Search PubMed⌕ Search

Biomedical subjects

J L Ascensao

Publications and source records attributed to J L Ascensao.

At least 19 recordsLinked to original sources
PubMed2000

In vitro infection of megakaryocytes and their precursors by human cytomegalovirus.

Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34(+) cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42(+) MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the beta-galactosidase (beta-gal) gene. Using chromogenic beta-gal substrates, HCMV was detected during MK differentiation of infected CD34(+) cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV. (Blood. 2000;95:487-493)

Antigens, CD↗
PubMed1999

Pre-treatment of transplant bone marrow cells with hydrocortisone and cyclosporin A alleviates graft-versus-host reaction in a murine allogeneic host-donor combination.

The aim of the study was to alleviate graft-versus-host reaction (GVHR) by pre-treatment of the bone marrow (BM) transplant with hydrocortisone (HC) and cyclosporin A (CsA) in C57BL/6J (donor) --> CBA/J (recipient) mouse combination. BM cells were exposed to HC and CsA for 1 h at 37 degrees C and then injected into lethally irradiated (9.5 Gy) mice at a dose of 2 x 10(6) BM cells/mouse. Haematopoietic recovery was assessed on day 12, and survival was followed for 100 days. Combinations of 1000 microg/ml HC and 100 microg/ml CsA, and 100 microg/ml HC and 10 microg/ml CsA significantly reduced MLR and additively mitigated GVHR in vivo, achieving 40% and 26% survival rates, respectively. However, HC and CsA altered neither the peripheral blood cell counts nor in vitro and in vivo BM cell clonogenic potential. Additional studies have shown that HC and CsA blocked con A-driven differentiation of CD8+ and CD4+ CD8+ lymph node cells (LNC) and progression of LNC to S + G2/M cell cycle phases, and inhibited IL-1, IL-2 and TGF-beta while enhancing GM-CSF gene expression in BM cells. Taken together, these data indicate that the pre-treatment of the BM transplant with HC and CsA results in inactivation of GVHR effector cells and mitigation of GVHR while sparing BM repopulating capacity.

Animals↗
PubMed1998

Human thymic stroma supports human natural killer (NK) cell development from immature progenitors.

NK cells are lymphocytes which exhibit spontaneous cytotoxicity against a variety of target cells, including cancer cells. Mature NK and T cells may derive from a common precursor which differentiates into T or NK cells depending on the microenvironment. We evaluated the effect of human fetal thymic stroma on human CD34+Lin- progenitors. The culture medium was supplemented with human AB serum with or without interleukin-2 (IL2; 1000 U/ml) and interleukin-7 (IL7; 1000 U/ml). After 3 weeks of culture, CD45/56 cells were detected by flow cytometry and their activity was tested against K562 targets. In cultures with IL2 the percentage of CD56-positive cells was much higher in the Transwell cultures (60.8 +/- 12.5% from CD34+Lin-DR+ and 51% from CD34+Lin-progenitors) than in adherent cultures (25 +/- 21.9% from CD34+Lin-DR+ and 25.3 +/- 9.5% from CD34+Lin-progenitors) or suspension cultures (23 +/- 21.4% from CD34+Lin-DR+ progenitors and 43.1 +/- 14.2% from CD34+Lin-progenitors). Cytolytic activity as measured by K562 lysis was also higher in Transwell cultures with IL2. NK cells were also obtained in cultures without factors or supplemented with IL7, but in smaller numbers. These data indicate that NK cells can be obtained in vitro by coculture of immature hematopoietic progenitors with thymic stromal cells and that IL2 appears to strongly favor their development in the absence of stromal contact. This would indicate a direct inhibitory effect of the thymic stroma on NK progenitors.

Cell Division↗
PubMed1998

Definition of early progenitors and functional maturation of human natural killer cells: requirements for cytocidal activity.

We examined the in vitro development of human natural killer (NK) cells to define the earliest hematopoietic progenitors which could give rise to NK cells. Our data indicates that NK cells arise from the Thy 1+ subpopulations of CD34+ marrow cells. In cultures with IL2 we generated up to 96.5% NK cells from CD34+ Thy 1+ and in cultures with IL7, cells bearing NK cell surface markers were also observed but were devoid of functional activity. We also addressed the functional integrity of these cultured cells by examining the appearance of granzyme A, serine esterase, Met-ase and perforin and correlating these with lytic activity. Granzyme A mRNA could be detected in IL2-cultured cells and serine esterase activity as well as perforin were also found at the single cell level. Human Met-ase (Hu-Met-1) was not detected at any time of analysis. Granzyme expression occurs early in cells cultured with IL2 but its presence did not correlate with lytic function; CD56+ cells grown in the presence of IL7 express these enzymes and perforin after longer periods of culture, but lack the ability to lyse NK targets.

Adult↗
PubMed1997

Transplantation of hematopoietic stem cells in utero.

Hematopoietic stem cell (HSC) transplantation in children and adults with congenital lymphohematopoietic disorders is limited by donor availability, graft failure, graft-versus-host disease (GVHD) and delayed immunological reconstitution. These problems may be circumvented by transplanting the patient before birth. Prenatal cellular therapy for the treatment of congenital diseases has tremendous theoretical appeal. Potential advantages of prenatal transplantation include: A) fetal immunologic immaturity and the potential for induction of donor-specific tolerance; B) available space in the developing bone marrow for engraftment of donor cells; C) the sterile, protective, fetal environment which provides isolation from environmental pathogens, and D) prevention of clinical manifestations of the disease. Normal hematopoietic and immunologic development during ontogeny creates a "window of opportunity" during which events favor the engraftment of transplanted allogeneic (and xenogeneic) HSC and their proliferation. This is a period in which the fetus is immunologically naive and thus incapable of rejecting the foreign HSC, and the expanding bone marrow spaces allow homing and engraftment of HSC without the need for myeloablation. Experiments in sheep have established the optimal age of the recipient, route of donor cell administration, sources of HSC, and other parameters necessary for the successful engraftment and long-term expression of donor HSC. In preclinical studies, transplantation of CD34-enriched or highly purified populations of human adult bone marrow cells in utero resulted in the long-term engraftment and expression of donor HSC without graft failure and GVHD. The strategies developed in allogeneic and xenogeneic fetal sheep models were used to successfully treat human fetuses with X-linked recessive severe combined immunodeficiency.

Animals↗
PubMed1997

A novel high-dose chemotherapy protocol with autologous hematopoietic rescue in patients with metastatic breast cancer or recurrent non-Hodgkin's lymphoma.

In this phase II trial, we used a double dose-intensive chemotherapy and stem cell rescue protocol to treat breast cancer (BCA) patients or non-Hodgkin's lymphoma patients (NHL). The first cycle consisted of high-dose melphalan followed by ABMT. The second cycle used a novel chemotherapy combination; thiotepa, etoposide, carboplatin and cyclophosphamide (TECC) followed by ABMT. We treated 12 patients in total, nine with BCA, three with NHL. All nine BCA patients were treated with the two cycle protocol. The three NHL patients were treated with the second cycle only. Bone marrow (BM, 1 patient), peripheral blood stem cells (PBSC, 10 patients) or both (1 patient) were reinfused 60-72 h after completion of each cycle of chemotherapy. Recovery was rapid; the ANC rose to greater than 500/microl on day +11 (+8 to + 20) and the platelet count to greater than 20000/microl on day +12 (+6 to +20). The toxicities included the expected neutropenic fevers, severe mucositis, diarrhea, and a low incidence of mild renal insufficiency. No patients developed veno-occlusive disease, hemorrhagic cystitis or overt bleeding. With a mean follow-up of 37 months, 83.3% of the patients are alive. Six patients are in complete remission; one patient with BCA relapsed and expired; one patient with NHL is in CR now over 18 months after relapse and subsequent treatment with interferon; one patient is too early to evaluate. Progression-free survival overall is 75%, which is at least equivalent to many other recent studies using similar regimens. In addition, we have also found that delayed addition of G-CSF during the mobilization of PBSC was feasible and resulted in excellent CD34+ cell counts and engraftments, and reduced treatment costs. These results indicate that this chemotherapy is effective with good remission rates and high progression-free survival rates. It is also well tolerated with acceptable toxicities that are manageable. Long-term follow-up of a larger cohort of patients will be needed to ascertain the overall efficacy of this type of therapy.

Adult↗
PubMed1996

The role of sheep stroma in human haemopoiesis in the human/sheep chimaeras.

We have previously reported on the successful engraftment and long-term expression of human HSC (haemopoietic stem cells) in sheep following transplantation into pre-immune fetuses. The mechanism(s) underlying the support/maintenance of human haemopoiesis in sheep is not understood. In these studies we examined the ability of sheep stroma to support human haemopoiesis in vitro and compared this with support provided by human stroma. We found that, overall, human stroma was significantly more effective than sheep stroma in supporting human haemopoiesis in vitro. Nevertheless, stroma established from bone marrow of normal sheep was capable of lower-level support of the proliferation and multilineage differentiation of highly purified (CD34+, CD38-) preparations of human HSC in vitro. At lower concentrations, sheep stroma conditioned media (CM) induced human bone marrow mononuclear cells enriched for the CD34-positive fraction to enter the cell cycle at levels comparable to that of human stroma CM. However, unlike human CM, at higher concentrations, sheep CM inhibited human cells from initiating the cell cycle. These results provide a possible explanation for the long-term maintenance of low levels of human haemopoietic cell activity in human/sheep chimaeras.

Animals↗
PubMed1995

Enhanced lymphokine-activated killer cell activity by an immunomodulator, Roquinimex.

Roquinimex (Roq) is an immunomodulator known to stimulate cellular immune responses. It is currently used for immunotherapy after bone marrow transplantation (BMT). One of the major features of this compound is an enhancement of natural killer (NK) cell activity and numbers. We studied the in vitro effect of Roq on human peripheral blood NK and adherent lymphokine-activated killer cell (ALAK) activities. In cultures supplemented with recombinant interleukin 2 (rIL-2) (1000 U ml-1) and Roq a significant increase in NK and LAK function was observed without a parallel increase in cell numbers. We also examined the generation of NK cells from human bone marrow (BM) immature progenitors, obtained by purging with 4-hydroperoxycyclophosphamide (4HC). NK cell numbers and activity were both increased when cultures with rIL-2 (10 U ml-1) were supplemented with Roq. These results confirm findings obtained in vivo and in vitro in the murine system and suggest that Roq is an active agent on these lymphoid populations. These properties and good tolerability make Roq an attractive tool for immunotherapy.

Adjuvants, Immunologic↗
PubMed1995

In vitro effects of etoposide purging on natural killer cells.

Autologous bone marrow transplantation (ABMT) is widely used to treat hematologic and non-hematologic malignancies. In an attempt to reduce potential neoplastic contamination of the graft, pharmacologic purging with cyclophosphamide derivatives and etoposide (VP16) is often used. Trilineage hemopoietic engraftment is seen following ABMT with VP16-purged bone marrow (BM), but little is known about immune reconstitution in this setting. We studied the in vitro development of natural killer (NK) cells from VP16-purged BM. These cells, defined by phenotypic and functional criteria, undergo rapid regeneration, expansion, and maturation in the presence of interleukin 2 (IL-2). VP16 seems to spare subsets of NK cells, and this cell lineage develops rapidly in VP16-treated BM cultures from patients with acute myelogenous leukemia (AML) and healthy donors. Since relapse rates remain high, immunotherapy following ABMT with purged BM may be useful and should be considered in these patients.

Adolescent↗
PubMed1995

Generation of human natural killer cells from pharmacologically purged bone marrow.

Human natural killer (NK) cells play an important role in first-line defence against primary and metastatic tumours. Their stimulation after autologous bone marrow transplantation (ABMT) may be useful to eradicate residual tumour cells. Human NK cells originate in bone marrow (BM) but little is known about their progenitors and lineage development. We studied NK cell ontogeny from BM progenitors obtained by 'purging' normal BM with 4-hydroperoxycyclophosphamide (4HC); this agent is known to destroy all but the most primitive BM progenitors. NK cells, defined by their phenotypic markers and cytolytic activity, could be generated from 4HC-treated BM cells during in vitro cultures over stromal BM feeder layers and in suspension cultures containing a mixture of soluble cytokines; Interleukine 2 appears to be essential for the full development of this population. A lag period exists before NK cells can be found in significant numbers in culture; this suggests that a delay in initiation of immunotherapy after ABMT ought to be considered, particularly when using purged marrow.

Antigens, CD↗
PubMed1994

Generation of human natural killer cells from immature progenitors does not require marrow stromal cells.

Human natural killer (NK) cells comprise 10% to 15% of peripheral blood mononuclear cells and have an important role in immune responses against tumors, viral infections, and graft rejection. NK cells originate in bone marrow (BM), but their progenitors and lineage development have not been completely characterized. We studied the generation of NK cells from purified CD34+HLADR- and CD34+HLADR+ BM progenitors and the influence of various cytokines on their production. We show that CD3-CD56+ cytotoxic NK cells can develop from both progenitors populations when interleukin-2 (IL-2) is present in an in vitro suspension culture system containing IL-1 alpha and stem cell factor. Up to 83.8% and 98.6% CD3-CD56+ cells were detected in CD34+HLADR- and CD34+DR+ cultures, respectively, after 5 weeks of culture; significant numbers of NK cells were first detected after 2 weeks. Cytotoxic activity paralleled NK cell numbers; up to 70% specific lysis at an effector:target ratio of 10:1 was observed at 5 weeks. IL-7 also triggered development of CD3-CD56+ cells from these immature progenitors (up to 24% and 55% appeared in CD34+HLADR- and CD34+HLADR+ cultures, respectively). Our data suggest that BM stromas are not necessary for NK cell development and that IL-2 remains essential for this lineage development and differentiation.

Adult↗
PubMed1994

Human cytomegalovirus alters interleukin-6 production by endothelial cells.

In an effort to study whether human cytomegalovirus (HCMV) can disrupt the balanced cytokine network that controls human hematopoiesis, we investigated the ability of a laboratory strain HCMV (AD169) to alter the production of interleukin-6 (IL-6) by cultured endothelial cells (HUVECs). ECs are important components of human bone marrow stroma and produce factors that stimulate the proliferation and differentiation of human hematopoietic progenitors. HCMV was able to greatly increase production of both mRNA and protein for IL-6 in unprimed HUVECs. When we discriminated between viral pellet and cleared viral supernatants, the supernatants induced an increase in mRNA at 30 minutes and protein by 2 hours, whereas an increase in IL-6 caused by virus itself did not become evident until 12 hours. The possibility that IL-6 induction was simply caused by the presence in the viral stock of endotoxin, IL-1 alpha, IL-1 beta, tumor necrosis factor alpha, or IL-4, all known inducers of IL-6 in HUVECs, was ruled out by the addition of polymyxin B and appropriate neutralizing antibodies. These findings show that HCMV is capable of directly and indirectly modulating the production by HUVECs of IL-6, one of the cytokines involved in the process of hematopoiesis.

Cells, Cultured↗
PubMed1994

Pancytopenia in allogeneic marrow transplant recipients: role of cytomegalovirus.

We describe the clinical course of three cytomegalovirus-antibody-positive allogeneic marrow graft recipients who developed progressive pancytopenia during the third month post-transplant. Bone marrow biopsy cores were hypocellular without evidence of disease recurrence. Haemopoietic progenitor assays demonstrated a decrease of all assayable progenitors. Cytomegalovirus was identified from the blood and urine of all three patients. However, none of the patients developed other manifestations of cytomegalovirus infection such as pneumonitis, hepatitis and enteritis. The therapeutic use of ganciclovir and intravenous immunoglobulins resulted in prompt resolution of both viraemia and viruria in all three patients, and of pancytopenia in two patients.

Adult↗
PubMed1994

Phase I/II study of cyclophosphamide, carboplatin, and etoposide and autologous hematopoietic stem-cell transplantation with posttransplant interferon alfa-2b for patients with lymphoma and Hodgkin's disease.

PURPOSE: To evaluate the safety and toxicity of interferon alfa-2b (IFN) following an intensive preparative transplantation regimen in patients with relapsed Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Thirty-two patients with NHL or HD underwent autologous transplantation following cyclophosphamide 7,200 mg/m2, carboplatin 1,600 mg/m2, and etoposide 1,600 mg/m2 (CCV). Fourteen patients received an escalating dose of IFN. IFN was started at 1 x 10(6) U/m2 subcutaneously (SC) three times per week with a monthly dose escalation to a maximum of 3 x 10(6) U/m2 SC three times per week. IFN was continued for a total of 6 months. RESULTS: The preparative regiment was well tolerated. Renal dysfunction was noted more frequently in patients with a history of pretransplant cisplatin treatment, and cardiac dysfunction was responsible for the single transplant-related death (3%). IFN was well tolerated with no serious complications. Transient neutropenia and thrombocytopenia were noted in several patients. The mean maximal-dose IFN achieved was 2 x 10(6) IU/m2. The median duration of treatment with IFN was 5.2 months. The overall probability of survival (OS) and event-free survival (EFS) at 36 months, with a median follow-up duration of 18 months, was 42% OS and 14% EFS in HD and 70% OS and 56% EFS in NHL. The EFS at 36 months was 73% for all NHL patients who received IFN and 50% for patients who refused IFN treatment (P = .12), with OS estimates of 100% in the IFN group and 35% in the untreated group (P = .0002). CONCLUSION: CCV is a safe, effective conditioning regimen in patients with NHL or HD. Posttransplant IFN can be safely administered at 2.0 x 10(6) U/m2 three times per week for 6 months and may have a meaningful antitumor effect.

Adolescent↗
PubMed1993

Liver-derived fetal hematopoietic stem cells selectively and preferentially home to the fetal bone marrow.

In the course of ontogeny, the homing site for the hematopoietic stem cells (HSC) moves with certain predictability from the yolk sac to the liver/spleen and then to the marrow. The pattern of this migration has thus far been established mostly on a morphologic basis. To delineate further the course of this migration and to gain insight into its possible mechanism, we used in utero transplantation of allogeneic or xenogeneic HSC in preimmune sheep fetuses. Sex chromosome, type of hemoglobin, and species-specific surface markers were used to follow the path of transplanted cells in the fetus. Before the development of the bone marrow, transplanted HSC (liver- or marrow-derived) homed exclusively to the liver/spleen. With the development of marrow, around day 60 of gestation (term, 145 days), homing occurred also in the nascent marrow and by day 80 transplanted cells homed exclusively to the marrow. This suggests that there may be a hierarchy in homing sites, with those of the marrow having higher affinity than those of liver/spleen. Interestingly, despite a change in homing that was followed by the expansion of the marrow compartment of HSC (ie, HSC proliferation), these cells did not participate actively in blood cell formation during most of the prenatal period. Liver remained the major hematopoietic organ throughout the gestation. It was only during the perinatal period that this organ assumed the function of hematopoiesis from the liver. This lack of expression of HSC in fetal marrow can possibly be attributable to the immaturity of marrow stroma required for differentiation and maturation of progenitors and the orderly egress of mature cells into the blood stream. The availability of this model allows us to begin studies in the molecular mechanism of stem cell homing in vivo during ontogeny.

Animals↗
PubMed1993

Hematopoietic origin of human natural killer (NK) cells: generation from immature progenitors.

Human natural killer (NK) cells originate from bone marrow, but little is known about NK cell progenitors and ontogeny. We studied the phenotype and functional activity of NK cells derived from highly purified human bone marrow CD34+ cells, which exhibited neither lytic activity nor expression of surface antigens characteristic of NK (CD56) or T (CD3) cells. However, when cultured with hematopoietic growth factors or feeder layers for up to 4 weeks, up to 86% functional CD56+ cells were seen in the absence of mature T cell development. CD56+ cells appeared in all cultures at 2 or 3 weeks, with the largest percentage in those exposed to IL-2. These studies demonstrated that NK cells arise 'in vitro' from immature bone marrow progenitors and also suggest a separate origin and differentiation pathway for NK and T cells.

Antigens, CD↗
PubMed1992

Ex vivo incubation with growth factors enhances the engraftment of fetal hematopoietic cells transplanted in sheep fetuses.

Hematopoietic stem cells (HSC) transplanted in utero are in competition with endogenous HSC; thus, ultimately the graft constitutes a relatively small fraction of total HSC pool. To enhance the engraftment of donor cells in sheep fetuses, we preincubated these cells, ex vivo, for 16 hours at 37 degrees C with the conditioned medium from phytohemagglutinin-stimulated lymphocytes (PHA-LCM) before in utero transplantation. PHA-LCM is a rich source of hematopoietic growth factors in sheep. Subsequent engraftment was significantly higher in cells preincubated with PHA-LCM compared with fresh cells or those incubated with control medium only. This was reflected in all markers of the donor cells (hemoglobin type, karyotype, and progenitor cell assays). Brief ex vivo incubation with PHA-LCM also increased viability of all marrow cells as well as total numbers of progenitors. Similar enhancement of engraftment was also noted in monkeys after a brief preincubation of donor cells with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We conclude that brief (16 hours) ex vivo incubation of donor cells with a source of such growth factors as IL-3 and GM-CSF enhances the subsequent engraftment of transplanted cells.

Animals↗