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1.
J Control Release ; 278: 110-121, 2018 05 28.
Article in English | MEDLINE | ID: mdl-29630987

ABSTRACT

Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Their proper delivery to dendritic cells (DCs) requires their protection against RNase degradation and more specificity for dose reduction. Lipid-Polymer-RNA lipopolyplexes (LPR) are attractive mRNA delivery systems and their equipment with mannose containing glycolipid, specific of endocytic receptors present on the membrane of DCs is a valuable strategy. In this present work, we evaluated the capacity of LPR functionalized with a tri-antenna of α-d-mannopyranoside (triMN-LPR) concerning (i) their binding to CD209/DC-SIGN and CD207/Langerin expressing cell lines, human and mouse DCs and other hematopoietic cell populations, (ii) the nature of induced immune response after in vivo immunization and (iii) their therapeutic anti-cancer vaccine efficiency. We demonstrated that triMN-LPR provided high induction of a local inflammatory response two days after intradermal injection to C57BL/6 mice, followed by the recruitment and activation of DCs in the corresponding draining lymph nodes. This was associated with skin production of CCR7 and CXCR4 at vaccination sites driving DC migration. High number of E7-specific T cells was detected after E7-encoded mRNA triMN-LPR vaccination. When evaluated in three therapeutic pre-clinical murine tumor models such as E7-expressing TC1 cells, OVA-expressing EG7 cells and MART-1-expressing B16F0 cells, triMN-LPR carrying mRNA encoding the respective antigens significantly exert curative responses in mice vaccinated seven days after initial tumor inoculation. These results provide evidence that triMN-LPR give rise to an efficient stimulatory immune response allowing for therapeutic anti-cancer vaccination in mice. This mRNA formulation should be considered for anti-cancer vaccination in Humans.


Subject(s)
Cancer Vaccines/administration & dosage , Dendritic Cells/immunology , Neoplasms/therapy , RNA, Messenger/administration & dosage , Animals , Antigens, Neoplasm/immunology , Cancer Vaccines/immunology , Cell Line, Tumor , Cell Movement/immunology , Female , Humans , Injections, Intradermal , Lipids/chemistry , Lymph Nodes/immunology , Mannose/chemistry , Melanoma, Experimental/immunology , Melanoma, Experimental/therapy , Mice , Mice, Inbred C57BL , Neoplasms/immunology , Ovalbumin/immunology , Vaccination
2.
Gene Ther ; 23(2): 144-50, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26488891

ABSTRACT

Targeting viral entry is the most likely gene therapy strategy to succeed in protecting the immune system from pathogenic HIV-1 infection. Here, we evaluated the efficacy of a gene transfer lentiviral vector expressing a combination of viral entry inhibitors, the C46 peptide (an inhibitor of viral fusion) and the P2-CCL5 intrakine (a modulator of CCR5 expression), to prevent CD4⁺ T-cell infection in vivo. For this, we used two different models of HIV-1-infected mice, one in which ex vivo genetically modified human T cells were grafted into immunodeficient NOD.SCID.γc⁻/⁻mice before infection and one in which genetically modified T cells were derived from CD34⁺ hematopoietic progenitors grafted few days after birth. Expression of the transgenes conferred a major selective advantage to genetically modified CD4⁺ T cells, the frequency of which could increase from 10 to 90% in the blood following HIV-1 infection. Moreover, these cells resisted HIV-1-induced depletion, contrary to non-modified cells that were depleted in the same mice. Finally, we report lower normalized viral loads in mice having received genetically modified progenitors. Altogether, our study documents that targeting viral entry in vivo is a promising avenue for the future of HIV-1 gene therapy in humans.


Subject(s)
CD4-Positive T-Lymphocytes/virology , Chemokine CCL5/genetics , Gene Transfer Techniques , HIV Infections/prevention & control , HIV-1 , Recombinant Fusion Proteins/genetics , Virus Internalization , Animals , Antigens, CD34 , CCR5 Receptor Antagonists/therapeutic use , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/transplantation , Female , Genetic Vectors , Humans , Lentivirus/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Receptors, CCR5/metabolism
4.
Blood Cancer J ; 2: e88, 2012 Sep 07.
Article in English | MEDLINE | ID: mdl-22961060

ABSTRACT

Waldenström's macroglobulinemia (WM) is a clonal B-cell lymphoproliferative disorder (LPD) of post-germinal center nature. Despite the fact that the precise molecular pathway(s) leading to WM remain(s) to be elucidated, a hallmark of the disease is the absence of the immunoglobulin heavy chain class switch recombination. Using two-dimensional gel electrophoresis, we compared proteomic profiles of WM cells with that of other LPDs. We were able to demonstrate that WM constitutes a unique proteomic entity as compared with chronic lymphocytic leukemia and marginal zone lymphoma. Statistical comparisons of protein expression levels revealed that a few proteins are distinctly expressed in WM in comparison with other LPDs. In particular we observed a major downregulation of the double strand repair protein Ku70 (XRCC6); confirmed at both the protein and RNA levels in an independent cohort of patients. Hence, we define a distinctive proteomic profile for WM where the downregulation of Ku70-a component of the non homologous end-joining pathway-might be relevant in disease pathophysiology.

5.
Bone Marrow Transplant ; 26(5): 497-503, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11019838

ABSTRACT

The aim of the present study was to evaluate the capacity to expand of hematopoietic stem cell (HSC) samples from eight patients with NHL, and to follow in parallel the fate of tumor cells in four of eight samples still containing bcl2/JH+ tumor cells after CD34+ or CD19-/20-/34+ cell selection. The presence of bcl2/JH+ cells was also investigated after expansion in four of eight samples, two of which were bcl2/JH at harvesting and two which were initially bcl2/JH+ but became bcl2/JH (below the level of PCR detection) after cell selection, to assess a possible reappearance of occult tumor cells after expansion culture. We used culture conditions that we previously had established to allow high level expansion of normal precursors, progenitors and LTC-ICs. In this study, particular attention was given to the role of Flt3-ligand, known to favor the growth of B cells. The expansion conditions were: 1.5 x 10(3) cells/ml in serum-free medium containing stem cell factor (SCF), interleukin-3 (IL-3), IL-6, granulocyte-stimulating factor (G-CSF), erythropoietin (Epo) +/- Flt3-ligand (Flt3-L) for 10 days. After culture, total cells, CFU-GMs, BFU-Es and LTC-ICs were expanded to a mean of 833-, 6.6-, 4.6-, and 1.8-fold, respectively with the cocktail of cytokines not including Flt3-L. When Flt3-L was added, the mean expansion values were 1095-, 31-, 15- and three-fold, respectively. Residual bcl2/JH+ cells present in four of eight samples before expansion were not detected after expansion. Similarly, no tumor cells reappeared after expansion of the two samples which had become negative after selection, as well as in the two samples which were bcl2/JH- at harvesting. These results suggest first that ex vivo expansion of hematopoietic stem cells in patients with non-Hodgkin's lymphoma is feasible without incurring the parallel risk of amplifying tumor cells; second, that Flt3-L did not stimulate the growth of tumor cells while it clearly favored the growth of normal progenitors.


Subject(s)
Antigens, CD34/blood , Cell Culture Techniques/methods , Hematopoietic Stem Cells/cytology , Lymphoma, Non-Hodgkin/blood , Lymphoma, Non-Hodgkin/genetics , Adult , Biomarkers, Tumor , Cell Division/drug effects , Cell Separation/methods , Female , Gene Rearrangement , Hematopoietic Stem Cells/immunology , Humans , Immunoglobulin Joining Region/genetics , Immunophenotyping , Lymphoma, Non-Hodgkin/pathology , Male , Membrane Proteins/pharmacology , Membrane Proteins/physiology , Middle Aged , Proto-Oncogene Proteins c-bcl-2/genetics , Transplantation, Autologous/methods
6.
Hum Gene Ther ; 11(8): 1189-200, 2000 May 20.
Article in English | MEDLINE | ID: mdl-10834620

ABSTRACT

This study was designed to retrovirally transduce T cells by a protocol that would be simple, short, cost effective, applicable for clinical use, and efficient enough to avoid further selection of transduced T cells. Because retrovirally mediated infection is depending on the cell cycle, we first optimized the conditions for activating T cells in the presence of immobilized CD3 monoclonal antibodies and recombinant interleukin 2. Cell cycle analysis indicated that CD8+ and total T cells reach a maximum of cycling within 4 days whereas CD4+ T cells attain their maximum of cycling only by day 6. Taking into account these data, CD4+, CD8+, and total T cells were preactivated for 5 and 3 days, respectively, and then infected for 24 hr with supernatant containing retrovirus pseudotyped with gibbon-ape leukemia virus envelope, using a cell centrifugation protocol. Results show that approximately 95% of CD4+, CD8+, and total T cells can be transduced, this transduction efficiency being significantly higher than that obtained with amphotropic retrovirus vectors. Furthermore, under permanent growth stimulation, transduced T cells can be expanded approximately 1,000-fold in 4 weeks of culture with maintenance of transgene expression. However, Immunoscope analysis revealed alterations of T cell repertoire diversity after 2-3 weeks in culture that was not due to retroviral transduction per se. Overall, these data provide evidence that T cells can be transduced at levels that may alleviate the need for both further selection of transduced cells and in vitro expansion, thereby preserving the repertoire diversity of the transduced T cells to be reinfused.


Subject(s)
Gene Transfer Techniques , Retroviridae/genetics , T-Lymphocytes/metabolism , Transduction, Genetic , Antibodies, Monoclonal/metabolism , CD3 Complex/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Cycle/genetics , Centrifugation , Galactosides/metabolism , Humans , Indoles/metabolism , Interleukin-2/metabolism , Kinetics , Leukemia Virus, Gibbon Ape/genetics , Lymphocyte Activation , Recombinant Proteins/metabolism , Time Factors , Tumor Cells, Cultured , beta-Galactosidase/metabolism
7.
Immunology ; 99(1): 8-15, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10651935

ABSTRACT

Dendritic cells (DC) are extremely efficient at generating both prophylactic and therapeutic anti-tumour immunity. We aimed to analyse the respective roles of humoral and cellular immune responses generated in mice vaccinated with bone marrow (BM)-derived DC in terms of in vivo anti-leukaemia effect. We used the murine L1210 B lymphocytic leukaemia genetically modified to express on the cell surface of human CD4 (hCD4) (L1210/hCD4) as a model tumour-associated antigen (TAA). DC cultures were loaded with either purified soluble hCD4 (shCD4) protein or unfractionated L1210/hCD4 extracts and injected as vaccine into mice. The efficacy of these vaccinations was compared with that of vaccination with shCD4 protein emulsified in Freund's adjuvant (FA). We evaluated the immune responses generated after these vaccinal protocols and the survival rate of vaccinated mice subsequently challenged with a lethal injection of L1210/hCD4 cells. Our results demonstrated that vaccination with shCD4 protein or tumour extract-loaded DC mainly generated an hCD4 antigen-specific cell-mediated cytotoxic immune response that was associated with a specific protection against leukaemia. In contrast, vaccination with the protein emulsified in FA only generated potent humoral immune responses that were not protective against leukaemia. Altogether, our results indicate that the unique property of loaded DC to trigger an anti-leukaemia protective effect is mainly associated with cellular immune responses.


Subject(s)
CD4 Antigens/immunology , Cancer Vaccines/administration & dosage , Dendritic Cells/immunology , Immunotherapy, Adoptive/methods , Leukemia, Experimental/prevention & control , T-Lymphocytes, Cytotoxic/immunology , Animals , CD4 Antigens/administration & dosage , Cancer Vaccines/immunology , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Histocompatibility Antigens Class I/immunology , Leukemia, Experimental/immunology , Mice , Mice, Inbred DBA , Specific Pathogen-Free Organisms , Tumor Cells, Cultured
8.
Exp Hematol ; 27(6): 1010-8, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10378890

ABSTRACT

Murine low-density bone marrow cells sorted from the blast cell window on the basis of high rhodamine-123 retention (Rh-bright), are highly enriched in histamine-, IL-4-, and IL-6-producing cells. We established by in situ hybridization that up to 50% of this population (around 0.25% of the whole bone marrow) coexpressed the transcripts for these molecules upon stimulation with 1L-3. Rh-bright cells were also positive for mRNA encoding the alpha, beta, and gamma chains of the Fc(epsilon)RI which was functional since aggregated IgE induced the same percentage of cells hybridizing with the HDC probe as IL-3. Clonogenic progenitors and histamine- and cytokine-producing cells copurified in the Rh-bright population, but could be distinguished by their c-kit expression, CFU-C being more frequent in the c-kit(high) fraction, while histamine and IL-6 producers were enriched in the kit(low) counterpart. Ultrastructural analysis of Rh-bright cells revealed essentially two subsets, namely undifferentiated blast cells and basophil precursors. No other lineage-committed population was enriched by this sorting procedure, and it can therefore be concluded that coexpression of HDC, IL-6, and IL-4 transcripts in response to IL-3 or aggregated IgE takes place mainly in hematopoietic precursors belonging to the basophil lineage.


Subject(s)
Basophils/metabolism , Hematopoietic Stem Cells/metabolism , Histidine Decarboxylase/genetics , Interleukin-3/pharmacology , Interleukin-4/genetics , Interleukin-6/genetics , Animals , Basophils/ultrastructure , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cytokines/biosynthesis , Female , Fluorescent Dyes , Gene Expression , Histamine/biosynthesis , In Situ Hybridization , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron , RNA, Messenger/analysis , Rhodamine 123
9.
Immunology ; 96(4): 569-77, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10233743

ABSTRACT

Dendritic cells (DC) are professional antigen-presenting cells that can be used as immune adjuvant for anti-tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or combined with interleukin-4 (IL-4) or Flt3 ligand (Flt3-L) on the number, immunophenotype and functions of bone marrow-derived DC. In GM-CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC-IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC-205+ F4/80+), and MHC-IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC-205-; F4/80+). The addition of Flt3-L to GM-CSF induced a twofold increase in MHC-IIhi DC number; besides, the MHC-IIlo cells lost all DC markers. In contrast, after addition of IL-4 to GM-CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1- DEC-205+ F4/80-), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty-four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM-CSF+Flt3-L. Finally, we showed that MHC-IIhi were more efficient than MHC-IIlo cells in an anti-tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro-generated DC before use in immunotherapy.


Subject(s)
Bone Marrow Cells/immunology , Cancer Vaccines/therapeutic use , Cytokines/immunology , Dendritic Cells/immunology , Animals , Cell Differentiation/immunology , Female , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Histocompatibility Antigens Class II/metabolism , Immunophenotyping , Interleukin-4/immunology , Leukemia L1210/prevention & control , Lymph Nodes/immunology , Lymphocyte Culture Test, Mixed , Male , Membrane Proteins/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Tumor Cells, Cultured , Vaccination
10.
Hum Gene Ther ; 10(2): 175-87, 1999 Jan 20.
Article in English | MEDLINE | ID: mdl-10022543

ABSTRACT

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.


Subject(s)
Antigens, CD34/immunology , Dendritic Cells/metabolism , Fetal Blood/cytology , Gene Transfer Techniques , Retroviridae/genetics , Base Sequence , Cell Division , Cell Line , DNA Primers , Dendritic Cells/immunology , Hematopoietic Stem Cell Mobilization , Humans , Immunophenotyping , T-Lymphocytes/cytology , Transduction, Genetic
11.
Hum Gene Ther ; 9(2): 225-34, 1998 Jan 20.
Article in English | MEDLINE | ID: mdl-9472782

ABSTRACT

The best methods for transducing hematopoietic progenitor cells usually involve either direct co-cultivation with virus-producing cells or human stromal supportive cells. However, these methods cannot be safely or easily applied to clinical use. Therefore, we aimed at improving retrovirus-mediated gene transfer into hematopoietic progenitors derived from cord blood CD34+ cells using viral supernatant to levels achieved at least with direct co-cultivation and under conditions that are suitable for clinical applications. In a first set of experiments, CD34+ cells were infected with supernatant containing amphotropic retroviral particles carrying the nls-lacZ reporter gene and the effects of centrifugation, cell adhesion to fibronectin, and Polybrene on the transduction of both clonogenic progenitors (CFC) and long-term culture initiating cells (LTC-IC) were studied. Transduction efficiency was evaluated on the percentage and total number of progenitors expressing the beta-galactosidase activity. Results show that a 48-hr infection of CD34+ cells with viral supernatant combining centrifugation at 1000 x g for 3 hr followed by adhesion to fibronectin allows transduction levels for both CFC and LTC-IC to be reached that are as good as using direct co-cultivation. In a second set of experiments, CD34+ cells were infected using this optimized protocol with pseudotyped retroviral particles carrying the gibbon ape leukemia virus (GALV) envelope protein. Under these conditions, between 50 and 100% of CFC and LTC-IC were transduced. Thus, we have developed a protocol capable of highly transducing cord blood progenitors under conditions suitable for a therapeutical use.


Subject(s)
Fetal Blood/virology , Gene Transfer Techniques , Genetic Vectors/therapeutic use , Leukemia Virus, Gibbon Ape/genetics , Stem Cells/virology , Antigens, CD34/analysis , Cell Line , Centrifugation , Coculture Techniques , Culture Media , Fetal Blood/cytology , Fetal Blood/drug effects , Fibronectins/pharmacology , Hexadimethrine Bromide/pharmacology , Humans , Leukemia Virus, Gibbon Ape/growth & development , Stem Cells/drug effects , Stem Cells/metabolism
12.
Blood ; 90(7): 2830-8, 1997 Oct 01.
Article in English | MEDLINE | ID: mdl-9326252

ABSTRACT

Human CD34+ selected cells are able to reconstitute hematopoiesis in patients receiving a myeloablative treatment. Although the role of reinfused tumor cells contaminating the grafts on the determination of postautograft relapses remains unclear, the major interest of CD34+ cell selection is to reduce the tumor contamination of the graft. This can be achieved if tumor cells do not express the CD34 antigen. We previously showed that this approach was effective with bone marrow (BM) collections in patients with non-Hodgkin's lymphoma (NHL). Because peripheral blood progenitor cells (PBPC) allow faster hematologic recovery than BM and are expected to contain less tumor contamination, we have compared the results of CD34+ cell selection in 35 BM and 16 PBPC from 48 patients with NHL. The PBPC were collected after a course of chemotherapy followed by granulocyte colony-stimulating factor (G-CSF) administration. The data showed that the final CD34+ cell purity achieved with PBPC was higher than with BM (medians, 70% v 50%; P = .02). The CD34+ cell recovery was also better for PBPC (medians, 42% v 24%; P = .001). Tumor contamination was assessed by detection of BCL2/JH rearrangement using polymerase chain reaction (PCR) in 38 of 48 patients (22 BM, 16 PBPC). In addition, immunoglobulin heavy chain gene (IgH) rearrangements were investigated using PCR with consensus IgH primers. At harvesting, 10 of 22 BM and two of 16 PBPC contained BCL2/JH+ cells, one of 22 BM and 14 of 16 PBPC contained abnormal IgH+ cells (one PBPC contained both BCL2/JH+ and abnormal IgH+ cells) at harvesting. However, because lymphoma tissue specimens from patients at diagnosis were not available, the malignant character of IgH rearrangements could not be confirmed by sequencing and probing with allele-specific nucleotides. After CD34+ cell selection, a reduction to below the level of detection of BCL2/JH+ cells of BM and PBPC was effective in seven of 12 informative selections. In contrast, a reduction to below the level of detection of abnormal IgH+ cells was effective in only three of 15 informative selections. However, the detection of cells with an abnormal IgH pattern in the context of chemotherapy plus G-CSF progenitor mobilization in patients with NHL and its correlation with actual tumor contamination needs further investigation.


Subject(s)
Antigens, CD34/analysis , Bone Marrow/pathology , Cell Separation/methods , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/cytology , Leukapheresis/methods , Lymphoma, Non-Hodgkin/therapy , Neoplastic Stem Cells/pathology , Adult , Biomarkers, Tumor/analysis , DNA, Neoplasm/genetics , Female , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Genes, Immunoglobulin , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin J-Chains/genetics , Immunophenotyping , Lymphoma, Non-Hodgkin/blood , Lymphoma, Non-Hodgkin/pathology , Male , Middle Aged , Neoplasm, Residual , Neoplastic Cells, Circulating , Oncogene Proteins, Fusion , Polymerase Chain Reaction , Transplantation, Autologous
13.
Stem Cells ; 15(3): 214-22, 1997.
Article in English | MEDLINE | ID: mdl-9170213

ABSTRACT

The cycling status of cord blood progenitors and the culture conditions triggering their activation into S-phase have been studied using flow cytometry and a 3H-thymidine suicide assay. Mononuclear cells cultured either in Iscove's modified Dulbecco's medium (IMDM) +/- 10% fetal calf serum ([FCS]; IMDM + FCS) or in Dulbecco's modified Eagle's medium (DMEM) +/- 10% newborn bovine serum ([NBS]; DMEM + NBS) were stimulated by various growth factors (GFs). Results showed that CD34+ cells, clonogenic progenitors (colony forming cells [CFCs]) and long-term culture initiating cells (LTC-IC) present in freshly harvested cord blood were quiescent. CFC numbers were maintained without cycling after 48-h cultures in serum-containing media without GFs. Addition of interleukin 3 (IL-3) + IL-6 + stem cell factor stimulated into S-phase approximately 40% of CFCs within 24-48 h, without modifying their number except in DMEM + NBS where erythroid progenitors decreased. When cells were stimulated in IMDM + FCS by these three GFs + insulin-like growth factor I and basic fibroblast growth factor used at high concentration, more than 50% of CFCs were in S-phase and their total number was maintained. The latter culture conditions also recruited up to 66% of LTC-IC into S-phase. Our data underline the importance of the combination of GFs and culture media used for optimizing the cycling and maintenance of CFCs and LTC-IC within two days.


Subject(s)
Fetal Blood/cytology , Growth Hormone/pharmacology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Antigens, CD34/analysis , Blood Proteins/pharmacology , Cell Culture Techniques/methods , Cell Cycle/drug effects , Cells, Cultured , Female , Growth Substances/pharmacology , Hematopoietic Stem Cells/chemistry , Humans , S Phase/drug effects
14.
Leuk Lymphoma ; 27(5-6): 487-94, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9477130

ABSTRACT

We have compared the effects of AcSDKP, Thymosin beta4 (Tbeta4), MIP1alpha and TGFbeta on acute myeloid leukemia (AML) and B-lineage acute lymphoid leukemia (B-ALL) cells using liquid cultures in the presence of GM-CSF, IL-3 and SCF for AML cells and IL-3 and IL-7 for ALL cells. Each molecule was added daily and cell proliferation was evaluated on day 3 by thymidine incorporation. Whereas TGFbeta was found inhibitory in all the AML and B-ALL cases studied, MIP1alpha was inhibitory in 6/12 AML cases and had no effect on B-ALL cells. AcSDKP and Tbeta4 showed an inhibitory effect in a few cases but only at high doses which were inactive on normal cells. Thus, our study not only confirms the effect of TGFbeta, MIP1alpha and AcSDKP on AML cells but also provides new data concerning their effect on B-ALL and the possible inhibitory effect of AcSDKP at high doses. Furthermore, we show for the first time the effect of Tbeta4 on leukemic cells. Altogether, our data indicate differences of sensitivity of leukemic cells to negative regulators, some leukemias being inhibited by one or several of these molecules whereas others were unresponsive to all used. The clinical relevance of these observations still remains to be determined.


Subject(s)
Burkitt Lymphoma/pathology , Cell Division/drug effects , Leukemia, Myeloid, Acute/pathology , Macrophage Inflammatory Proteins/pharmacology , Oligopeptides/pharmacology , Thymosin/pharmacology , Transforming Growth Factor beta/pharmacology , Adolescent , Adult , Aged , Chemokine CCL4 , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Tumor Cells, Cultured/drug effects
15.
Blood ; 88(4): 1277-83, 1996 Aug 15.
Article in English | MEDLINE | ID: mdl-8695845

ABSTRACT

We successfully infected fluorescence-activated cell-sorted CD34+ cells from normal cord blood by the human cytomegalovirus (HCMV) laboratory strain Towne. An inhibitory effect of HCMV on clonogenic myeloid progenitors was observed in primary methylcellulose cultures. After an initial 7-day liquid culture of CD34(+)-infected cells, this inhibition was further amplified in secondary methylcellulose cultures, then involving both the myeloid and erythroid lineages. Under these conditions, viral DNA was detected both in erythroid and myeloid colonies using the polymerase chain reaction (PCR), but reverse transcription PCR (RT-PCR) failed to detect viral RNA. In contrast, when CD34(+)-infected cells were maintained in liquid suspension, both immediate, early, and late transcripts were detected as soon as day 3. In addition, viral production was demonstrated in the culture supernatants, thus confirming that a complete viral cycle occurred under liquid conditions. Furthermore, by resorting cells into CD34+ and CD34- fractions, we showed by RT-PCR that viral replication took place in cells still expressing CD34 antigen, whereas no RNA was found in more differentiated cells that had subsequently lost their CD34 antigen. These findings suggest that HCMV replication can occur at the early steps of progenitor differentiation and may be involved in the viral-induced myelosuppression.


Subject(s)
Cytomegalovirus/pathogenicity , Hematopoiesis , Hematopoietic Stem Cells/microbiology , Antigens, CD34/analysis , Antigens, Viral/analysis , Base Sequence , Cells, Cultured , DNA Primers/chemistry , DNA, Viral/analysis , Fetal Blood , Gene Expression Regulation, Viral , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Humans , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Viral/analysis , Virus Replication
16.
Exp Hematol ; 24(7): 776-82, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8647227

ABSTRACT

Thymosin beta4 (Tbeta4), isolated from the calf thymus fraction 5, has a ubiquitous localization and plays a pleiotropic role in both the immune and nonimmune systems. Because it contains at its N-terminal end the sequence of a known inhibitor of hematopoiesis, the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP, Goralatide), we have assayed Tbeta4 on human hematopoietic cells. We demonstrate that it inhibits normal bone marrow progenitor cell growth; indeed, it decreased the growth of both granulo-macrophagic and erythroid progenitors and reduces their percentage in S phase. Furthermore, we show that Tbeta4 reduces both the clonogenicity and the cell proliferation of purified CD34+ cells induced by a combination of seven growth factors. Although Tbeta4's inhibitory effect is very similar to that of AcSDKP, we demonstrate, using neutralizing antibodies and a truncated form of Tbeta4 devoid of the AcSDKP sequence, that the inhibitory effect of Tbeta4 is not mediated by the sequence AcSDKP. These data indicate that Tbeta4 is a novel inhibitor for human normal hematopoietic progenitors.


Subject(s)
Growth Inhibitors/pharmacology , Hematopoietic Stem Cells/drug effects , Thymosin/pharmacology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Cattle , Cells, Cultured , Colony-Forming Units Assay , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/drug effects , Hematopoietic Cell Growth Factors/pharmacology , Humans , Molecular Sequence Data , Oligopeptides/antagonists & inhibitors , Oligopeptides/immunology , Oligopeptides/pharmacology , Recombinant Proteins/pharmacology , Thymosin/antagonists & inhibitors , Thymosin/immunology
17.
Leuk Lymphoma ; 21(3-4): 267-80, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8726408

ABSTRACT

The spontaneous proliferation and the effects of 8 various growth factors (GF) were evaluated on leukemic cells from 27 patients with B-lineage ALL. Two groups of ALLs were distinguished. ALLs from group I (21 patients) exhibited a low spontaneous proliferative rate and were stimulated by IL-3 + IL-7 +/- SCF and/or LIF, while ALLs from group II (6 patients) had a high spontaneous proliferative rate and did no longer require this combination of GFs for proliferation. No effect of bFGF, IGF-I, IL-10 and IL-11 alone or in combination, was observed. Such differences in the behaviour of B-ALLs indicated that the GF requirement of ALL blasts was not related to the presence of serum in the culture nor to the pattern of reactivity of ALL blasts for B lymphoid markers or CD34 antigen. Furthermore, we showed in 1/9 cases that high proliferation might be due to an overexpression of the bcl-2 proto-oncogene and to the acquisition of an autocrine secretion.


Subject(s)
Burkitt Lymphoma/metabolism , Burkitt Lymphoma/pathology , Cytokines/pharmacology , Growth Substances/pharmacology , Proto-Oncogene Proteins/biosynthesis , Adult , Apoptosis/drug effects , Cell Division/drug effects , Cell Division/physiology , Cell Survival/drug effects , Child , Child, Preschool , Female , Humans , Infant , Male , Proto-Oncogene Mas , Proto-Oncogene Proteins c-bcl-2 , RNA, Messenger/metabolism
18.
Blood ; 87(8): 3161-9, 1996 Apr 15.
Article in English | MEDLINE | ID: mdl-8605330

ABSTRACT

The calcium ionophore A23187 promotes histamine synthesis in murine bone marrow cells by increasing the expression of mRNA encoding histidine decarboxylase (HDC), the histamine-forming enzyme. The cells responsible for this biological activity copurify with hematopoietic progenitors in terms of density, light scatter characteristics, and rhodamine retention, similar to interleukin (IL) 3-induced histamine-producing cells. Yet, the effect of calcium ionophore is not mediated by IL-3. The most purified rhodamine-bright bone marrow subset contains 80% cells that respond to calcium ionophore by increased HDC mRNA expression. This high frequency makes the involvement of one particular progenitor subset in histamine synthesis unlikely. The finding that all IL-3-dependent cell lines tested so far exhibit increased histamine production and HDC mRNA expression in response to calcium influx lends further support to this notion. Cell lines requiring other growth factors or proliferating spontaneously lack this ability. Finally, it should be noted that IL-3-dependent cell lines do not produce histamine in response to their growth factor. It might, therefore, be suggested that the pathway transducing the signal for increased histamine synthesis after IL-3 receptor binding in normal hematopoietic progenitors is modified in these cell lines.


Subject(s)
Calcimycin/pharmacology , Calcium/metabolism , Hematopoietic Stem Cells/drug effects , Histamine/biosynthesis , Interleukin-3/pharmacology , Ionophores/pharmacology , Animals , Base Sequence , Biological Transport , Bone Marrow/drug effects , Bone Marrow Cells , Cell Division/drug effects , Cell Line , Cells, Cultured , Enzyme Induction/drug effects , Female , Gene Expression Regulation/drug effects , Histidine Decarboxylase/biosynthesis , Histidine Decarboxylase/genetics , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , RNA, Messenger/biosynthesis , Recombinant Proteins/pharmacology
19.
Blood ; 86(2): 531-9, 1995 Jul 15.
Article in English | MEDLINE | ID: mdl-7605988

ABSTRACT

We examined various murine hematopoietic cell populations for their capacity to interact with radiolabeled histamine. Only bone marrow cells (BMC) retained substantial amounts of radioactivity, in contrast to thymus, spleen, and peritoneal cells. The characteristics of this interaction are consistent with histamine uptake rather than receptor binding. Indeed, this process is temperature and sodium dependent and reduced by various metabolic inhibitors. Furthermore, the effect of antagonists or agonists of the H1, H2, and H3 receptor subtypes is not in accordance with the involvement of either of these receptors in histamine binding. The target cells of histamine copurify with hematopoietic progenitors in the low-density BM population. They are most enriched in the subset sorted from the blast cell window on the basis of high rhodamine retention. This fraction contains on the average 80% to 90% immature cells and is highly enriched for several clonogenic progenitor subsets. Sixty percent of the Rh-bright cells are labeled by 3H-histamine, as assessed by autoradiography, suggesting that a variety of immature cells participates in this phenomenon. Furthermore, in all sorting procedures used here, the cells capable of histamine uptake coenrich with those producing histamine in response to interleukin-3, indicating at least a partial identity between these cells.


Subject(s)
Hematopoietic Stem Cells/metabolism , Histamine/metabolism , Animals , Antimetabolites/pharmacology , Bone Marrow Cells , Cell Separation , Cells, Cultured , Female , Flow Cytometry , Histamine Agonists/pharmacology , Histamine Antagonists/pharmacology , Interleukin-3/pharmacology , Male , Mice , Mice, Inbred C57BL , Organ Specificity , Peritoneal Cavity/cytology , Receptors, Histamine/drug effects , Receptors, Histamine/metabolism , Specific Pathogen-Free Organisms , Spleen/cytology , Thymus Gland/cytology
20.
Exp Hematol ; 23(6): 551-6, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7539383

ABSTRACT

The aim of this study was to compare the inhibitory effect of the tetrapeptide AcSDKP, tumor necrosis factor-alpha (TNF-alpha), which contains the sequence of the peptide, transforming growth factor-beta (TGF-beta), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) on sorted CD34+ cells using both proliferation and clonogenic assays. Although a short treatment with any of the molecules decreased the growth of colony-forming unit granulocyte/macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) progenitors (except for TNF-alpha as it is a greater inhibitor for CFU-GM), further experiments using a 6-day liquid culture in the presence of a combination of growth factors (recombinant human interleukin-3 [rhIL-3], IL-6, IL-1 beta, GM colony-stimulating factor [GM-CSF], G-CSF, erythropoeitin [Epo], and stem cell factor [SCF]) allowed us to determine a number of differences between their effects: 1) TGF-beta and TNF-alpha induced a stronger decrease in the proliferation and clonogenicity of CD34+ subsets than MIP-1 alpha and AcSDKP, 2) the dose-response curves appeared different, and 3) contrary to TGF-beta and TNF-alpha, AcSDKP and MIP-1 alpha required repeated addition to induce inhibition. Therefore, our data clearly show that while the inhibitory effect of TNF-alpha and AcSDKP appeared to be different, there is a close similarity in the effect of AcSDKP and MIP-1 alpha on normal human progenitor response to the combination of growth factors used.


Subject(s)
Bone Marrow/drug effects , Cytokines/pharmacology , Hematopoiesis/drug effects , Hematopoietic Stem Cells/drug effects , Monokines/pharmacology , Oligopeptides/pharmacology , Transforming Growth Factor beta/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Antigens, CD/analysis , Antigens, CD34 , Cell Division/drug effects , Cell Separation , Cells, Cultured , Chemokine CCL4 , Humans , Macrophage Inflammatory Proteins
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