Biomedical subjects

G Chiocchia

Publications and source records attributed to G Chiocchia.

At least 19 recordsLinked to original sources

PubMed2004

[What place for DNA microarray in inflammatory diseases?].

PURPOSE: DNA chip is a recently developed technique allowing analysis of thousands of genes at the same time in multiple biological samples. In few years it has become an obligatory step in massive gene expression study. The enormous quantity of results generated and the new way of thinking allowed make this kind of study a true revolution. KEY MESSAGE AND RECENT FACTS: The enormous discovery potential permitted by the accomplishment of multiple genomes sequencing and the advent of technologies allowing massive gene expression analyses have totally modified the diseases approach. Considering the obtainment of a real full picture of the transcriptional activity in an organ, tissue or cell is now legitimate. DNA microarray is obviously not the only technique allowing such type of analysis but it is without contest the technology which is the most popular and the one which has been recently the subject of the most important developments. It is certainly the technology which brought the main advances in tumour classification and discovery of new biomarkers. The first results based on this technology in inflammatory diseases have recently been reported. PERSPECTIVE AND PROJECTS: The optimal use of DNA microarrays will necessitate a powerful statistical analysis and an high quality biological experimentation. Strict standard and quality criteria are developing. Obviously, the DNA chips have a role to play in multifactorial inflammatory diseases mainly through their potential to bring new answers to diagnostic and pathophysiological problems. One potential development of the technique in such diseases will be the definition of disease specific gene profiles and the generation of chips allowing the detection of few targeted genes with all the known mutations of these genes. The correlation of global or targeted gene expression with clinical and pathological data will allow a new step forward in the understanding and taking care of inflammatory diseases.

Animals↗

PubMed2004

DNA microarray allows molecular profiling of rheumatoid arthritis and identification of pathophysiological targets.

This study was undertaken to evaluate the possibility to obtain a molecular signature of rheumatoid arthritis (RA) comparatively osteoarthritis (OA), and to lay the bases to develop new diagnostic tools and identify new targets. Microarray technology was used for such an analysis. The gene expression profiles of synovial tissues from patients with confirmed RA, and patients with OA were established and compared. A set of 63 genes was selected, based, more specifically, on their overexpression or underexpression in RA samples compared to OA. Results for six of these genes have been verified by quantitative PCR using both samples identical to those used in the microarray experiments and entirely separate samples. Expression profile of the 48 known genes allowed the correct classification of additional RA and OA patients. Furthermore, the distinct expression of three of the selected genes was also studied by quantitative RT-PCR in cultured synovial cells. Detailed analysis of the expression profile of the selected genes provided evidence for dysregulated biological pathways, pointed out to chromosomal location and revealed novel genes potentially involved in RA. It is proposed that such an approach allows valuable diagnosis/prognostics tools in RA to be established and potential targets for combating the disease to be identified.

Adult↗

PubMed2001

Transgenic expression of CD95 ligand on thyroid follicular cells confers immune privilege upon thyroid allografts.

Constitutive Fas ligand (FasL) expression by specialized cells in the body participates in the immune privilege status of tissues containing these cells. This property has been used to prevent rejection of allogeneic grafts. Nevertheless, the mechanism responsible for such protection has not been fully elucidated. Unfortunately, grafting of FasL transgenic (TG) tissues has been unsuccessful. We have generated TG mice expressing FasL (soluble + membrane bound) on thyroid follicular cells (TFC), and used them to show that ectopic FasL expression prevents thyroid allograft rejection. FasL expression on TFC led to markedly decreased anti-allogeneic, cytotoxic, and helper T lymphocyte activities. The alloantibody response in TG thyroid recipients was either completely inhibited or switched toward a T2-Ab response. Surprisingly, the beneficial effect of FasL on TG thyroid grafts was abolished by host CD4(+) T cell depletion. Host CD8(+) T cell depletion improved nontransgenic (NTG), but not TG graft survival. Altogether, our results suggest that FasL-induced tolerance is concomitant with a move away from a T1 type response, and a CD4 T cell-mediated regulation of the allocytotoxic T cell response. These results were dependent upon the level of FasL expression on TFC, in that low expression of FasL led to a less marked effect compared with the effect observed with high expression of FasL. These results provide some insight into the role of FasL in regulating destructive alloimmune responses in the case of whole organ grafting, and they have important implications for the development of FasL-based immunotherapy in organ transplantation.

Animals↗

PubMed2001

Collagen II-pulsed antigen-presenting cells genetically modified to secrete IL-4 down-regulate collagen-induced arthritis.

We explored the possibility that pulsed antigen-presenting cells (APC) provide a model vector system for site-specific delivery of immunosuppressive proteins during collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. Thus, mice were treated with either B cells or macrophages engineered to secrete IL-4 and loaded (or not) with type II collagen (CII). Systemic injection of an IL-4-producing B cell hybridoma resulted in a reduction of arthritis severity which was further improved when APC were incubated with CII before their transfer. Unmanipulated B cells loaded with CII also exerted a potent suppressive effect. Likely, clinical amelioration was observed in mice given at priming syngeneic bone marrow-derived macrophages producing IL-4 and pulsed with CII in comparison to the other groups. When the same dose of cells was transferred at disease onset, a moderate beneficial effect was observed. Whatever the APC inoculated, the beneficial effect did not rely upon an IL-4-driven shift towards Th2 phenotype. Systemic administration of fluorescent dye labeled macrophages to arthritic mice has shown that some of these cells rapidly migrate to joints. Moreover, IL-4 transfected macrophages retained their potent capacity to present CII peptides to T cells. These findings validate the use of CII peptide-loaded engineered APC as therapeutic vector cells in CIA and allow consideration of this strategy for the administration of various anti-inflammatory proteins.

Animals↗

PubMed2000

Transgenic expression of Fas ligand on thyroid follicular cells prevents autoimmune thyroiditis.

"Immune privilege" is defined as tissue resistance to aggression by specifically activated lymphocytes, and involves the interaction between Fas expressed on infiltrating cells and Fas ligand (FasL) constitutively expressed on the target tissue. To test whether ectopic expression of FasL on thyrocytes could prevent autoimmune aggression of the thyroid by activated lymphoid cells, three lines of transgenic mice expressing low, intermediate, and high levels of functional FasL on thyroid follicular cells were generated. Experimental autoimmune thyroiditis was induced by immunization with mouse thyroglobulin. In all of the experiments, the effects were dependent on the level of FasL expression. Low and intermediate expression had no or only weak preventive effects, respectively, whereas high FasL expression strongly inhibited lymphocytic infiltration of the thyroid. Anti-mouse thyroglobulin-proliferative and cytotoxic T cell responses, as well as autoantibody production, were diminished in transgenic mice expressing high levels of FasL relative to controls. Furthermore, in these latter mice Th1 responses to mouse thyroglobulin were profoundly down-regulated, uncovering a new potential role for FasL in peripheral tolerance to organ-specific Ags. In sum, the prevention of experimental autoimmune thyroiditis by FasL on thyrocytes is dependent on the level of FasL expression.

Animals↗

PubMed2000

Expression of Fas ligand improves the effect of IL-4 in collagen-induced arthritis.

The present study was aimed at investigating whether the expression of Fas ligand (FasL) by CHO cells transfected with IL-4 (CHO/IL-4) or IL-10 (CHO/IL-10) genes would improve the effect of the cytokine. DBA/ 1 mice immunized with type II collagen were treated with suboptimal doses of transfected CHO cells (a single s. c. injection of 2 x 10(5) cells) around onset of arthritis. Severe collagen-induced arthritis (CIA) developed in the control groups injected with PBS, CHO /beta-galactosidase/FasL, CHO/IL-4 or CHO/IL-10 cells. In contrast, administration of CHO/IL-4/FasL, but not CHO/IL-10/FasL, cells significantly reduced the clinical severity and resulted in rapid and sustained suppressive effect. Amelioration of CIA was not due to a prolonged in vivo secretion of IL-4 since expression of FasL by CHO cells shortened the in vivo survival of the xenogeneic cells. In fact, administration of FasL(+) cells was associated with a decreased proportion of Mac1(+) neutrophils in the blood and an increased expression of myeloperoxidase at the site of engineered cell engraftment. These findings suggest that the mechanism underlying the beneficial effect of IL-4 delivered by cells expressing FasL involves the combination of the anti-inflammatory properties of IL-4 and the apoptosis of Fas(+) Mac1(+) granulocytes participating in the pathogenic process.

Animals↗

PubMed1999

Gene therapy of experimental autoimmune thyroiditis by in vivo administration of plasmid DNA coding for Fas ligand.

Fas-Fas ligand (FasL) interaction is required for the maintenance of immune homeostasis and seems to be responsible for the privileged immune status of some tissues. Experimental autoimmune thyroiditis (EAT), which is characterized by autoreactive T and B cell responses and a marked lymphocytic infiltration of the thyroid, is a model of choice to study the therapeutic effects of FasL. Here, we provide evidence that direct injection of DNA expression vectors encoding FasL into the inflamed thyroid inhibited development of lymphocytic infiltration of the thyroid and induced death of infiltrating T cells. These results were paralleled by a total abrogation of anti-Tg cytotoxic T cell response in FasL-treated animals vs controls. In summary, our results show that FasL expression on thyrocytes may have a curative effect on ongoing EAT by inducing death of pathogenic autoreactive infiltrating T lymphocytes.

Animals↗

PubMed1999

Curative treatment of experimental autoimmune thyroiditis by in vivo administration of plasmid DNA coding for interleukin-10.

The autoimmune response in experimental autoimmune thyroiditis (EAT) is characterized by a lymphocyte infiltration of the thyroid gland and by the appearance of circulating autoantibodies to thyroglobulin (Tg). Cytokines play a crucial role in the immunoregulation and pathology of EAT. Systemic administration of IL-10 has curative effects on EAT, but requires high doses and iterative injections due to the rapid turnover of this molecule. We have designed an original in vivo gene transfer using a mixture of liposomes and poly-L-Lysine that greatly enhanced the transfection yield, and induced a fast and long-lasting expression of IL-10 on mouse thyroid follicular cells (TFC). IL-10 expression on TFC of mice wit EAT dramatically wipe out the lymphocytic infiltration in the thyroids. A significant diminution in the proliferative anti-Tg T cell response was observed, along with a trend towards a Th2 response characterized by decreased production of IFN-gamma and by increased anti-Tg IgG1/IgG2a Ab ratios. In conclusion, local IL-10 gene therapy using non-viral vectors is a novel and promising approach for the treatment of thyroid autoimmune disorders.

Animals↗

PubMed1999

A recurrent valpha17/vbeta10 TCR-expressing T cell clone is involved in the pathogenicity of collagen-induced arthritis in DBA/1 mice.

Collagen-induced arthritis (CIA) is an experimental model that mimics clinical and histological features of rheumatoid arthritis. In this disease, a crucial role in initiating the pathological changes has been assigned to T lymphocytes expressing the Th1 phenotype. Aiming at identifying type II collagen (CII)-specific T cells involved in CIA, T cell clones were generated in vitro from the lymph nodes (LN) of CII-immunized DBA / 1 mice. In three independent experiments, we repeatedly isolated CD4(+) Th1 clones recognizing the immunodominant epitope in the CB11 fragment of bovine CII and expressing a unique alpha betaTCR produced by the rearrangement of Valpha17/Jalpha20 and Vbeta10/Dbeta1.1/Jbeta2.5 gene segments. By reverse transcriptase-PCR, we demonstrated the presence of mRNA transcripts specific for the beta complementary-determining region 3 of this clonotype in the LN of the majority (73%) of mice with CIA whereas it was never detected in control animals. When transferred to CII-immunized DBA/1 mice, this recurrent Th1 clone augmented the incidence, aggravated significantly the clinical signs of CIA and greatly enhanced the anti-CII antibody response. Altogether, these results provide evidence that a CD4(+) Th1 clone belonging to the public arm of the response toward the immunodominant epitope of CII is involved in the cascade of events leading to CIA.

Animals↗

PubMed1998

Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-alpha)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13.

TNF-alpha is one of the major proinflammatory cytokines involved in the pathogenesis of chronic inflammatory joint disease, in human rheumatoid arthritis as well as in murine models of this disease. It was previously described that a highly destructive chronic spontaneous inflammatory arthritis develops in mice expressing a human TNF-alpha transgene modified with the 3' untranslated region of beta-globin. The present study investigates in this mouse model the effects of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 administered in vivo on proinflammatory cytokine expression. Groups of TNF-alpha-transgenic mice were engrafted with xenogeneic transfected Chinese hamster ovary (CHO) fibroblasts secreting murine IL-4, IL-10 or IL-13. In vivo treatments consisted of 3 or 4 weekly engraftments, starting when the mice were 4weeks old. Control groups of transgenic mice were engrafted with beta-galactosidase gene-transfected CHO cells or injected with medium. A significant decreased expression of TNF-alpha transgene, endogenous mouse TNF-alpha and IL-1 mRNA was observed in splenocytes of mice treated for 3 or 4 weeks with CHO/IL-4 and CHO/IL-13, and, to a lesser extent, with CHO/IL-10, compared with controls. Finally, attenuation of histological scores of arthritides was statistically significant only in the group of CHO/IL-4-treated mice after 3weeks of treatment (P<0.05), and was not significant in any other group. These results show that IL-4, IL-10 or IL-13, administered by gene therapy, can decrease the mRNA steady state levels of both endogenous and transgenic cytokines in human TNF-alpha transgenic mice. In addition, IL-4 can slightly attenuate the development of arthritides in this model.

Animals↗

PubMed1997

High susceptibility to collagen-induced arthritis in mice lacking IFN-gamma receptors.

Collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis, is induced in DBA/1 (H-2q) mice following immunization with type II collagen (CII) in CFA. Since we have previously shown that IFN-gamma exerts a biphasic effect during the evolution of CIA in DBA/1 mice, we analyzed the development of this disease in mice with a disruption of the IFN-gamma receptor gene (IFN-gammaR(0/0)). Mutant mice were interbred with the DBA/1 strain to yield IFN-gammaR(0/0) mice expressing the H-2q haplotype. In three consecutive experiments, IFN-gammaR(0/0) male mice were found to exhibit severe clinical and histologic arthritis with an average incidence of 88.5 vs 94.1% for the wild DBA/1 strain. Notably, onset of clinical symptoms occurred significantly earlier than in DBA/1 mice. Although of a lower magnitude than in males, CIA also developed early in IFN-gammaR(0/0) female mice and with higher clinical severity than in control DBA/1 females. Immunization of knockout mice with CII resulted in the generation of CII-specific T cells belonging to the Th1 phenotype that recognize the same immunodominant peptides as do DBA/1 mice. CIA in IFN-gammaR(0/0) mice was associated with a down-regulation of the CII-specific IgG response, and this impairment was essentially due to a strong reduction of Abs of the IgG2a isotype. Taken together, our findings provide evidence that IFN-gammaR deficiency in DBA/1 mice leads to the occurrence of severe CIA with an accelerated onset compared with that in wild-type mice, indicating that the proinflammatory action of IFN-gamma has been bypassed in the IFN-gammaR(0/0) mice.

Animals↗

PubMed1997

Selective increased presentation of type II collagen by leupeptin.

Type II collagen (CII) is an arthritogenic self antigen in DBA/1 (H-2q) mice. To analyze the intracellular processing of this fibrillar protein in the context of I-Aq molecules, we have generated hybrid antigen-presenting cells (APC) by fusion of B lymphoma (A20 and M12) cells with CII-primed spleen cells from DBA/1 mice. Efficient presentation of CII by these APC to specific T cell hybridomas required prior cleavage of the antigen and intracellular handling of the peptides. Inhibition of protein transport by brefeldin A prevented the presentation of CII peptides to T cell hybridomas, indicating that the intracellular presentation of CII was dependent on neo-synthesis of I-Aq molecules. In contrast, exposure of hybrid B lymphomas to leupeptin, a protease inhibitor, induced a dose-dependent increase of CII-specific T cell response, while abrogating the I-Aq-restricted presentation of ovalbumin. The enhancing effect of leupeptin was also observed when immune B cells were used as APC. In contrast, leupeptin inhibited the presentation of CII peptides by macrophages or total spleen cells. Pulse-chase analysis of metabolically labeled hybrid APC and immunoprecipitation with antibodies specific for class II molecules or invariant (li) chain revealed that leupeptin did not affect the li chain processing or the formation of stable class II dimers. The stimulatory effect of leupeptin observed on CII presentation suggests that leupeptin protects CII epitopes by interfering with proteases involved in the intracellular degradation of CII.

Animals↗

PubMed1997

Highly sensitive method to detect mRNAs in individual cells by direct RT-PCR using Tth DNA polymerase.

A new method for detecting the expression of low-abundance mRNA molecules has been developed that combines the sensitivity of PCR, the high efficiency and specificity of reverse transcription (RT) using Tth DNA polymerase at high temperature, and the enhancement of sensitivity and specificity of nested PCR. This method is highly sensitive, reproducible and allows the detection of mRNAs in individual cells by direct RT-PCR.

Animals↗

PubMed1995

Biphasic effect of interferon-gamma in murine collagen-induced arthritis.

Interferon-gamma (IFN-gamma) exerts both enhancing and suppressing influences on collagen-induced arthritis (CIA), depending on the route and protocol of administration. To study the role of IFN-gamma on the autoimmune process of CIA, we treated DBA/1 mice with two different rat monoclonal antibodies (mAb) to murine IFN-gamma. Treatments, given twice weekly for 4 weeks, consisted of intraperitoneal injections of either mAb. In early treatments, starting from the day of immunization with type II collagen (CII), the severity of arthritis was reduced in both groups of anti-IFN-gamma-treated mice compared with control groups. Moreover, anti-CII antibody levels decreased in the sera of these mice. CIA was also down-regulated in mice treated from days 14 or 28 post immunization. In contrast, late treatments with anti-IFN-gamma mAb either induced aggravating effects, or did not affect the course of the disease. On the other hand, administration of high doses (8 x 10(4) U three times/week) of rat recombinant IFN-gamma exerted a transient increase of CIA severity. These findings suggest that IFN-gamma may play a critical role during both the induction and the course of CIA, first enhancing the immune response, and then regulating the arthritis process.

Animals↗

PubMed1995

Processing and presentation of type II collagen, a fibrillar autoantigen, by H-2q antigen-presenting cells.

Native type II collagen (CII) is a high molecular-weight fibrillar molecule which induces a chronic polyarthritis in mice expressing the H-2q haplotype. The present study was initiated to analyze the processing and the presentation of this nonglobular protein by H-2q antigen-presenting cells (APC). Efficiency of presentation was assessed by the ability of antigen-pulsed APC to activate collagen-specific CD4+ T cell hybridomas. Fixation of APC or the presence of chloroquine completely blocked the reactivity of the T cell hybrids to native, denatured and cyanogen bromide (CB) degraded CII, thus indicating the requirement of intracellular processing for adequate presentation of CII peptides to T cells. In the presence of various processing inhibitors (brefeldin A, leupeptin and N-tosyl-L-phenylalanine chloromethylketone) stimulation of T hybrids by CII-pulsed APC was reduced, pointing to the need of newly synthesized class II molecules, the use of several intracellular compartments and the implications of different proteases in the generation of CII peptides. Peritoneal macrophages and, to a lesser extent, total spleen cells, presented native and denatured CII with higher efficiency than purified splenic dendritic cells, naive or even immune B cells from CII-primed mice. In contrast, these dendritic and B cells were fully competent to present intact ovalbumin to a specific T cell hybrid. The stimulation by dendritic cells and immune B cells was greater when CB peptides of CII were added instead of the native molecule. Similarly, the cleavage of CII was an absolute requirement for its presentation by epidermal cells and B cell lymphomas to the T cell hybridomas. Taken together, these findings emphasize the crucial role of intracellular processing for recognition of soluble CII, similar to most antigens. However, in contrast to ovalbumin, the size and fibrillar nature of the native CII molecule influences its capture by the APC, thus limiting the type of APC able to present this antigen.

Animals↗

PubMed1994

T cell regulation of collagen-induced arthritis in mice. III. Is T cell vaccination a valuable therapy?

Since T cells play a critical role in collagen-induced arthritis (CIA), CD4+ T cell hybridomas were derived from DBA/1 mice immunized with bovine type II collagen (CII). The hybrid clones selected were Thy-1-2+, CD4+, CD8-, T cell receptor (TcR) alpha beta + and produced interleukin-2 in response to CII peptides presented by I-Aq molecules. The clones were collagen type-specific and recognized CII from many species except the mouse. More precisely, the reactivity was directed against the immunodominant cyanogen bromide-cleaved fragment CB11(II). Analysis of the TcR carried by the T cell hybridomas showed that they used identical V alpha and J alpha (V alpha BMB, J alpha 20) gene segments and two distinct V beta (V beta 1 and V beta 4) associated with the J beta 2.5 gene segment. Interestingly, the junctional regions were highly conserved in structure and length. These findings may indicate a strong in vivo selection by the antigen for a particular combination of both alpha and beta chains of the TcR. Inoculation of irradiated anti-CII T cell hybrids into DBA/1 mice, before priming with CII, altered the course of the disease resulting in either a long-lasting suppression or an exacerbation of CIA whereas a control CD4+ hybridoma with an unrelated specificity did not influence the development of arthritis. However, the regulatory effect of anti-CII T cell clones was unpredictable, suggesting that the TcR structure may not solely account for the modulation of CIA and that T cell vaccination is not a reliable method for inducing suppression of CIA.

Amino Acid Sequence↗

PubMed1993

T cell regulation of collagen-induced arthritis in mice. II. Immunomodulation of arthritis by cytotoxic T cell hybridomas specific for type II collagen.

Injection of native type II collagen (CII) to susceptible strains of mice (H-2q) induces a rheumatoid arthritis-like disease. To study the role of CD8+ T cells in the collagen-induced arthritis (CIA), we generated CII-specific T cell hybridomas by fusion of cells from arthritic C3H. Q mice and an AKR thymoma. Two hybrid clones (P3G8 and P2D9) were selected for their ability to lyse syngeneic CII-pulsed macrophages and recognize different antigenic epitopes in association with Kq molecules. When these T cell clones were irradiated and inoculated into (C3H.Q x AKR)F1 mice 21 days prior to priming with native CII/complete Freund's adjuvant, the incidence and the duration of CIA were significantly reduced in comparison to groups receiving saline or control T cell hybridoma. Furthermore, both anti-CII T cell hybridomas were able to attenuate CIA in highly susceptible inbred strains of mice and this suppression was antigen and disease specific. The protective activity seems to require intact cells as neither membrane fractions nor cytosolic preparations of the hybridoma T cells retained the vaccinating activity. Most importantly, one of the hybrid clones (P3G8) had a therapeutic effect on CIA since its administration to arthritic DBA/1 mice on day 30 after priming down-regulated the ongoing disease. Taken together, these findings suggest that anti-CII cytotoxic T cell clones can vaccinate against CIA and even reverse the disease.

Adjuvants, Immunologic↗

PubMed1992

Combination of cyclosporine A and calcitriol in the treatment of adjuvant arthritis.

We investigated combination treatment with cyclosporine A (CsA) and calcitriol (CT), 2 immunomodulatory agents, in vivo in the adjuvant arthritis (AA) model in rats. Treatment initiated at priming with complete Freund's adjuvant consisted of daily administration of CsA and CT during the prearthritic phase (14 days). CsA given alone delayed the onset of the disease, whereas the severity was either not affected or paradoxically enhanced. Injection of CT alone reduced the severity of arthritis. The association of low doses of CsA (5 mg/kg) and CT (0.2 micrograms/kg) further delayed the onset of AA and abolished the aggravating effect. Our findings suggest that low doses of CsA and CT in association exhibit the additive beneficial effects of either agent alone.

Administration, Oral↗