In vitro and in vivo evaluation of the haematopoietic potential of skeletal muscle in a non-human primate model.

This study was aimed at evaluating the in vitro and in vivo haematopoietic potential in macaque skeletal muscle cells. Biopsy samples showed the presence of CD34(+) (7.6%), CD90(+) (8.4%), CD117(+), CD31(+), side population (SP) cells (7-10%) and a low number of CD45(+) cells. In clonogenic and long-term culture-initiating cell assays, no haematopoietic potential could be detected in either total mononuclear cells or SP cells. Regarding in vivo studies, two animals were transplanted with unfractionated fresh muscle cells after lethal irradiation. Both animals died early after transplant without any evidence of haematopoietic reconstitution. In two other monkeys, harvested muscle cells were frozen and secondarily marked using a green fluorescent protein (GFP)-lentiviral vector. After sublethal irradiation, both animals were transplanted with GFP-expressing muscle cells followed by a bone marrow rescue. Both animals had haematopoietic reconstitution at days 22 and 25, but no GFP-expressing haematopoietic cells could be detected by flow cytometry, either in the blood or in clonogenic cells from marrow aspirates. Using PCR assays, GFP(+) cells were detected in a single marrow sample of one animal at 41 days after transplantation. These results strongly suggest that as opposed to murine muscle, the non-human primate skeletal muscle does not harbour cells with a straightforward haematopoietic potential.

Expression of a foreign protein in human megakaryocytes and platelets by retrovirally mediated gene transfer.

Recent progress in the culture of human megakaryocytes (MKs) has led to the capacity to produce platelets in vitro. This capability enables investigation into the possibility of modifying platelet structure and/or function by genetically altering the MK. To this end, a cDNA for the murine CD9 (mCD9) cell surface protein was introduced into MK progenitors by retrovirally mediated gene transfer and subsequently detected in cultured MKs with a monoclonal antibody (MoAb) that specifically recognizes the murine protein. CD34+ human peripheral blood or marrow progenitors, enriched by immunomagnetic bead selection, were cultured for 5 days in the presence of growth factors, including stem cell factor and thrombopoietin, to induce MK progenitors into the cell cycle. The stimulated cells were then cocultured with the mCD9 retroviral producer cell line for 3 days, followed by culture in serum-depleted medium for 3 to 7 additional days. Flow cytometry analysis using the anti-CD9 MoAb and TAB, a MoAb recognizing human GPIIb, revealed that a large proportion (40-100%) of the MKs expressed mCD9. To ascertain whether these cells were capable of producing mCD9+ platelets, flow cytometry analysis was performed at a time when proplatelets were observed in the culture. mCD9 was detected in up to 59% of the TAB+ platelet-sized particles. Because deteriorating MKs can produce platelet-sized particles in vitro, experiments were performed to determine whether mCD9+ TAB+ particles were functionally active. Addition of phorbol myristate acetate resulted in the redistribution of P-selectin (CD62) from the alpha granule to the platelet surface as detected by MoAbs S12 and G5 in three-color flow cytometry analyses. These studies showed that up to 76% of the mCD9+ TAB+ particles were functionally active. The data show that retrovirally mediated gene transfer is a viable approach for genetically altering MK progenitors, resulting in platelets that express heterologous proteins.

Hepatitis C virus infection and allogeneic bone marrow transplantation.

Serum antibodies to hepatitis C virus (HCV) were tested for inpatients undergoing allogeneic BMT to determine the risk of acquiring HCV infection and the role of HCV in posttransplant liver complications. The HCV seroconversion rate was evaluated according to the date of BMT and blood donor screening at the time. Anti-HCV antibodies (anti-HCV) were detected with a second-generation ELISA and confirmed with a second-generation radioimmunoblot assay. All patients received leukocyte-depleted blood products and most received apheresis platelet concentrates. One hundred twenty of 181 consecutive patients transplanted from January 1987 to December 1991 were anti-HCV-negative before BMT, had at least 6 months of follow-up, and were thus evaluated for the seroconversion rate. Before screening for non-A, non-B hepatitis, 14% of the patients seroconverted to HCV (0.44% per unit transfused). After introduction of screening for alanine aminotransferase and antibodies to hepatitis B core antigen the risk of seroconversion was 4% per patient (0.26% per unit). When, in addition, blood was screened for anti-HCV the risk fell to 1.6% (0.03% per unit). Positive anti-HCV status before and after BMT was not predictive of veno-occlusive disease, liver graft-versus-host disease (GVHD), or death due to liver dysfunction. In contrast, the risk of chronic hepatitis was significantly increased.

Reverse seroconversion of hepatitis B after allogeneic bone marrow transplantation: a retrospective study of 37 patients with pretransplant anti-HBs and anti-HBc.

BACKGROUND: Reverse seroconversion to hepatitis B virus (HBV), i.e., HBV reactivation in patients with pretransplant antibodies to hepatitis B surface antigen (anti-HBs) and to hepatitis B core antigen (anti-HBc), is rarely re-ported after allogeneic bone marrow transplantation. METHODS: To determine this risk, we studied clinical outcome and serological changes in 37 patients with pretransplant anti-HBs and anti-HBc. RESULTS: In 33 cases, no change in HBV markers was observed in the posttransplant period. In four cases, anti-HBs and anti-HBc were lost, and hepatitis B surface antigen, hepatitis B e antigen, and HBV DNA emerged together with acute hepatitis, after cessation of immunosuppression. The actuarial risk of reactivation in the 37 patients was 20.5% (median follow-up 20 months). No reactivation occurred in patients with anti-HBs-positive donors. CONCLUSION: Although few cases of postallogeneic bone marrow transplantation reverse seroconversion to HBV have been reported, this study demonstrates that the actuarial risk is relatively high and suggests that donor vaccination might be proposed prophylactically or that HBs-specific immunoglobulin infusions might be warranted.

Comparison of autografting using mobilized peripheral blood stem cells with and without granulocyte colony-stimulating factor in malignant lymphomas.

Peripheral blood is becoming widely used as the only source of hematopoietic stem cells to support marrow ablative therapy in advanced lymphoma. We report data from 23 patients with high risk non-Hodgkin's (n = 19) and Hodgkin's lymphoma (n = 4) who underwent high-dose therapy with mobilized peripheral blood stem cell (PBSC) autografting. Peripheral blood progenitors were recruited using cytotoxic chemotherapy followed by administration of recombinant human G-CSF (filgrastim 5 micrograms/kg/day). Myeloablative treatment with autologous PBSC support was administrated to the 23 patients and followed by G-CSF at the same dose after cell reinjection. Hematopoietic reconstitution was compared with a control group of lymphoma patients who received chemotherapy mobilized PBSC transplantation but without G-CSF prior to leukaphereses or after high-dose therapy. The median time to neutrophil recovery > 0.5 x 10(9)/l was significantly shorter in study patients compared with the control patients (10 days and 17 days respectively) (p < 0.05). Self sustaining platelet counts of > 50 x 10(9)/l occurred at a median time of 17 days in both groups. Stable hemopoietic reconstitution was seen with a follow-up of 6 months after PBSC transplantation. In addition, a significant relationship was observed between the number of CFU-GM infused and the time to platelet recovery. We confirm the effectiveness of G-CSF given prior to PBSC harvesting in generating high numbers of progenitor cells. Hematologic recovery following high-dose therapy was improved after PBSC rescue and G-CSF.

[Ineffectiveness of platelet transfusions in the course of thrombocytopenia of central origin. Work Group coordinated by ANDEM]. / Inefficacité des transfusions de plaquettes au cours des thrombopénies d'origine centrale. Groupe de Travail Coordonné par l'ANDEM.

This study, conducted under the auspices of ANDEM, was aimed at determining the reasons for the inefficacy of platelet transfusions for thrombocytopenia of central origin, and to establish recommendations for therapy and prevention. It was based on a critical review of the literature. Three main causes of inefficacy were identified: an immunological conflict (mainly anti-HLA alloimmunization), clinical status, and the quality of platelet preparations (especially storage time). Our recommendations depend on several factors, such as the degree of urgency, the cause of platelet refractoriness, clinical status and the availability of platelet preparations. The mainstay of the recommendations is prevention, based on the quality of platelet preparations (as fresh as possible, in adequate number preferably obtained by apheresis and ABO compatible) and on the prevention of anti-HLA alloimmunization.

[Transfusion and alloimmunization in sickle cell anemia patients]. / Transfusion et alloimmunisation chez les patients drépanocytaires.

Transfusion therapy for sickle cell anemia is limited by the development of antibodies to red cell antigens. The aim of this study was to evaluate whether transfusion of blood matched for antigens Rh and Kell would reduce the incidence of alloimmunization. We determined the transfusion history, red cell phenotype and development of alloantibodies in 173 patients with sickle all anemia who received transfusions. Forty nine patients were transfused exclusively with frozen red blood cells (RBL) matched for antigens Rh and Kell; the rate of alloimmunization was 8.2%; antibodies to the Jkb, Jka, Fya and S were developed; 1 patient developed 2 antibodies. In a control group of 124 patients who received standard red blood cells, the rate of alloimmunization was significantly increased to 30.6% (p < 0.05); antibodies against C, E, K, Fya were the most frequently developed and 19 patients (16%) developed antibodies reacting with different antigens. In the 2 groups, alloimmunization occurred after receiving a significantly different number of transfusions: mean 9 in the patients transfused with matched RBC and 32 in the control group. The influence of the kinetics of transfusion was not demonstrated. To assess the effect that racial differences might have on alloimmunization, comparison of the red cell phenotype of patients with that of a panel of unselected blood bank donors was performed: the patients had a significant decrease in the frequency of red cell antigens corresponding to most of the detected alloantibodies JkB, C, S. Fyb, Fya and Kell.(ABSTRACT TRUNCATED AT 250 WORDS)

Extensive chronic GVHD is associated with donor blood CD34+ cell count after G-CSF mobilization in non-myeloablative allogeneic PBSC transplantation.

The correlation between the incidence of GVHD and the number of infused CD34(+) cells remains controversial for PBSC transplantation after a reduced-intensity-conditioning (RIC) regimen. We evaluated 99 patients transplanted with an HLA-identical sibling after the same RIC (2-Gy-TBI/fludarabine). Donor and recipient characteristics, donor's blood G-CSF-mobilized CD34(+) cell count, and number of infused CD34(+) and CD3(+) cells were analyzed as risk factors for acute and chronic GVHD There was a trend for an increased incidence of extensive chronic GVHD in the quartile of patients receiving more than 10 × 10(6) CD34(+) cells/kg (P = 0.05). Interestingly, the number of donor's blood CD34(+) cells at day 5 of G-CSF mobilization was closely associated with the incidence of extensive chronic GVHD, that is, 48% (95% CI: 28-68) at 24-months in the quartile of patients whose donors had the highest CD34(+) cell counts versus 24.3% (95% CI: 14-34) in the other patients (P = 0.007). In multivariate analysis, the only factor correlating with extensive chronic GVHD (cGVHD) was the donor's blood CD34(+) cell count after G-CSF (HR 2.49; 95% CI: 1.16-5.35, P = 0.019). This study shows that the incidence of cGVHD is more strongly associated with the donor's ability to mobilize CD34(+) cells than with the number of infused CD34(+) cells.

Fully functional NK cells after unrelated cord blood transplantation.

Promising results of umbilical cord blood transplantation (UCBT) from unrelated donors have been reported in patients with hematologic disorders. These transplants, having potential to trigger beneficial donor-versus-recipient natural killer (NK) cell-mediated alloreaction, we have conducted the first extensive analysis of the phenotypic and functional properties of NK cells after UCBT. NK cells from 25 patients with high-risk hematologic malignancies were compared with cells derived from both healthy adult and CB cells. We found that following UCBT, NK cells display not only some phenotypic features associated with maturity but also unique characteristics that make them fully functional against leukemic blasts. We propose that this full functionality of alloreactive donor-derived NK may drive graft-versus-leukemia reactions after UCBT.

[Thrombopoietin and megakaryocyte differentiation]. / Thrombopoïétine et différenciation mégacaryocytaire.

Regulation of megakaryocytopoiesis and platelet production is a complex phenomenon. It has been demonstrated that numerous pleiotropic cytokines act in vivo and in vitro on megakaryocytopoiesis. Historically, studies on the regulation of megakaryocytopoiesis were largely dominated by the concept of humoral regulation. Despite 35 years of work, the factor responsible for these effects (thrombopoietin, TPO) could not be purified to homogeneity. The proto-oncogene c-mpl has been demonstrated to encode a protein which is the receptor for a humoral cytokine regulating megakaryocytopoiesis. Its ligand was isolated by three independent groups. The purified protein was termed Mpl-L (Mpl-Ligand), thrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF). Strong evidence that Mpl-L is the homeostatic regulator of platelet production has been provided by c-mpl or Mpl-L knock-out mice which have a severe but not lethal thrombocytopenia. In vitro experiments have indicated that the Mpl-L acts both as a proliferative and differentiative factor as many other CSF. At the unicellular level, Mpl-L acts on MK progenitors inducing their proliferation. Mpl-L induces polyploidisation of the MKs and cytoplasmic maturation leading to an in vitro platelet production. As a single cytokine Mpl-L is the most potent growth factor for the MK lineage in vitro. However, a combination of cytokines can totally replace the effects of Mpl-L both on proliferation of MK progenitors and their maturation. In addition, Mpl-L has a major effect on primitive hematopoietic progenitors.

Apheresis experience with a continuous flow cell separator.

The Dideco Vivacell separator is a continuous-flow centrifugation system that has only recently been used for cytapheresis. The authors' experience with this separator in 451 plateletpheresis and 164 leukapheresis procedures is presented. Platelet collection provided high platelet yields (9.53 +/- 2.85 X 10(11) with a collection efficiency of 74 +/- 14 percent for about 6 liters of total blood processed. Functional integrity was confirmed by normal in vitro tests (aggregation and response to hypotonic stress) and good in vivo recovery (55%). In leukapheresis, white cell yields were high (3.42 +/- 1.2 X 10(10) with 85 percent polymorphonuclear neutrophil cells. Their oxidative metabolism functions (generation of free oxygen radicals), investigated by chemiluminescence, were increased over donor values. Donor reactions, all of the mild citrate type, were rare.

Peripheral blood stem cell collection with a blood cell separator.

Forty-three patients with malignant nonmyeloid diseases underwent peripheral blood stem cell collections on an apheresis system (Spectra, COBE BCT, Lakewood, CO). Collections took place during the white cell (WBC) recovery phase following conditioning chemotherapy. One hundred two procedures were done after chemotherapy alone, and 72 procedures after chemotherapy plus granulocyte-colony-stimulating factor (G-CSF). Four centrifugal separation factors were tested. One and one-half patient blood volumes were processed in each procedure. The mean volume of the collected component was 158 +/- 16 mL. After chemotherapy alone, the procedures provided a mean of 0.8 x 10(8) WBCs per kg and 2.3 x 10(4) colony-forming units-granulocyte macrophage (CFU-GM) per kg of recipient body weight. The mononuclear cell percentage in the components increased with the centrifugal separation factor from 85 to 96 percent. In parallel, platelet contamination increased from 2.1 to 3.8 x 10(11). The collect hematocrit ranged from 1.0 to 2.5 percent (0.01-0.025). The collection efficiency for mononuclear cells and CFU-GM also increased with the centrifugal separation factors from 52 to 70 percent for mononuclear cells and from 55 to 68 percent for CFU-GM. Collections performed after G-CSF-stimulated mobilization were characterized by a higher neutrophil contamination independent of centrifugal separation factor, which gave a mean mononuclear cell percentage of 64 percent in the collected component. The average yield for these procedures was 2 x 10(8) WBCs per kg and 28 x 10(4) CFU-GM per kg.(ABSTRACT TRUNCATED AT 250 WORDS)

[Value of filariopheresis in the treatment of Loa loa filariasis]. / Intérêt de la filariophérèse dans le traitement de la filariose A Loa loa.

To prevent the side effects as encephalitis, related with diethylcarbamazine treatment in loiasis, an exchange blood transfusion was proposed. To avoid the disadvantage of this heavy technique, the authors are proposing filariopheresis to trap the microfilariae using cytapheresis technique and filtration. They obtained a medium 75 p. cent microfilariae extraction: 55-70 p. cent by cytapheresis and 5-20 p. cent by filtration. The number of blood cells must be supervised as an important decrease of the platelets may occur temporary. This simple and not expensive technique is efficient, but diethylcarbamazine treatment must be instituted very quickly after filariopheresis session. Furthermore, the millions of microfilariae collected can provide useful antigen for immuno-parasitological tests.

Cell cycle activation of peripheral blood stem and progenitor cells expanded ex vivo with SCF, FLT-3 ligand, TPO, and IL-3 results in accelerated granulocyte recovery in a baboon model of autologous transplantation but G0/G1 and S/G2/M graft cell content does not correlate with transplantability.

Ex vivo expansion is a new strategy for hematopoietic stem and progenitor cell transplantation based on cytokine-induced amplification to produce grafts of controlled maturity. If the cell cycle position of CD34(+) cells has been reported to govern their engraftment potential, the respective role of stem and progenitor cells in short- and long-term hematopoietic recovery remains debated. Studies focused on long-term engraftment potential suggest impairment when using cultured grafts, but the capacity to sustain short-term recovery is still controverted. The aim of this study was: A) to evaluate the consequences of cell cycle activation on short and long-term engraftment capacity, and B) to determine if cell cycle status of grafts could predict hematopoietic recovery. We showed in a nonhuman primate model of autologous peripheral blood stem and progenitor cell transplantation that cell cycle activation of CD34(+) cells in the presence of stem cell factor + FLT3-ligand + thrombopoietin + interleukin 3 (six days of culture) which induced G1 and S/G2/M cell amplification (G0: 6.1% +/- 2.8%; G0/G1: 64.2% +/- 7.2%; S/G2/M: 30.4% +/- 7.3% respectively of expanded CD34(+) cells on average) resulted in the acceleration of short-term granulocyte recovery. By contrast, G0/G1 and S/G2/M cell content of expanded grafts did not correlate with short- or long-term engraftment.

Characteristics of peripheral blood progenitor cells frozen after 24 hours of liquid storage.

Peripheral blood progenitor cells (PBPC) harvested for autologous transplantation are usually cryopreserved within 2-4 h of collection. However, there are conditions in which it would be useful to freeze PBPC after a liquid storage period. This study was performed to evaluate whether prior storage for just 24 h damages frozen PBPC. First, leukapheresis products were obtained from 9 patients and divided into three fractions. The first fraction was frozen within 2 h and used as a control. The second and the third fractions were stored either at 4 degrees C or at 22 degrees C for 24 h before freezing. Cell counts, CFU-GM values, and pH were studied after collection, after storage, and after cryopreservation. Similar results were obtained at 4 degrees C and 22 degrees C. However, pH decreased most markedly at 22 degrees C. Mean postcryopreservation CFU-GM recoveries were not significantly different and were, respectively, 74.03% (control), 96.39% (4 degrees C), and 80.33% (22 degrees C). These observations were confirmed in an additional study of frozen PBPC collected from 12 patients and previously stored for 24 h at 4 degrees C. These data indicate that blood progenitors may be stored for 24 h and subsequently frozen without quantitative and qualitative impairment.

[Changes in serum lipids and lipoproteins in 3 cases of heterozygous familial hypercholesterolemia treated by LDL apheresis on dextran sulfate-cellulose columns]. / Evolution des lipides et lipoprotéines sériques dans 3 cas d'hypercholestérolémie familiale hétérozygote traités par aphérèse des LDL sur colonne de sulfate de dextran-cellulose.

Three patients with heterozygote type IIa hyperlipoproteinemia were treated by specific LDL apheresis with a dextran sulfate-cellulose column every two weeks. Each apheresis resulted in a fall in total cholesterolemia of about 50 p. 100 with a parallel fall in apoprotein B. The average drop of 15 p. 100 in HDL-cholesterol appeared to be due to the hemodilution induced at the end of apheresis. After two months, cholesterol and apoprotein B concentrations before removal were 15 p. 100 lower than baseline values. Treatment was well tolerated. These findings appear encouraging for the treatment of heterozygote forms of familial hyperlipoproteinemia resistant to chemotherapy.