Increase in intracellular PGE2 induces apoptosis in Bax-expressing colon cancer cell.

BACKGROUND: NSAIDs exhibit protective properties towards some cancers, especially colon cancer. Yet, it is not clear how they play their protective role. PGE2 is generally shown as the only target of the NSAIDs anticancerous activity. However, PGE2 known targets become more and more manifold, considering both the molecular pathways involved and the target cells in the tumour. The role of PGE2 in tumour progression thus appears complex and multipurpose. METHODS: To gain understanding into the role of PGE2 in colon cancer, we focused on the activity of PGE2 in apoptosis in colon cancer cell lines. RESULTS: We observed that an increase in intracellular PGE2 induced an apoptotic cell death, which was dependent on the expression of the proapoptotic protein Bax. This increase was induced by increasing PGE2 intracellular concentration, either by PGE2 microinjection or by the pharmacological inhibition of PGE2 exportation and enzymatic degradation. CONCLUSIONS: We present here a new sight onto PGE2 in colon cancer cells opening the way to a new prospective therapeutic strategy in cancer, alternative to NSAIDs.

Regulatory, effector, and cytotoxic T cell profiles in long-term kidney transplant patients.

Animal studies have suggested a potential role for regulatory T cells (Tregs) in allograft tolerance, but these FOXP3+ cells seem to be an inherent component of acute rejection (AR) in human recipients of renal transplants. The balance between regulatory cells and effector/cytotoxic cells may determine graft outcome; this balance has not been described for chronic allograft injury. We investigated the expression of key regulatory, effector, and cytotoxic transcripts (i.e., FOXP3, T-bet, and granzyme B, respectively) in the grafts and peripheral blood of long-term-surviving renal transplant patients. We found that, whereas neither intragraft nor peripheral blood FOXP3 or T-bet mRNA could distinguish between rejection and nonrejection status, granzyme B (GrzB) mRNA could: It was significantly increased in the graft and significantly decreased in the peripheral blood of patients with chronic antibody-mediated rejection (CAMR). Quantifying peripheral blood GrzB mRNA demonstrated potential to aid in the noninvasive diagnosis of CAMR. In summary, these data affirm GrzB as a marker not only for AR but also for CAMR. In addition, we identified several previously unreported clinical or demographic factors influencing regulatory/effector/cytotoxic profiles in the peripheral blood, highlighting the necessity to consider confounding variables when considering the use of potential biomarkers, such as FOXP3, for diagnosis or prognosis in kidney transplantation.

Tribbles-1 as a novel biomarker of chronic antibody-mediated rejection.

Diagnosis of the specific cause of late allograft injury is necessary if more personalized and efficient immunosuppressive regimens are to be introduced. This study sought previously unrecognized biomarkers for specific histologic diagnoses of late graft scarring by comparison of gene sets from published microarray studies. Tribbles-1 (TRIB1), a human homolog of Drosophila tribbles, was identified to be a potentially informative biomarker. For testing this, mRNA expression in 76 graft biopsies, 71 blood samples, and 11 urine samples were profiled from independent cohorts of renal transplant patients with different histologic diagnoses recruited at two European centers. TRIB1 but not TRIB2 or TRIB3 was found to be a potential blood and tissue biomarker of chronic antibody-mediated rejection, an active immune-mediated form of chronic allograft failure associated with a poor prognosis. TRIB1 mRNA levels in peripheral blood mononuclear cells discriminated patients with chronic antibody-mediated rejection from those with other types of late allograft injury with high sensitivity and specificity. TRIB1 was also upregulated in a rodent model of chronic cardiac vasculopathy, suggesting that this biomarker may be useful in other solid-organ transplants and across species. It was determined that TRIB1 is expressed primarily by antigen-presenting cells and activated endothelial cells. Overall, these data support the potential use of TRIB1 as a biomarker of chronic antibody-mediated allograft failure.

Immunosuppressive drug-free operational immune tolerance in human kidney transplants recipients. Part II. Non-statistical gene microarray analysis.

Kidney transplant is the reference treatment for patients with end-stage renal disease, but patients may develop long-term rejection of the graft. However, some patients do not reject the transplant, but instead are operationally tolerant state despite withdrawal of immunosuppressive treatment. In this second article we outline a microarray-based identification of key leader genes associated respectively to rejection and to operational tolerance of the kidney transplant in humans by utilizing a non/statistical bioinformatic approach based on the identification of "key genes," either as those mostly changing their expression, or having the strongest interconnections. A uniquely informative picture emerges on the genes controlling the human transplant from the detailed comparison of these findings with the traditional statistical SAM (Tusher et al. 2001 Proc Natl Acad Sci USA 98:5116-5121) analysis of the microarrays and with the clinical study carried out in the accompanying part I article.

Immunosuppressive drug-free operational immune tolerance in human kidney transplant recipients: Part I. Blood gene expression statistical analysis.

Survival of solid organ grafts depends on life-long immunosuppression, which results in increased rates of infection and malignancy. Induction of tolerance to allografts would represent the optimal solution for controlling both chronic rejection (CR) and side effects of immunosuppression. Although spontaneous "operational tolerance" can occur in human kidney transplantation, the lack of noninvasive peripheral blood biological markers of this rare phenomenon precludes the identification of potentially tolerant patients in whom immunosuppression could be tapered as well as the development of new tolerance inducing strategies. Here, the potential of high throughput microarray technology to decipher complex pathologies allowed us to study the peripheral blood specific gene expression profile and corresponding EASE molecular pathways associated to operational tolerance in a cohort of human kidney graft recipients. In comparison with patients with CR, tolerant patients displayed a set of 343 differentially expressed genes, mainly immune and defense genes, in their peripheral blood mononuclear cells (PBMC), of which 223 were also different from healthy volunteers. Using the expression pattern of these 343 genes, we were able to classify correctly >80% of the patients in a cross-validation experiment and classified correctly all of the samples over time. Collectively, this study identifies a unique PBMC gene signature associated with human operational tolerance in kidney transplantation by a classical statistical microarray analysis and, in the second part, by a nonstatistical analysis.

Contrasted blood and intragraft toll-like receptor 4 mRNA profiles in operational tolerance versus chronic rejection in kidney transplant recipients.

BACKGROUND: Deciphering the mechanisms of tolerance and chronic rejection (CR) remains a major goal in transplantation. Data in rodents suggest that Toll-like receptors (TLR), regulators of innate immune responses, play a role in determining graft outcome. However, few studies have focused on TLR expression in human kidney transplant recipients. METHODS: Here, we analyzed the expression of TLR4 in peripheral blood mononuclear cells from kidney recipients with contrasted clinical situations: operational tolerance and CR, compared with patients with stable graft function, nontransplant patients with renal failure and healthy volunteers. RESULTS: We report that myeloid differentiation factor 88 and TLR4 are significantly contrasted in the peripheral blood mononuclear cells, and in particular in monocytes, of patients with CR versus operational tolerance. Chronic rejection patients have significantly increased TLR4 and myeloid differentiation factor 88 compared with operationally tolerant patients, who resemble healthy volunteers and nontransplant patients with renal failure. Interestingly, analysis of TLR4 transcripts in graft biopsies from patients with normal histology or CR reflected the blood findings, with a significant increase of TLR4 in CR. CONCLUSIONS: These data support a link between TLR4 expression and long-term graft outcome. Moreover, whereas absence of TLR signaling may be a feature of tolerance, increased TLR4 signaling may be implicated in CR.

Alterations of the synovial T cell repertoire in anti-citrullinated protein antibody-positive rheumatoid arthritis.

OBJECTIVE: The association of HLA-DRB1 alleles with anti-citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) suggests the potential involvement of T lymphocytes in ACPA-seropositive disease. The purpose of this study was to investigate this hypothesis by systematic histologic and molecular analyses of synovial T cells in ACPA+ versus ACPA- RA patients. METHODS: Synovial biopsy samples were obtained from 158 RA patients. Inflammation was determined histologically and immunohistochemically. RNA was extracted from peripheral blood mononuclear cells and synovial tissues obtained from 11 ACPA+ RA patients, 7 ACPA- RA patients, and 10 spondylarthritis (SpA) patients (arthritis controls). T lymphocyte clonality was studied by combined quantitative and qualitative T cell receptor CDR3 length distribution (LD) analysis and direct sequencing analysis. RESULTS: ACPA+ and ACPA- RA patients were similar at both the clinical and histologic levels. At the molecular level, however, patients with ACPA+ synovitis displayed a marked elevation of qualitative CDR3 LD alterations as compared with those with ACPA- synovitis and with the SpA controls. These differences in CDR3 LD were not observed in the peripheral blood, indicating a selective recruitment and/or local expansion of T cells in the synovial compartment. The CDR3 LD alterations reflected true monoclonal or oligoclonal expansions, as confirmed by direct sequencing of the T cell receptor. The CDR3 LD alterations in RA synovium did not correlate with B cell clonal expansions but were inversely associated with synovial lymphoid neogenesis. CONCLUSION: The T cell repertoire is specifically restricted in RA patients with ACPA+ synovitis. Whereas the origin and role of these clonal alterations remain to be determined, our data suggest the preferential involvement of T lymphocytes in ACPA-seropositive RA.

Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance.

Long-term allograft survival generally requires lifelong immunosuppression (IS). Rarely, recipients display spontaneous "operational tolerance" with stable graft function in the absence of IS. The lack of biological markers of this phenomenon precludes identification of potentially tolerant patients in which IS could be tapered and hinders the development of new tolerance-inducing strategies. The objective of this study was to identify minimally invasive blood biomarkers for operational tolerance and use these biomarkers to determine the frequency of this state in immunosuppressed patients with stable graft function. Blood gene expression profiles from 75 renal-transplant patient cohorts (operational tolerance/acute and chronic rejection/stable graft function on IS) and 16 healthy individuals were analyzed. A subset of samples was used for microarray analysis where three-class comparison of the different groups of patients identified a "tolerant footprint" of 49 genes. These biomarkers were applied for prediction of operational tolerance by microarray and real-time PCR in independent test groups. Thirty-three of 49 genes correctly segregated tolerance and chronic rejection phenotypes with 99% and 86% specificity. The signature is shared with 1 of 12 and 5 of 10 stable patients on triple IS and low-dose steroid monotherapy, respectively. The gene signature suggests a pattern of reduced costimulatory signaling, immune quiescence, apoptosis, and memory T cell responses. This study identifies in the blood of kidney recipients a set of genes associated with operational tolerance that may have utility as a minimally invasive monitoring tool for guiding IS titration. Further validation of this tool for safe IS minimization in prospective clinical trials is warranted.

Phenotypically and functionally distinct CD8+ lymphocyte populations in long-term drug-free tolerance and chronic rejection in human kidney graft recipients.

A substantial proportion of long-term kidney graft recipients, including those with a stable renal function in the absence of immunosuppressive therapy, present a skewed T cell receptor (TCR) Vbeta chain usage, essentially in the CD8+ subset. This study analyzed in more detail phenotypical and functional alterations of CD8+ lymphocytes in drug-free tolerant patients (DF-Tol) compared with recipients with chronic rejection (CR). Phenotyping revealed a significant increase in central memory and a decrease in effector CD8+ lymphocytes in DF-Tol versus CR. The expression of CD28+ and CD27+ on these effector cells was significantly decreased in CR. These profiles were stable over time and independent of treatment. Functionally, the CD8+CD28- lymphocytes were less sensitive to apoptosis than their CD8+CD28+ counterparts, without differences in polyclonal proliferation. The CD8+CD28- cells did not express GITR and FoxP3 but were characterized by high levels of preformed perforin and granzyme A, pointing toward a cytotoxic rather than a suppressor function. CD8+CD28- lymphocytes did not show antigen-specific degranulation when co-cultured with targets that bear donor HLA class I antigens, suggesting that the cytotoxicity is directed either to other determinants of the graft or to nongraft epitopes. Of interest, CD8+ cells from DF-Tol displayed the same profile as healthy individuals, indicating an increase in CD8+CD28- effector lymphocytes in CR rather than a decrease in DF-Tol. CD8+ lymphocytes from stable kidney recipients under conventional maintenance immunosuppression displayed a mixed profile, independent of treatment and time of sampling. Taken collectively, these data show a strong cytotoxicity-associated CD8+CD28- signature in CR and suggest a suppression of pathologic cytotoxicity in DF-Tol. Further prospective studies should assess whether serial CD8+ phenotyping may help to identify patients who are at risk for CR when immunosuppression is tapered.