PubMed Health⌕ Search

Biomedical subjects

J L Pauly

Publications and source records attributed to J L Pauly.

At least 37 records · Page 2Linked to original sources
PubMed1988

Long-term cultivation of functional human macrophages in Teflon dishes with serum-free media.

Reported herein are the results of studies demonstrating the utility of a chemically defined, serum-free medium designated as AIM-V (GIBCO) for the long-term (greater than 2 weeks) cultivation of functionally-defined human macrophages. The AIM-V medium is a mixture of HEPES-buffered Dulbecco's Modified Eagle Medium and Ham's Nutrient Mixture F12 that had been supplemented with purified human albumin, transferrin, insulin, and a proprietary mixture of purified factors. Nonadherent macrophages for serial studies were generated in petri dishes with a Teflon liner that had been seeded with a heterogeneous population of peripheral blood mononuclear cells of healthy human adults. For comparison, cultures were initiated with serum-free medium AIM-V and medium supplemented with AB/Rh+ serum or freshly collected autologous serum. Viability was defined by trypan blue dye, glass adherence, and phagocytosis of yeast. Macrophages were characterized by light microscopy, cytochemistry, and phenotypic analysis. Ultrastructural morphology was defined by scanning electron microscopy. These studies demonstrate that functionally defined human macrophages can be sustained in long-term culture with the use of serum-free medium that has not been augmented with mitogenic stimulants, growth-promoting lymphokines/monokines, or differentiation-inducing agents. Serum-free medium AIM-V, which has been approved for generating lymphokine, (i.e., interleukin-2; IL-2)-activated killer cells (LAK) for IL-2/LAK adoptive immunotherapy modalities, may also prove useful for studies defining and isolating regulatory proteins produced by activated monocytes and macrophages and for generating cytolytic macrophages for different antitumor regimens.

Adult↗
PubMed1988

A simple radioimmunoassay of human interleukin-2.

We have developed a liquid phase radioimmunoassay of human interleukin-2 which uses inexpensive commercially available reagents exclusively. The assay is simple, reproducible and specific in detecting different batches of human interleukin-2 of natural as well as recombinant origin, but not detecting recombinant murine interleukin-2. The assay is sensitive to a concentration of approximately 0.05 ng/ml and can be used in measurement of IL-2 in serum containing culture media.

Antibodies↗
PubMed1988

Identification of the interleukin-2 receptor (IL-2R) on human leukemic T cells using colloidal gold and scanning electron microscopy.

Results of studies demonstrating the identification of the interleukin-2 receptor (IL-2R; i.e., anti-Tac) on the membrane ultra-structure of human leukemic T cells with an antibody carrying an electron dense colloidal gold microsphere (e.g., immunogold) that was visualized using a scanning electron microscope (SEM) are reported. Our IL-2R model system employed HTLV-1 retrovirus-infected lymphoblastoid cells of the long-term human leukemic T cell line HUT-102B2. The presence of the IL-2R on these cells was defined using a double antibody procedure that employed as the primary antibody a purified mouse monoclonal anti-Leu-IL-2R antibody (mIgGlk, anti-Tac, CD25), and used as the secondary antibody a goat anti-mouse IgG (gamma-chain specific) antibody that had been covalently bonded to a 40 nm colloidal gold particle. More than 95% of the HUT-102B2 were IL-2R+, and there was a uniform distribution of the IL-2R over the surface of the cells. Corresponding controls were employed in all examinations and included IL-2R- Jurkat human leukemic T cells and isotype identical immunoglobulins. The primary and secondary antibody reagents contained whole human serum and bovine serum albumin, and there was no evidence of the non-specific binding of these antibodies. These studies are the first to demonstrate the presence of a lymphokine receptor on the surface architecture of a cell. We anticipate no difficulty in applying the immunogold/SEM technology to define both normal and malignant cell membrane receptors for other cytokines.

Cell Line↗
PubMed1987

Influence of the administration schedule on the therapeutic effect of interleukin-2.

The local and systemic therapeutic effects of multiple peritumoral injections of recombinant human interleukin-2 (IL-2) were tested against the syngeneic, immunogenic mammary carcinoma MC2 implanted subcutaneously into C3H/He mice. Multiple (12) low-dose injections had a greater local therapeutic effect than the same total amount of IL-2 given in 2 injections. Peri-tumoral injections were equally effective in inhibiting the growth of the injected tumors in normal and in immunized mice. The systemic therapeutic effect was manifested by the inhibition of tumors implanted contra-laterally to the injected tumors, and was only discernible in non-immunized mice.

Animals↗
PubMed1987

Activation and long-term proliferation of human cord blood T cells with human recombinant interleukin-2.

We report here the results of in vitro studies that demonstrate that purified human recombinant interleukin-2 (hrIL-2) is a potent stimulant for freshly isolated cord blood lymphocytes of healthy full-term infants. Different culture parameters were studied to define the optimal conditions for eliciting maximal levels of activation. Highest levels of hrIL-2-induced TdR[3H] uptake were recorded on day 7 for cultures containing 100 U hrIL-2/ml. Results of comparative studies demonstrated that the reactivity of cord blood lymphocytes to hrIL-2 was equal to, if not greater than, that of healthy adult lymphocytes. Cord blood cells that had been activated with hrIL-2 could be propagated in long-term (greater than 30 days) cultures as hrIL-2-dependent lines, and these lines could be initiated with a high degree of success. Phenotypic analysis was performed using different monoclonal antibodies and cytofluorometry, and studies characterizing cells of the long-term lines have shown that they consisted of a heterogenous population of T4 helper and T8 cytotoxic/suppressor cells; in some instances, natural killer (NK) cells were also present. Other experiments demonstrated that hrIL-2-activated and hrIL-2-propagated T cells expressed the IL-2 receptor (IL-2R; defined by monoclonal antibody anti-Tac) and the number of IL-2-R-positive cells could be increased two-fold or more by exposing the cells to a phorbol ester. This report provides additional information to support the hypothesis that hrIL-2 not only sustains T cell proliferation (i.e., second signal) but also induces T cell activation (i.e., first signal).(ABSTRACT TRUNCATED AT 250 WORDS)

Culture Media↗
PubMed1986

Effect of human recombinant tumor necrosis factor on the growth of different human and mouse long-term hematopoietic cell lines.

Previous studies have demonstrated that one of the most salient features of tumor necrosis factor (TNF) is its ability to induce tumor necrosis in vivo, and the specificity of its cytotoxic/cytostatic activity for tumor cells has been demonstrated in in vitro studies in which this lymphokine has been shown to kill cultured cells of malignant lines and to have no effect on cells of normal diploid lines. Studies described herein defined the effect of highly purified human recombinant TNF on cells of 34 different human and murine hematopoietic cell lines, particularly human leukemic T and B cells of long-term lymphoblastoid cultures. Results of these studies demonstrated that TNF at concentrations of 3,600 U/ml had no significant effect on the growth of these cells as defined by cytotoxicity, measured with the use of the trypan blue dye-exclusion assay and as defined by cytostasis, assayed by the enumeration of cells and the uptake of [3H]-thymidine and -uridine. In contrast, positive control cultures of TNF-sensitive cells from a murine tumor (L-M/clone L-929, connective tissue) displayed at 50% (LD50) reduction in growth by TNF at approximately 5 U/ml. Likewise, human tumors (MCF-7, breast, and HT-29, colon) were also highly sensitive (LD50 less than 100 U/ml). These studies demonstrate that T and B cells of lymphoblastoid lines as well as cells of other hematopoietic lines display little or no sensitivity to TNF.

Animals↗
PubMed1986

Immunofluorescent patterns of spectrin in lymphocyte cell lines.

Spectrin, a membrane-associated cytoskeletal protein, has been observed in all of 45 lymphoid and myeloid cell lines examined. For these experiments, formalin-fixed cells from randomly selected lines propagated by using conventional tissue culture procedures were examined by immunofluorescence, using an antibody directed against chicken erythrocyte alpha-spectrin. Two distinct immunofluorescent patterns of spectrin distribution were identified. In most lines examined (16 mouse and 18 human lymphoid or myeloid lines), spectrin was symmetrically distributed near the submembranous region of the plasma membrane. In the remainder of the cell lines examined, a second pattern was observed; in these cultures, the cells contain a polar submembranous aggregate of spectrin with little staining at the rest of the plasma membrane. Long-term T lymphocyte cell lines in which greater than 60% of the cells expressed a polar submembranous aggregate of spectrin (PSA-S) include mouse cell lines EL-4, LBRM-33, CT-6X, NIXT, 22CM-37, and 7ON-2 and human lines JM and PEER. Other established cultures in which PSA-S were observed included the human macrophage-like line U-937 and gibbon T cell line MLA-144. Phorbol myristate acetate or mezerin caused a reversible alteration in the distribution of spectrin in these cell lines. These drugs, which increase membrane fluidity, caused a complete but temporary symmetrical redistribution of the spectrin aggregate. Our results indicate that the pattern of spectrin distribution, either aggregated or evenly dispersed, is a stable characteristic (but one that can be altered) in various cell lines, and that because similar variations in pattern have been noted in situ, it is likely that the pattern present in any given cell line reflects a characteristic associated with a particular stage of a cell's maturation. It is anticipated that these cell lines, positive and negative for the expression of natural polarity of spectrin distribution, will provide useful models for future studies to define further the role of spectrin in lymphocyte plasma membrane functions.

Animals↗
PubMed1985

Human recombinant interleukin-2 is mitogenic to human lymphocytes.

Reported herein are the results of studies demonstrating that purified recombinant human interleukin-2 (hrlL-2) is a potent mitogen for lymphocytes of healthy human donors. The specificity of the hrlL-2-induced response was defined in experiments in which mitogenicity of this T cell growth-promoting lymphokine was completely abrogated by blocking the T cell membrane receptor for IL-2 with the anti-Tac monoclonal antibody. Depletion of adherent mononuclear leukocytes markedly reduced lymphocyte reactivity to hrlL-2, but the response could be fully recovered by the addition of interleukin-1 (IL-1). Increased proliferative responses were observed using a combination of hrlL-2 and a monoclonal antibody OKT3 that defines a T cell membrane antigen. These studies demonstrate that hrlL-2, as with antigens and phytomitogens, may serve as the first signal of T cell proliferation.

Antibodies, Monoclonal↗
PubMed1985

Isolation of mouse T-cell lymphoma lines from different long-term interleukin 2-dependent cultures.

A number of different biological properties have been ascribed to the hormone-like protein interleukin 2 (IL-2). However, the most salient feature of this lymphokine is its ability to sustain the long-term proliferation of T-cells from humans and mice. Reported herein are the results of studies demonstrating the isolation of growth factor-independent cell lines from the long-term IL-2-dependent murine T-cell line CTLL-2 that is used frequently as the source of target cells in IL-2 bioassays. Sustained log-phase growth of these T-cells in vitro has been achieved using Petri dishes of polymethylpentene; growth could not be sustained in similar dishes of glass, untreated polystyrene, polystyrene that had been treated for cell culture, or polycarbonate. The IL-2-independent line grew as a T-cell lymphoma when injected i.p. into pristane-treated, but not untreated, syngeneic C57BL/6 mice. In contrast, cells from the IL-2 parental line CTLL-2 did not grow in vivo. Characterization of the IL-2-independent lines propagated in vitro (denoted as line CEC) or in vivo (denoted as line CEP) demonstrated that they retained their dependency for 2-mercaptoethanol and expressed phenotypic profiles of their parental line CTLL-2 (Thy 1.2+, Lyt-1-; Lyt-2-). Isolation of an IL-2-independent T-cell lymphoma from a CTLL-2 line obtained from another investigator using a protocol that has proven reproducible under carefully controlled laboratory conditions and defined phenotypic traits of the syngeneic T-cell isolates provided evidence that the tumors were not a cross-culture contaminant arising as a result of a laboratory accident. Moreover, karyotypic analysis using a quinacrine:Hoechst banding technique revealed similar marker chromosomes in the IL-2-dependent and -independent lines. IL-2-independent lines have also been established from the IL-2-dependent murine T-cell line CT-6. Accordingly, the results of these studies suggest that, during prolonged cultivation that has included exposure to crude IL-2 preparations known to contain phorbol ester, possibly viruses, and other contaminants, the IL-2-dependent lines have developed subpopulations that are thought to have undergone malignant transformation of unknown etiology to generate IL-2-independent murine T-cell lymphomas that can be passaged repetitively either in vitro or in vivo.

Animals↗
PubMed1984

Isolation of interleukin 2 (IL-2) from human and mouse lymphocyte culture supernatants by batch adsorption onto silicic acid.

Described herein is a simple, efficient and inexpensive batch adsorption procedure for the isolation and partial purification of the hydrophobic T cell growth-promoting lymphokine interleukin 2 (IL-2) from crude culture supernatants (SN) of freshly isolated human lymphocytes and leukemic T cells of established lines including human Jurkat J6.2, Gibbon ape MLA-144 and mouse EL-4. In this method, IL-2 was isolated by batch adsorption onto microparticulate silicic acid (SA) by stir-mixing the SA with SN (10 mg/ml; 30 min; 37 degrees C). Thereafter, the SA was pelleted by centrifugation and washed twice with phosphate-buffered saline (PBS). The IL-2 was eluted by adding to the pelleted IL-2-binding SA 5 vols. of ethylene glycol (EG; 50%, v/v) in PBS (pH 7.2) with high salt (1.4 M NaCl). The lymphokine-rich concentrate was then dialyzed (6 kDa MWCO) against PBS to remove the EG and low molecular weight growth inhibitors. The application of the proposed procedure was further defined in experiments in which SA was successfully employed for recovering IL-2 from SN of cultures in which the medium had been supplemented with fetal calf (FCS) or human serum to achieve maximal lymphokine production. Also presented are the results of experiments defining the SA adsorption of proteins from whole sera (e.g., FCS, calf, human and horse) and the relative affinity of different purified proteins for this matrix (e.g., bovine serum albumin, human serum albumin, casein hydrolysate, bovine gamma-globulin and bovine beta-lactoglobulin). The proposed SA procedure may prove useful for isolating other hydrophobic immunoregulatory molecules, and a 2-step purification scheme is anticipated in which the SA adsorption procedure will be used as a preparative method preceding reverse phase high performance liquid chromatography and monoclonal antibody affinity chromatography.

Adult↗
PubMed1982

Studies of cultured human T lymphocytes. I. Production of the T cell growth-promoting lymphokine interleukin-2.

Described herein is a large-scale procedure that has been successfully employed for producing 62 lots (800-3000 ml) of supernatants containing the T cell growth-promoting factor Interleukin-2 (IL-2). The efficiency of these crude, unconcentrated supernatants was documented in studies in which 70 human long-term (greater than 100 days) IL-2-dependent T cell lines were established from 50 different donors. These included lines initiated from the peripheral blood of healthy subjects (N = 54), blood of children with active acute lymphoblastic leukemia (N = 6) and the thymus of children undergoing surgery to correct congenital heart defects (N = 10). The underlying concept used in constructing this method emphasizes the requirement of the monocyte-derived macrophage and its Interleukin-1 (IL-1) product to mediate IL-2 production by activated T cells. The most salient feature of this technique is the utilization of buffy coat leukocytes that had been pooled from several blood donors and sustained in spinner cultures for several days prior to polyclonal activation with phytohemagglutinin and pooled B cells of established human lymphoblastoid lines.

Adolescent↗
PubMed1978

Neuraminidase alteration of human lymphocyte reactivity to mitogens, antigens and allogenic lymphocytes.

Lymphocyte reactivity to phytohemagglutinin (PHA) was reduced significantly in whole-blood cultures initiated with nontoxic concentrations of Vibrio cholerae neuraminidase (VCN). Reduced responsiveness was not due to degradation or inactivation of PHA by this enzyme, and the reaction was dependent upon the dose and time at which this mitogen and enzyme were added to the cultures. In cultures from some subjects, VCN also inhibited reactivity to concanavalin A. In contrast, lymphocyte reactivity to mitogens from pokeweed or Staphylococcus aureus as well as to antigens or allogeneic lymphocytes was enhanced by VCN. At concentrations required to elicit these effects, VCN itself was not stimulatory.

Adult↗
PubMed1976

A simple screening assay for substances affecting lymphocyte reactivity.

A simple method is described for assaying substances modifying lymphocyte reactivity in vitro to mitogens and antigens. This procedure employes whole blood, microtiter plates and an automated cell harvester. The simplicity and expediency afforded by this assay, which requires very little blood, enables the large-scale screening of different test materials which may be of experimental or clinical merit.

Culture Techniques↗
PubMed1976

Elaboration of pyrimidine-specific nucleosidases by human lymphoblastoid cells of established cultures.

Pyrimidine-specific nucleosidases were released rapidly by human lymphoblastoid cells of established cultures when incubated under certain culture conditions having no adverse affect on their viability or morphology. Nucleosidase production was not restricted to any particular type of lymphoblastoid line; enzymes with a high level of activity were elaborated by cells of cultures initiated from healthy subjects and patients with uncontrolled lymphocytic or myelocytic leukemia, as well as by cells of cultures exhibiting mostly B- or T-cell properties. Tritiated thymine and uracil, which were not incorporated to any appreciable extent by DNA- and RNA-synthesizing cells, were identified by paper chromatography as the primary products arising from nucleosidase degradation of radiolabeled thymidine, uridine, and cytidine. Neither adenosine nor guanosine was catabolized. These heat-labile and ultraviolet-sensitive enzymes with a molecular weight of 5 to 10 X 10(4) did not affect the viability, morphology, or proliferation of lymphocytes in mitogenactivated cultures, lymphoblastoid cells in long-term cultures, or fibroblasts in monolayer cultures.

Cells, Cultured↗