David S. Gordon

Coordinate and independent effects of heroin, cocaine, and alcohol abuse on T-cell E-rosette formation and antigenic marker expression

Simultaneous and independent use of cocaine and alcohol by heroin addicts was shown to variably modulate the ability of their T cells to form E-rosettes with sheep erythrocytes (E). As reported previously, the percentages of E-rosette-forming T cells of both active and total types were depressed in association with heroin addiction. We show here that the kinetic curve of the rate of E-rosette formation is also depressed by heroin use and that the use of cocaine but not alcohol by heroin addicts reverses depression of E-rosette formation by heroin. The percentages of E-rosette-forming T cells from the bloods of heroin addicts who used both alcohol and cocaine, as well as the kinetic rate curves of E-rosette formation, were intermediate between the essentially normal levels found for heroin addicts who used cocaine and the severely depressed levels evident for users of heroin alone or heroin plus alcohol. Modulation of the levels of E-rosette formation by alcohol used in conjunction with cocaine and/or heroin was variably dose dependent. Polydrug effects evident by analyses of E-rosette formation were not seen when the percentages of lymphocytes reactive with LYT-3 (anti-E-receptor, 9.6 epitope) and OKT-3 (anti-total T cell) monoclonal antibodies were assessed cytofluorometrically, although the data suggested that subnormal percentages of LYT-3+ T cells were present when heroin addicts also used cocaine. These findings are relevant to basic understanding of T-cell physiology from a neuroimmunological perspective and also suggest ways that addictive drugs may modulate the immunocompetence of drug addicts.

Treatment of acute myelogenous leukemia: influence of three induction regimens and maintenance with chemotherapy or BCG immunotherapy.

The effect of a synchronizing‐recruiting drug schedule vs. myelotoxic therapy on remission rate and of Bacillus Calmette‐Guerin on remission duration and survival of adults with acute myelogenous leukemia were studied in a prospective cooperative trial. After randomized remission induction with Arabinosyl Cytosine + vincristine + methotrexate + leucovorin (AVML), thioguanine + Ara‐C + Daunorubin (TAD), or Daunorubicin + Ara‐C (DA), complete remissions (CR) were consolidated with TAD or AVML, CRs were maintained with BCG vaccination (Tice strain) by the tine technique, or BCNU plus Ara‐C (B/A), or no further therapy (NFT). Of 209 evaluable TAD patients, 105 (50%) achieved CR; of 187 DA, 97 (52%) achieved CR. AVML yielded only 15 CR among 59 patients (25%). The time to remission was significantly shorter with DA compared with TAD. Ninety‐seven patients were randomized to maintenance therapy (35 B/A, 30 BCG, 32 NFT). There were no differences in remission duration (7, 8, 6 months) or survival (16, 22, 16 months, respectively). Manipulation of the cell cycle, as employed in this study, was not helpful. There may be a marginal effect of BCG, but our data fail to show a statistically significant benefit.

Reproducibility, efficacy and methodology of mitogen-induced lymphocyte transformations by the whole blood assay

For the past few years, the whole blood culture method has been scrutinized for its usefulness and reproducibility in evaluating the immunologic status of patients. This method, used in our laboratory mainly screening specimens for evidence of immunodeficiencies, has been systematically evaluated for reproducibility, and factors affecting normal response through testing specimens from over 70 normal persons. Three mitogens (phytohemagglutinin, Concanavalin A, and pokeweed) were studied; peak time responses occurred later than in the separated cell culture method, and they were different for each mitogen. The dose response curves also depended on the mitogen tested, with pokeweed giving a sharp curve and the others a broad plateau. The harvesting procedure was studied by varying reagents and was optimized for speed and efficiency. The mononuclear cell count of the specimen had little effect on the results as long as it remained less than 3.5 X 10(6)/ml. The results for all mitogens followed a similar distribution curve irrespective of whether the results were expressed as cpm or cpm divided by the mononuclear cell count. The use of the stimulation ratio method to express results was less satisfactory. The mean coefficient of variation for all triplicate samples remained 20% or less and for all conditions tested. In evaluating the immunodeficient patient, the whole blood culture method was found to be equally as informative and easier to perform than the separated cell method.

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity: II. Reversibility of the heat inactivation of K cell killing and relationship of Fc receptor capping to lysis

Abstract The heat inactivation of human blood mononuclear cells active in antibody-dependent cellular cytotoxicity (ADCC) is largely reversed after 24 hr in culture at 37 °C. The reactivation process is inhibited by actinomycin D, cycloheximide, and emetine but not by mitomycin-C, indicating that recovery requires RNA and protein synthesis but not DNA synthesis. The ability of lymphocytes to cap surface immunoglobulin (SIg) and IgG-Fc receptors (FcR) was also studied. As with ADCC effector cell activity, both SIg and FcR capping were abolished by heating, and the kinetics of inactivation was similar to that of the inactivation of ADCC effector activity. In addition, the heat inactivation of capping was reversible in culture and followed kinetics of reactivation similar to that of K cell reactivation. These results suggest the participation of heat-labile proteins at or near the surface of the effector cell, which are also apparently involved in the capping of surface receptors. Presumably these heat-labile proteins are membrane-associated enzymes, but they may also be cytoskeletal structures such as microfilaments or microtubules whose heat-sensitivity is currently unknown. The mounting of lethal hits may involve the same membrane machinery which is responsible for capping or a capping process itself.

Serial immunologic assessment during a randomized trial of chemoimmunotherapy in acute myelogenous leukemia

SummaryThirty-one adults who had acute myelogenous leukemia and in whom remission had been induced and consolidated with chemotherapy were randomized to receive one of three maintenance schedules: (A) BCG + chemotherapy [1, 3-bis-(2-chlorethyl)-1-nitrosourea (BCNU) and cytosine arabinoside]; (B) splenectomy, followed 1 week later with BCG and chemotherapy; or (C) allogeneic leukemic cells, BCG, and chemotherapy. Serial immunologic assessments were performed at the onset of maintenance and every 3 months.No differences were found in duration of remission (median 209 days) or survival (median 454 days) among the three schedules. Six patients remain in remission after from 2−4 + years. Skin test responses, mitogen responses, mixed lymphocyte culture responses, antibody responses, and T and B lymphocyte numbers were depressed at the onset of maintenance therapy. Therapy clearly improved the state of anergy as defined by recall antigen responsiveness, and induced in vivo and in vitro PPD reactivity. However, immunotherapy resulted in a reduction of the number of T or B cells and of the in vitro lymphocyte response to mitogens and allogeneic cells. Serum obtained at diagnosis and during remission inhibited in vitro blastogenic responses in more than half the patients. These data indicate that chemoimmunotherapy given as described tended to be more immunosuppressive than stimulatory.

Antibody-dependent cellular cytotoxicity to virus-infected target cells: Role of nylon wool-adherent T cells as effectors

Abstract This study was designed to elucidate whether populations of human blood lymphocytes other than non-T, non-B, Fc receptor-positive K cells can mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells acutely infected with type 1 herpes simplex virus. With appropriate technical precautions, a subset of E rosette-positive, nylon wool-adherent T cells were found to be effective in killing antibody-coated target cells. Thus, there appears to be at least two relatively distinct populations of lymphocytes in human peripheral blood that function as effectors in ADCC: one that consists of E rosette-negative cells, the other of E rosette-positive cells. These findings suggest a possible relationship between “classical” E rosette-negative K cells and E rosette-positive T cells.

The use of 2-deoxy-D-glucose to assess changes in tumor target cell membranes in vitro

Following short-term suspension culture, cells from the Balb/C sarcoma Meth A were allowed to incorporate both [14C] leucine and 2-deoxy-D-glucose-1-[3H] (2DG). The 2DG is trapped as a small anionic marker of the cytosol. Deviation from the kinetics of spontaneous efflux of the markers is interpreted as reflecting perturbation of the target cell membrane. In the presence of guinea pig complement and a rabbit antiserum to Meth A, enhanced 2DG efflux was effected in a titer comparable to that detected with a 51Cr-release assay. With a number of alloantisera and syngeneic immune sera, 2DG efflux was enhanced while 51Cr-release was unaffected. Only in the presence of syngeneic immune sera from mice bearing a low tumor mass, syngeneic splenic leukocytes effect a retardation in the spontaneous 2DG efflux. Sera from animals with a large tumor mass were ineffective. Effux of proteins labeled with [14C] leucine was not altered. The phenomenon was not dependent on the presence of a heat-inactivatable syngeneic complement source. The method described provides a sensitive probe of target cell membrane permeability in the tumor model studied. The phenomenon detected is the capacity of serum, sampled relatively early in syngeneic oncogenesis, to direct syngeneic splenic leukocytes to interact with the target cell membrane differentially altering its permeability to the small cytosol marker.

CHARACTERIZATION OF THE EFFECTOR CELL IN AN ANTIBODY DEPENDENT CELL MEDIATED CYTOTOXICITY SYSTEM

Publisher Summary This chapter discusses the characterization of the effector cell in an antibody-dependent cell-mediated cytotoxicity system. A study described in the chapter utilized herpes simplex virus (HSV) type-I-infected Chang liver cells coated with human anti-HSV antibody as targets and human mononuclear cell preparations as effectors. Human peripheral blood mononuclear cells (MC), after Ficoll/Hypaque separation, were further purified by a variety of techniques, including carbonyl iron depletion, plastic adherence, nylon wool adherence, and E rosette depletion, alone and in combination. Evaluation of membrane markers was done at all stages of the separation procedures. Plastic adherence routinely removed 80–90% of the latex positive monocytes from the MC preparations but did not lead to depletion of the ADCC activity, suggesting that the major effector cell was not a monocyte. Carbonyl iron depletion experiments also suggested that the effector cell was not a monocyte, though this technique is even more efficient in removing latex-positive cells.

IN VITRO INDUCTION AND ASSAY OF HELPER T-CELLS USING UNPRIMED PURIFIED MOUSE CELL POPULATIONS

Publisher Summary This chapter describes in vitro induction and assay of helper T cells using unprimed purified mouse cell populations. 15 × 106 nylon-wool-purified, cortisone-resistant thymocytes and 5 × 105 peritoneal exudate macrophages purified by glass adherence, scraping, and dead-cell removal are cocultured with carrier protein in Marbrook–Diener culture chambers. After 4 days, 5–10 × 105 viable cells (or supernatant in graded doses) are added with haptencarrier conjugate to cultures containing 15 × 106 spleen cells from mice primed with DNP–ficoll 3–4 months earlier. After an additional 4 days, triplicate cultures are assayed for high affinity DNP-specific plaque-forming cells (PFC) using a hapten inhibitable hemolytic plaque assay technique. The capacity of cells (or supernatant) from the helper cell culture to enhance the anti-DNP PFC response of the recipient spleen cell culture to DNP-carrier conjugates is the assay for helper cell induction. The induction of helper cells requires that the T-cells, but not macrophages, proliferate prior to the manifestation of helper cell function. Once generated, however, the active cell is functionally resistant to the anti-mitotic effect of mitomycin C.