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A D Donnenberg

Publications and source records attributed to A D Donnenberg.

71 records · Page 4Linked to original sources
PubMed1987

Implementation of a semiclosed large scale counterflow centrifugal elutriation system.

A semiclosed counterflow centrifugal elutriation system that minimizes the risks of contamination and cell loss is presented. A detailed description of the configuration of the system and its sterilization and assembly is provided along with examples of its application to large-scale separation of leukapheresis buffy coat and cadaveric bone marrow. We are currently using this system to deplete lymphocytes from human bone marrow prior to use in allogeneic bone marrow transplantation. The implementation of a semiclosed system increases the safety, ease of operation, and reproducibility of a technique that has the potential for a wide range of clinical applications.

Cell Separation↗
PubMed1987

BK virus specific humoral and cell mediated immunity in allogeneic bone marrow transplant (BMT) recipients.

BKV specific humoral and cell mediated immunity was examined weekly in 19 allogeneic BMT recipients and correlated with the occurrence of BKV shedding. Responses to staphylococcal antigen (SPL) and tetanus toxoid (TET) were measured as specificity controls. Responses and virus shedding were assayed weekly for up to 15 weeks after BMT. Fourteen of 19 patients enrolled in this study were immunized with TET on the day of transplant. Significant rises in TET specific IgG were observed in TET immunized, but not in unimmunized, subjects. Similarly, TET specific lymphoproliferative responses were observed only in TET immunized subjects when they emerged from aplasia 3 to 4 weeks later. In contrast, all subjects had positive lymphoproliferative responses to SPL, an antigen derived from a ubiquitous agent All subjects were assayed weekly for the presence of BKV in the urine as detected by ELISA. By 15 weeks, 11 (58%) of these patients shed detectable quantities of BKV antigen at least once. Subjects who excreted BKV (BKV+) maintained BKV specific IgG at pretransplant levels, whereas levels steadily declined in those who did not (BKV-) (p less than 0.001). Lymphoproliferative responses to BKV, TET, and SPL antigens were not significantly different across time or between excretion groups. The temporal relationship of BKV shedding and the expression of BKV specific immunity was investigated by examining average responses measured in 5-week intervals surrounding positive and negative antigen detection assays. Both BKV specific IgG and BKV specific lymphoproliferative responses were higher (1.8 fold, p less than 0.005 and 2.5 fold, p = 0.06, respectively) surrounding positive detection assays. These findings indicate a strong association between BKV excretion and the development of indicators of virus specific immunity following BMT.

Adolescent↗
PubMed1986

Large scale separation of human bone marrow by counterflow centrifugation elutriation.

Counterflow centrifugation elutriation (CCE) has been used to separate moderate quantities of bone marrow (BM) into distinct cell populations for further in vitro investigation. Recently, this technique was employed to reduce the incidence of graft-versus-host disease (GVHD) in human allogeneic bone marrow transplantation (BMT) by removing the majority of mature lymphocytes from the graft. Unfortunately, current methods employing the small (J6-B) elutriator rotor require time consuming preseparation steps and multiple runs. We report our experience with a fixed rotor speed, two-flow rate elutriation procedure using the new Beckman JE-10X rotor system which can separate more than 10(10) nucleated BM cells in 30 min. Three fractions were obtained which differed in size, morphology, and immunologic capacity. The large cell fraction is suitable for allogeneic BMT since it is radically depleted of mature T lymphocytes and retains high clonogeneic capacity.

Bone Marrow Cells↗
PubMed1986

Development of a simplified counterflow centrifugation elutriation procedure for depletion of lymphocytes from human bone marrow.

Graft-versus-host disease (GVHD) remains a major complication of allogeneic bone marrow (BM) transplantation. Techniques that effectively purge BM of mature T lymphocytes should reduce the incidence of GVHD and improve survival. We have developed a simplified, two-flow rate, fixed rotor speed counterflow centrifugation-elutriation (CCE) procedure that reproducibly depletes 99% of lymphocytes from Ficoll-Hypaque(F/H)-separated BM or BM buffy-coat. Two predetermined flow rates (24 and 28 ml/min) were used to purge small and intermediate-to-large lymphocytes, respectively, whereas faster sedimenting cells were recovered at the termination of the run. Lymphocyte depletion was substantiated by pan-T monoclonal antibody analysis as well as by complete loss of responsiveness to alloantigens and mitogens. Despite the lack of mature T cells, the depleted marrow fraction retained lymphoid colony-forming ability. Lymphocyte-purged marrow was obtained in high yield (72%), and retained high viability (greater than 97%) and hematopoietic colony-forming ability (greater than 99%). The ratio of total myeloid/erythroid colony-forming cells to T lymphocytes was 73-fold higher in the lymphocyte-depleted fraction than in unseparated BM. We concluded that a two-step CCE procedure can be used to rapidly deplete lymphocytes from both F/H-separated and buffy-coat BM inocula without altering hematopoietic capacity as measured by the in vitro clonogenic assays. It may be possible to adapt this procedure to the separation of the large number of marrow cells required for human BM transplantation.

Antibodies, Monoclonal↗
PubMed1986

Thymic regulation of hematopoiesis. III: Isolation of helper and suppressor populations using counterflow centrifugal elutriation.

We have evidence that thymic regulatory cells can either enhance or inhibit the growth of hematopoietic progenitors in vitro. We have suggested that two separate populations are responsible for this regulatory interaction but isolation of the cell types has proven difficult. We now report the isolation by counterflow centrifugal elutriation (CCE) of two separate populations of thymocytes which regulate erythropoiesis in coculture. We demonstrate that a minority population (less than 10%) of slow sedimenting elutriated thymocytes provide a helper function whereas the suppressor population is the majority population. Furthermore, some thymocytes of intermediate cell volume neither enhance nor inhibit erythroid colony growth. We conclude that isolation of thymic subsets can lead to identification of populations which induce cell-cell regulation of hematopoietic progenitors resulting in both a positive and negative feedback control of growth.

Animals↗
PubMed1986

Immunological heterogeneity of human monocyte subsets prepared by counterflow centrifugation elutriation.

Human peripheral blood mononuclear cells were separated into three fractions by means of counterflow centrifugation elutriation (CCE). The first fraction, eluted at a flow rate of 24 ml/min, was composed of lymphocytes with less than 2% contaminating esterase-positive cells. The cells in this fraction were incapable of responding to either soluble antigen (tetanus toxoid) or particulate antigen (cytomegalovirus-infected fibroblasts) unless recombined with accessory cells. The second fraction, eluted at a flow rate of 28 ml/min, was composed predominantly (72%) of small Ia, leu M3, and esterase-positive monocytes, which stained weakly with leu 10 antibody. Cells in this fraction efficiently presented soluble and particulate antigens to monocyte-depleted lymphocytes. Of the remaining cells, 87% were large esterase-positive monocytes that labelled strongly with Ia, leu M3, and leu 10. These cells were less efficient in antigen presentation than the small monocytes. However, lymphocytes activated with antigen-pulsed large monocytes exhibited more suppressor cell activity than those activated with antigen-pulsed small monocytes.

Antigen-Presenting Cells↗
PubMed1985

Cell-mediated immune responses to BK virus in normal individuals.

A lymphoproliferative assay was developed to study cell-mediated immunity (CMI) to BK virus (BKV), a human papovavirus, in healthy volunteer subjects. Responses to ultraviolet-inactivated antigen prepared from BKV-infected fibroblasts were compared to those elicited against a mock antigen preparation and an unrelated control antigen (tetanus toxoid, TET). CMI to BKV and TET were contrasted with humoral immunity as measured by enzyme-linked immunosorbent assay (ELISA). Specificity of the assay was confirmed by absence of response to mock antigen in all subjects studied. Positive response to BKV antigen was observed in all of 15 seropositive individuals but not in 5 neonates or 1 seronegative child. Similarly, all TET seropositive (n = 13) but no seronegative subjects (n = 2) responded to TET. The magnitude of lymphoproliferation to either antigen did not correlate with antibody titer. Additionally, the frequency of peripheral blood BKV-specific proliferating lymphocytes was determined by limiting dilution analysis (LDA). The frequency was approximately tenfold less than that observed for TET in the same group of subjects (1/30,300 vs 1/2,700). This may be due to differences in route and frequency of antigen exposure, both of which are unknown, at present, for BKV.

Adult↗
PubMed1985

Cinemicroscopic studies of lymphocyte behavior in semi-solid medium: the polyclonal origin of lymphocyte colonies.

Time-lapse cinemicroscopy was used to observe the development of lymphocyte colonies in a phytohemagglutinin-dependent one-step, two-layer agar culture system. More than 1000 h of culture time were recorded in a total of 14 independent experiments. Blast formation and organization of lymphocytes into highly motile pairs and clusters occurred early in culture (0-3 days). An increase in thymidine uptake also preceded the first detectable proliferation by 24 h. Mature lymphocyte colonies were found to be dynamic entities characterized by the continuous influx and egress of highly motile cells. Cell clusters and entire colonies were observed to locomote and on several occasions fuse to form larger structures. Macrophagelike cells located centrally within colonies appeared to play a major role in such behavior. Taken together these results conclusively demonstrate that, in the present system, lymphocyte colonies are the product of a complex pattern of cell interaction, proliferation, and cell motility and, as such, are polyclonal in origin.

Adult↗
PubMed1984

Cell-mediated immunity to cytomegalovirus (CMV) and herpes simplex virus (HSV) antigens in the acquired immune deficiency syndrome: interleukin-1 and interleukin-2 modify in vitro responses.

Peripheral blood lymphocytes (PBL) were obtained from five patients with the acquired immune deficiency syndrome (AIDS), six homosexual males with lymphadenopathy, and five normal heterosexual controls. Modulation of virus-specific immunity was assayed in vitro by measuring the lymphocyte blastogenic response and the production of lymphokine (leukocyte inhibition factor; LIF) by PBL stimulated with herpes simplex virus (HSV) or cytomegalovirus (CMV) antigens in the presence or absence of interleukin-1 (IL-1) and interleukin-2 (IL-2). PBL from the control and lymphadenopathy subjects responded to both antigens in the lymphocyte transformation assay (LT) measured on day 7, and the responses were significantly enhanced in cultures grown in the presence of antigen and IL-2 (1 U/ml). PBL from the AIDS patients were unresponsive, but responsiveness was restored by the addition of IL-2. The addition of IL-1 (0.02 micrograms/ml) to antigen-stimulated PBL cultures failed to enhance the proliferative responses in all three study groups. LIF production was assayed in the supernatants from day 1 PBL cultures. LIF was not produced by PBL from AIDS patients grown in the presence of viral antigens, whereas three of five patients from the lymphadenopathy group, and three of five control subjects gave rise to positive responses. The addition of IL-1 to the antigen-stimulated cultures enhanced LIF production in the control and lymphadenopathy groups but not in the AIDS patients. The addition of IL-2 did not modulate LIF production by antigen-stimulated PBL from the control or AIDS patients while suppressing the LIF response of the similarly stimulated PBL from the lymphadenopathy patients.

Acquired Immunodeficiency Syndrome↗
PubMed1984

Secondary immunization with a protein antigen (tetanus toxoid) in man. Characterization of humoral and cell-mediated regulatory events.

Tetanus toxoid (TET) was used as an immunogen to explore regulatory events occurring after secondary immunization in man. Changes in dose-response patterns, kinetics, and frequency of antigen-reactive cells in the peripheral blood, and the serum antibody titres were studied. The most striking feature of these studies was the finding that immediately after immunization, a brief period of decreased responsiveness preceded the expected amplification of antigen-specific humoral and cell-mediated responses. One day after immunization serum antibody levels declined to approximately half their initial values before the expected increase in titre. These results could not be explained by the formation of antigen-antibody complexes. Lymphoproliferative responses were also depressed 1 day after immunization but increased in both magnitude and sensitivity from 7 to 28 days after challenge. A decrease in thymidine incorporation evident at supraoptimal antigen concentrations also became progressively more prominent during this interval. Cell mixing experiments documented the presence of a radioresistant population present 1 day after immunization capable of suppressing TET-specific lymphoproliferative responses of autologous lymphocytes obtained before immunization. These suppressors are preferentially activated at high antigen concentrations. Limiting dilution analysis revealed a 30-fold increase in TET-reactive proliferating cells by 28 days after immunization. Expansion occurred first in cells recognizing high antigen concentrations and subsequently in cells recognizing lower doses. Taken together, these findings present a consistent if somewhat counterintuitive picture of some of the regulatory processes accompanying booster immunization. The earliest detectable event appears to be the application of an active suppressor mechanism, release from which signals initiation of those processes classically associated with an anamnestic response.

Adult↗
PubMed1982

Immunity to herpes simplex virus type 2. IV. Impaired lymphokine production during recrudescence correlates with an imbalance in T lymphocyte subsets.

Cell-mediated immunity (CMI) to herpes simplex virus type 2 (HSV-2) was studied in patients with recurrent disease. Peripheral blood lymphocytes (PBL) obtained during recrudescence (0 to 3 days post-onset of lesion), convalescence (4 to 14 days), and quiescence (greater than 14 days), which were cultured in vitro in the presence of UV-inactivated HSV-2 (G) antigen, demonstrated similar, virus-specific lymphoproliferative responses. However, the production of the lymphokine, leukocyte migration-inhibition factor (LIF), was completely suppressed during recrudescence. During convalescence, LIF production returned to levels similar to those of PBL from seropositive controls (without a history of recurrent subsets revealed a significant increase in the proportion of T8+ and Ia+ cells during recrudescence and at prodrome (24 to 48 hr before onset of lesions). Glass adherence reduced the proportion of both T8+ and Ia+ cells and restored early (1 day) LIF activity, and recrudescent PBL suppressed the proliferation of autologous T cells in presence of UV-HSV-2 (G) antigen. The expression of both phenotypes and LIF production returned to control levels during convalescence.

Antibodies, Monoclonal↗
PubMed1982

Cell-cell cooperation in lymphocyte colony formation: studies in human allogeneic marrow transplantation.

The pace of restoration of phytohemagglutinin- (PHA) stimulated lymphocyte colony-forming response was investigated in 50 bone marrow transplant (BMT) recipients (49 allogeneic, 1 syngeneic) studied on 86 occasions. On the majority of occasions (28 of 48), patients studied early (3 to 8 wk) after BMT failed to make detectable lymphocyte colonies, whereas patients studied late (greater than 6 mo) after BMT displayed responses comparable to those of healthy adult volunteers. The sequential study of 10 of the 15 patients with positive lymphocyte colony-forming responses early after BMT revealed that this response is short-lived. The results of recipient donor mixing experiments and experiments involving the addition of the lymphokine interleukin 2 (IL 2) to recipient cells indicated that lymphocytes obtained from unresponsive recipients evaluated early after BMT can be driven to colony formation, and suggest that the major limiting component in such subjects is functional T help.

Bone Marrow Transplantation↗
PubMed1980

Immunity to herpes simplex virus type 2: cell-mediated immunity in latently infected guinea pigs.

Cell-mediated (CMI) and humoral immunity to herpes simplex virus type 2 (HSV-2) were evaluated in infected strain 13/N guinea pigs with (45%) and without a history of recurrent herpetic disease. Virus was isolated by cocultivation from active herpetic lesions (9 of 10) as well as from the footpads (17 of 38), sacral ganglia (7 of 21), and sciatic nerves (1 of 21) of asymptomatic animals. Viral isolates grew in cells of human origin and were neutralized by hyperimmune anti HSV-2 sera. Humoral immunity measured by the presence of virus-neutralizing antibody was similar in both experimental groups. The involvement of CMI in recurrent disease was assessed by comparing lymphocyte transformation (LT) and leukocyte migration inhibition factor (LIF) responses in animals with a history of recurrent disease studied while asymptomatic (quiescent) and in animals without clinical evidence of recurrent disease (seropositive controls). Spleen cells from quiescent animals evidenced significant impairment of both LIF and LT responses as reflected in the requirement of higher antigen concentrations (up to 58-fold) and longer in vitro culture periods (up to 2.5 days) to mount responses comparable in magnitude to those observed in the seropositive control groups. Peripheral blood lymphocyte cultures obtained from quiescent animals showed similar impairment of LIF responses but displayed intact LT response. The data suggest that recurrent disease is associated with an impairment in the generation of anamnestic effector functions as reflected by altered kinetics and dose response patterns in in vitro secondary responses.

Animals↗