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To address the role of chromatin structure in the establishment of hematopoietic stem cell (HSC) multilineage potential and commitment to the lymphoid lineage, we have analyzed histone modifications at a panel of lymphoid- and myeloid-affiliated genes in multipotent and lineage-committed hematopoietic cells isolated from human cord blood. Our results show that many B- and T-lymphoid genes, although silent in HSCs, are associated with acetylated histones H3 and H4. We also detected histone H3 lysine 4 methylation but not repressive lysine 9 or 27 methylation marks at these loci, indicative of an open chromatin structure. Interestingly, the relative level of H3 lysine 4 dimethylation to trimethylation at B-specific loci was high in multipotent CD34(+)CD38(lo) progenitors and decreased as they become actively transcribed in B-lineage cells. In vitro differentiation of CD34(+) cells toward the erythroid, granulocyte, and T-cell lineages resulted in a loss of histone acetylation at nonlineage-associated genes. This study provides evidence that histone modifications involved in chromatin decondensation are already in place at lymphoid-specific genes in primary human HSCs, supporting the idea that these genes are "primed" for expression before lineage commitment. This permissive chromatin structure is progressively lost as the stem cell differentiates.

The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34(+) hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.

In humans, the CD4 molecule is expressed on a subset of T-cells and at various levels on myeloid and lymphoid cells. The mechanisms regulating human CD4 gene expression are yet poorly understood. We speculated that the CD4 silencer, which operates in CD8+ T-cells to repress CD4 expression, could be responsible for CD4 repression in human lymphoid non-T-cells. To test this possibility, we used lentiviral vectors carrying CD4 regulatory sequences, with or without the silencer element, to express an eGFP reporter gene. We observed that (i) in the absence of the silencer element, eGFP expression was detected in CD34+-derived B- and NK-cells that otherwise lacked endogenous CD4 mRNA, indicating active repression of the CD4 regulatory sequences and (ii) the addition of the CD4 silencer could repress eGFP expression in these same cells, as well as in human B-cells generated in vivo in NOD/SCID mice. Collectively, our results suggest that beyond its well-characterized function in T-cells, the CD4 silencer also regulates CD4 gene expression in human lymphoid non-T-cells.

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells, we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells, whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover, long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage, strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether, these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors, and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis.

Here, we identify fetal bone marrow (BM)-derived CD34hiCD45RAhiCD7+ hematopoietic progenitors as thymus-colonizing cells. This population, virtually absent from the fetal liver (FL), emerges in the BM by development weeks 8-9, where it accumulates throughout the second trimester, to finally decline around birth. Based on phenotypic, molecular, and functional criteria, we demonstrate that CD34hiCD45RAhiCD7+ cells represent the direct precursors of the most immature CD34hiCD1a- fetal thymocytes that follow a similar dynamics pattern during fetal and early postnatal development. Histological analysis of fetal thymuses further reveals that early immigrants predominantly localize in the perivascular areas of the cortex, where they form a lymphostromal complex with thymic epithelial cells (TECs) driving their rapid specification toward the T lineage. Finally, using an ex vivo xenogeneic thymus-colonization assay, we show that BM-derived CD34hiCD45RAhiCD7+ progenitors are selectively recruited into the thymus parenchyma in the absence of exogenous cytokines, where they adopt a definitive T cell fate.

The fate of hematopoietic stem cells (HSCs) is regulated through a combinatorial action of proteins that determine their self-renewal and/or their commitment to differentiation. Stem cell leukemia/T-cell acute lymphoblastic leukemia 1 (SCL/TAL1), a basic helix-loop-helix (bHLH) transcription factor, plays key roles in controlling the development of primitive and definitive hematopoiesis during mouse development but its function in adult HSCs is still a matter of debate. We report here that the lentiviral-mediated enforced expression of TAL1 in human CD34+ cells marginally affects in vitro the differentiation of committed progenitors, whereas in vivo the repopulation capacity of the long-term SCID (severe combined immunodeficient) mouse-repopulating cells (LT-SRCs) is enhanced. As a consequence, the production of SRC-derived multipotent progenitors as well as erythroid- and myeloid-differentiated cells is increased. Looking at the lymphoid compartment, constitutive TAL1-enforced expression impairs B- but not T-cell differentiation. Expression of a mutant TAL1 protein that cannot bind DNA specifically impairs human LT-SRC amplification, indicating a DNA-binding dependent effect of TAL1 on primitive cell populations. These results indicate that TAL1 expression level regulates immature human hematopoietic cell self-renewal and that this regulation requires TAL1 DNA-binding activity.

The early stages of human lymphopoiesis are poorly characterized. Here, we compared the lymphoid potential of a novel umbilical cord blood CD34(+)CD45RA(hi)CD7(+) hematopoietic progenitor cell (HPC) population with that of CD34(+)CD45RA(hi)Lin(-)CD10(+) HPCs, previously proposed as candidate common lymphoid progenitors. Limiting-dilution and clonal analysis, fetal thymic organ cultures, and culture onto Notch ligand Delta-like-1-expressing OP9 cells, showed that although CD34(+)CD45RA(hi)CD7(+) HPCs could generate cells of the 3 lymphoid lineages, their potential was skewed toward the T/natural killer (T/NK) lineages. In contrast, CD34(+)CD45RA(hi)Lin(-)CD10(+) HPCs predominantly exhibited a B-cell potential. Gene expression profiling with DNA microarrays confirmed that CD34(+)CD45RA(hi)CD7(+) HPCs selectively expressed T-lymphoid and NK lineage-committed genes while retaining expression of genes affiliated to the granulomonocytic lineage, whereas CD34(+)CD45RA(hi)Lin(-)CD10(+) HPCs displayed a typical pro-B-cell transcription profile and essentially lacked genes unrelated to the B lineage. In addition, both populations could be generated in vitro from CD34(+)CD45RA(int)CD7(-) and CD34(+)CD45RA(hi)Lin(-) HPCs with mixed lymphomyeloid potential, from which they emerged independently with different growth/differentiation factor requirements. These findings indicate that CD34(+)CD45RA(hi)CD7(+) and CD34(+)CD45RA(hi)Lin(-)CD10(+) HPCs correspond to multipotent early lymphoid progenitors polarized toward either the T/NK or B lineage, respectively.

The transcription factor TAL1 has major functions during embryonic hematopoiesis and in adult erythropoiesis and megakaryocytopoiesis. These functions rely on different TAL1 structural domains that are responsible for dimerization, transactivation, and DNA binding. Previous work, most often done in mice, has shown that some TAL1 functions do not require DNA binding. To study the role of TAL1 and the relevance of the TAL1 DNA-binding domain in human erythropoiesis, we developed an approach that allows an efficient enforced wild-type or mutant TAL1 protein expression in human hematopoietic CD34(+) cells using a lentiviral vector. Differentiation capacities of the transduced cells were studied in a culture system that distinguishes early and late erythroid development. Results indicate that enforced TAL1 expression enhances long-term culture initiating cell (LTC-IC) potential and erythroid differentiation of human CD34(+) cells as shown by increased beta globin and porphobilinogen deaminase (PBGD) gene expressions and erythroid colony-forming units (CFU-Es), erythroid burst-forming units (BFU-Es), and glycophorin A-positive (GPA(+)) cell productions. Enforced expression of a TAL1 protein deleted of its DNA-binding domain (named Delta bTAL1) mimicked most TAL1 effects except for the LTC-IC enhancement, the down-regulation of the CD34 surface marker, and the GPA(+) cell production. These results provide the first functional indications of DNA-binding-dependent and -independent roles of TAL1 in human erythropoiesis.

In rodents, bone marrow-derived cells enter the brain during adult life. Allogeneic bone marrow transplantation is used to treat genetic CNS diseases, but the fate of human bone marrow and CD34(+) cells within the brain remains to be elucidated. The present study demonstrates that cells derived from human CD34(+) cells, isolated from either cord blood or peripheral blood, migrate into the brain after infusion into nonobese diabetic/severe combined immunodeficient mice. Both types of CD34(+)-derived cells differentiate into perivascular and ramified microglia. The lentiviral transfer of genes into CD34(+) cells before infusion does not modify the differentiation of human CD34(+) cells into microglia, allowing new transgenic proteins to be expressed in these cells. The transplantation of CD34(+) cells could thus be used for the treatment of CNS diseases.

Expansion of human hematopoietic stem cells (HSCs) is a major challenge in cellular therapy, and currently relies on the use of recombinant cytokines or on gene transfer of transcription factors. Of these, the HOXB4 homeoprotein protein is of particular interests as it promotes the expansion of mouse HSCs without inducing the development of leukemia. To eliminate any deleterious effects that might be associated with stable HOXB4 gene transfer into human cells, we took advantage of the ability of HOX proteins to passively translocate through cell membranes. Here we show that when cultured on stromal cells genetically engineered to secrete HOXB4, human long-term culture-initiating cells (LTC-ICs) and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse repopulating cells (SRCs) were expanded by more than 20- and 2.5-fold, respectively, over their input numbers. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. This method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

The association of hepatitis C and certain hemotological diseases (non-Hodgkin B-cell lymphoma, villous splenic lymphoma.) is a debated question which remains open due to discordant epidemiological data. We report the case of a new patient with chronic hepatitis C and tricholeukocyte leukemia.

X-linked adrenoleukodystrophy (ALD), an inherited demyelinating disorder of the central nervous system, can be corrected by allogeneic bone marrow transplantation, likely due to the turnover of brain macrophages that are bone marrow derived. ALD is characterized by an accumulation of very long chain fatty acids (VLCFA) due to the deficiency of an ATP binding cassette transporter that imports these fatty acids in peroxisomes. Murine retroviral transduction results in metabolic correction of ALD CD34(+) cells in vitro but reinfusion of these cells into ALD patients would not provide clinical benefit owing to the absence of selective advantage conferred by transgene expression. High-efficiency transduction of ALD CD34(+) peripheral blood mobilized cells was achieved using an HIV-based vector driving ALD gene expression under the elongation factor 1 alpha promoter and a protocol without prestimulation of CD34(+) cells with cytokines prior to transduction to preserve their stem cell properties. Efficient expression of the ALD gene was demonstrated in monocytes/macrophages derived from cultures of transduced ALD CD34(+) cells and in long-term culture initiating cells. VLCFA metabolism was corrected in transduced CD34(+), CFU-derived, and LTC-derived cells, indicating that the vector-encoded ALD protein was fully functional. Transplantation of transduced ALD CD34(+) cells into NOD/SCID mice resulted in long-term expression of ALD protein in monocytes/macrophages derived from engrafted stem cells.

Background/aims: Adult Still's disease is one of the febrile disorders of unknown etiology, characterized by high fever, transient cutaneous rash and leukocytosis. Liver dysfunction in adult Still's disease has been described in some case reports. The objective of this study was to analyze the pattern and the frequency of liver abnormalities in a monocenter series of adult Still's disease patients., Methodology: Data of 17 patients with adult Still's disease (fulfilling Yamaguchi's diagnostic criteria) were retrospectively reviewed. These patients were followed in an Internal Medicine Department over a period of 7 years., Results: The median age was 27 years with a sex ratio M/F of 1.4. Fever was present in 100% of the cases and hepatomegaly occurred in 47% of the cases. Abnormalities in liver biochemistry, apparent in 76% of the subjects were characterized from moderate (elevation of transaminases between 2 and 5 N) (65%) to severe cytolysis (level of transaminases > 5 N) (12%), cholestasis (elevation of gamma GT and/or PAL) (65%), and increase in the level of LDH (35%). All of these symptoms disappeared either spontaneously or under treatment (83%), within a median period of 18 days., Conclusions: This study confirms the high frequency of liver dysfunction in adult Still's disease patients. Although it is moderate and asymptomatic in most cases, severe cytolytic hepatitis has been described. This study especially puts forward the need for exploring the possibility of adult Still's disease in the presence of a fever and hepatic cytolysis.

Gene therapy using permanent modifications of hematopoietic stem cells (HSC) has increasing potential applications for both genetic and acquired diseases. Considerable progress has been made recently in gene transfer to HSC by the use of lentivirus-derived vectors, which have the capacity to transduce noncycling cells. However, overall efficiency of HSC transduction reported so far is still not sufficient for numerous applications. We describe here an improved HSC transduction protocol, using the previously described lentiviral vector, that leads to more than 90% transduction of human CD34+ cells from cord blood, including NOD-LtSz-scid/scid repopulating cells. Moreover, under the same conditions, we transduce more than 75% and 80% of CD34+ cells mobilized in peripheral blood and from bone marrow, respectively. We further show that transgene expression is stable through time and hematopoietic cell differentiation in vitro as well as in vivo. Such a high HSC transduction efficiency opens new opportunities for both gene therapy applications and functional studies of regulator proteins of hematopoiesis.

The ability of ex vivo expansion to increase the long-term repopulating capacity of a graft is still unknown. One problem is the most reliable way to quantify transplantable cells. We addressed this point in a baboon model based on autologous transplantation of serial limiting doses of non-manipulated or ex vivo-expanded mobilized CD34+ cells and determined the threshold doses of non-manipulated and expanded cells which supported long-term multilineage engraftment. In the expansion group, CD34+ cells were cultured for 6 d with a combination of early acting cytokines (Flt3-ligand, stem cell factor, thrombopoietin and interleukin 3). Grafted cells were characterized by their surface antigens and biological properties [semisolid assays, long-term culture-initiating cells (LTC-IC) and non-obese diabetic severe combined immunodeficient reconstituting cells (SRC)]. Animals were followed for at least 12 months post transplantation. The expansion protocol yielded 12.3-fold, 16.9-fold, 3.7-fold, 3.5-fold and 2.2-fold increases in CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte CFU (CFU-MK), LTC-IC and SRC respectively. It induced a modest increase in the long term reconstitutive ability of the graft; the threshold value for long-term engraftment was 0.5 x 10(6)/kg CD34+ cells in the control group and 0.3 x 10(6)/kg CD34+ cells in the expansion group, although one animal in this latter group remained hypoplastic. Frequencies of SRC had a high predictive value of long-term engraftment (r > 0.80). The main advantage of the protocol was the acceleration of granulocyte recovery, achieved at the different doses tested. In conclusion, these experiments suggest that this ex vivo expansion protocol marginally amplifies long-term reconstituting cells.

Hematopoietic stem cells (HSC) are subject to great interest because of their medical importance and their biological properties. Therefore, the possibility of genetically modifying human HSC is a major concern in several inherited pathologies. In this study, we aimed to demonstrate that a murine oncoretroviral vector can transduce multipotential cord blood (CB) stem cells. Sorted CB CD34(+)CD38(low) cells were transduced with a Moloney-based MFG retroviral vector containing the coding sequence of the murine CD2 (mCD2). CD34(+)mCD2(+) cells were sorted by flow cytometry and cultured either in bulk or at one cell per well in culture conditions that allow differentiation along lymphoid (T, B, and NK) and myeloid (M) lineages. Phenotypic analysis of cells generated in culture showed that CD34(+)mCD2(+) cells could give rise to all lymphoid and myeloid progeny, indicating that the MFG/mCD2 vector had transduced progenitors of all tested lineages. Moreover, clonal cultures of 660 CD34(+)mCD2(+) cells showed that approximately 5% of these cells were able to generate both myeloid and lymphoid (B + NK) progenies; for 25% of them, this included the production of lymphoid T cells. We also demonstrate that transduced CD34(+)CD38(low) CB cells with lymphoid and myeloid potentials were capable of engraftment into the bone marrow (BM) of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice during several months. These results show that MFG retroviral vectors can transduce multipotent (T, B, NK, M) human hematopoietic progenitors with in vivo repopulating activity.

Our aim was to compare the sensitivity of various diagnostic criteria for adult Still's disease in 17 patients with established adult Still's disease who were followed in a department of internal medicine over a mean period of 7 years. The median age of the 17 patients was 27 years and the sex ratio M:F was 1:4. The patients had essentially systemic manifestations with fever (n=14), inflammation and leukocytosis (n=16), and moderate liver dysfunction (n=13) that disappeared after a mean of 21 days (range 8-48 days). In the first week of hospitalization, the sensitivities of the criteria proposed by Yamaguchi, Reginato, and Kahn were 94, 18, and 23%, respectively. One month later, the sensitivity was 100% for Yamaguchi's diagnostic criteria versus only 88% for Reginato's and Kahn's criteria. This study confirms that Yamaguchi's diagnostic criteria are more sensitive and are met earlier than Reginato's and Kahn's criteria in patients followed in internal medicine.

The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34(+) cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3' LTR in the TRIP-CMV vector resulted in a safer vector (TRIPDeltaU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1alpha (EF1alpha). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34(+)-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPDeltaU3-EF1alpha, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34(+)-derived T, B, NK, and myeloid hematopoietic cells.

CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. This study compared the expansion capacities of CD133(+) and CD34(+) cells isolated from the same cord blood (CB) samples. After 14 days culture in stroma-free, serum-free medium in the presence of stem cell factor (SCF), Flt3-1, megakaryocyte growth and development factor (MGDF), and granulocyte colony-stimulating factor (G-CSF), the CD133(+) and CD34(+) fractions displayed comparable expansion of the myeloid compartment (CFC, LTC-IC, and E-LTC-IC). The expansion of CD133(+) CB cells was up to 1262-fold for total cells, 99-fold for CD34(+) cells, 109-fold for CD34(+) CD133(+) cells, 133-fold for CFU-GM, 14.5-fold for LTC-IC, and 7.5-fold for E-LTC-IC. Moreover, the expanded population was able to generate lymphoid B (CD19(+)), NK (CD56(+)), and T (CD4(+) CD8(+)) cells in liquid or fetal thymic organ cultures, while expression of the homing antigen CXCR4 was similar on expanded and nonexpanded CD133(+) or CD34(+) cells. Thus, the CD133(+) subset could be expanded in the same manner as the CD34(+) subset and conserved its multilineage capacity, which would support the relevance of CD133 for clinical hematopoietic selection.

The glycoprotein (Gp) IIb/IIIa integrin, also called CD41, is the platelet receptor for fibrinogen and several other extracellular matrix molecules. Recent evidence suggests that its expression is much wider in the hematopoietic system than was previously thought. To investigate the precise expression of the CD41 antigen during megakaryocyte (MK) differentiation, CD34(+) cells from cord blood and mobilized blood cells from adults were grown for 6 days in the presence of stem cell factor and thrombopoietin. Two different pathways of differentiation were observed: one in the adult and one in the neonate cells. In the neonate samples, early MK differentiation proceeded from CD34(+)CD41(-) through a CD34(-)CD41(+)CD42(-) stage of differentiation to more mature cells. In contrast, in the adult samples, CD41 and CD42 were co-expressed on a CD34(+) cell. The rare CD34(+)CD41(+)CD42(-) cell subset in neonates was not committed to MK differentiation but contained cells with all myeloid and lymphoid potentialities along with long-term culture initiating cells (LTC-ICs) and nonobese diabetic/severe combined immune-deficient repopulating cells. In the adult samples, the CD34(+)CD41(+)CD42(-) subset was enriched in MK progenitors, but also contained erythroid progenitors, rare myeloid progenitors, and some LTC-ICs. All together, these results demonstrate that the CD41 antigen is expressed at a low level on primitive hematopoietic cells with a myeloid and lymphoid potential and that its expression is ontogenically regulated, leading to marked differences in the surface antigenic properties of differentiating megakaryocytic cells from neonates and adults. (Blood. 2001;97:2023-2030)

Objective: Certain liver test abnormalities have been described in adult Still's disease. The objective of the present study was to analyze their type and frequency., Patients: In a 10 year retrospective study, patients were included if they fulfilled Kahn's and/or Yamaguchi's diagnostic criteria (median follow-up: 6.5 years)., Results: Twelve patients were selected. The median age was 25 years old and the sex ratio H/F was 2.7. Fever was present in 100% of patients and hepatomegaly in 41%. Liver test abnormalities were identified in 92% of patients: moderate cytolysis (level of transaminases between 2 and 5 N) (83%), severe cytolysis (level of transaminases > 5 N) (17%), cholestasis (elevated levels of GGT and/or alkaline phosphatase) (75%), and an increase in the LDH level (41%). All these liver abnormalities resolved spontaneously or during treatment (83%), within a median of 18 days., Conclusion: Our study confirms the high frequency of liver test abnormalities (> 2/3 of the patients) in adult Still's disease. These abnormalities are generally moderate and asymptomatic (3/4 of the cases), but severe cytolysis may exist. This emphasizes the need to consider a diagnosis of adult Still's disease in the presence of fever and elevated transaminase activity.

Retroviral vectors have become the primary tool for gene delivery into hematopoietic cells, including T lymphocytes. Lentiviral vectors offer an advantage over Moloney murine leukemia virus (MuLV) vectors because of their ability to translocate across an intact nuclear membrane and integrate into the genome of nonproliferating cells. We have recently demonstrated that a central strand displacement event, controlled by the central polypurine tract (cPPT) and the central termination sequence (CTS), results in the formation of a central DNA flap which acts as a cis-determinant of HIV-1 genome nuclear import. Here, we show that insertion of this DNA determinant in a classical lentiviral vector resulted in a significantly higher level of transduction in activated T cells (51 +/- 12.7% versus 15 +/- 1.4%). CD4(+) and CD8(+) T cells were transduced at equivalent levels. Importantly, freshly isolated T cells stimulated only during the 12-h transduction period could be efficiently transduced with this new flap-containing lentiviral vector, but not with the parental lentiviral vector nor an MuLV vector. Transgene expression in the flap-containing lentiviral vector, under the control of either an internal cytomegalovirus or the elongation factor-1 alpha (EF1 alpha) promoter, was significant and expression remained elevated in resting T cells. Thus, this system allows stable expression of transgenes in T lymphocytes following a short ex vivo transduction protocol.

Introduction: The ex vivo expansion of hematopoietic grafts could be an important therapeutic tool for accelerating hematopoietic recovery after administration of high-dose chemotherapy regimens. The fate of the long-term repopulating cells during the ex vivo manipulation of grafts is a critical issue and will ultimately define the clinical applicability of this technology to hematopoietic transplantation., Materials and Methods: To study the effects of a clinically applicable ex vivo expansion protocol in the proliferative potential of the most primitive human hematopoietic cells, both LTC-IC and NOD/SCID-RC assays were used to determine LTC-IC and NOD/SCID-RC contents of hematopoietic grafts, both before and after expansion (SCF, IL-3, PEG-MGDF Flt3-L and 5% AB serum), in four children with non-hematological malignancies., Results: The mean percentage of CD34+ cells after expansion was 16%. The numbers of nucleated cells increased 20-fold with a mean three-fold increase in the numbers of CD34+ cells during the expansion period. The CFC content of the samples showed a mean 11-fold increase (range: 5-17) after ex vivo expansion. The primitive hematopoietic stem cell content of the expanded cell fraction evaluated by LTC-IC assays was found to be increased in two patients out of three, with maintenance of the LTC-IC frequency in the third patient. The NOD/SCID-RC potential, evaluated in five experiments from four patients using 109 mice injected 5-6 weeks earlier with human hematopoietic cells, increased from a mean percentage of 36% (range: 7-75%) before expansion, to a mean percentage of 70% (range: 37-100%) after expansion (P < 0.00001). The frequency of NOD/SCID-RC calculated with pooled data from all patients was 1/80,000 at day 0 and 1/40,000 after seven days of culture. The full phenotypic analysis of human hematopoietic cells obtained in NOD/SCID mice injected with expanded cells showed the presence of significant numbers of CD34+, CD19+ and CD15+ cells, suggesting the persistent lympho-myeloid potential of the expanded hematopoietic cells., Conclusion: Our results suggest that efficient expansion of NOD/SCID-RC with lympho-myeloid potential can be achieved not only in cord blood or normal marrow as previously reported, but also in hematopoietic grafts obtained from children exposed to high-dose chemotherapy.

Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34(+) hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity. Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture. These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.

Background: Rare cases of venous thrombosis associated with celiac disease have been reported., Case Report: We report a case of 40-year-old woman with splenic infarction and splenic venous thrombosis associated with celiac disease. This patient was homozygous for the C677T mutation of the methyltetrahydrofolate reductase (MTHFR) gene and had moderately elevated homocysteinemia., Discussion: We discuss the link between celiac disease and thrombosis as well as the interest and appropriate duration of anticoagulation and hypothesize a mechanism of thrombotic disease in this setting with hyperhomocyseinemia.

Transplantation of genetically marked donor cells in mice have unambiguously identified individual clones with full differentiative potential in all lymphoid and myeloid pathways. Such evidence has been lacking in humans because of limitations inherent to clonal stem cell assays. In this work, we used single cell cultures to show that human cord blood (CB) contains totipotent CD34(+) cells capable of T, B, natural killer, and granulocytic cell differentiation. Single CD34(+) CD19(-)Thy1(+) (or CD38(-)) cells from fresh CB were first induced to proliferate and their progeny separately studied in mouse fetal thymic organotypic cultures (FTOCs) and cocultures on murine stromal feeder layers. 10% of the clones individually analyzed produced CD19(+), CD56(+), and CD15(+) cells in stromal cocultures and CD4(+)CD8(+) T cells in FTOCs, identifying totipotent progenitor cells. Furthermore, we showed that totipotent clones with similar lymphomyeloid potential are detected in the bone marrow of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted 4 mo earlier with human CB CD34(+) cells. These results provide the first direct demonstration that human CB contains totipotent lymphomyeloid progenitors and transplantable CD34(+) cells with the ability to reconstitute, in the marrow of recipient mice, the hierarchy of hematopoietic compartments, including a compartment of functional totipotent cells. These experimental approaches can now be exploited to analyze mechanisms controlling the decisions of such primitive human progenitors and to design conditions for their ampification that can be helpful for therapeutic purposes.

Factors that may improve retroviral transduction of primitive human hematopoietic cells were studied using MFG-based vectors containing a LacZ gene and produced either by a murine (psi-Crip) or a human (Tasaf) cell line. Cord blood (CB) or bone marrow (BM) CD34+ cells were stimulated and transduced in the presence of three cytokines (interleukin 3 [IL-3], IL-6, and stem cell factor [SCF; c-Kit Ligand]). In the supernatant infection protocol, hematopoietic progenitor cells as measured by X-Gal staining of colony-forming unit cells (CFU-Cs) were transduced more effectively with Tasaf (20%) than with psi-Crip (8%). In contrast, there was no difference between these two cell lines in a coculture protocol. However, gene transfer into more primitive CD34+CD38- subsets and in LTC-IC-derived colonies was low. The use of a large number of cytokines including FLT3-L and PEG-rhMGDF increased the transduction efficiency into CD34+CD38(-)-derived CFU-Cs (35% by PCR) or LTC-ICs (10%). A virus pseudotyped with gibbon ape leukemia virus (GALV) envelope further improved gene transfer to 60 and 48% for LacZ+ CFU-C- and LTC-IC-derived colonies, respectively. These conditions of transduction allowed multilineage engraftment of primitive cord blood cells in NOD-SCID mice. Moreover, 10% (at least) of the human hematopoietic cells recovered from the marrow of these immunodeficient animals were transduced. These data suggest that the efficiency of transduction of human hematopoietic primitive cells can be significantly improved by judicious combinations of recombinant cytokines and high retroviral titers.

Adenoviral vectors may be useful tools to deliver a cytokine in vivo. A single intravenous injection of an adenovirus vector containing the human thrombopoietin (TPO) cDNA (AdRSVhuTPO) was able to induce a thrombocytosis for more than 6 weeks in SCID mice, associated with a megakaryocyte (MK) hyperplasia in different organs. A marrow and spleen fibrosis was observed at 6 weeks. In immunocompetent mice, a single AdRSVhuTPO injection led to a moderate and transient thrombocytosis without myelofibrosis. To evaluate the usefulness of TPO for the prevention of secondary side-effects during an aplastic period, mice were subjected to a myeloablative regimen 7 days after the intravenous AdRSVhuTPO injection. In this setting, TPO prevented mortality by accelerating hematological recovery. Survival was essentially related to an improvement in the leukopenia since all control mice died from septicemia. However, the effects of TPO may be potentiated by the release of inflammatory cytokines following the adenovirus infection; AdRSV beta galactosidase injected-mice had higher numbers of BFU-E and CFU-GM in the marrow than PBS-injected mice. Myelosuppression induced transient immunosuppression responsible for a sustained expression and elevation of platelet numbers for at least 5 months. These results further suggest that TPO may be an effective therapy in diminishing hematological complications related to myeloablative regimens, but emphasize that immunosuppression secondary to myelosuppression may lead to sustained expression associated with a risk of thrombosis and myelofibrosis when delivered by adenovirus vectors.

Identification of human hematopoietic stem cells and analysis of molecular mechanisms regulating their function require biological assays that permit differentiation in all hematopoietic lineages simultaneously. In this study, we established conditions that permit the joint expression of the B-lymphoid and myeloid potential from cord blood-derived CD34+CD38lowCD19-/CD10- primitive progenitors that lack B-specific markers and transcripts. When cocultured during 6 weeks with the murine stromal cells MS-5 in the absence of exogenous human cytokines, CD34+CD38low-CD19-CD10- cells generated a high number of CD19+ B cells. Virtually all of these cells expressed a CD34-CD10+- CD19+cIgM- phenotype of late pro-B cells and transcripts of Pax-5, lambda-like, and mu chain were detected. We further show that 7% of CD34+CD38lowCD19- cells from cord blood, when grown individually with MS-5 cells, generated both CD19+ and CD11b+ cells after 6 weeks. Efficient B-cell differentiation was also observed in vivo after transplantation of human cord blood-derived unfractionated mononuclear cells or CD34+CD19+CD10- cells into immune-deficient mice. In contrast to the in vitro situation, all stages of B-cell differentiation were observed in vivo, including pro-B, pre-B, and sIgM+ B cells. Interestingly, human progenitors with the ability to differentiate along both B-lymphoid and granulocytic pathways were also detected among human CD34+CD38low cells in the marrow of chimeric mice 6 to 7 weeks after transplantation. Both in vitro and in vivo systems will offer an invaluable tool to further identify the lymphoid and myeloid potentialities of primitive progenitor cells isolated from fetal as well as adult human hematopoietic tissues and characterize stromal-derived signals that regulate their function.

In an attempt to understand better the regulation of stem cell function in chimeric immunodeficient mice transplanted with human cells, and the filiation between progenitor cells identified in vitro and in vivo, we assessed the different compartments of hematopoietic progenitors found in the marrow of CB17-severe combined immunodeficiency (SCID) mice (34 mice, 9 experiments) after intravenous injection of 2 to 3 x 10(7) cord blood mononuclear cells. On average 6.3 +/- 4 x 10(5) human cells were detected per four long bones 4 to 6 weeks after the transplant predominantly represented by granulomonocytic (CD11b+) and B lymphoid (CD19+) cells. Twenty five percent of these human cells expressed the CD34 antigen, of which 90% coexpressed the CD38 antigen and 50% the CD19 antigen. Functional assessment of progenitor cells (both clonogenic and long-term culture-initiating cells [LTC-IC]) was performed after human CD34+ cells and CD34+/CD38- cells have been sorted from chimeric CB17-SCID marrow 3 to 10 weeks after intravenous (IV) injection of human cells. The frequency of both colony-forming cells and LTC-IC was low (4% and 0.4%, respectively in the CD34+ fraction) when compared with the frequencies of cells with similar function in CD34+ cells from the starting cord blood mononuclear cells (26% +/- 7% and 7.2% +/- 5%, respectively). More surprisingly, the frequency of LTC-IC was also low in the human CD34+ CD38- fraction sorted from chimeric mice. This observation might be partly accounted for by the expansion of the CD34+ CD19+ B-cell precursor compartment. Despite their decreased frequency and absolute numbers, the differentiation capability of these LTC-IC, assessed by their clonogenic progeny output after 5 weeks in coculture with murine stromal cells was intact when compared with that of input LTC-IC. Furthermore the ratio between clonogenic progenitor cells and LTC-IC was similar in severe combined immunodeficiency (SCID) mice studied 4 weeks after transplant and in adult marrow or cord blood suspensions. Results generated in experiments where nonobese diabetic (NOD)-SCID mice were used as recipients indicate a higher level of engraftment but no change in the distribution of clonogenic cells or LTC-IC. These results suggest that the hierarchy of hematopoietic differentiation classically defined in human hematopoietic tissues can be reconstituted in immunodeficient SCID or NOD-SCID mice.

A non Hodgkin's lymphoma strictly located in the bone marrow, was discovered in a patient presenting with asymetric muscle weakness of upper and lower limbs. Both the lymphoma and the neurological syndrome were successfully treated with chemotherapy.

Background/aims: Resection and liver transplantation are currently considered as the most useful treatments for hepatocellular carcinoma. However, transcatheter oily chemoembolization may be favourably compared with these two surgical treatments in patients with anatomically operable tumors., Methods: Between 1985 and 1991, 122 patients with an Okuda stage I tumor were hospitalized in two French hospitals. Among these patients, 33 remained untreated, 42 were treated by transcatheter oily chemoembolization, 30 by resection and 17 by liver transplantation. The four groups were closely comparable except for age, the patients in the two surgical groups being significantly younger. Moreover, the frequency of pTNM II tumor was significantly higher in the resection group., Results: The 5-year probability of survival was close to 45% in each of the three treated groups and was significantly higher than in the untreated group (0% at 4 years, p < 0.0001). The probability of cancer recurrence and/or metastatic dissemination was lower after transcatheter oily chemoembolization than after surgery., Conclusion: Thus, transcatheter oily chemoembolization seems comparable at 5 years with resection or transplantation for the treatment of resectable hepatocellular carcinoma.

Synthetic antimalarial agents can cause exacerbation of latent or patent psoriatic skin lesions. A case of psoriatic onychoperiostitis precipitated by hydroxychloroquine therapy is reported. The patient had primary Sjögren's syndrome, raising questions about the incidence and causation of the ungual abnormalities associated with this condition.

Assembly of antigen receptor V (variable), D (diversity), and J (joining) gene segments requires lymphocyte-specific genes and ubiquitous DNA repair activities. Severe combined immunodeficient (SCID) mice are defective in general double-strand (ds) DNA break repair and V(D)J coding joint formation, resulting in arrested lymphocyte development. A single treatment of newborn SCID mice with DNA-damaging agents restored functional, diverse, T cell receptor beta chain coding joints, as well as development and expansion of thymocytes expressing both CD4 and CD8 coreceptors, but did not promote B cell development. Thymic lymphoma developed in all mice treated with DNA-damaging agents, suggesting an interrelation between V(D)J recombination, dsDNA break repair, and lymphomagenesis.

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo-erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood-derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.

Cord blood is increasingly used as an alternative stem cell source for autologous and allogeneic transplantation, particularly in pediatric patients. We therefore adopted our protocol for transplanting human adult bone marrow cells into severe combined immunodeficient (SCID) mice [1] to develop an in vivo model for cord blood hematopoiesis. Intravenous injection of unfractionated or Ficoll-separated cord blood cells into sublethally irradiated SCID mice led to high levels of human hematopoiesis in the majority of the recipients [2]. Multilineage human hematopoiesis including committed and multipotential myeloerythroid progenitors as well as CD19+ B-lymphoid cells were observed in the murine bone marrow for at least 18 weeks. Together, these data indicate that the SCID mice were engrafted with an immature cell that was able to maintain multiple progenitor lineages in vivo. In contrast to our experiences with adult bone marrow, high levels of human cell engraftment in the mouse could be achieved without exogenous cytokine treatment, suggesting that the cord blood cells respond differently to the murine microenvironment. Alternatively, the cord blood cells might have been able to provide themselves with the necessary growth factors in a paracrine fashion. This model will be useful in gaining new insights into the biology of immature human cord blood progenitors and cord blood transplantation.

Although there has been considerable progress in transplanting normal human hematopoietic cells into immune-deficient mice, the establishment of a functional human immune system has proved to be difficult. Current methods of introducing mature human lymphoid cells into adult SCID mice lead to oligoclonal engraftment with restricted dissemination to various organs. We have attempted to improve human lymphoid cell engraftment in mice, both qualitatively and quantitatively, by injecting human bone marrow cells and peripheral blood leukocytes intraperitoneally into newborn SCID mice. Newborn mice were used as recipients because certain immune functions such as natural killer cell activity do not develop until several weeks after birth and the numerous growth factors secreted in young mice may facilitate the engraftment and proliferation of transplanted human cells. At various times after transplantation, the presence of human cells in different organs was determined by Southern blot analysis using a human specific probe. Within 4 weeks, 70% of the mice were engrafted with human cells. Human cell engraftment of the bone marrow, spleen, lungs, kidneys, liver, and thymus exceeded 10% in at least 40% of the transplanted mice; most of these highly engrafted mice were sick. Flow cytometry and immunocytochemistry indicated these organs were heavily infiltrated with mature T and B lymphocytes. Histologic and molecular analysis showed massive human cell infiltrates within the liver, lung and spleen. The presence of human IgG and IgM antibodies against mouse red blood cells provided evidence that the engrafted human cells retained some immune function. Mice transplanted with peripheral blood leukocytes from donors that were allergic to mouse antigens engrafted to the same extent as normal cells but in addition developed the classical symptoms of acute allogeneic graft-versus-host disease (GVHD) including infiltrates of the skin, gut, and liver. The newborn SCID system provides a new in vivo model to study human xenoreactivity and GVHD.