fine specificity of idiotype-specific suppressor T cells (Tsid) were studied. Spleen cells from C57BL/6 mice, immunized 4 wk previously with NP-KLH, failed to express NPb3 idiotype-bearing PFC when challenged in vitro with NP-Ficoll or NP-Brucella abortus. After treatment of NP-primed responder cultures with anti-Thy-1.2 anti-serum + C, NPb idiotype-bearing B cells could be detected. This B cell subset was preferentially suppressed by the addition of T cells from NP-primed mice. With this reconstitution protocol, it was determined that suppression of the NPb idiotype-bearing portion of the B cell response was mediated by a specifically induced T cell population (Tsid) that directly suppressed NPb-bearing B cells. As with a previously described suppressor population induced with hapten-modified syngeneic spleen cells (Ts2), the Tsid population bound and was lysed by NPb idiotype-bearing serum antibodies. However, the Tsid could be distinguished from the Ts2 population because it lacked I-J determinants and functioned as an effector T cell, not an intermediary suppressor cell. Furthermore, fine specificity studies with monoclonal NP-specific antibodies expressing various levels of serologically detectable NPb idiotypic determinants indicated that unlike the Ts2, the Tsid population reacts with conventional, serologically detected members of the NPb family. The combined idiotype binding studies for the Tsid and Ts2 populations demonstrate that the fine specificity of suppressor T cell populations reflects their independent mechanisms of regulation.