[Therapeutic applications and promises of cellular and tissue engineering. What strategic choice for the Etablissement Français du Sang?]. / Domaines d'application et enjeux de l'ingénierie et de la thérapie cellulaires et tissulaires. Quel choix stratégique pour l'EFS ?

A new medical field, known as regeneration medicine, is developing and attracting more and more researchers and practitioners. Whereas hematopoietic cell-based therapies have already proven their efficacy in numerous--malignant or not--diseases, non-hematopoietic cell-based therapies have not. They can be useful to dozens, if not hundreds, of patients with various disorders, such as cardiopathy, diabetes, some types of cancer, osteoarticular and neurodegenerative disorders. In these fields, numerous clinical applications are possible for mesenchymal stem cells. Cell and tissue (corneas, bone, skin) therapy products require the definition of pharmaceutical standards with new European requirements in terms of quality and safety. The legitimacy of the Etablissement Français du Sang (EFS) in cell and tissue engineering activities is established, it is recognized by most specialists and by regulatory authorities and has been asserted by the orientations of its "contrat d'objectifs et de moyens". An independent committee has been set up by the EFS President to define an EFS-specific strategy. This committee made up of qualified specialists was required to draw up a rational organization plan for these activities, in order for EFS to be in a position to produce cells and tissues according to pharmaceutical standards. The committee proposals are based on economic data and an inventory of existing cell and tissue engineering activities. Public/private partnerships are required and efforts must focus towards the industrial valorization of EFS expertise in R&D activities and staff know-how. Implementing such a new organization requires national management and the cooperation of institutional actors (university hospitals, cancer treatment centers, universities). For the success of this approach, EFS personnel must be convinced of its legitimacy and new skills must be encouraged. With its numerous assets, EFS can be ambitious and assert itself as a major actor in cell and tissue engineering in Europe.

Urinary cytotoxic molecular markers for a noninvasive diagnosis in acute renal transplant rejection.

Perforin (P), Granzyme B (GB) and Fas-Ligand (FAS-L) are cytotoxic molecules involved in acute rejection (AR) after renal transplantation. A noninvasive diagnostic test to monitor AR and other complications could improve clinical management. We investigated the predictive and diagnostic interest of target mRNA measurements, with a quantitative PCR assay, in AR, as well as in other clinical complications recurrent in kidney transplantation. One hundred and sixty-two urine specimens from 37 allograft recipients were investigated. Clinical settings were AR, urinary tract infection (UTI), cytomegalovirus infection (CMVi) or disease (CMVd), chronic allograft nephropathy (CAN), delayed graft function (DGF) and stable graft course (controls). In the case of AR, mRNA levels of all three molecules were significantly higher than in recipients not showing any clinically evident signs of complication. Indeed, it was observed that expression levels of P, GB and Fas-L mRNA also increase in other clinical situations such as UTI, CMV and DGF. Finally, kinetic studies in three patients with AR revealed that increased P, GB and Fas-L mRNA levels could precede or were concomitant with increased serum creatinin levels. P, GB and Fas-L gene expression in urine specimens were upregulated in AR episodes but also in UTI, CMV infection and DGF. Therefore, this technique would appear to be of limited clinical value as a noninvasive method of diagnosing AR.

Endothelial cells in organ transplantation: Friends or foes?

Vascular endothelial cells are the first interface between donor and recipient in organ transplantation. Endothelial cells and smooth muscle cells are key actors of acute and chronic rejection processes in organ allografting, but they also have the capacity to protect themselves from allograft-induced injury. Recent advances in our understanding of the precise mechanisms leading to endothelial dysfunction or, on the contrary, to endothelial protection, suggest that therapeutic interventions targeting endothelial cells could improve allograft survival and have even raised the question of whether such manipulations can be considered with a view to inducing immunological tolerance.

Differential sensitivity of T lymphocytes and hematopoietic precursor cells to photochemotherapy with 8-methoxypsoralen and ultraviolet A light.

The combination of 8-methoxypsoralen (8-MOP) and long wave ultraviolet radiation (UV-A) has immunomodulatory effects and might abolish both graft-vs-host and host-vs-graft reactions after allogeneic hematopoietic stem cell transplantation. In the present study, we have confirmed the sensitivity of T lymphocytes to 8-MOP treatment plus UV-A exposure as evidenced by the abrogation of the alloreactivity in mixed lymphocyte cultures as well as the inhibition of the response to phytohemagglutinin A. However, the clonogenic capacity of the bone marrow hematopoietic progenitors was inhibited with UV-A doses lower than the doses needed to inhibit T-lymphocytes alloreactivity. Moreover, long-term bone marrow cultures showed that 8-MOP plus UV-A treatment had detrimental effects on the more immature bone marrow stem cells. These data were confirmed when murine bone marrow graft was treated with 8-MOP, exposed to UV-A, then transplanted into semiallogeneic recipient mice. The treated cells could not maintain their clonogenic capacity in vivo resulting in death of all animals. Taken together, these data show that ex vivo 8-MOP plus UV-A treatment of the marrow graft cannot be used to prevent post-bone marrow transplantation alloreactivity.

In vitro functional defects of bone marrow-derived CD34+ progenitors from newly diagnosed mature B-cell malignancies with bone marrow tumor involvement.

OBJECTIVE: We hypothesized that the presence of tumor cells in bone marrow (BM) could alter hematopoietic progenitor cell functions. Therefore, we evaluated phenotypic and in vitro functional properties of BM-derived CD34+ progenitors issued from untreated and newly diagnosed patients presenting a mature B-lymphoproliferative disorder (LPD) involving the BM (Inv+). PATIENTS AND METHODS: In vitro proliferation and differentiation capacities of primitive and committed progenitors were evaluated by cobblestone area-forming cell (CAFC) and colony-forming cell (CFC) assays, and ex vivo cell expansion. Migratory capacities of CD34+ cells were explored by chemotaxis assays using a CXCL12alpha gradient. RESULTS: Our results showed that CD34+ cells from Inv+ patients overexpressed CD117 and had a significant decrease of week-3 and -6 CAFC, and CFC frequencies, compared to cells obtained from healthy volunteers and LPD patients without BM involvement (Inv-). In addition, progenitors from Inv+ patients maintained a significantly decreased CFC capacity after ex vivo cell expansion, compared to healthy volunteers. However, the former cells held their migratory capacity in response to CXCL12alpha. CONCLUSION: Functional defects of primitive and committed CD34+ progenitors detected among LPD patients with BM tumor involvement suggest either that tumor cells may induced bystander effects on progenitors or that "unusual" CD34+ cells may exist in the BM that could belong to the proliferating tumor tissue.

The French Haemovigilance System: organization and results for 2003.

In 1993 by law, in France, haemovigilance became a national system of surveillance and alert, from blood collection to the follow-up of the recipients, gathering and analysing all adverse events of blood transfusion in order to prevent their recurrences. In 2003, 2911 incidents with strong imputability have been specially analysed, among them seven confirmed cases of bacterial contamination, 137 incorrect blood components transfused with 12 cases of ABO incompatibility, 15 adverse reactions diagnosed as TRALI and 12 deaths. The analysis of information provided by haemovigilance has led to the implementation of new guidelines.

Retrovirus-mediated gene transfer in human primary T lymphocytes induces an activation- and transduction/selection-dependent TCR-B variable chain repertoire skewing of gene-modified cells.

In a clinical trial that we recently reported, a suicide gene transfer in human primary T cells required 12 days of ex vivo culture, including activation of peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody (CD3 mAb), retrovirus-mediated transduction, and selection of gene-modified cells (GMC) by G418. The aim of the present study was to determine the impact of the initial T cell activation and of the transduction/selection on T cell receptor beta variable chain (TCRBV) repertoire of GMC by using the spectratyping method. The TCRBV repertoires of nontransduced, nonselected control (Co) cells and of GMC generated after an initial stimulation with CD3 mAb, CD3/CD28 beads, or allogeneic PBMC or Epstein-Barr virus-transformed B (B-EBV) cells were compared to the ones of their corresponding PBMC. The TCRBV repertoires were skewed in Co cells generated after CD3 mAb or after allogeneic stimulation, and even more so in their corresponding GMC, demonstrating that both culture-dependent and transduction/selection-dependent events led to TCRBV repertoire alterations. However, TCRBV repertoires were not altered, or to a lesser extent, in Co cells or GMC produced after CD3/CD28 bead activation, demonstrating a protective effect on both culture-dependent and transduction/selection-dependent repertoire alterations. Thus, we suggest to replace the initial CD3 mAb stimulation by CD3/CD28 beads for the production of clinical-grade GMC in the setting of future gene therapy trials.

Regulatory (suppressor) T cells in peripheral allograft tolerance and graft-versus-host reaction.

Among the mechanisms capable of inducing peripheral tolerance, regulatory (suppressor) T cells (Treg) probably play a key role in the control of both reactivity to self-antigens and alloimmune response. Augmentation or manipulation of Treg could improve organ allograft survival or control graft-versus-host disease, thus resulting in operational tolerance. The role of this immunomanipulation as one method of inducing tolerance has yet to be clearly defined.

[Allorecognition of the recipient after hematopoietic transplantation]. / La réactivité allogénique dirigée contre le receveur lors d'une greffe hématopoïétique.

Donor allorecognition of the recipient after hematopoietic transplantation can result in graft-versus-host disease, a potent graft-vs-leukemia effect as well as a graft facilitation effect. Danger signals, host Ag-presenting cells and minor histocompatibility Ag have recently emerged as major determinants of such an alloreactivity. A better understanding of the involved immune mechanisms, the development of novel immunomonitoring tools and cell engineering approaches should result in a significantly increased therapeutic index of allogeneic alloreactivity.

Mesenchymal progenitor cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a rat cellular cardiomyoplasty model.

BACKGROUND: Cellular cardiomyoplasty is a promising approach to improve postinfarcted cardiac function. The differentiation pathways of engrafted mesenchymal progenitor cells (MPCs) and their effects on the left ventricular function in a rat myocardial infarct heart model were analyzed. METHODS AND RESULTS: A ligation model of left coronary artery of Lewis rats was used. MPCs were isolated by bone marrow cell adherence. Seven days after ligation, MPCs labeled with 4',6-diamidino-2'-phenylindole were injected into the infarcted myocardium (n=8). Culture medium was injected in the infarcted myocardium of control animals (n=8). Thirty days after implantation, immunofluorescence studies revealed some engrafted cells expressing a smooth muscle phenotype (alpha SM actin+), as similarly observed in culture. Other engrafted cells lost their smooth muscle phenotype and acquired an endothelial phenotype (CD31+). Furthermore, vessel density was augmented in the MPC group in comparison with the control group. After 30 days, echocardiography showed an improvement on left ventricular performance in the MPCs compared with the control group. CONCLUSIONS: In vivo administration of syngenic MPCs into a rat model of myocardial infarcted heart was safety demonstrated. Some engrafted cells appeared to differentiate into endothelial cells and loss their smooth muscle phenotype. MPC engraftment might to contribute to the improvement on the cardiac function in such a setting.

Direct selection of human bone marrow mesenchymal stem cells using an anti-CD49a antibody reveals their CD45med,low phenotype.

Human bone marrow mesenchymal stem cells (MSC) generate, via a fibroblast colony-forming unit (CFU-F), osteo-chondroblastic cells as well as adipocytes and stromacytes. To date, these stem cells are isolated indirectly using a cell culture method and phenotyped as CD45 negative while the in vivo counterparts are undetermined. Our aim was to develop a direct selection method and to determine the phenotype of the MSC isolated in this way. Mesenchymal cells were selected with anti-CD49a and/or anti-CD45 antibodies using either flow cytometry or a magnetic beads method. All CFU-F were always detected in the small population of CD49a-positive cells. These CFU retained their differentiation potential and gave rise to osteo-chondroblastic cells, adipocytes and stromacytes. Phenotypic studies on uncultured cells revealed a CD45med,low, CD34low, HLA-II- cell population. Flow cytometry cell sorting showed that MSC with CFU-F potential were obtained only from a CD49a+/CD45med,low population. In addition, when cultured, they clearly became CD45-, CD34-, HLA-II-, CD49a+. These results confirmed that MSC can be directly selected easily from human bone marrow using magnetic beads without altering their differentiation potential. These cells expressed mildly the haematopoietic marker CD45, which was dramatically downregulated by in vitro culture. The expression of CD45 coupled to CD49a thus enabled direct selection of the MSC.

Donor-derived hematopoietic stem cells in organ transplantation: technical aspects and hurdles yet to be cleared.

The use of hematopoietic stem-cell (HSC) therapy in organ transplantation is a challenge to promote chimerism with the aim of enhancing organ tolerance. Several HSC sources are available, including bone marrow (most of the time), peripheral blood after stem-cell mobilization, and placental blood. HSC collection techniques from vertebral bodies or iliac crests require a number of complex manipulations. The best yield of HSC is obtained from vertebral bodies. HSC harvesting by cytapheresis after cell mobilization with a cytokine such as granulocyte colony-stimulating factor should be preferred with a live donor. The number of CD3+ T cells is more than 10-fold higher in peripheral blood than in bone marrow. Cell separation by the immunoselection technique (positive selection of the CD34+ cell population) eliminates erythrocytes, granulocytes, and T cells, thus preventing the possible occurrence of acute graft-versus-host disease. In the future, an accreditation will be required for HSC collection and processing. In Europe, the reference tool is the Joint Accreditation Committee of Ishage-Europe or the Foundation for the Accreditation of Haematopoietic Cell Therapy manual, which provides standards for every technical step of these procedures.

Retrovirus-mediated gene transfer in polyclonal T cells results in lower apoptosis and enhanced ex vivo cell expansion of CMV-reactive CD8 T cells as compared with EBV-reactive CD8 T cells.

To modulate alloreactivity after hematopoietic stem cell transplantation, "suicide" gene-modified donor T cells (GMCs) have been administered with an allogeneic T-cell-depleted marrow graft. We previously demonstrated that such GMCs, generated after CD3 activation, retrovirus-mediated transduction, and G418 selection, had an impaired Epstein-Barr virus (EBV) reactivity, likely to result in an altered control of EBV-induced lymphoproliferative disease. To further characterize the antiviral potential of GMCs, we compared the frequencies of cytomegalovirus (CMV)-specific CD8+ T (CMV-T) cells and EBV-specific CD8+ T (EBV-T) cells within GMCs from CMV- and EBV-double seropositive donors. Unlike anti-EBV responses, the anti-CMV responses were not altered by GMC preparation. During the first days of culture, CMV-T cells exhibited a lower level of CD3-induced apoptosis than did EBV-T cells. In addition, the CMV-T cells escaping initial apoptosis subsequently underwent a higher expansion rate than EBV-T cells. The differential early sensitivity to apoptosis could be in relation to the "recent activation" phenotype of EBV-T cells as evidenced by a higher level of CD69 expression. Furthermore, EBV-T cells were found to have a CD45RA-CD27+CCR7- effector memory phenotype, whereas CMV-T cells had a CD45RA+CD27-CCR7- terminal effector phenotype. Such differences could be contributive, because bulk CD8+CD27- cells had a higher expansion than did bulk CD8+CD27+ cells. Overall, ex vivo T-cell culture differentially affects apoptosis, long-term proliferation, and overall survival of CMV-T and EBV-T cells. Such functional differences need to be taken into account when designing cell and/or gene therapy protocols involving ex vivo T-cell manipulation.

Bacterial contamination of a cornea tissue bank: implications for the safety of graft engineering.

PURPOSE: To analyze the difficulties involved in managing an episode of bacterial contamination in a cornea bank. We describe (1) the circumstances of bacterial contamination discovery, (2) the methods used to investigate the outbreak, (3) the corrective measures adopted, and (4) the method introduced to improve the reaction capacity in case of bacterial contamination. METHODS: All the samples collected were cultured in an attempt to identify the environmental reservoir of the contaminated epidemic clone. Bacteria were identified by Gram stain, oxidase test, and biochemical characteristics. The clonality of the strains was assessed by pulsed-field gel electrophoresis. RESULTS: The bacterial contamination was confirmed for 28 corneas, and 70 additional corneas were discarded. The source of the contamination was identified 17 days after the beginning of the episode. It consisted of a clonal bacterial strain that was found in trypan blue, the dye, used to examine all the tissues. The contaminating bacterium was Burkholderia cepacia, a well-known nosocomial pathogen. A total of 169 grafted corneas had been checked with the contaminated reagent. No cases of post-graft infection were recorded. CONCLUSION: Trypan blue played a major role in this outbreak. The mode and chronology of contamination remain unresolved. This exceptional event emphasizes the risk of bacterial contamination in tissue/cell banks, the necessity to improve methods for its prevention, and procedures to limit its consequences.

[Tolerance in transplantation: potential contribution of haematopoietic transplantation and cell therapy]. / Tolérance en transplantation: apport potentiel de la greffe hématopoïétique et de la thérapie cellulaire.

The ultimate objective of organ transplantation is to obtain a state of tolerance, i.e. long-term acceptance of the graft without immunosuppressive therapy in order to limit the complications of these treatments (viral infections, tumours, etc.). The various immunological mechanisms allowing a state of tolerance will be described in this review. Among these various experimental strategies, combined bone marrow (or haematopoietic stem cell) transplantation and organ transplantation, made possible by the development of non-myeloablative or less intensive conditioning, appears to be one of the most promising lines of research. This approach leads to colonization of the recipient by donor cells. This state is described as "macro-chimerism" and achieves a real state of central tolerance in relation to an organ derived from the bone marrow donor. We have shown recently that intravenous injection of apoptotic cells in combination with allogeneic bone marrow cells increases the success rate of bone marrow transplantation. In a model of combined bone marrow/solid organ transplantation, these apoptotic cells induce tolerance limited to the donor's bone marrow cell antigens without inducing auto-immunization. We therefore propose a new approach to cell-based therapy (using the immunomodulating properties of apoptotic cells) to promote the success of haematopoietic stem cell transplantation. This approach can be particularly useful in combined haematopoietic stem cell and organ transplantation in order to induce a state of macro-chimerism.

Hemovigilance network in France: organization and analysis of immediate transfusion incident reports from 1994 to 1998.

BACKGROUND: Hemovigilance networks have been introduced in several countries to improve knowledge of blood transfusion-related morbidity and mortality. The general organization of the French network and its results from 1994 through March 1999 are presented here. STUDY DESIGN AND METHODS: The hemovigilance network relies on blood transfusion centers and hospital correspondents, who analyze unexpected and untoward blood transfusion-related effects and transmit a Transfusion Incident Report (TIR) to a national database (Transfusion Incident Reports Electronic Data Management [GIFIT]). RESULTS: As of March 1, 1999, the GIFIT database contained 24,234 TIRs related to incidents that occurred from the start of the hemovigilance network until December 31, 1998. The network was not fully implemented until 1996; but the reporting rate seems to have since stabilized at approximately 7000 per year (2.5 reports per 1000 blood components). The highest reporting rate is observed with platelet concentrates (4.02/1000), followed by RBCs (1.71/1000) and FFP (0.34/1000). Bacterial contamination quickly appeared as a major cause of morbidity and mortality (185 cases and 18 fatalities). However, a general trend of reduction in this type of incident was observed over time, which can be attributed to adoption of several preventive measures. In contrast, major ABO mismatchings during RBC transfusion remained at a constant rate throughout this period and accounted for six fatalities. After the implementation of universal WBC reduction, some incidents known to be related to WBCs, such as nonhemolytic febrile transfusion reactions (NHFTR) and HLA immunization, were dramatically reduced. CONCLUSION: Hemovigilance is an important tool not only to analyze blood transfusion incidents, but also to measure the effects of new processes or corrective actions at a national level.

Polybrene and interleukin-4: two opposing factors for retroviral transduction of bone-marrow-derived dendritic cells.

BACKGROUND: Gene transfer using retroviral transduction offers the advantage of long-term transgene expression in developing strategies that use dendritic cells (DCs) for immunotherapy. The goal of this study was to infect DCs in an immature state in order to take advantage of their proliferating and tolerogenic potential. METHODS: Immature DCs were generated from murine bone marrow (BM) using either GM-CSF alone or GM-CSF plus IL-4. The cells were transduced directly with retroviral supernatants or by co-culture with the GP + E-86 retroviral packaging cell line in the presence of two different cationic polymers: polybrene and protamine sulfate. Phenotypic and functional characterization of the transduced cells were then performed. RESULTS: Our results show a low efficiency of retroviral infection of DCs in the presence of polybrene. This cationic polymer was found to be directly cytotoxic to murine DCs and thus favored the growth of contaminating macrophages. This effect was not observed using protamine sulfate. Furthermore, stimulation by IL-4 early in the culture increased DC differentiation, proliferation and transduction. However, we found that DCs generated in GM-CSF plus IL-4 presented a more mature phenotype with an enhanced allogeneic stimulating activity. Finally, we showed that DCs themselves down-regulated transgene expression in the co-cultured packaging cell line in a promoter-dependent manner. CONCLUSIONS: We have defined optimal conditions to generate and transduce murine BM-derived DCs. This included: the use of protamine sulfate during exposure to retroviral infectious supernatant and the addition of IL-4 at an early stage of the culture. Nevertheless, this cytokine also induced DC maturation. These findings have potential implications in experimental gene therapy.