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Biomedical subjects

Christian Jorgensen

Publications and source records attributed to Christian Jorgensen.

At least 19 recordsLinked to original sources
PubMed2006

Earlier onset of syngeneic tumors in the presence of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSC) are widely investigated for cell therapy purposes as support of hematopoietic cell transplantation, skeletal tissue regeneration, or as a cell delivery system of therapeutic agents in cancer. However, because of their immunosuppressive capacities, we investigated the effect of MSC on the development of syngeneic tumors. METHODS: The murine MSC line C3H10T1/2 was coinjected with the Renca adenocarcinoma or the B16 melanoma cell lines in BALB/c mice. RESULTS: The injection of MSC permitted the growth of the allogeneic B16 tumor cells and reduced the delay of tumor appearance when Renca cells were implanted, without modifying the kinetics of tumor growth. This effect was observed even with a low ratio of cancer cells, mimicking minimal residual disease. In this last case, no MSC were detected in the tumor mass, suggesting that cell contact was not necessary. The presence of MSC did not enhance the development of lung metastasis after systemic injection of Renca cells. Because the proliferative rate of Renca cells was not affected by in vitro coculture with MSC, this observation is likely due to a systemic suppressive effect on the host immune system. CONCLUSIONS: Altogether, these data suggest that MSC did not interfere with the kinetics of tumor development but may reduce the delay for tumor occurrence. An important finding of this study is that a low but relevant amount of MSC may induce tumor rejection.

Animals↗
PubMed2006

Immature dendritic cells suppress collagen-induced arthritis by in vivo expansion of CD49b+ regulatory T cells.

Dendritic cells (DCs) are specialized APCs with an important role in the initiation and regulation of immune responses. Immature DCs (iDCs) reportedly mediate tolerance in the absence of maturation/inflammatory stimuli, presumably by the induction of regulatory T cells. In this study, we show for the first time that repetitive iDC injections trigger the expansion of a novel regulatory population with high immunomodulatory properties, able to protect mice from collagen-induced arthritis. These regulatory T cells are characterized by the expression of the CD49b molecule and correspond to a CD4+ alpha-galactosylceramide/CD1d-nonrestricted T cell population producing IL-10. Adoptive transfer of < 10(5) TCRbeta+ CD49b+ cells isolated from the liver of iDCs-vaccinated mice, conferred a complete protection against arthritis. This protection was associated with an attenuation of the B and T cell response associated with a local secretion of IL-10. Thus, together these data demonstrate that iDCs can expand and activate a novel regulatory population of CD49b+ T cells, with high immunosuppressive potential able to mediate protection against a systemic autoimmune disease.

Adoptive Transfer↗
PubMed2006

Chondrogenic differentiation of murine and human mesenchymal stromal cells in a hyaluronic acid scaffold: differences in gene expression and cell morphology.

Chondrogenesis is a complex process characterized by a sequence of different steps that start with the condensation of the cells, followed by the expression of specific components, such as collagens and proteoglycans. We evaluated in vitro chondrogenic differentiation of C3H10T1/2 murine mesenchymal cells and compared them with human mesenchymal stromal cells (h-MSCs) in a hyaluronic acid scaffold. We analyzed (from day 0 to day 28) cellular morphology, proliferation, and chondrogenic/osteogenic gene expression at different time points. Our data demonstrate that, during chondrogenic differentiation, murine cells proliferate both in the absence and presence of TGFbeta, while h-MSCs require the presence of this activating factor. Murine cells, even if viable, differentiate on hyaluronan scaffold, maintain a fibroblastic morphology, and form a capsule outside the scaffold. At the mRNA level, murine cells showed a decrease in collagen type I combined with a significant increase in collagen type II (from day 0), and aggrecan (on day 28), as found for h-MSCs. Immunohistochemical data confirmed that chondrogenic differentiation of murine cells, induced by TGFbeta, occurred only in some restricted areas inside the scaffold that were positive to collagen type II, but did not show a cartilage-like tissue structure, as we had found using h-MSCs. These data demonstrate that C3H10T1/2 murine cells, widely used as a chondrogenic model, show a different sequence of chondrogenic events in hyaluronic acid scaffold, compared with primary h-MSCs.

Animals↗
PubMed2006

Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor alpha in experimental arthritis.

OBJECTIVE: Tumor necrosis factor alpha (TNFalpha) is among the most prominent cytokines in rheumatoid arthritis (RA) and is secreted mainly by macrophages. A direct method for restoring the immunologic balance in RA is use of small interfering RNA (siRNA) for silencing the TNFalpha transcript. The aim of this study was to determine the therapeutic effect of systemic administration of TNFalpha siRNA in an experimental model of RA, optimizing its delivery using new liposome formulations. METHODS: Murine macrophages were transfected with siRNA targeting TNFalpha, and expression was measured. The therapeutic effect in collagen-induced arthritis (CIA) was assessed after intravenous delivery of TNFalpha siRNA. Delivery was optimized using a carrier DNA for complexation with the cationic liposome RPR209120/DOPE. Levels of TNFalpha and other cytokines were measured in sera and joint tissue-conditioned media. Biodistribution was determined using a fluorescent siRNA. RESULTS: In vitro, TNFalpha siRNA efficiently and specifically modulated the expression of TNFalpha at both the messenger RNA and protein levels. In vivo, complete cure of CIA was observed when TNFalpha siRNA was administered weekly, complexed with the liposome and combined with carrier DNA. Inhibition (50-70%) of articular and systemic TNFalpha secretion was detected in the siRNA-injected groups, which correlated with a decrease in the levels of interleukin-6 and monocyte chemotactic protein 1. The main organs targeted by siRNA were the liver and spleen; the addition of liposome RPR209120 and carrier DNA significantly increased organ uptake. CONCLUSION: We demonstrated the efficiency of systemic delivery of siRNA designed to silence TNFalpha in CIA, using a liposome carrier system as a way to address the methodologic limitations in vivo.

Animals↗
PubMed2005

Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells.

Previous studies have reported that mesenchymal stem cells (MSC) may be isolated from the synovial membrane by the same protocol as that used for synovial fibroblast cultivation, suggesting that MSC correspond to a subset of the adherent cell population, as MSC from the stromal compartment of the bone marrow (BM). The aims of the present study were, first, to better characterize the MSC derived from the synovial membrane and, second, to compare systematically, in parallel, the MSC-containing cell populations isolated from BM and those derived from the synovium, using quantitative assays. Fluorescent-activated cell sorting analysis revealed that both populations were negative for CD14, CD34 and CD45 expression and that both displayed equal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to be characteristic of BM-MSC. Comparable with BM-MSC, such MSC-like cells isolated from the synovial membrane were shown for the first time to suppress the T-cell response in a mixed lymphocyte reaction, and to express the enzyme indoleamine 2,3-dioxygenase activity to the same extent as BM-MSC, which is a possible mediator of this suppressive activity. Using quantitative RT-PCR these data show that MSC-like cells from the synovium and BM may be induced to chondrogenic differentiation and, to a lesser extent, to osteogenic differentiation, but the osteogenic capacities of the synovium-derived MSC were significantly reduced based on the expression of the markers tested (collagen type II and aggrecan or alkaline phosphatase and osteocalcin, respectively). Transcription profiles, determined with the Atlas Human Cytokine/Receptor Array, revealed discrimination between the MSC-like cells from the synovial membrane and the BM-MSC by 46 of 268 genes. In particular, activin A was shown to be one major upregulated factor, highly secreted by BM-MSC. Whether this reflects a different cellular phenotype, a different amount of MSC in the synovium-derived population compared with BM-MSC adherent cell populations or the impact of a different microenvironment remains to be determined. In conclusion, although the BM-derived and synovium-derived MSC shared similar phenotypic and functional properties, both their differentiation capacities and transcriptional profiles permit one to discriminate the cell populations according to their tissue origin.

Activins↗
PubMed2005

Reversal of the immunosuppressive properties of mesenchymal stem cells by tumor necrosis factor alpha in collagen-induced arthritis.

OBJECTIVE: Adult mesenchymal stem cells (MSCs) represent promising tools for therapeutic applications such as tissue engineering and cellular therapy. Recent data suggest that, due to their immunosuppressive nature, MSCs may be of interest to enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease. Using a murine model of rheumatoid arthritis (RA), this study investigated whether the immunosuppressive properties of MSCs could be of therapeutic value to inhibit reactive T cells in autoimmune diseases such as RA. METHODS: In mice with collagen-induced arthritis (CIA), we injected various doses of C3 MSCs at the time of immunization or booster injection, and subsequently evaluated the clinical and immunologic parameters. The immunosuppressive properties of MSCs were determined in vitro in mixed lymphocyte reactions with or without the addition of tumor necrosis factor alpha (TNFalpha). RESULTS: In the CIA model of arthritis, MSCs did not confer any benefit. Both the clinical and the immunologic findings suggested that MSCs were associated with accentuation of the Th1 response. Using luciferase-expressing MSCs, we were unable to detect labeled cells in the articular environment of the knee, suggesting that worsening of the symptoms was unlikely due to the homing of MSCs in the joints. Experiments in vitro showed that the addition of TNFalpha was sufficient to reverse the immunosuppressive effect of MSCs on T cell proliferation, and this observation was associated with an increase in interleukin-6 secretion. CONCLUSION: Our data suggest that environmental parameters, in particular those related to inflammation, may influence the immunosuppressive properties of MSCs.

Animals↗
PubMed2004

Antigen-specific immunomodulation of collagen-induced arthritis with tumor necrosis factor-stimulated dendritic cells.

OBJECTIVE: Dendritic cells (DCs) are crucial for the initiation of T cell immunity and therefore play an important role in the initiation and regulation of immune responses in arthritis. Full mobilization of effector T cells depends on the proper maturation of DCs. Current evidence indicates that the type of T cell response induced is crucially dependent on the activation status of the DCs. In this study, we explored the immunologic effects of differentially matured DCs on the development of collagen-induced arthritis (CIA). METHODS: Bone marrow-derived DCs were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Before immunization with bovine type II collagen (CII) protein, mice were repeatedly injected with DCs that had been pulsed with CII. Immature, semimature, or fully mature DCs were injected. Mice were boosted on day 21 after CII immunization, and the disease course was monitored. RESULTS: While vaccination with immature or lipopolysaccharide-activated DCs had no significant effect on the disease course, administration of antigen-loaded, tumor necrosis factor (TNF)-modulated DCs propagated in GM-CSF with or without interleukin-4 resulted in a delayed onset of arthritis and a lower clinical score. The response was antigen-specific, since TNF-treated DCs pulsed with a control antigen did not modify the disease course. A specific decrease in the collagen-specific "Th1-associated" IgG2a response was observed, whereas IgG1 titers were unaffected. CONCLUSION: CIA can be prevented through vaccination with TNF-matured DCs in an antigen-specific manner. These findings provide a rationale for immunotherapy using DCs in rheumatoid arthritis.

Animals↗
PubMed2004

Tissue engineering through autologous mesenchymal stem cells.

The regeneration of damaged cartilage in different pathological situations is a major goal for the future and could be achieved through cell and/or gene therapy. Mesenchymal stem cells (MSCs) are the progenitors of multiple lineages, including bone, cartilage, muscle, fat, and astrocytes. MSCs seem to be the best candidates for cell therapy to regenerate injured tissue, as they are easily isolated from bone marrow and can be rapidly amplified. Full healing is extremely demanding, however, and includes integration of the regenerated tissue within the surrounding host tissue and true differentiation through pathways involved in embryonic development. This goal might be reached through the combined use of scaffolds, MSC-mediated therapy and the expression of selective differentiating factors. The long-term behavior of MSCs associated with biomaterials and implanted in pathological joints remains to be investigated before clinical application in osteoarthritis or rheumatoid arthritis.

Animals↗
PubMed2004

Gene therapy platform for bone regeneration using an exogenously regulated, AAV-2-based gene expression system.

Viral delivery of the therapeutic gene bone morphogenetic protein-2 (BMP-2) is a promising approach for bone regeneration. The human parvovirus adeno-associated virus (AAV) type 2 is considered one of the most encouraging viral vector systems because of its high transduction rates and biosafety ratings. Bone morphogenetic protein-2 is a highly potent osteoinductive protein, which induces bone formation in vivo and osteogenic differentiation in vitro. The exogenous regulation of BMP-2 expression in bone-regenerating sites is required to control BMP-2 protein secretion, thus promoting safe and controlled bone formation and regeneration. We have therefore constructed a dual-construct vector for the recombinant AAV (rAAV)-based recombinant human BMP-2 (rhBMP-2) gene delivery system, which is regulated by the tetracycline-sensitive promoter (TetON). Each vector was encapsidated separately, yielding two recombinant viruses. We evaluated the efficiency of rAAV-hBMP-2 to induce bone formation in ectopic and orthotopic sites. Doxycycline (Dox), an analogue of tetracycline, was orally administered to mice via their drinking water to induce rhBMP-2 expression. Bone formation was measured using quantitative imaging-microcomputerized tomography and cooled charge-coupled device imaging-to detect osteogenic activity at the cellular level, detecting osteocalcin expression. The rAAV-hBMP-2-treated mice that were given Dox demonstrated bone formation in both in vivo models compared to none in mice prevented from receiving Dox. Thus, the Tet-regulated rAAV-hBMP-2 vector is an effective means of induction and regulation of bone regeneration and repair.

Animals↗
PubMed2004

Leflunomide-induced aseptic meningitis.

Drug-induced aseptic meningitis is uncommon and occurs primarily in patients with autoimmune disease. We report the first known case of leflunomide-induced aseptic meningitis, in a patient with rheumatoid arthritis.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2004

Feline immunodeficiency virus vectors for efficient transduction of primary human synoviocytes: application to an original model of rheumatoid arthritis.

The potential of gene therapeutics is hindered by the limitations of the delivery systems presently available. Recently, human immunodeficiency virus (HIV) vectors have been developed that allow the efficient and stable transduction of primary nondividing cells in vivo. Because of the safety concerns raised by HIV vectors, we developed a gene delivery system derived from the ungulate lentivirus feline immunodeficiency virus (FIV). We describe in the present study the optimization of the safety and efficiency of this system that proved to be as potent as HIV vectors to transduce nondividing cells, with titers over 10(8) transducing units per ml. We used this tool to transduce TNF-alpha into human primary synoviocytes, and showed a high efficiency of transduction. TNF-alpha-transduced synoviocytes injected into the knee joints of severe combined immunodeficient (SCID) mice induced cell proliferation, as well as cartilage and bone erosion as soon as day 7, creating a standardized, humanized animal model relevant for rheumatoid arthritis. FIV vectors appear to be promising tools for biologic research and gene therapy.

Animals↗
PubMed2004

Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells.

Currently available murine models to evaluate mesenchymal stem cell (MSC) differentiation are based on cell injection at ectopic sites such as muscle or skin. Due to the importance of environmental factors on the differentiation capacities of stem cells in vivo, we investigated whether the peculiar synovial/cartilaginous environment may influence the lineage specificity of bone morphogenetic protein (BMP)-2-engineered MSCs. To this aim, we used the C3H10T1/2-derived C9 MSCs that express BMP-2 under control of the doxycycline (Dox)-repressible promoter, Tet-Off, and showed in vitro, using the micropellet culture system that C9 MSCs kept their potential to differentiate toward chondrocytes. Implantation of C9 cells, either into the tibialis anterior muscles or into the joints of CB17-severe combined immunodeficient bg mice led to the formation of cartilage and bone filled with bone marrow as soon as day 10. However, no differentiation was observed after injection of naïve MSCs or C9 cells that were repressed to secrete BMP-2 by Dox addition. The BMP-2-induced differentiation of adult MSCs is thus independent of soluble factors present in the local environment of the synovial/cartilaginous tissues. Importantly, we demonstrated that a short-term expression of the BMP-2 growth factor is necessary and sufficient to irreversibly induce bone formation, suggesting that a stable genetic modification of MSCs is not required for stem cell-based bone/cartilage engineering.

Animals↗
PubMed2003

Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals.

Mesenchymal stem cells (MSCs) are largely studied for their potential clinical use. Recently, they have gained further interest after demonstration of an immunosuppressive role. In this study, we investigated whether in vivo injection of MSCs could display side effects related to systemic immunosuppression favoring tumor growth. We first showed in vitro that the murine C3H10T1/2 (C3) MSC line and primary MSCs exhibit immunosuppressive properties in mixed lymphocyte reaction. We demonstrated that this effect is mediated by soluble factors, secreted only on "activation" of MSCs in the presence of splenocytes. Moreover, the immunosuppression is mediated by CD8+ regulatory cells responsible for the inhibition of allogeneic lymphocyte proliferation. We then demonstrated that the C3 MSCs expressing the human bone morphogenetic protein 2 (hBMP-2) differentiation factor were not rejected when implanted in various allogeneic immunocompetent mice and were still able to differentiate into bone. Importantly, using a murine melanoma tumor model, we showed that the subcutaneous injection of B16 melanoma cells led to tumor growth in allogeneic recipients only when MSCs were coinjected. Although the potential side effects of immunosuppression induced by MSCs have to be considered in further clinical studies, the usefulness of MSCs for various therapeutic applications still remains of great interest.

Animals↗
PubMed2002

Tetracycline transcriptional silencer tightly controls transgene expression after in vivo intramuscular electrotransfer: application to interleukin 10 therapy in experimental arthritis.

The doxycycline (Dox)-inducible reverse tetracycline transactivator (rtTA) is often used to control gene expression. However, the Tet-on system displays a high background activity. To overcome this unregulated expression we used the tetracycline-dependent transcriptional silencer (tTS), which binds the tetO inducible promoter in the absence of Dox. Controlled gene expression was analyzed in vivo by delivering combinations of Dox-regulated luciferase reporter construct, rtTA, and tTS expression plasmids into mouse muscle, using electrotransfer. Elevated luciferase expression levels were observed in the absence of doxycycline, and a 10-fold induction was obtained after drug administration. In contrast, when tTS was added, background expression was dramatically lowered by three to four orders of magnitude, and induction was maintained. The tTS system was then used to control expression of a therapeutic gene in experimental arthritis. DBA/1 mice were coinjected with plasmids encoding the antiinflammatory interleukin-10 cytokine under the control of the tetO promoter, the rtTA, and the tTS. Electrotransfer resulted in a dose-dependent increase in IL-10 expression, maintained over a 3-month period, and significant inhibitory effects on collagen-induced arthritis. We conclude that the use of tTS significantly improves the utility of the rtTA system for somatic gene transfer by reducing background activity.

Animals↗
PubMed2002

Tetracycline-inducible interleukin-10 gene transfer mediated by an adeno-associated virus: application to experimental arthritis.

The adeno-associated viruses (AAV) offer new perspectives for cytokine gene transfer in rheumatoid arthritis (RA) because they are nonpathogenic and allow long-term transgene expression in vivo. Moreover, the use of a tetracycline-inducible promoter allows regulation of therapeutic gene expression. This study assessed the potential long-term gene regulation of a recombinant AAV vector expressing viral interleukin-10 (vIL-10) in human rheumatoid synovium and the therapeutic efficiency in a mouse model of RA. We constructed a recombinant AAV vector in which the transcription of vIL-10 cDNA is controlled by the TetON system. Transduction of human primary RA synovial cells with AAV-tetON-vIL10 conferred in vitro controlled vIL-10 expression. After intramuscular injection, both incidence and severity of collagen-induced arthritis were significantly reduced at macroscopic, radiological, and histological levels in the group of DBA1 mice treated with AAV-TetON-vIL10 vector plus doxycycline after immunization and boosting compared to control groups. When coinjecting two separate AAV vectors, one encoding the inducible vIL-10 and the other the transcriptional activator, a 10 times excess of the transactivator vector dose allowed efficient control of vIL-10 secretion by doxycycline administration or withdrawal, over an 8-week period. Our results supported, for the first time, the utility of AAV-tetON-vIL10 as a therapeutic tool for gene therapy in RA.

Animals↗
PubMed2002

CD4 coating, but not CD4 depletion, is a predictor of efficacy with primatized monoclonal anti-CD4 treatment of active rheumatoid arthritis.

OBJECTIVE: Double blind studies were conducted with the anti-CD4 monoclonal antibody (Mab) keliximab in patients with active, stable rheumatoid arthritis (RA), to confirm preliminary evidence of efficacy and safety from open. uncontrolled studies. METHODS: We enrolled 136 and 186 patients into 2 consecutive, randomized, double blind trials, with similar populations [apart from inclusion of disease modifying antirheumatic drug (DMARD)-naïve patients in Study 2]. Patients received 4 weeks intravenous placebo or keliximab [40, 80, 120, or 140 mg twice weekly (bw), or 240 mg once weekly (ow)]. The primary endpoint was the American College of Rheumatology (ACR) 20 response criteria, one week after the end of treatment. RESULTS: ACR 20 response rates in Study I were 19%, 42%, 51%*, and 69%* (*p < 0.05 compared to placebo), with placebo, 40, 80, or 140 mg keliximab bw, respectively. The response rates in Study 2 were 30%, 39%, 46% and 47% with placebo, 80 or 120 mg bw, or 240 mg keliximab ow, respectively. In the 2 studies, there was a dose dependent increase in peripheral blood CD4+ T cell coating with keliximab, but a different pattern of CD4 depletion was seen. While only 12% of keliximab treated patients in Study I had CD4 counts below 250 cells/mm3 at the end of the treatment period, 47% fell below this level in Study 2. Clinical response was not correlated with CD4 depletion, but was correlated with CD4+ T cell coating with keliximab. CONCLUSION: Coating of peripheral blood CD4+ T cells with keliximab, but not CD4 depletion, is a determinant of clinical response.

Adolescent↗