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Human immunodeficiency virus type 1 (HIV-1) R5 isolates that predominantly use CCR5 as a coreceptor are frequently described as macrophage tropic. Here,we compare macrophage tropism conferred by HIV-1 R5 envelopes that were derived directly by PCR from patient tissue. This approach avoids potentially selective culture protocols used in virus isolation. Envelopes were amplified (i) from blood and semen of adult patients and (ii) from plasma of pediatric patients. The phenotypes of these envelopes were compared to those conferred by an extended panel of envelopes derived from brain and lymph node that we reported previously. Our results show that R5 envelopes vary by up to 1,000-fold in their capacity to confer infection of primary macrophages. Highly macrophage-tropic envelopes were predominate in brain but were infrequent in semen,blood,and lymph node samples. We also confirmed that the presence of N283 in the C2 CD4 binding site of gp120 is associated with HIV-1 envelopes from the brain but absent from macrophage-tropic envelopes amplified from blood and semen. Finally,we compared infection of macrophages,CD4(+) T cells,and peripheral blood mononuclear cells (PBMCs) conferred by macrophage-tropic and non-macrophage-tropic envelopes in the context of full-length replication competent viral clones. Non-macrophage-tropic envelopes conferred low-level infection of macrophages yet infected CD4(+) T cells and PBMCs as efficiently as highly macrophage-tropic brain envelopes. The lack of macrophage tropism for the majority of the envelopes amplified from lymph node,blood,and semen is striking and contrasts with the current consensus that R5 primary isolates are generally macrophage tropic. The extensive variation in R5 tropism reported here is likely to have an important impact on pathogenesis and on the capacity of HIV-1 to transmit. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

OBJECTIVE: Identification of a clinical grade method for the ex vivo generation of donor-derived T cells cytotoxic against both myeloid and lymphoblastic cells still remains elusive. We investigated rapid generation and expansion of donor derived-allogeneic T-cell lines cytotoxic against patient leukemic cells. MATERIALS AND METHODS: Acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) blasts were cultured 5 days in Stem Span,granulocyte macrophage colony-stimulating factor,interleukin-4,and calcium ionophore. All B-precursor ALL (N22) and AML (N13),but not T-cell ALL (N3),differentiated into mature leukemia-derived antigen-presenting cells (LD-APC). All but one LD-APC generated cytotoxic T lymphocyte (CTL) from adult human leukocyte antigen (HLA)-identical (N8) or unrelated donors (N2). RESULTS: Upon in vitro culture,donor-derived CTL acquired a memory T phenotype,showing concomitant high CD45RA,CD45RO,CD62L expression. CD8(+) cells,but not CD4(+) cells,were granzyme,perforine,and interferon-gamma-positive. Pooled CD4(+) and CD8(+) cells were cytotoxic against leukemic blasts (32%,30:1 E:T ratio),but not against autologous or patient-derived phytohemagglutinin blasts. LD-APC from five ALL patients were used to generate CTL from cord blood. A mixed population of CD4(+) and CD8(+) cells was documented in 54% of wells. T cells acquired classical effector memory phenotype and showed a higher cytotoxicity against leukemia blasts (47%,1:1 E:T ratio). Adult and cord blood CTL showed a skewing from a complete T-cell receptor repertoire to an oligo-clonal/clonal pattern. CONCLUSIONS: Availability of these cells should allow clinical trials for salvage treatment of leukemia patients relapsing after allogeneic stem cell transplantation. View Publication

The ability of acute myeloid leukemic (AML) blasts to differentiate into leukemic dendritic cells (DC) thus acquiring the potential to present known and unknown leukemic antigens efficiently,holds promise as a possible new treatment for AML patients with minimal residual disease. Recent advances in culture methods have made the clinical use of leukemic DC feasible. However,additional measures appear to be essential in order to potentiate vaccines and to overcome the intrinsic tolerant state of the patients immune system. This review describes ways to improve AML-DC vaccines and discusses critical aspects concerning the development of clinical vaccination protocols. View Publication

BACKGROUND AND OBJECTIVES: Campath-1H is used in conditioning regimens and more recently as an anti-leukemic therapy in acute lymphoblastic leukemias (ALL). We therefore investigated CD52 expression and campath-1H-mediated lysis of ALL cells in vitro. DESIGN AND METHODS: Complement-mediated cytotoxicity assays were performed on freshly isolated neoplastic cells and cell lines using human serum. Antibody-dependent cellular cytotoxicity (ADCC) was performed by calcein-AM release assays. RESULTS: CD52 was expressed in four out of eight ALL cell lines studied. Among 61 freshly isolated ALL samples CD52 was expressed at varying levels in 87% of cases. Whereas ADCC was equivalent in different CD52+ lines,complement-dependent cytotoxicity (CDC) was variable. The REH cell line bearing the t(12;21) translocation showed 47-60% lysis when treated with 10 microg/mL campath-1H compared to 0-6% for the other cell lines expressing equivalent amounts of CD52. Furthermore all nine ALL samples with t(12;21) showed very high CDC (mean 97%) compared to the other 24 CD52+cases (mean 24%)(ptextless0.0001). In t(12;21) samples,efficient CDC was obtained with as little as 1 microg/mL campath-1H. CDC correlated in part with CD52 levels,suggesting that CD52 expression and other yet undefined factors contribute to the particular sensitivity of t(12;21) cells. The resistance of non t(12;21) ALL cases could be overcome to a limited extent by increasing the concentration of campath-1H,blocking the CD55 and CD59 complement inhibitors,and more effectively by combining campath-1H with fludarabine. INTERPRETATION AND CONCLUSIONS: We conclude that most ALL samples express CD52 to a variable level and that campath-1H has cytotoxic activity against CD52+ALL,alone or in combination with cytotoxic drugs. View Publication

Dendritic cell (DC) maturation state is a key parameter for the issue of DC-T cell cognate interaction,which determines the outcome of T cell activation. Indeed,immature DCs induce tolerance while fully mature DCs generate immunity. Here we show that,in the absence of any deliberate activation signal,DCs freshly isolated from mouse spleen spontaneously produce IL-12 and tumor necrosis factor-alpha and up-regulate co-stimulation molecules,even when directly re-injected into their natural environment. Furthermore,after their isolation,these cells acquire the capacity to induce specific T(h)1 responses in vivo. These results demonstrate that the sole isolation of spleen DCs leads to the full maturation of these cells,which therefore cannot be considered as immature DCs. Moreover,we also show that the kinetics of DC activation do not influence the polarization of T(h) response in vivo challenging the idea that exhausted DCs induce preferentially T(h)2 response. Altogether,these observations should be taken into account in all experiments based on the transfer of ex vivo purified DCs. View Publication

The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice. Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis. However,the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched for MaSCs by transplantation. Here we show that a single cell,marked with a LacZ transgene,can reconstitute a complete mammary gland in vivo. The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer,the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV-wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing,properties that define them as MaSCs. View Publication

Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation,which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time,attesting to a selective advantage of transduced progenitors. Notably,a high transgene copy number was detected in all lymphoid organs,and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether,these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases. View Publication

IkappaB kinase (IKK) complex plays a key regulatory role in macrophages for NF-kappaB activation during both innate and adaptive immune responses. Because IKK1-/- mice died at birth,we differentiated functional macrophages from embryonic day 15.5 IKK1 mutant embryonic liver. The embryonic liver-derived macrophage (ELDM) showed enhanced phagocytotic clearance of bacteria,more efficient antigen-presenting capacity,elevated secretion of several key proinflammatory cytokines and chemokines,and known NFkappaB target genes. Increased NFkappaB activity in IKK1 mutant ELDM was the result of prolonged degradation of IkappaBalpha in response to infectious pathogens. The delayed restoration of IkappaBalpha in pathogen-activated IKK1-/- ELDM was a direct consequence of uncontrolled IKK2 kinase activity. We hypothesize that IKK1 plays a checkpoint role in the proper control of IkappaBalpha kinase activity in innate and adaptive immunity. View Publication

HIV infection leads to a state of chronic immune activation and progressive deterioration in immune function,manifested most recognizably by the progressive depletion of CD4+ T cells. A substantial percentage of natural killer (NK) cells from patients with HIV infection are activated and express the natural cytotoxicity receptor (NCR) NKp44. Here we show that a cellular ligand for NKp44 (NKp44L) is expressed during HIV-1 infection and is correlated with both the progression of CD4+ T cell depletion and the increase of viral load. CD4+ T cells expressing this ligand are highly sensitive to the NK lysis activity mediated by NKp44+ NK cells. The expression of NKp44L is induced by the linear motif NH2-SWSNKS-COOH of the HIV-1 envelope gp41 protein. This highly conserved motif appears critical to the sharp increase in NK lysis of CD4+ T cells from HIV-infected patients. These studies strongly suggest that induction of NKp44L plays a key role in the lysis of CD4+ T cells by activated NK cells in HIV infection and consequently provide a framework for considering how HIV-1 may use NK cell immune surveillance to trigger CD4+ T cells. Understanding this mechanism may help to develop future therapeutic strategies and vaccines against HIV-1 infection. View Publication

Reduced-intensity conditioning regimens for transplant recipients have heightened awareness of immunologic resistance to allogeneic bone marrow transplants (BMT). Although T cell-mediated cytotoxicity has been assumed to play a role in the resistance against donor allogeneic hematopoietic stem and progenitor cell grafts,several studies have reported relatively unimpaired resistance by recipients who lack perforin,Fas ligand (FasL),and other cytotoxic mediators. This study compared the early kinetics of T cell-mediated resistance in B6 (H2b) cytotoxically normal versus deficient recipients after transplantation with major histocompatibility complex-matched,minor histocompatibility antigen (MiHA)-mismatched allogeneic marrow grafts. Wild-type B6 or cytotoxic double-deficient perforin-/-/ gld+/+ (B6-cdd) mice were sensitized against major histocompatibility complex-matched BALB.B or C3H.SW (H2b) MiHA and transplanted with a high dose (1 ?? 107) of T cell-depleted bone marrow. CD8 T memory cells were shown to be present in recipients before BMT,and anti-CD8 monoclonal antibody infusion abolished resistance,thus demonstrating that CD8 T cells are the host effector population. Donor-committed and high proliferative potential progenitor numbers were markedly diminished by 48 hours after transplantation in both wild-type B6 and B6-cdd anti-donor MiHA-sensitized recipients. These observations indicate that the resistance pathway used in the cytotoxic deficient mice was both potent and rapidly induced - consistent with a CD8 memory T-cell response. To examine the role of Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)- and TL1A-mediated cytotoxicity in this strong resistance,newly generated monoclonal antibodies specific for these ligands were administered to B6-cdd recipients sensitized to donor antigens. Recipients of syngeneic B6-gfp bone marrow exhibited significant donor colony-forming unit numbers after BMT. In contrast,low or absent colony-forming unit levels were detected in allogeneic recipients,including those that lacked perforin and FasL and that received anti-TWEAK,anti-tumor necrosis factor-related apoptosis-inducing ligand,and anti-TL1A monoclonal antibodies. These findings extend previous observations by demonstrating the existence of a rapidly effected resistance pathway mediated by memory CD8 effector T cells independent of the 2 major pathways of cytotoxicity. Together with previous findings,these results support the notion that effector cells derived from memory CD8 T-cell populations can mediate strong resistance against donor allogeneic MiHA-disparate hematopoietic engraftment by using a mechanism that is independent of the contribution of perforin,FasL,and the known death ligand receptor pathways. ?? 2005 American Society for Blood and Marrow Transplantation. View Publication

Hereditary hemochromatosis (HH),an iron overload disease associated with mutations in the HFE gene,is characterized by increased intestinal iron absorption and consequent deposition of excess iron,primarily in the liver. Patients with HH and Hfe-deficient (Hfe-/-) mice manifest inappropriate expression of the iron absorption regulator hepcidin,a peptide hormone produced by the liver in response to iron loading. In this study,we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe-/- mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading,accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely,wt mice reconstituted with Hfe-deficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression. View Publication