Synoviocytes assist in modulating the effect of Ross River virus infection in micromass-cultured primary human chondrocytes.

Introduction. Ross River virus (RRV) is a mosquito-borne virus prevalent in Australia and the islands of the South Pacific, where it causes an arthritogenic illness with a hallmark feature of severe joint pain. The joint space is a unique microenvironment that contains cartilage and synovial fluid. Chondrocytes and synoviocytes are crucial components of the joint space and are known targets of RRV infection.Hypothesis/Gap statement. Understanding the relationship between synoviocytes and chondrocytes during RRV infection will provide further insights into RRV-induced joint pathology.Methodology. To better understand the unique dynamics of these cells during RRV infection, we used primary chondrocytes cultured in physiologically relevant micromasses. We then directly infected micromass chondrocytes or infected primary fibroblast-like synoviocytes (FLS), co-cultured with micromass chondrocytes. Micromass cultures and supernatants were collected and analysed for viral load with a PCR array of target genes known to play a role in arthritis.Results. We show that RRV through direct or secondary infection in micromass chondrocytes modulates the expression of cellular factors that likely contribute to joint inflammation and disease pathology, as well as symptoms such as pain. More importantly, while we show that RRV can infect micromass-cultured chondrocytes via FLS infection, FLS themselves affect the regulation of cellular genes known to contribute to arthritis.Conclusion. Single-cell culture systems lack the complexity of in vivo systems, and understanding the interaction between cell populations is crucial for deciphering disease pathology, including for the development of effective therapeutic strategies.

Cellular and Tissue Selectivity of AAV Serotypes for Gene Delivery to Chondrocytes and Cartilage.

Background: Despite several studies on the effect of adeno-associated virus (AAV)-based therapeutics on osteoarthritis (OA), information on the transduction efficiency and applicable profiles of different AAV serotypes to chondrocytes in hard cartilage tissue is still limited. Moreover, the recent discovery of additional AAV serotypes makes it necessary to screen for more suitable AAV serotypes for specific tissues. Here, we compared the transduction efficiencies of 14 conventional AAV serotypes in human chondrocytes, mouse OA models, and human cartilage explants obtained from OA patients. Methods: To compare the transduction efficiency of individual AAV serotypes, green fluorescent protein (GFP) expression was detected by fluorescence microscopy or western blotting. Likewise, to compare the transduction efficiencies of individual AAV serotypes in cartilage tissues, GFP expression was determined using fluorescence microscopy or immunohistochemistry, and GFP-positive cells were counted. Results: Only AAV2, 5, 6, and 6.2 exhibited substantial transduction efficiencies in both normal and OA chondrocytes. All AAV serotypes except AAV6 and rh43 could effectively transduce human bone marrow mesenchymal stem cells. In human and mouse OA cartilage tissues, AAV2, AAV5, AAV6.2, AAV8, and AAV rh39 showed excellent tissue specificity based on transduction efficiency. These results indicate the differences in transduction efficiencies of AAV serotypes between cellular and tissue models. Conclusions: Our findings indicate that AAV2 and AAV6.2 may be the best choices for AAV-mediated gene delivery into intra-articular cartilage tissue. These AAV vectors hold the potential to be of use in clinical applications to prevent OA progression if appropriate therapeutic genes are inserted into the vector.

Mayaro Virus Infects Human Chondrocytes and Induces the Expression of Arthritis-Related Genes Associated with Joint Degradation.

Mayaro virus (MAYV) is an emerging arthritogenic alphavirus belonging to the Togaviridae family. Infection leads to a dengue-like illness accompanied by severe polyarthralgia. However, the molecular and cellular mechanisms of arthritis as a result of MAYV infection remain poorly understood. In the present study, we assess the susceptibility of human chondrocytes (HC), fibroblast-like synoviocytes and osteoblasts that are the major cell types involved in osteoarthritis, to infection with MAYV. We show that these cells are highly permissive to MAYV infection and that viral RNA copy number and viral titers increase over time in infected cells. Knowing that HC are the primary cells in articular cartilage and are essential for maintaining the cartilaginous matrix, gene expression studies were conducted in MAYV-infected primary HC using polymerase chain reaction (PCR) arrays. The infection of the latter cells resulted in an induction in the expression of several matrix metalloproteinases (MMP) including MMP1, MMP7, MMP8, MMP10, MMP13, MMP14 and MMP15 which could be involved in the destruction of articular cartilage. Infected HC were also found to express significantly increased levels of various IFN-stimulated genes and arthritogenic mediators such as TNF-α and IL-6. In conclusion, MAYV-infected primary HC overexpress arthritis-related genes, which may contribute to joint degradation and pathogenesis.

Chondrocytes Contribute to Alphaviral Disease Pathogenesis as a Source of Virus Replication and Soluble Factor Production.

Arthritogenic alphavirus infections often result in debilitating musculoskeletal disorders that affect the joints, muscle, and bone. In order to evaluate the infection profile of primary human skeletal muscle and chondrocyte cells to Ross River virus (RRV) in vitro, cells were infected at a multiplicity of infection (MOI) of 1 over a period of two days. Viral titers were determined by plaque assay and cytokine expression by Bio-Plex® assays using the supernatants harvested. Gene expression studies were conducted using total RNA isolated from cells. Firstly, we show that RRV RNA is detected in chondrocytes from infected mice in vivo. Both human primary skeletal muscle and chondrocyte cells are able to support productive RRV infection in vitro. We also report the production of soluble host factors including the upregulation of heparanase (HPSE) and inflammatory host factors such as interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), RANTES (regulated on activation, normal T cell expressed and secreted), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), which are also present during clinical disease in humans. Our study is the first to demonstrate that human chondrocyte cells are permissive to RRV infection, support the production of infectious virus, and produce soluble factors including HPSE, which may contribute to joint degradation and the pathogenesis of disease.

Human endogenous retrovirus W activity in cartilage of osteoarthritis patients.

The etiology of viruses in osteoarthritis remains controversial because the prevalence of viral nucleic acid sequences in peripheral blood or synovial fluid from osteoarthritis patients and that in healthy control subjects are similar. Until now the presence of virus has not been analyzed in cartilage. We screened cartilage and chondrocytes from advanced and non-/early osteoarthritis patients for parvovirus B19, herpes simplex virus-1, Epstein Barr virus, cytomegalovirus, human herpes virus-6, hepatitis C virus, and human endogenous retroviruses transcripts. Endogenous retroviruses transcripts, but none of the other viruses, were detected in 15 out the 17 patients. Sequencing identified the virus as HERV-WE1 and E2. HERV-W activity was confirmed by high expression levels of syncytin, dsRNA, virus budding, and the presence of virus-like particles in all advanced osteoarthritis cartilages examined. Low levels of HERV-WE1, but not E2 envelope RNA, were observed in 3 out of 8 non-/early osteoarthritis patients, while only 3 out of 7 chondrocytes cultures displayed low levels of syncytin, and just one was positive for virus-like particles. This study demonstrates for the first time activation of HERV-W in cartilage of osteoarthritis patients; however, a causative role for HERV-W in development or deterioration of the disease remains to be proven.

Overexpression of human IGF-I via direct rAAV-mediated gene transfer improves the early repair of articular cartilage defects in vivo.

Direct therapeutic gene transfer is a promising tool to treat articular cartilage defects. Here, we tested the ability of an recombinant adeno-associated virus (rAAV) insulin-like growth factor I (IGF-I) vector to improve the early repair of cartilage lesions in vivo. The vector was administered for 3 weeks in osteochondral defects created in the knee joints of rabbits compared with control (lacZ) treatment and in cells that participate in the repair processes (mesenchymal stem cells, chondrocytes). Efficient IGF-I expression was observed in the treated lesions and in isolated cells in vitro. rAAV-mediated IGF-I overexpression was capable of stimulating the biologic activities (proliferation, matrix synthesis) both in vitro and in vivo. IGF-I treatment in vivo was well tolerated, revealing significant improvements of the repair capabilities of the entire osteochondral unit. IGF-I overexpression delayed terminal differentiation and hypertrophy in the newly formed cartilage, possibly due to contrasting effects upon the osteogenic expression of RUNX2 and ß-catenin and to stimulating effects of this factor on the parathyroid hormone/parathyroid hormone-related protein pathway in this area. Production of IGF-I improved the reconstitution of the subchondral bone layer in the defects, showing increased RUNX2 expression levels in this zone. These findings show the potential of directly applying therapeutic rAAVs to treat cartilage lesions.

Rapid baculovirus titration based on regulatable green fluorescent protein expression in mammalian cells.

Baculovirus is a promising gene delivery vector and can be titrated by constitutive EGFP expression in HeLa cells, which, however, might interfere with target transgene expression and impart cytotoxicity. Here we constructed Bac-ME accommodating egfp under the inducible metallothionein promoter and Bac-MECB harboring an additional BMP-2 gene. Bac-ME effectively transduced HeLa cells with minimal leaky expression, but expressed EGFP robustly upon induction with ZnSO(4), hence allowing for virus titration by transducing HeLa cells with serially diluted virus, subsequent ZnSO(4) induction and flow cytometry analysis of EGFP-positive cells. The titration protocol enabled the generation of discernable titration curves, determination of transducing titers, and discrimination of the transducing abilities of different virus batches. After titration, cell transduction with pre-determined Bac-ME dose revealed consistent transduction efficiency dependence on the dose, regardless of virus batch and cell type. Bac-MECB was similarly titrated by inducible EGFP expression and used to transduce de-differentiated articular chondrocytes without EGFP induction. BMP-2 expression was proportional to the Bac-MECB dose and promoted cartilage-specific matrix synthesis, implicating the potential of Bac-MECB in restoring chondrocyte differentiation. These data confirmed that regulatable EGFP expression enabled rapid, reliable baculovirus titration without interference with subsequent applications.

Primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 BMPs and Sox9 maintain appropriate phenotype.

OBJECTIVE: To confirm that primary intervertebral disc cells cultured in monolayer transduced with adenovirus maintained their phenotype, hence is an appropriate system to test gene therapy agents. DESIGN: Adult bovine nucleus pulposus and anulus fibrosus cells cultured in monolayer were transduced with adenoviruses expressing human bone morphogenetic proteins (AdBMPs) or Sox9 (AdSox9), or green fluorescence protein (AdGFP, as control). Chondrocyte phenotypic markers (e.g., type II collagen and aggrecan) and the chondrocyte hypertrophy marker (type X collagen) were measured 6 days after viral transduction by reverse-transcription polymerase chain reaction. RESULTS: Primary nucleus pulposus and anulus fibrosus cells transduced with AdBMPs, AdSox9, or adenovirus-expressing green fluorescence protein only (AdGFP, as control) continue to express healthy chondrocyte phenotypic markers and showed no evidence of the expression of the chondrocyte hypertrophy marker (type X collagen gene). Thus, we have shown that bovine nucleus pulposus and anulus fibrosus cells transduced with adenovirus overexpressing 12 different bone morphogenetic proteins or Sox9 maintain their phenotype in short-term culture. CONCLUSIONS: In this study, primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 bone morphogenetic proteins or Sox9 preserved their phenotype in short-term culture. These cells did not express the type X collagen gene, an undesirable chondrocyte hypertrophic gene that could lead to ossification. Therefore, low-passage intervertebral disc cells cultured in monolayer is an appropriate culture system to test therapeutic genes. We further suggest that these cells may also be appropriate for engineering tissues or for cell therapy for degenerative disc diseases.

The ubiquitin-proteasome pathway and viral infections in articular cartilage of patients with osteoarthritis.

Many viruses can evolve different strategies to exploit the ubiquitin-proteasome pathway (UPP) for their own benefit. Some data have recently established connections between UPP and osteoarthritis (OA). The aim of this study was to determine the possible involvement of viral infections linked with the UPP in the physiopathology of OA. Samples of human cartilage were obtained from 12 patients with clinical and radiological features of OA and from 12 normal controls. DNA was extracted from cultured chondrocytes from these patients, and quantitative real-time PCR was performed to analyse the DNA/RNA prevalence and viral loads of HSV, EBV, HCMV, enterovirus, and HTLV-1. The prevalence of total viral DNA/RNA among patients with OA was 16.7% (mean viral load of 7.86 copies/mug DNA), EBV being responsible for the two positive samples, while the prevalence in controls was 0%. We did not detect any positive samples for HSV, CMV, enterovirus, and HTLV-1 among patients with OA and controls. This first approach to the study of the prevalence of viruses linked to the UPP in articular cartilage of end-stage OA patients provides evidences supporting the risk of EBV transmission or reactivation in a subset of patients with disorders requiring tissue regeneration.

Detection of reticuloendotheliosis virus by immunohistochemistry and in situ hybridization in experimentally infected Japanese quail embryos and archived formalin-fixed and paraffin-embedded tumours.

Reticuloendotheliosis virus (REV) infection can result in immunosuppression, a runting syndrome, high mortality, acute reticulum cell neoplasia, or T-cell and/or B-cell lymphomas, in a variety of domestic and wild birds. Histopathological changes in REV infection are not sufficient to differentiate it from avian lymphoid leukosis and Marek's disease, and currently there are no available in situ diagnostic methods for detection of active REV presence in pathologic specimens. To develop immunohistochemistry and in situ hybridization assays for detection of REV active infections, experimentally inoculated Japanese quail embryos, and archived formalin-fixed paraffin-embedded tissues from natural and experimental reticuloendotheliosis cases in chickens and turkeys, were examined. The in situ hybridization and immunohistochemistry assays proved to be efficient for the detection of several REV strains in Japanese quail embryos during active infection, whereas these assays were much less sensitive when applied to archived tissue samples from chronically infected birds with lymphoid tumours. The diagnostic assays developed in this study have potential as diagnostic tools for detection of active REV infections.

The location in cartilage of infectious retrovirus in cats infected with feline leukemia virus.

BACKGROUND: Previous studies have suggested that articular cartilage allografts were not likely to transmit infectious retrovirus since viral DNA could not be isolated from chondrocytes of infected individuals. However, the ability of the extracellular matrix of articular cartilage to harbor and transmit a retrovirus has not been examined. We hypothesized that articular cartilage fragments, but not isolated chondrocytes, from cats systemically infected with feline leukemia virus (FeLV) are capable of transmitting infectious retrovirus. METHODS: Fresh cartilage segments and chondrocytes isolated from cats systemically infected with feline leukemia virus were used in this study. Feline embryonic fibroblast cells were cocultured with segments of cartilage, isolated chondrocytes, or fragments of cortical bone from each infected cat. The FeLV p27 antigen was measured in the coculture media by enzyme-linked immunosorbent assay. In addition, FeLV proviral nucleic acids were quantified by real-time quantitative polymerase chain reaction with use of DNA extracted from feline embryonic fibroblast cell cocultures as well as isolated chondrocytes. Immunohistochemistry was used to assess for FeLV p27 antigen in both intact cartilage fragments and isolated chondrocytes. RESULTS: Feline embryonic fibroblast cells cocultured with cartilage fragments from each of the five FeLV-infected cats all demonstrated high levels of proviral DNA, indicating transmission of infective virus. In addition, media from all cocultures of feline embryonic fibroblast cells and chondral fragments became positive for p27 antigen, indicating active viral replication. In contrast, cocultures of feline embryonic fibroblast cells and isolated chondrocytes from all FeLV-infected cats were negative for proviral DNA and p27 antigen. Likewise, no proviral nucleic acids could be detected in isolated chondrocytes from any infected cats. Cocultures of feline embryonic fibroblast cells with cortical bone fragments were positive for proviral DNA and p27 antigen. Immunohistochemical staining of cartilage fragments from FeLV-infected cats demonstrated the presence of p27 antigen throughout the extracellular matrix, but the p27 antigen was not detected in isolated chondrocytes. CONCLUSIONS: Articular cartilage fragments can readily transmit infectious retrovirus, but isolated chondrocytes were likely not the source of the infectious virus because they did not harbor proviral DNA or p27 antigen.

Cytomegalovirus (AD169) infection in human chondrocyte cell cultures.

Human chondrocytes are susceptible to productive human cytomegalovirus (HCMV, AD169 strain) infection. Infection of chondrocytes resulted in pronounced cytopathic effects including cell rounding and aggregation, fusions, and lysis. Immunohistochemical staining reveals that a portion of chondrocytes expressed antigens reactive to monoclonal antibodies against early HCMV proteins. The interaction of HCMV with cartilage cells may be one key for elucidating the etiopathogenesis of articular diseases.

Basophilic intracytoplasmic viral matrix inclusions distributed widely in layer hens affected with avian-leukosis-virus-associated tumours.

Layer hens (310 days old) affected with subcutaneous tumours were investigated pathologically. Basopholic intracytoplasmic viral matrix inclusions (MIs) were widely distributed in the chickens affected with subcutaneous myxoma rhabdomyosarcoma, perineuroma, glioma, intra-abdominal adenocarcinoma, and nephroblastoma. MIs were observed in the myocardial cells and the impulse-conducting-system cells. They were also present in the smooth muscle cells of the arteries in the spleen and lungs, in the smooth muscle of the digestive tract muscular layer (crop, oesophagus, proventriculus, gizzard, duodenum, jejunum, ileum, caecum, and large intestine), and in the smooth muscle of the capsule in the ovary and pancreas. They were also observed in the podocytes of glomeruli and renal epithelium in the kidneys, tumour cells of nephroblastoma, chondrocytes of the trachea, squamous-epithelial cells of the pharynx, and nerve cells of the cerebrum and tumour cells of the glioma in the cerebellum. Histochemically, MIs were stained with RNA, but not with DNA. MIs in the various tissues were strongly positive for the avian leukosis virus (ALV) antigen. Ultrastracturally, MIs were found in the cytoplasm of myocardial cells and podocytes of renal glomeruli. They consisted of electron-dense small granules and ring-shaped particles. Viral particles were observed in the vesicles of the cytoplasm of myocardial cells and glomerular podocytes. The gene product specific for subgroup A of ALV was detected in the livers or tumours by reverse transcription-polymerase chain reaction. This result suggests that MIs can be formed in organs rather than muscular systems in the chickens naturally affected with ALV-associated tumours.

Evaluation of permissiveness and cytotoxic effects in equine chondrocytes, synovial cells, and stem cells in response to infection with adenovirus 5 vectors for gene delivery.

OBJECTIVE: To evaluate host cell permissiveness and cytotoxic effects of recombinant and modified adenoviral vectors in equine chondrocytes, synovial cells, and bone marrow-derived mesenchymal stem cells (BMD-MSCs). SAMPLE POPULATION: Articular cartilage, synovium, and bone marrow from 15 adult horses. PROCEDURES: Equine chondrocytes, synovial cells, and BMD-MSCs and human carcinoma (HeLa) cells were cultured and infected with an E-1-deficient adenovirus vector encoding the beta-galactosidase gene or the green fluorescent protein gene (Ad-GFP) and with a modified E-1-deficient vector with the arg-gly-asp capsid peptide insertion and containing the GFP gene (Ad-RGD-GFP). Percentages of transduced cells, total and transduced cell counts, and cell viability were assessed 2 and 7 days after infection. RESULTS: -Permissiveness to adenoviral vector infection was significantly different among cell types and was ranked in decreasing order as follows: HeLa cells > BMD-MSCs > chondrocytes > synovial cells. Morphologic signs of cytotoxicity were evident in HeLa cells but not in equine cells. Numbers of transduced cells decreased by day 7 in all cell types except equine BMD-MSCs. Transduction efficiency was not significantly different between the Ad-GFP and Ad-RGD-GFP vectors. CONCLUSION AND CLINICAL RELEVANCE: Sufficient gene transfer may be achieved by use of an adenovirus vector in equine cells. High vector doses can be used in equine cells because of relative resistance to cytotoxic effects in those cells. Greater permissiveness and sustained expression of transgenes in BMD-MSCs make them a preferential cell target for gene therapy in horses.

Inhibition of NF-kappaB renders human juvenile costal chondrocyte cell lines sensitive to TNF-alpha-mediated cell death.

BACKGROUND: Recently, therapeutics employing knowledge on various signaling pathways are being developed, with NF-kappaB being one of the most promising targets. NF-kappaB has been suggested to play a role not only in the induction of inflammatory mediators, but also in the protection from cell death. OBJECTIVES: This study pursued the role of the NF-kappaB pathway in the regulation of chondrocyte death induced by tumor necrosis factor alpha (TNF-alpha) and of the pertinent target molecules involved. METHODS: The human chondrocyte cell line C28/I2 was used for the experiment. Chondrocytes were transduced with adenovirus-encoding IkappaB (IkappaB) superrepressor which inhibits NF-kappaB activation, and treated with TNF-alpha. The proportion of cell death was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazdium bromide (MTT) assay. Activation of p38 mitogen activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3K) by TNF-alpha was inhibited with SB202190 and Ly 294002 respectively. The expression of apoptosis related protein was analyzed with western blot assay, and the activation of c-Jun N-terminal kinase (JNK) by solid-phase kinase assay. RESULTS: Treatment with TNF-alpha led to cell death in 23% and 50% of ad-IkappaB-SR infected chondrocytes after 24 and 72 h respectively. The expression of Bcl-XL, Bcl-2, and XIAP significantly decreased, and activation of JNK was prolonged for up to 6 h in infected cells treated with TNF-alpha. Preincubation with p38 inhibitor or PI3K inhibitor before TNF-alpha led to a significant increase in cell death in ad-IkappaB-SR transduced chondrocytes, resulting in 53% and 30% cell death after 24 h for p38 inhibitor and PI3K inhibitor respectively. CONCLUSION: In our experimental system, specific inhibition of NF-kappaB activation rendered chondrocytes susceptible to cell death induced by TNF-alpha. The cell death was enhanced by inhibition of another signaling pathway such as p38 MAP kinase or PI3K. The expression of Bcl-XL, Bcl-2 and XIAP and activation of JNK were affected by ad-IkappaB-SR transduction, implying a role in the NF-kappaB regulated cell survival signaling in human chondrocytes.

Culture of immortalized chondrocytes and their use as models of chondrocyte function.

Immortalization of chondrocytes increases life span and proliferative capacity but does not necessarily stabilize the differentiated phenotype. Expansion of chondrocyte cell lines in continuous monolayer culture may result in the loss of phenotype, particularly if high cell density is not maintained. This chapter describes strategies for maintaining or restoring differentiated phenotype in established chondrocyte cell lines involving culture in serum-free defined culture medium, in suspension over agarose or polyHEMA, or within alginate or collagen scaffolds. Chondrocyte cell lines have been used successfully to develop reproducible models for studying the regulation of gene expression in experiments requiring large numbers of cells. Thus, approaches for studying transcriptional regulation by transfection of promoter-driven reporter genes and cotransfection of expression vectors for wild-type or mutant proteins are also described.

Immortalization of human articular chondrocytes for generation of stable, differentiated cell lines.

Immortalized chondrocytes of human origin have been developed to serve as reproducible models for studying chondrocyte function. In this chapter, methods for immortalization of primary human chondrocytes with SV40-TAg, HPV-16 E6/E7, and telomerase by retrovirally mediated transduction and selection for neomycin resistance are described. However, stable integration of an immortalizing gene stabilizes proliferative capacity, but not the differentiated chondrocyte phenotype. Thus, strategies for selection of chondrocyte cell lines, involving the maintenance of high cell density and moderation of cell proliferation, are also described. The methods for immortalization and selection are applicable to the development of chondrocyte cell lines using any immortalizing agent. Although immortalized chondrocytes should not be considered as substitutes for primary chondrocytes, they may be useful tools for evaluating and further validating mechanisms relevant to cartilage biology.

Immune response against gene therapy vectors: influence of synovial fluid on adeno-associated virus mediated gene transfer to chondrocytes.

Intraarticular gene transfer with adeno-associated virus (AAV) vectors may allow efficient therapeutic transgene expression within the joint. In an effort to understand potential obstacles (particularly immunity against AAV vectors) to intraarticular gene therapy better, our objective was to determine whether synovial fluid (SF) influenced AAV-mediated gene transfer to chondrocytes. SF and sera from 21 patients with joint diseases were collected. Neutralizing activity against AAV/interleukin-4 (IL-4) was determined by assessing the ability of SF or serum to inhibit AAV/IL-4 transduction to the C20A4 chondrocytes. IgGs were purified from SF by salt-dependent chromatography. Anti-AAV IgG levels were determined by ELISA in the SF. SF and sera from all the patients inhibited AAV-mediated gene transfer to chondrocytes. Six SF out of 21 exerted a stronger inhibition. Serum from healthy patients were also inhibitory. Purified IgGs from SF exhibited inhibition patterns similar to those seen with whole SF. Anti-AAV IgG were found in SF from 13 patients out of 18. Moreover, in the SF, anti-AAV IgG level was correlated with the neutralizing activity (p < 0.001, r = 0.716). A correlation was observed between levels of inhibition by the SF and serum (P < 0.0001, r = 0.813). Inhibition of AAV/IL-4 infection of C20A4 cells by SF and sera was abolished by increasing the number of AAV/IL-4 particles. SF from patients with joint disease consistently inhibited AAV infection of chondrocytes in vitro. This effect was ascribable to IgG, most probably directed against AAV. In the future, these data may be useful for tailoring intraarticular AAV-mediated gene therapy to individual patients.

Light-activated gene transduction enhances adeno-associated virus vector-mediated gene expression in human articular chondrocytes.

OBJECTIVE: To evaluate the effects of ultraviolet (UV) light as an adjuvant for recombinant adeno-associated virus (rAAV) transduction in human articular chondrocytes. METHODS: Primary articular chondrocytes and immortalized chondrocytes (tsT/AC62) were exposed to various doses of UV light (0-1,000 J/m(2)) and infected at various multiplicities of infection (MOIs) with rAAV containing the enhanced green fluorescent protein (EGFP) gene. Cells were analyzed for viability and EGFP expression by fluorescence-activated cell sorting on days 2, 4, and 8 following infection. To evaluate the transduction efficiency in intact articular cartilage, full-thickness explants were exposed to UV light (0-200 J/m(2)), infected with rAAV-eGFP, and analyzed for transduction via immunohistochemistry. RESULTS: Toxicity from UV exposure was observed at doses > or =500 J/m(2) and > or =200 J/m(2) in primary and immortalized chondrocyte cultures, respectively. Transduction efficiency was dependent on the UV dose, MOI, and time. In the cell line, the adjuvant effect of UV on the percentage of cells transduced was modest, but 100 J/m(2) increased the mean fluorescence intensity (MFI) of the transduced cells 4-fold. In contrast, UV treatment had a profound effect on the transduction efficiency of primary chondrocytes, which reached approximately 100% after exposure to 100 J/m(2) of UV light and 10(3) MOIs for 8 days. Under the same conditions, 200 J/m(2) of UV light enhanced the MFI 7-fold. In cartilage explants, there was no difference in the number of transduced chondrocytes at the edge of the explants in the superficial, intermediate, or basal zones; however, 200 J/m(2) of UV light increased the transduction efficiency 2-fold at a low MOI. In the center of the explants, the superficial chondrocytes were efficiently transduced; those in the intermediate and basal zones could not be efficiently transduced under any condition. In the superficial chondrocytes, a low MOI and 200 J/m(2) of UV light increased the transduction efficiency 3-fold (to 100%). CONCLUSION: UV light at doses of up to 200 J/m(2) (which do not significantly affect cell viability) significantly enhances the transduction efficiency and expression of the transduced gene in cultures of rAAV-infected primary chondrocytes and in chondrocytes in the superficial zone of intact articular cartilage. These findings support the concept that UV-activated gene transduction could be used as an adjuvant for in vivo rAAV articular cartilage gene therapy with low viral titers to prevent and/or treat arthritis.