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Biomedical subjects

A D Donnenberg

Publications and source records attributed to A D Donnenberg.

At least 37 records · Page 2Linked to original sources
PubMed1997

The use of counterflow centrifugal elutriation for the depletion of T cells from unrelated donor bone marrow.

Transplantation of marrow from unrelated donors is associated with an increased incidence and severity of graft-versus-host disease (GVHD). In an attempt to minimize GVHD without compromising engraftment, unrelated donor marrow was depleted of lymphocytes by counterflow centrifugal elutriation (CCE), and a fixed dose of 0.5 x 10(6) CD3+ T cells/kg, as measured in real time by flow cytometry, was added back to the graft. Patients received cyclosporine (CYA) and corticosteroids for GVHD prophylaxis and to facilitate engraftment. In the first cohort (study I), 7 patients received busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (CY) and one patient received CY (200 mg/kg) + 1260 cGy fractionated TBI. Of 6 who were evaluable for both engraftment and rejection, 4 rejected their graft. The study was terminated, and the protocol was modified (study II) by the addition of antithymocyte globulin (ATG) to the pre-BMT and post-BMT therapy. Twelve patients received CY + TBI as above plus ATG given pre-BMT and post-BMT. Ten of twelve who received ATG engrafted. Twelve patients from studies I and II were evaluable for acute GVHD. Two developed grade I acute GVHD. Two patients developed grade II acute GVHD, 2 patients developed grade III GVHD, and 1 patient developed grade IV acute GVHD. Two of three cases of acute GVHD (> grade II) occurred later than day 100 after BMT concomitant with reduction of immunosuppressive therapy. The rate of engraftment was significantly higher in study II (p = .054). In numbers of CD34+ cells infused, numbers of CFU-GM infused, and numbers of nucleated cells did not correlate with engraftment. We conclude that (a) in contrast to the results seen in recipients of marrow from HLA-matched sibling donors, the depletion of unrelated donor marrow of all but 0.5 x 10(6) CD34+ cells/kg together with CYA + corticosteroids was not sufficient to facilitate engraftment. The use of a more immunosuppressive regimen containing TBI and ATG appeared to improve engraftment. (b) The reduction of the graft T cell dose to 0.5 x 10(6) CD34+ cells/kg resulted in a higher incidence of acute GVHD than that seen in recipients of marrow from genotypically identical donors whose marrow was similarly processed.

Adolescent↗
PubMed1996

Evaluation of monoclonal antibody-mediated anti-acute myeloid leukemia immunotherapy in a SCID/hu model.

The therapeutic potential of the IgM complement-fixing murine monoclonal antibody (mAb) PM-81 (anti-CD15) against acute myeloid leukemia (AML) was assessed in a SCID/hu leukemia model. Intraperitoneal (i.p.) injection of NB4 leukemia cells resulted in aggressive growth of leukemia cells in the peritoneal cavity of irradiated SCID/CB-17 mice. Flow cytometric analysis of human CD15, 33 and 45 expression, as well as cytologic examination, revealed that leukemia cells disseminated into the peripheral blood and multiple tissues of the mice. The approximately linear relationship between the injected leukemia cells and the subsequent leukemia cell proliferation provided a reliable model for monitoring the therapeutic effects of immunotherapy. Intraperitoneal injection of the mAb PM-81 markedly suppressed leukemia cell growth in this SCID/leukemia model. Most of the untreated mice died within 35-50 days of leukemia cell inoculation. Four weeks after inoculation of NB4 cells, five of nine mAb PM-81 treated mice had no solid tumor growth and six of nine had no detectable peritoneal exudate leukemia cells as determined by flow cytometry. In contrast, 100% of the mice in the untreated or control mAb groups were found to have both solid and peritoneal leukemia growth. In further experiments designed to evaluate the effects of therapy on survival, 50% (4/8) of PM-81 treated mice survived to 150 days, and had no detectable solid or suspension leukemia cells detectable at necropsy. In contrast, the median survival of untreated or negative control antibody-treated mice was 40 days (comparison to PM-81 treated; p = 0.006 and p = 0.03, respectively). The mechanism of leukemia cell suppression is not likely due to complement fixation since we could not demonstrate in vitro any cytotoxicity mediated by SCID mouse plasma. Further study is required to understand the mechanism of the antileukemia effect of PM-81 in this model.

Animals↗
PubMed1996

Generation of activated natural killer (A-NK) cells in patients with chronic myelogenous leukaemia and their role in the in vitro disappearance of BCR/abl-positive targets.

Activated natural killer (A-NK) cells, a subset of CD56(dim)CD3- lymphocytes, are obtained from PBMC of normal donors by adherence to plastic and culture in the presence of IL2. In this study we tested the feasibility of generating A-NK cells in patients with Ph+ chronic myeloid leukaemia (CML). Cultures obtained from patients with early chronic phase (ECP; n=7) contained a mean (+/-SD) of83 +/- 7% of CD3- cells, and those from patients with advanced chronic phase (ACP; n=7) contained 27+/-33% CD56+CD3- cells. In three patients with leukaemia in a blastic phase (BP) it was only possible to obtain one culture enriched in CD56+CD3- cells (81%). Cellular aggregates of myeloid cells and large granular lymphocytes were observed in early A-NK cell cultures. Paired freshly-adherent and cultured A-NK cells were tested for the presence of BCR/abl mRNA by RT-PCR. The BCR/abl+ cells were detected in all 12 preparations of the freshly adherent A-NK cells tested. In 6/12 the BCR/abl+ cells were no longer detectable by RT-PCR on day 14 of culture. Both proliferation and antileukaemic cytotoxicity were significantly higher (P=0.002 and P=0.029, respectively) in the BCR/abl- cultures than those in the six BCR/abl+ cultures. 5/6 BCR/abl- cultures were highly enriched in A-NK cells on day 14, and 1/6 contained predominantly CD56+CD3+ cells. Only 2/6 BCR/abl + cultures were enriched in A-NK cells on day 14, but they had poor cytotoxicity and a low proliferative index. Myeloid cells (CD33+) were more frequently detected in the BCR/abl+ than BCR/abl- A-NK cell cultures (P=0.028). These observations suggest that: (1) populations of benign A-NK cells can be generated from the peripheral blood of CML patients; (2) the ability to generate A-NK cells is impaired in patients with advanced CML; and (3) the ability to generate A-NK cells with antileukaemic activity correlates with the disappearance of BCR/abl+ cells from these cultures.

Antigens, CD↗
PubMed1996

The appearance of Thy-1- donor T cells in the peripheral circulation 3-6 weeks after bone marrow transplantation suggests an extrathymic origin.

Donor and host T cells were distinguished by T cell antigen marker Thy-1 isotype and cytoplasmic isozyme Gpi-1 in this study of bone marrow transplantation between congenic mice. During the first 3-6 weeks after irradiation and marrow transfer, percentages of cells bearing the donor Thy-1 isotype in the periphery are much lower than percentages of T cells bearing the donor Gpi-1 marker. Apparently a population of Thy-1- donor T cells exists for several weeks after bone marrow transplantation. Further study showed that this population of CD3+, Thy-1- donor T cells expressed CD4+ or CD8+ and was found in peripheral blood and spleen but not in the thymus. This finding suggests their extrathymic origin.

Animals↗
PubMed1995

Lymphopoiesis, apoptosis, and immune amnesia.

Bone marrow transplant recipients have a functional T-cell deficit long after T-cell counts have returned to normal levels. Early after BMT, T-cell phenotype is predominantly CD45RO+/CD29high/HLA-DR+/CD38high. This profile is associated with activated memory cells in healthy subjects, but also appears on the earliest mature naive T-cells in times of lymphopoietic stress. Most of these cells apoptose in short-term unstimulated culture, suggesting that they would have had a similar fate in vivo. Twelve to 24 months after BMT, CD45RA+/CD29low/HLA-DR-/CD38low T cells increase, apoptosis decreases, and T-cell function normalizes. We hypothesize that in the adult, mature memory T cells regulate their own replacement by rescuing a proportion of newly generated naive cells from apoptosis. Ablation of memory cells consequent to high dose therapy disrupts this process, resulting in a protracted period of high lymphocyte turnover with few cells surviving to make the antigen-driven transition to memory cells. Infection with HIV-1 also eventuates in immune deficiency associated with a loss in CD4+ T cells and dominance of the phenotypic/apoptotic profile which we have associated with lymphopoietic stress. Recent data independently confirm that T-cell turnover is greatly elevated in HIV infection. Catastrophic or chronic depletion of memory T cells due to marrow ablative therapy or HIV-1 infection interferes with memory replacement, substituting short-lived hypofunctional naive T cells which characterize the state of immune amnesia.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Apoptosis parallels lymphopoiesis in bone marrow transplantation and HIV disease.

Apoptosis has been implicated in a variety of physiological processes ranging from tissue modeling to deletion of autoreactive T lymphocytes during thymic development. The recent finding that a large proportion of peripheral T cells from HIV-infected subjects (corrected from subjects) apoptose in culture raises an important issue: does this represent a pathologic mechanism by which the virus disrupts the immune system, or a normal physiologic response to virus-mediated T-cell loss? To study the potential relationship between apoptosis and lymphopoiesis, we compared apoptosis rates in unstimulated lymphocyte cultures from healthy subjects, HIV+ gay men, and bone marrow transplant (BMT) recipients undergoing immune reconstruction. BMT recipients were chosen because they undergo massive regeneration of lymphocytes following marrow ablation and graft infusion. The data obtained in BMT recipients suggests that elevated apoptosis accompanies, and is the consequence of, elevated lymphopoiesis. We also found a strong inverse relationship between in vitro T-cell apoptosis rates and peripheral T-cell counts. These results provide new interpretation for elevated apoptosis observed in HIV-infected individuals--that it reflects increased T-cell turnover consequent to virus-mediated destruction of CD4+ T-cells.

Apoptosis↗
PubMed1995

Failure of T-cell homeostasis preceding AIDS in HIV-1 infection. The Multicenter AIDS Cohort Study.

We and others have postulated that a constant number of T lymphocytes is normally maintained without regard to CD4+ or CD8+ phenotype ('blind' T-cell homeostasis). Here we confirm essentially constant T-cell levels (despite marked decline in CD4+ T cells and increase in CD8+ T cells) in homosexual men with incident human immunodeficiency virus, type 1 (HIV-1), infection who remained free of acquired immunodeficiency syndrome (AIDS) for up to eight years after seroconversion. In contrast, seroconverters who developed AIDS exhibited rapidly declining T cells (both CD4+ and CD8+) for approximately two years before AIDS, independent of the time between seroconversion and AIDS, suggesting that homeostasis failure is an important landmark in HIV disease progression. Given the high rate of T-cell turnover in HIV-1 infection, blind T-cell homeostasis may contribute to HIV pathogenesis through a CD8+ T lymphocytosis that interferes with regeneration of lost CD4+ T cells.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Autologous peripheral blood stem cell transplantation and adoptive immunotherapy with activated natural killer cells in the immediate posttransplant period.

Relapse after high-dose chemotherapy supported by peripheral blood stem cell transplantation (HDC-PBSCT) is the main cause of therapeutic failure in patients with lymphoma and breast cancer. Adoptive immunotherapy with activated natural killer (A-NK) cells and interleukin 2 might eliminate surviving residual tumor without adding to toxicity. Eleven patients with relapsed lymphoma and one with metastatic breast cancer were entered on a pilot clinical trial of HDC-PBSCT followed on day 2 after transplant by infusion of cultured autologous A-NK cells. Simultaneously, recombinant human interleukin 2 (rhIL-2) was initiated as a 4-day continuous i.v. infusion at 2 x 10(6) IU/m2/day, referred to as high-dose rhIL-2. Therapy with high-dose rhIL-2 was followed by a 90-day continuous i. v. infusion at 3 x 10(5) IU/m2/day, referred to as low-dose rhIL-2. All patients engrafted and nine completed treatment. Posttransplant days to a neutrophil count of 500/microliter and to a platelet count of 50,000/microliter were similar to comparable patients treated with HDC-PBSCT alone. Generation of A-NK cells for therapy was feasible in all patients except the three patients with Hodgkin's disease, whose cells did not proliferate in culture. Overall toxicity associated with early posttransplant transfer of A-NK cells and interleukin 2 did not differ from that observed with peripheral blood stem cell transplantation alone in comparable patients. There was early amplification of natural killer cell activity in the peripheral blood of four patients that appeared to result from the transfused A-NK cells. Adoptive transfer of A-NK cells and rhIL-2 during the pancytopenic phase after HDC-PBSCT was feasible and well tolerated, did not adversely affect engraftment, and resulted in amplified natural killer activity in the peripheral blood during the immediate posttransplantation period.

Adoptive Transfer↗
PubMed1994

Differential effect of 4-hydroperoxycyclophosphamide and antimyeloid monoclonal antibodies on T and natural killer cells during bone marrow purging.

Autologous bone marrow (BM) transplantation after high dose therapy is widely used to treat acute leukemia, lymphoma, and selected solid tumors. In studies of BM purging with chemical agents, monoclonal antibodies (MoAbs), or other agents, the emphasis has been on the efficacy of tumor cell removal and sparing of hematopoietic progenitor cells. Two commonly used methods of BM purging for patients with acute myeloid leukemia have been the drug 4-hydroperoxycyclophosphamide (4-HC) and (MoAbs) directed to myeloid antigens such as CD14, CD15, and CD33. Although both methods of BM purging have potent activity against leukemia cells, 4-HC is also quite toxic to normal hematopoietic progenitor cells in the same concentrations that are used to deplete leukemia cells. To further characterize the cellular composition of BM after purging, we examined the effects of MoAbs plus complement and 4-HC on cells of the lymphoid lineage in the BM. 4-HC exerted a concentration-dependent cytotoxicity on clonogenic T lymphocytes, natural killer (NK) cells, and lymphokine (interleukin-2)-activated killer (LAK) cells, whereas the anti-CD14 and anti-CD15 MoAbs had little effect. At a concentration of 4-HC commonly used for BM purging (60 micrograms/mL), there were 4 to 5 logs of T-cell depletion and almost complete elimination of NK- and LAK-cell activity. In contrast, 4-HC at low concentrations (eg, 3 micrograms/mL) spared the majority of lymphoid cells suggesting that low concentration 4-HC combined with MoAb purging may be a desirable alternative to higher concentration 4-HC. These data indicate that purging with antimyeloid MoAbs, but not with 4-HC, spares the function of mature graft lymphocytes. Infusion of viable lymphocytes may be important for the transfer of immune memory against microbial and neoplastic antigens and may hasten immune reconstitution. In addition, mature graft lymphocytes may also be selectively activated and expanded in conjunction with interleukin-2 administration after BM transplantation.

Antibodies, Monoclonal↗
PubMed1994

Granulocyte-macrophage colony-stimulating factor (GM-CSF), given concurrently with induction therapy for acute myelogenous leukemia (AML), augments the syndrome of T-lymphocyte recovery.

A transient lymphocytosis precedes myeloid recovery in many patients with AML treated with intensive chemotherapy. We describe the kinetics, clinical features, and immunology of lymphocyte recovery which is markedly augmented by the inclusion of GM-CSF in induction therapy. Lymphocyte recovery from 19 patients receiving GM-CSF as part of induction therapy was compared to a historical control of 25 patients treated with identical chemotherapy in the absence of cytokine. Kinetics and clinical features of lymphocyte recovery were analyzed. Peripheral blood was studied by flow cytometry, chromium release assays, and Southern analysis of the T-cell antigen receptor, beta chain gene. Patients treated with GM-CSF to recruit cells into cycle, exhibit markedly increased peaks of lymphocyte recovery. Recovering lymphocytes demonstrated an activated memory T-cell phenotype suggestive of a cytokine release syndrome. Lymphoid recovery was often associated with rash, fever, and lymphadenopathy. Study patients who developed peak lymphocyte counts > or = 1000 microliters were more likely to achieve remission than those with a lower peak. Recovery lymphocytes did not lyse pretreatment autologous bone marrow cells. Southern analysis demonstrated dominant potentially clonal rearrangements in the majority of patients studied. Lymphocyte recovery, which appears to include oligoclonal expansion of memory T cells is markedly augmented by administration of GM-CSF during chemotherapy. This may represent a non-specific response by a limited repertoire of T cells surviving therapy, or a specific clonal response to a powerful exogenous or endogenous antigen. Possible antileukemic activity of these cells remains to be elucidated.

Adult↗
PubMed1993

CD34+ progenitors and colony-forming units-granulocyte macrophage are recruited during large-volume leukapheresis and concentrated by counterflow centrifugal elutriation.

The recruitment of mononuclear cells (MNCs), colony-forming units-granulocyte macrophage (CFU-GM), lymphocyte subpopulations, and CD34+ progenitor cells was studied during large-volume (15-25 L blood processed) peripheral blood stem cell (PBSC) harvests. Normal donors (n = 13) underwent a 4-hour leukapheresis designed to maximize PBSC yield (blood flow rate, 85 mL/min). Mean (+/- SD) volume processed was 17.7 +/- 0.4 L, and yield was 2.4 +/- 0.7 x 10(10) white cells containing 99 percent MNCs and 1.3 mL red cells per L of blood processed. Postapheresis hematocrit, platelets, and MNCs were reduced from preapheresis values by 7, 35, and 23 percent, respectively (p < 0.05). In nine donors, the component was collected as four 1-hour samples, and culturing of CFU-GM and flow cytometric analysis of lymphocyte subpopulations and CD34+/HLA-DR+ cells were done in individual samples. Total CFU-GM were 2.4 +/- 1.4 x 10(6) (3.0 +/- 1.8 x 10(4) CFU-GM/kg) and lymphocytes were 20.8 x 10(9), with 75 percent CD3+ T cells, 10 percent CD19/CD20+ B cells, and 17 percent natural killer cells. A more than twofold increase in CFU-GM and CD34+ cells was noted over the course of the 4-hour procedure (p < 0.05). In four donors, the leukapheresis component underwent counterflow centrifugal elutriation (CCE), which separated it into four fractions in an attempt to concentrate CD34+ and CFU-GM progenitors and to deplete T-lymphocytes on a large scale. There was a 1.8-, 4.6-, 3.9-, and 0.32-fold increase in CFU-GM in the four fractions relative to the unseparated component.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Relationship between IL-2 receptor expression and proliferative responses in lymphocytes from HIV-1 seropositive homosexual men.

Previous studies have shown that exogenous IL-2 does not correct the reduction in phytohaemagglutinin (PHA)-induced proliferation of lymphocytes from HIV-1 infected (HIV+) individuals. We investigated the mechanism of this reduction to determine if reduced expression of the complete IL-2 receptor (IL-2R) was responsible. In a series of experiments, PHA-stimulated lymphocytes from a total of 89 HIV- and 93 HIV+ homosexual men from the Baltimore Multicentre AIDS Cohort Study (MACS) were studied to determine the expression of messages for the alpha and beta subunits of the IL-2R, the binding of 125I-IL-2 to high affinity IL-2R, and the effect of IL-2 on cell proliferation. Compared to HIV- donors, PHA-stimulated lymphocytes from most HIV+ donors demonstrated (i) a reduction in high affinity IL-2R expression that correlated with the reduction in the IL-2-induced proliferative response; and (ii) a reduction in expression of both IL-2R alpha- and beta-chain mRNA which may be responsible for decreased high affinity IL-2R expression. However, lymphocytes from some HIV+ individuals had borderline low IL-2-induced proliferation despite normal or elevated expression of high affinity IL-2R. These results suggest that decreased expression of IL-2R may account, at least in part, for the lower proliferative response of cells from HIV+ donors.

CD4-Positive T-Lymphocytes↗
PubMed1993

Changes in T and non-T lymphocyte subsets following seroconversion to HIV-1: stable CD3+ and declining CD3- populations suggest regulatory responses linked to loss of CD4 lymphocytes. The Multicenter AIDS Cohort Study.

We investigated changes in lymphocyte subsets (total, CD4+ and CD8+ T cells, as well as non-T cells) associated with human immunodeficiency virus type I (HIV-1) seroconversion in 321 homosexual or bisexual men in the Multicenter AIDS Cohort Study (MACS). These subjects had serial lymphocyte characterizations for up to 4 years before and 5 years after seroconversion. CD4+ lymphocyte levels declined rapidly in the first 18 months following seroconversion and less rapidly thereafter, while CD8 lymphocytes increased with similar kinetics. In contrast, total T (CD3+) lymphocytes declined only slightly in the first 18 months following seroconversion, and then remained stable. These results support the hypothesis of physiologic regulation of the total number of circulating T cells, such that lost CD4+ lymphocytes are replaced by newly generated CD4+ and CD8+ lymphocytes; over time, continued loss of CD4+ lymphocytes due to HIV-1 infection would result in net replacement of lost CD4+ lymphocytes with CD8+ lymphocytes. Non-T (CD3-) lymphocytes also declined after seroconversion, and this decline paralleled that of CD4+ lymphocytes. Thus, changes in both T and non-T lymphocytes after HIV-1 seroconversion may reflect the operation of homeostatic or regulatory mechanisms. Whether these mechanisms contribute to the development of immune deficiency requires further study.

CD3 Complex↗
PubMed1993

Counterflow centrifugal elutriation: present and future.

Counterflow centrifugal elutriation (CCE) has been proposed as a method for separating heterogeneous cell populations into distinct subpopulations on the basis of different sedimentation characteristics, without impairment of cell function or yield. The advantages of this technique are the high recovery and viability of fractionated cells and the rapidity and reproducibility of results. CCE alone or in combination with other separation methods can provide homogeneous populations of cells for further investigations. Recently, CCE has been employed in clinical studies aimed at preventing GVHD in BMT recipients by depleting lymphocytes prior to BM infusion. Furthermore, ongoing studies are concentrating on the use of negative selection procedures on the fractions currently excluded from the graft with the purpose of adding these depleted fractions to the graft preparation to augment the number of stem cells, accessory cells and unselected lymphocyte subsets. In the experimental field, CCE combined with negative and positive selection techniques may be useful in the study of hematopoiesis by separating 'pure' stem cell populations from more committed hematopoietic progenitors. We review here the present and possible future applications of elutriation in the clinical and experimental field.

Cell Separation↗
PubMed1992

Characterization of YB2/0 cell line by counterflow centrifugation elutriation.

The non-secreting rat myeloma cell line YB2/0 could be separated into different cell fractions by counterflow centrifugal elutriation. The obtained fractions are analyzed by morphology studies, morphometrics, clonogenic assays and flow cytometry. The methodology is extensively described. A separation of different cell fractions according to cell cycle stages was achieved. This implies further application possibilities for clinical use like the in vitro fractionation of autologous bone marrow prior to transplantation in patients with multiple myeloma.

Animals↗
PubMed1992

Counterflow centrifugal elutriation: experimental and clinical applications.

Counterflow centrifugal elutriation (CCE) separates mixed cell populations into distinct subpopulations, on the basis of different sedimentation characteristics, without impairment of cell function or yield. The advantages of this technique are the high recovery and viability of fractionated cells, as well as the rapidity and the reproducibility of results. CCE alone, or in combination with other separation methods, can provide homogeneous populations of cells for further investigations. Recently, CCE was employed in clinical studies aimed at preventing graft-versus-host disease in bone marrow transplant recipients, by depleting lymphocytes prior to bone marrow infusion. This article reviews the principles of elutriation and describes the possible experimental and clinical applications of this technique, which seems suitable for both peripheral blood (PB) and bone marrow (BM) separation.

Bone Marrow Transplantation↗