[Control of the bacterial risk of transfusion in France in 2013]. / Maîtrise du risque bactérien transfusionnel en France en 2013.

Bacterial contamination of blood products (BP) remains the most important infectious risks of blood transfusion in 2013. Platelet concentrates (PC) are the blood products the most at risk, whether CPA or MCPS. In France, the residual risk has been steadily declining since 1994. For the platelets, the frequency of transfusion reaction due to bacterial contamination (TRBC) is now about at one per 50,000 CP distributed. The number of deaths has remained stable since 1994 with one death per year (300,000 distributed CP). The progressive decrease in the number of cases of TRBCs is the result of steady improvement of practices and prevention methods at all stages from collection to the transfusion of BP. But if all these improvements have significantly reduced the incidence of TRBCs, mortality is not changed with the CP and the reduction of this risk is a priority for the French Blood Establishment (EFS). Detection methods of CP contaminated or pathogen inactivation are two approaches available and can provide a significant reduction (for the former) or deletion (for seconds) of the risk of transfused contaminated CP. Currently, the choice is in favor of the detection of bacteria. New detection "rapid tests" methods were added to the panel of candidates and are being evaluated. Inactivation of pathogens remains the safest prospect of eliminating this adverse effect of transfusion. Implementation of one method for bacterial detection is probably a transitional measure.

[Prevention of bacterial risk: pathogen inactivation/detection of bacteria]. / Prévention du risque bactérien: inactivation des pathogènes/détection des bactéries.

Bacterial contamination of blood products remains the most important infectious risk of blood transfusion in 2013. Platelet concentrates (PC) are in cause in the majority of the transfusion reaction due to bacterial contaminations. A lot of prevention methods have been developed over the last 10 years (pre-donation interview, skin decontamination, diversion of the first 30 mL of the donation, leuko-reduction...), they have focused on limiting the contamination of the donations and prevent the bacterial growth in donations and/or in the blood products. These measures were effective and led to significantly reducing the risk of adverse effects associated with bacterial growth. However, every year there are about six accidents (with a high level of imputability) and one death. The reduction of the bacterial risk remains a priority for the French Blood Establishment (EFS). The procedure for skin disinfection is going to be improved in order to further strengthen this crucial step to avoid the contamination of donation. Methods of pathogen inactivation applied to plasma and PC are available in France and their effectiveness is demonstrated on the bacterial risk. Methods for bacterial detection of PC are used in many countries now. Automated culture is the most common. Alternatives are now available in the form of rapid tests able to analyze the PC just before the delivery and avoid false negatives observed with automated culture. Assessments are under way to confirm these benefits in 2013.

[Bacterial detection leading to pathogen inactivation]. / De la détection bactérienne à l'inactivation des pathogènes.

Bacterial contamination of blood components remains the highest infectious risk in blood transfusion, the risk is particularly high when it affects platelet concentrates (PC). In France, the residual risk of transfusion reaction due to bacterial contamination of PC has been decreasing slowly since 1994 but for all severity 1 case occurs with about 25,000 distributed PC and one death occurs with 200,000 distributed units. This reduction of the risk may be due to the measures which were implemented during the last 10 years in order to prevent contamination during donation. Improving strategies for reducing the risks of bacterial contamination is one of the priorities of the French National Blood Transfusion Service (l'Etablissement Français du sang - EFS). The main target remains PC. Bacterial detection or pathogens inactivation are now available and are able to reduce (for detection) or prevent (for inactivation) the occurrence of reaction due to bacterial contamination of PC. Up to now, the choice is in favour of bacterial detection. A national study was carried out in seven regional EFS at the end of 2004. It aims at confirming the feasibility of a systematic bacterial screening of PC before their delivery. The first conclusions show that this screening can be implemented with acceptable modifications in term of platelets availability. We can expect in a next future that new pathogens reduction technique and/or new detection systems will be available, certainly more efficient to prevent reaction due to bacterial contamination. Implementation of actual detection methods is probably a temporary solution.

[Transfusion-transmitted bacterial infection: residual risk and perspectives of prevention]. / Transfusion et bactéries: risque résiduel et perspectives de prévention.

Bacterial contamination of blood components represents today the highest infectious risk of blood transfusion, the risk is particularly high when it affects platelet concentrates. The residual risk of transfusion reaction due to bacterial contamination of platelets concentrates remains stable. For all severity 1 case occurs with 25,000 distributed platelets concentrates and 1 death occurs with 200,000 distributed units. In France, efforts have focused on the prevention of contamination during donation--involving measures such as rejecting the first few millilitres of donated blood and improving skin disinfection--and the prevention of bacterial proliferation in platelets concentrates--notably by removing leukocytes and ensuring high-quality storage of donated blood. Improving strategies for reducing the risks of bacterial contamination is one of the priorities of the French National Blood Transfusion Service (l'Etablissement français du sang-EFS). There is currently considerable debate about the relative importance of bacterial screening methods and methods for inactivating pathogens present in PC. Automated culture (Biomérieux) and the ScanSystem (Hemosystem) and BDS (Pall) method are the most advanced detection systems available, to our knowledge. In term of pathogen inactivation system for platelets, Intercept (Baxter) is nearing the commercial market. These new prevention have logistic and/or functional consequences that will require close scrutiny methods. A national study group is currently considering the consequences of each of these methods and should give its opinion at the end of the first half of 2003.

[Detection of bacterial contamination in platelet concentrates: perspectives]. / Dépistage des bactéries dans les concentrés de plaquettes: perspectives.

Bacterial contamination of blood components represents today the highest infectious risk of blood transfusion, the risk is particularly high when it affects platelet concentrates. In France the prevention methods developed over the past six years (donor selection, phlebotomy site preparation, first 30 ml diversion, systematic leuko-reduction...) aimed at limiting the introduction of bacteria in blood and bacterial proliferation. Several methods have been tested for the detection of bacterial contamination in platelet concentrates but none have been generalised. Difficulties were met, due to the necessity of 1) detecting only the platelet concentrates presenting a real infectious risk, when the presence of bacteria is observed in 2.2% (2-4%) of donated blood and 2) guaranteeing the availability of platelet concentrates. New methods have been developed which seem able to bring responses to these difficulties. Several processes are being (or will be) assessed, including automated blood culture, bacterial genomic detection with or without amplification, flow cytometric methods. In parallel, an indirect method able to detect the presence of bacteria, based on oxygen consumption, will also be evaluated. One (or several) of these processes should allow, in the short-term, to detect platelet concentrates presenting an infectious risk. In the future, the interest of bio-chips for bacterial detection in biological fluids must be investigated.

Resistance/susceptibility to Echinococcus multilocularis infection and cytokine profile in humans. I. Comparison of patients with progressive and abortive lesions.

To clarify the role of Th1- and Th2-type cytokines in the various outcomes of human alveolar echinococcosis (AE), the cytokine immune response of self-cured patients was studied and compared with those of progressive AE patients and healthy subjects. Self-cured patients were divided into two groups according to the following clinical features: subjects who had positive Echinococcus multilocularis serologies and hepatic calcifications typical of AE were classified as 'abortive AE' patients, and those who had positive E. multilocularis serologies but no hepatic lesions or calcifications detectable by ultrasonography were classified as 'positive serology' subjects. Secretions of IL-5, IL-10 and interferon-gamma, and expression of IL-5 mRNA were evaluated in peripheral blood mononuclear cells (PBMC) stimulated in vitro with the mitogen phytohaemagglutinin-C or specific E. multilocularis antigenic preparations. The cytokine profile of abortive AE patients was the opposite of that observed in progressive AE patients. An intermediate profile was observed in positive serology subjects. PBMC from abortive AE patients, whether non-stimulated or stimulated with PHA and antigenic preparations, secreted significantly lower levels of IL-10 than those isolated from progressive AE patients. Our observations seem to confirm the regulatory role of IL-10 in the immunopathology of human AE.

Low dose T-cell lymphocyte infusion combined with marrow T-cell depletion as prophylaxis of acute graft vs host disease for HLA identical sibling bone marrow transplantation.

T-cell depletion (TCD) of the bone marrow graft remains the most effective method to prevent severe graft versus host disease after allogeneic bone marrow transplantation. Early studies of HLA-identical sibling transplants showed that although T-cell depletion decreased GVHD, T-cell depleted transplants had higher risks of graft failure and leukemia relapse, leukemia free survival (LFS) was not improved compared to non-T-cell depleted transplants. In order to avoid graft failure and increased risk of relapse associated with this approach, we initiated a pilot study of T-cell depletion of the marrow graft combined with reinfusion of a fixed quantity of CD2+ peripheral blood T-cells. Depletion technique consisted in negative purging using CD2 and CD7 monoclonal antibodies (MoAbs) followed by rabbit complement cytolysis. This approach was associated with an intensified conditioning regimen using total body irradiation, high-dose cytosine arabinoside and melphalan (TAM) for all but one patient. Twenty-one patients were included with a mean age of 40 years. Only one acute severe Graft Versus Host Disease (GVHD) was observed and all patients engrafted. At 63 months, probability of survival is 42.86% with a relapse risk of 19.89%, two patients died from B-cell lymphoproliferative disease, seven other died from the procedure partially because of the use of the TAM as pretransplant regimen. This approach is being pursued by a gene therapy trial using herpes-simplex - 1 thymidine kinase gene expressing peripheral donor T-cells.

Vascular smooth muscle differentiation of murine stroma: a sequential model.

Previous studies by our group showed that stromal cells from human long-term marrow cultures were mesenchymal cells following a vascular smooth muscle pathway. The present study using 58 immortalized stromal lines from different hematopoietic sites was conducted to verify whether this hypothesis also held true for murine stroma. Principal components analysis performed using cytoskeletal and extracellular matrix proteins allowed the segregation of five factors explaining more than 70% of the variance. Factor I, including osteopontin and vimentin, and factor II, laminins and fibronectins, were representative of the mesenchyme. The remaining three factors were representative of vascular smooth muscle: factor III, including alphaSM actin, SM alpha actinin, SM22alpha, EDa+ fibronectin, and thrombospondin-1; factor IV, metavinculin and h-caldesmon; and factor V, smooth muscle myosin SM1 and desmin. All lines expressed factors I and II; 53 lines expressed factor III, 35 lines expressed factor IV; and 11 lines expressed factor V. A second principal components analysis including membrane antigens indicated the cosegregration of vascular cell adhesion molecule-1 with osteopontin and that of Ly6A/E with vimentin, whereas CD34 and Thy-1 appeared to be independent factors. The heterogeneity of vascular smooth muscle markers expression suggests that harmonious maintenance of hematopoiesis depends on the cooperation between different stromal cell clones.

Are complicated forms of celiac disease cryptic T-cell lymphomas?

We assessed the clonality of duodenal mucosal T cells in patients with celiac disease and controls. Fifteen adult patients were studied. Four patients had a complicated celiac disease, 3 did not respond to a gluten-free diet, and 2 had an ulcerative jejunitis (including 1 patient with nonresponsive celiac disease). Seven patients had an untreated celiac disease responsive to a gluten-free diet. Histological examination of duodenal biopsies of these 11 patients showed benign-appearing celiac disease without evidence of lymphoma. Four patients with nonulcer dyspepsia and normal duodenal biopsies served as controls. TCRgamma gene rearrangements were analyzed by multiplex polymerase chain reaction on DNA extracted from duodenal biopsies. Major clonal rearrangements of the T-cell receptor were found in 4 cases, all with complicated celiac disease. Monoclonality was confirmed by DNA sequencing of the junctional region in 3 cases and by hybridization with clone-specific oligoprobes. Patients with celiac disease responsive to gluten-free diet had mainly a polyclonal pattern, with 1 of them having an oligoclonal rearrangement. An oligoclonal pattern was also observed in 2 control patients. Three patients with complicated celiac disease evolved to T-cell lymphoma with liver (n = 2) or bone marrow (n = 1) invasion. Identical clones were found in the enteropathic duodenojejunum and peripheral blood in the patient with large-cell lymphoma with bone marrow invasion. This study suggests that complicated celiac disease is a cryptic T-cell lymphoma.

Prolonged remission and autologous recovery in two patients with chronic myelogenous leukemia after graft failure of allogeneic bone marrow transplantation.

Two patients with Philadelphia-positive chronic myelogenous leukemia underwent allogeneic bone marrow transplantation from a related HLA mismatched donor (patient 1) or from an unrelated HLA-identical donor (patient 2). Following bone marrow transplantation partial engraftment (patient 1) or graft failure (patient 2) occurred followed by autologous Philadelphia negative hematopoietic recovery either spontaneously (patient 1) or after infusion of autologous bone marrow rescue (patient 2). Neither Philadelphia chromosome, nor bcr-abl rearrangement was detectable by PCR analysis up to 7 years (patient 1) and 9 years (patient 2) post-transplantation. These two observations indicate that sustained engraftment of allogeneic bone marrow stem cells following a myeloablative regimen is not necessary to cure chronic myelogenous leukemia. It is hypothesized that the proliferative advantage of Philadelphia-negative progenitors and the anti-leukemic effect of lymphocytes in the graft have resulted in prolonged remission of the patients.

The broad spectrum of cytokine gene expression by myoid cells from the human marrow microenvironment.

Nontransformed stromal colony-derived cell lines (CDCLs) consist of a pure stromal cell population that differentiates following a vascular smooth muscle cell repertoire, and whose in vivo counterpart is that of myoid cells found in adult and fetal human bone marrow cords. We studied the cytokine expression by reverse-transcriptase polymerase chain reaction (RT-PCR) from pooled fast-growing clones from 10 different bone marrow samples. RT-PCR indicated that 30 cytokines (out of 42 studied) were expressed by CDCLs (20 after medium renewal and hydrocortisone renewal, three after addition of interleukin I beta (IL-1 beta) and seven in only part of the CDCL layers examined). The cytokines expressed comprised mediators known to be involved in the maintenance of early and late hematopoiesis (IL-1 alpha and IL-beta, IL-6, IL-7, IL-8, IL-11 and IL-13; colony-stimulating factors, thrombopoietin, erythropoietin, stem cell factor, fit 3-ligand, hepatocyte cell growth factor, tumor necrosis factor alpha, leukemia inhibitory factor, transforming growth factors beta 1 and beta 3; and macrophage inflammatory protein 1 alpha), angiogenic factors (fibroblast growth factors 1 and 2, vascular endothelial growth factor) and mediators whose usual target (and source) is the connective tissue-forming cells (platelet-derived growth factor A, epidermal growth factor, transforming growth factors alpha and beta 2, oncostatin M and insulin-like growth factor 1), or neuronal cells (nerve growth factor). The cytokines not expressed were lymphokines (IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, and IL-12 and interferon gamma) or mediators synthesized by macrophages (inhibin, activin, platelet-derived growth factor B, and IL-1 receptor antagonist). This study complements the description of the phenotype of the myoid cells, confirming that these cells are the marrow connective tissue-forming cells; moreover, this work suggests that stromal control of hematopoiesis is multifactorial and that myoid cells are involved in the control of marrow angiogenesis and innervation.

Increased basal production of interleukin-10 by peripheral blood mononuclear cells in human alveolar echinococcosis.

The secretion of IL-10 by peripheral blood mononuclear cells (PBMC) and the expression of IL-10 mRNA in fractionated CD4+ and CD8+ lymphocyte subsets and non-B-non-T cells, with and without stimulation by the mitogen phytohemagglutinin-C (PHA-C) and specific Echinococcus multilocularis (E. multilocularis) antigens, were assessed in 7 patients with alveolar echinococcosis (AE) and 6 healthy subjects. Results of studies on IL-10 were compared to those on IFN-gamma, IL-4 and IL-5 in the same patients and control subjects. IL-10 production was significantly higher in patient PBMC-culture supernatants than in the control group supernatants, both at the basal level and after mitogen or specific E. multilocularis antigen stimulation. Both CD4+ and CD8+ lymphocyte populations and non-B-non-T cells of AE patients and controls expressed IL-10 mRNA. Semi-quantification of IL-10 mRNA revealed a significantly higher transcript level in unstimulated-CD8+ T cells from AE patients in comparison with CD8+ T cells of healthy donors. PBMC from patients produced very low levels of IL-4 but the production of IFN-gamma was not significantly depressed compared to the controls. PBMC, isolated from 4 AE patients and 4 control subjects stimulated with specific E. multilocularis antigens, secreted IL-5; IL-5 mRNA was only detected in the CD4+ lymphocyte subset. The secretion of IL-5 and the expression of IL-5 mRNA in healthy subjects could be due to the presence of non-specific mitogenic parasitic factors. This non-specific mitogenic activity of the parasite, besides inducing a high secretion of IL-10 in patients with evolutive AE, may contribute to the lack of host control of parasite growth and to the persistence of granulomatous lesions, due to the inhibition of an efficient Th1 immune response.

Comparative preclinical study of three bone marrow purging methods using PCR evaluation of residual t(14;18) lymphoma cells.

The t(14;18) chromosomal translocation occurring in most follicular lymphomas can be exploited by a Bcl2/JH polymerase chain reaction (PCR) to detect residual disease and to monitor the effectiveness of ex-vivo tumor cell immunological purging. We first demonstrated the 10(-5) Bcl2/JH PCR sensitivity with serial dilutions of OCY-LY8 lymphoma cell lines in normal mononuclear cells; and then the specificity and reproductibility of this technique by analysing follicular and non follicular lymphoma samples. With the Bcl2/JH PCR, we tested the efficiency of three marrow purging protocols with an experimentally contaminated bone marrow either treated by three anti-B cell monoclonal antibodies (mAb) followed by three rounds of rabbit complement or two rounds of immunomagnetics beads. Samples obtained after each purging were amplified by Bcl2/JH PCR and hybridized with PFL3 probe. We were able to produce a 2 to 3 log tumor cell reduction after three rounds of complement and a 4 to 5 log reduction after two rounds of beads. This study showed that it is feasible to use the Bcl2/JH PCR technique for residual cell lymphoma detection in patients undergoing intensive chemotherapy or BM transplantation. These results indicate that ex-vivo immunomagnetic BM purging is probably superior to complement mediated lysis for the eradication of B lymphoma cells from the marrow of patients undergoing autologous transplantation.

Herpes virus-related lymphoproliferative disorders following allogeneic bone marrow transplantation: clinical and biological characteristics of six cases.

The present paper describes six cases of lymphoproliferative disorders (LPD) occurring after bone marrow transplantation. Treatments were ineffective and disease was rapidly fatal in all patients, although immunotyping of cells in blood, bone marrow or cerebrospinal fluid was helpful to establish the diagnosis of LPD. Monoclonality was demonstrated in the 4 cases which it was possible to analyse. Herpes virus genome was present in tumoral cells of 4 in 4 cases tested for EBV, one in 3 cases tested for CMV, one in 3 cases tested for HHV6 and 3 in 3 cases tested for HSV. Patients developing LPD should benefit from earlier diagnosis and new therapeutic approaches such as donor lymphocyte infusions, while further studies are necessary to elucidate the role of Herpes viruses in the pathogenesis of LPD.

Cellular localisations of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha mRNA in a parasitic granulomatous disease of the liver, alveolar echinococcosis.

Alveolar echinococcosis (AE), an uncommon and very severe parasitic liver disease, can be considered as an "infectious model" of granulomatous disease, where cellular immunity plays a key role in the defence against Echinococcus multilocularis, the larval cestode responsible for the disease. We analysed the localisation of the expression of the pro-inflammatory cytokines IL-1 beta, IL-6 and TNF-alpha mRNA in human AE liver lesions, in the periparasitic granulomas and in the hepatic parenchyma, as well as the phenotypic characteristics of the cells on serial sections. In situ hybridizations, using anti-sense 35S dUTP-labeled IL-1 beta, IL-6 and TNF-alpha riboprobes, were performed on cryostat liver sections; the sense probes were used as negative controls. IL-1 beta, IL-6 and TNF-alpha mRNA were observed in macrophages located at the extreme periphery of the granuloma, between the lymphocytic ring and the liver parenchyma, in patients with active AE. No cytokine mRNA expression was observed in a patient with an abortive case. Only TNF-alpha mRNA was located in the periparasitic area, in cells morphologically identified as macrophages but exhibiting an unusual phenotype (CD 11b-, CD 25+); this particular expression was observed only in those patients with very fertile lesions, associated with centro-granulomatous necrosis. These results show that pro-inflammatory cytokines are consistently produced by macrophages at the periphery of the periparasitic granuloma and can serve as mediators of acute-phase protein secretion and of fibrogenesis in that location.(ABSTRACT TRUNCATED AT 250 WORDS)

Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T-cell depletion after bone marrow transplantation?

Allogeneic bone marrow transplantation (BMT) is associated with a severe complication--graft-versus-host disease (GVHD). Although effectively preventing GVHD, ex vivo T-lymphocyte marrow depletion unfortunately increases graft rejection and reduces the graft-versus-leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir (GCV) if subsequent GVHD was to occur. Thus, one could preserve the beneficial effects of the T cells on engraftment and tumor control in patients not experiencing severe GVHD. To obtain T cells specifically depleted by GCV, we transduced primary T cells with a retroviral vector containing the HS-tk and neomycin resistance (NeoR) genes. Gene transfer was performed by coculturing PHA +/- CD3- or alloantigen-stimulated purified T cells on an irradiated retroviral vector producer cell line or by incubating the T cells in supernatant from the producer. Subsequent culture in G418 for 1 week allowed for the selection of transduced cells. GCV treatment of interleukin-2-responding transduced and selected cells resulted in greater than 80% growth inhibition, whereas GCV treatment of control cells had no effect. Similarly, the allogeneic reactivity of HS-tk-transduced cells was specifically inhibited by GCV. Combining transduced and nontransduced T cells did not show a bystander effect, thus implying that all of the cells inhibited by GCV were indeed transduced. Lastly, studies involving the transduction of the HUT-78 (T-lymphoma) cell line suggest that stable expression of HS-tk can be maintained over 3 months in vitro in the absence of G418. In summary, we have established the feasibility of generating HS-tk-transduced T cells for subsequent in vivo transfer with hematopoietic stem cells and, if GVHD occurs, specific in vivo GCV-induced T-cell depletion in allogeneic BMT recipients.

In situ hybridization for the detection of cytomegalovirus in blood or bone marrow leucocytes after allogeneic bone marrow transplantation.

We report a study of CMV detection by a nonradioactive in situ hybridization (ISH) technique using a biotinylated CMV-DNA probe. The method was applied to blood and bone marrow cells and its results were compared with those of other currently used techniques. First, we analysed peripheral leucocytes on blood donors, and, after comparison with PCR, we obtained 75% sensitivity and 87% specificity rates. We then studied four BMT recipients during the first 105 d post-transplant. A positive signal was detected as a predominantly nuclear staining. The detection of an ISH-positive signal occurred at least 8 d before CMV isolation from viral culture. The number of positive cells was variable according to the patients' clinical evolution, showing a dramatic increase of labelled cells in viraemic patients and then a decrease until complete disappearance of labelled cells when an efficient anti-viral treatment was initiated. This study suggests that the detection of CMV-DNA by ISH performed on peripheral leucocytes is a valuable tool for the early diagnosis of CMV infection and could lead to the initiation of optimal ganciclovir treatment.

The detection of colony-stimulating factors and steel factor in adherent layers of human long-term marrow cultures using reverse-transcriptase polymerase chain reaction.

In human long-term marrow cultures granulomonopoiesis is maintained for several weeks. Studies on granulomonocytic progenitors (CFU-GM) and their progeny have shown that survival, proliferation, differentiation and maturation of these cells are controlled by a set of glycoproteins, the colony-stimulating factors (CSFs) and the Steel factor. We have studied the expression of these factors using reverse transcriptase polymerase chain reaction (RT-PCR) in 17 adherent layers of normal bone marrow at 3, 5 or 7 weeks of culture. We have taken the 5637 bladder carcinoma cell line as a control for expression of GM-CSF, M-CSF, G-CSF and Steel factor, and PHA-activated T lymphocytes as a control for expression of multi-CSF (interleukin 3, IL-3). We have found that GM-CSF was expressed in the 17 adherent layers without induction by interleukin 1 beta (IL-1 beta). M-CSF was also detected in all cases, but in two early-stage (week 3 and week 5) cultures only after stimulation by IL-1 beta. G-CSF was detected in only 11 cases (three without IL-1 beta, and eight after addition of IL-1 beta). Steel factor was detected in 14 cases (ten without IL-1 beta, and four after addition of IL-1 beta). IL-3 was not detected even by means of nested RT-PCR. These data indicate in six late-stage (week 5 or week 7) cultures G-CSF messenger concentrations 10(3)-fold less than in 5637 control cells (for an identical amount of total cellular RNA). A similar conclusion may be drawn for Steel factor in three late-stage cultures. For IL-3 our negative results indicate a messenger concentration 10(5)-fold less than in activated T lymphocytes. These results suggest a crucial role for GM-CSF and M-CSF in the maintenance of granulomonopoiesis in human long-term cultures. The role of G-CSF and Steel factor may be more marginal. Eventually IL-2 may not be involved in the regulatory process.

Graft failure after T cell depleted HLA identical allogeneic bone marrow transplantation: risk factors in leukemic patients.

In a retrospective analysis of T cell depleted bone marrow transplantation, we have looked at different parameters in order to determine risk-factors of graft-failure after allogeneic bone marrow transplantation for leukemia. Fifty-one patients with acute leukemia or chronic myeloid leukemia have been analysed. For 33 of them, the pretransplant conditioning regimen consisted of fractionated total body irradiation (TBI) at 12 Gy prior to cyclophosphamide (120 mg/kg). The other patients received various reinforced preparative regimens. T-cell depletion consisted of treating marrow cells with pan-T monoclonal antibodies (CD2+CD3 or CD2-CD5-CD7) followed by complement mediated cytolysis. No post-transplant immunosuppressive prophylaxis was administered except for the first nine patients who received Methotrexate alone. In this group of 51 patients, 12 died within 3 months from graft-related complications and 10 developed graft failure (no engraftment or rejection). Among the possible risk factors associated with this failure, two graft-related parameters appeared significant: the number of CFU-GM progenitors and the number of viable T cells injected with the marrow inoculum. No correlation with graft failure was found with other parameters including diagnosis, disease status at transplant, conditioning regimen, age, sex, and CMV status of donor/host pairs. However, the interpretation must remain cautious because of the relatively small samples in each group.