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Biomedical subjects

R E Longley

Publications and source records attributed to R E Longley.

29 records · Page 2Linked to original sources
PubMed1991

A rapid colorimetric microassay to detect agonists/antagonists of protein kinase C based on adherence of EL-4.IL-2 cells.

A rapid, colorimetric, microassay for detection of agents which are known agonists/antagonists of protein kinase C (PKC) was developed, utilizing their effects on adherence of EL-4.IL-2 cells. Cells that were incubated with agents which are known inducers of PKC activation, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), mezerein and indolactam V, readily adhered to wells of 96 well microtiter plates within 1-2 hours, whereas cells incubated with the negative PKC activator, 4 alpha-phorbol 12-myristate 13-acetate (4 alpha-PMA), which is structurally related to PMA (4 beta-PMA), did not adhere. The adherent cells withstood repeated vigorous washings with tissue culture medium. Adherence of EL-4.IL-2 cells in the presence of PMA could be blocked by the addition of two known inhibitors of PKC, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride and staurosporine. Detection of the presence of adherent cells was accomplished by the addition of a tetrazolium salt to culture wells and determination of the remaining viable cells by scanning using a multiwell spectrophotometer (ELISA reader). The EL-4.IL-2 adherence assay meets several important criteria for use as a primary screen in the detection of potential PKC agonists/antagonists, i.e. its selectivity, simplicity, rapid performance through automation and reproducibility.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Recovery of functional human lymphocytes from Leukotrap filters.

We report here a method by which functional human PBL may be recovered from a blood product preparation unit (Leukotrap) which is normally employed by blood banks to prepare a leukocyte-poor blood product and which is commonly discarded after use. Blood filters which were back-flushed with cold, phosphate-buffered saline (PBS) and the resulting buffy coats collected and separated on Histopaque gradients yielded an average of 2.67 x 10(8) (+/- 1.17 x 10(8] cells per filter processed (n = 38), with viability greater than 95%. The percentages of cell types recovered before or after Histopaque separation closely approximated those reported for freshly drawn blood when examined using differential staining and morphological criteria. Fluorescent antibody analysis revealed a normal distribution of Leu-4 (T), Leu-12 (B), Leu-3a (T helper), Leu-2a (T suppressor) and Leu-M3 (macrophage) positive cell subsets. Recovered lymphocytes were fully responsive to phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed (PWM) mitogens. Random donor lymphocytes which were co-cultured in a two-way mixed lymphocyte response (MLR) were fully capable of proliferative responses compared to the same populations cultured alone. The results from our study indicate that Leukotrap filters provide a readily available, easily processed and convenient source of functional human PBL for use in the in vitro study of the human immune system.

Cell Separation↗
PubMed1989

Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells.

The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated. Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions. Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay. PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period. In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets. Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity. The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity. This suppression can be reversed both in vitro and in vivo by indomethacin. This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.

Animals↗
PubMed1986

Characterization of a newly established feline lymphoma-derived cell line (BKD) lacking T and B cell surface markers.

A new feline lymphoma-derived cell line, designated BKD, was isolated from an anterior mediastinal tumor. Cells of this line were characterized as lymphoid based on morphology, the lack of intracellular esterase and peroxidase activity, and absence of phagocytic function. In contrast with other established feline lymphoma-derived cell lines, cells of the BKD line lack characteristics of both feline T-cells and B-cells in that they neither form rosettes with guinea pig erythrocytes nor have demonstrable surface or cytoplasmic immunoglobulin. Approximately one third of BKD cells form EAC rosettes, a significant number of rosette forming cells (p = 0.0001) when compared to background sheep E-rosetting activity. In addition, a consistently titratable level of interleukin-2-like activity was produced when BKD cells were coincubated with concanavalin A and phorbol-12-myristate-13-acetate. Chromosome analysis showed that a majority of BKD cells are diploid. This new cell line has been continuously replicating in culture for over one year and produces feline leukemia virus as demonstrated by several analyses.

Animals↗
PubMed1986

Leukemia prevention and long-term survival of AKR mice transplanted with MHC-matched or MHC-mismatched bone marrow.

The current studies were designed to evaluate the effectiveness of marrow transplantation within and outside the major histocompatibility complex (MHC) on the long-term survival and occurrence of spontaneous leukemia in AKR mice. AKR mice, which were lethally irradiated and received MHC-matched marrow from CBA/J mice (CBA----AKR), never developed leukemia and were alive and remained healthy for up to 280 days post-transplant. These long-term surviving chimeras possessed substantial immune vigor when both cell-mediated and humoral responses were tested. Lethally irradiated AKR mice, which had received MHC-mismatched marrow (anti-Thy-1.2 treated or nontreated) from C57BL/6J mice (B6----AKR), never developed leukemia and survived up to 170 days post-transplant. However, both groups of these chimeras began dying 180 to 270 days post-transplant due to a disease process which could not be readily identified. Histological analysis of B6----AKR chimeras revealed severe lymphoid cell depletion in thymus and spleen; however, none of these chimeras exhibited classical features of acute graft versus host disease. Concanavalin A mitogenesis, primary antibody responses to sheep red blood cells and the production of interleukin 2 (IL-2) were suppressed in B6----AKR chimeras. IL-2 treatment of B6----AKR chimeras was shown to partially correct these deficiencies without stimulating mixed lymphocyte responsiveness to donor or host lymphocytes. These studies indicate that the use of MHC-mismatched marrow for the prevention of spontaneous AKR leukemia may rely on augmentative IL-2 therapy for complete immune reconstitution of leukemia-free chimeras.

AKR murine leukemia virus↗
PubMed1986

Natural killer cells in the blood and bone marrow of the rhesus monkey.

Natural killer (NK) cells in peripheral blood lymphocytes (PBL) and bone marrow (BM) cells of the rhesus monkey were detected by their functional activity against K562 cells. Animals could be grouped into "high" or "low" NK responders, a trait found to be consistent over a period of 2 years. NK active cells in PBL were in the nonadherent population, with the majority bearing Fc receptors and a further subdivision of these into CR+ (complement receptor) and CR- NK cells. Of 10 monoclonal antibodies directed against different epitopes of human lymphocytes, OKT11, OKT10, and Leu 11 showed reactivity with rhesus NK cells. Only OKT10 was reactive with the effector site of the cell, as shown by its capability to block NK function. Of the Leu 11 monoclonal antibodies (a, b, c), Leu 11c was nonreactive while Leu 11a and Leu 11b were shown by immunofluorescence to bind to 7 to 21% of PBL; Leu 11b was also cytotoxic to the NK cells. Leu 11b did not prevent binding of Leu 11a to PBL, suggesting reactivity of these antibodies with different epitopes. Percoll fractionation of PBL and BM revealed a greater enrichment of NK activity with BM; also, with PBL peak NK activity occurred in fractions 4 and 5 while this occurred in fraction 5 with BM. Although Percoll PBL fractions contained a higher percentage of Leu 11b cells, the NK activity of the BM fractions was proportionately greater. The majority of PBL cells with NK activity were FcR+ while significant activity could be attributed to FcR- cells of BM, in both the unseparated and Percoll fractions of each tissue. The data suggest NK active cells of BM may be distinct from those found in PBL.

Animals↗
PubMed1981

The influence of dietary fat and non-specific immunotherapy on carcinogen-induced rat mammary adenocarcinoma.

The incidence of mammary adenocarcinoma in Sprague-Dawley female rats, caused by the carcinogen 7,12-dimethylbenz(alpha)anthracene, was influenced by the level of dietary fat fed after exposure to carcinogen. Carcinogen was given by stomach tube to 50-day-old rats, and tumors were evaluated when rats were 9 months old. Rats on diets containing 20% unsaturated fat had a tumor incidence of 97%, while rats changed to a low-fat diet (2% unsaturated fat) three or four weeks after exposure to the carcinogen had an incidence of 45%. Some rats on each diet were given two treatments with the methanol extraction residue of Bacillus Calmette-Guerin, either three and five weeks after carcinogen or four and six weeks after carcinogen. Tumor incidence in the treated group and the untreated group was the same when rats were maintained on the high-fat diet, but tumors in the treated group were larger and the disease was more severe by histological criteria. These tumors were more anaplastic and many were extensively infiltrated with lymphocytes compared to the untreated group. Tumor incidence was significantly lower in rats changed to the low-fat diet (45%) than in those on the high-fat diet (97%), and tumor incidence was reduced to 20% when rats changed to the low-fat diet were treated with methanol extraction residue. The treated group had less severe disease than the untreated group on the low-fat diet. Only half the tumor-bearing rats in this group had malignant tumors, and none were invasive. Methanol extraction residue protected most rats on the low-fat diet against mammary adenocarcinoma, and reduced the severity of disease in those rats that did develop tumors. Methanol extraction residue treatment provided no protection, and even increased the severity of disease, in rats on the high-fat diet.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1979

Thymosin restoration of cellular immunity to Blastomyces dermatitidis in T-cell-depleted mice.

The immunopotentiating properties of thymosin in thymectomized, lethally irradiated, bone marrow-reconstituted mice (ThyXBM) were characterized, using footpad sensitivity to Blastomyces dermatitidis. Normal mice were shown to exhibit increasing delayed-type hypersensitivity responses to killed yeast cells of B. dermatitidis after injections of the organism on days 0 and 7, as measured by footpad swelling tests. The footpad response of normal thymosin-treated mice was similar to that of normal, non-thymosin-treated mice. ThyXBM mice were unable to elicit a footpad response when similarly injected and footpad tested with B. dermatitidis. Thymosin-treated ThyXBM mice responded to footpad testing at a level that was 62% greater than the response seen in non-thymosin-treated ThyXBM mice. This peak response occurred on day 12. The results indicated that thymosin was unable to enhance immune responses of normal intact mice but could restore immunocompetence in a T-cell-depleted host, as measured by footpad sensitivity to B. dermatitidis.

Animals↗
PubMed1979

Inhibition of lymphocyte function in rats fed higher-fat diets.

Concanavalin A-induced blastogenesis of spleen lymphocytes was significantly inhibited when lymphocytes from rats on a high-polyunsaturated-fat diet were compared to lymphocytes from rats on a low-fat diet. Responsiveness was dependent on source of serum since lymphocytes from rats fed a low-fat diet were suppressed in serum from rats fed a high-polyunsaturated-fat diet. Alternatively, lymphocytes from rats on a high-polyunsaturated-fat diet were more responsive in serum from low-fat-fed rats compared to their response in autologous serum. One of the inhibiting factors in serum was the lipoprotein fraction; however, rats on a high-polyunsaturated-fat diet probably had additional inhibitors in their serum. While tumor incidence was highest in rats with the least responsive lymphocytes was highest in rats with the least responsive lymphocytes and lowest in rats with the most responsive lymphocytes, the significance of the observation is not known.

9,10-Dimethyl-1,2-benzanthracene↗
PubMedDate not supplied

Immunosuppressive compounds from a deep water marine sponge, Agelas flabelliformis.

Two immunosuppressive compounds, 4 alpha-methyl-5 alpha-cholest-8-en-3 beta-ol and 4,5-dibromo-2-pyrrolic acid were isolated from a deep water marine sponge, Agelas flabelliformis. Their structures were determined by comparison of their spectral data with those of samples isolated from other organisms. Both compounds were highly active in suppression of the response of murine splenocytes in the two-way mixed lymphocyte reaction (MLR) with little to no demonstrable cytotoxicity at lower doses. In addition, 4,5-dibromo-2-pyrrolic acid suppressed the proliferative response of splenocytes to suboptimal concentrations of the mitogen, concanavalin A (Con A). These results describe for the first time compounds isolated from the marine sponge A. flabelliformis that possess potent in vitro immunosuppressive activity.

Animals↗