Enhanced interferon production and lymphokine-activated cytotoxicity of human placental cells

The immunologic competence of human placental mononuclear cells was compared to that of adult and cord blood mononuclear cells. Mononuclear cells were isolated from fresh placentas by digestion with collagenase and DNase, followed by Ficoll-Hypaque and discontinuous Percoll separation. Placental cells incubated with phytohemagglutinin (PHA) synthesized significantly more interferon-gamma (IFN-gamma) at 2 days (29 +/- 5.5 IU/ml) and 5 days (46 +/- 8.5 IU/ml) than PHA-activated cord cells (3.6 +/- 0.6 IU/ml at 2 days and 2.7 +/- 0.7 IU/ml at 5 days) but less than PHA-activated adult cells (81 +/- 20 IU/ml at 2 days and 270 +/- 161 IU/ml at 5 days). Placental and adult cells, but not cord cells, also synthesized significant quantities of IFN-gamma following incubation with interleukin-2 (IL-2). There was synergism between IL-2 and PHA activation for IFN-gamma production for some cord samples. After a 5- to 7-day incubation with IL-2, the lymphocyte-activated killer (LAK cell) cytotoxicity of placental cells (measured in a 3-hr chromium-release assay at an E:T ratio of 40:1) was enhanced 13-fold against K562 target cells (6 +/- 2% to 77 +/- 4%) compared to a 4-fold increase in cord cells (16 +/- 4% to 68 +/- 3%) and a 2-fold increase in normal adult cells (35 +/- 4% to 65 +/- 3%. Against the natural killer (NK)-resistant Raji target, placental cells increased their LAK cytotoxic activity (3 +/- 1% to 59 +/- 7%) compared to a 7-fold increase with cord cells (6 +/- 1% to 43 +/- 3%) and a 3-fold increase with adult cells (11 +/- 2% to 38 +/- 4%). A notable degree of cytotoxic activity in the absence of IL-2 against Molt targets was noted in 11 of 14 (79%) placental cell samples at 5 days. Only 10 of 24 (42%) adult and 17 of 37 (40%) cord samples showed spontaneous cytotoxic activity equal to or greater than 10%. Some placental samples actually showed an increase in cytotoxic activity when incubated without IL-2. The ability of placental cells to produce significant levels of IFN-gamma, to develop considerable LAK activity, and to maintain or develop cytotoxic activity in the absence of IL-2 suggests a vigorous, active immune system of the placenta compared to the relatively dormant immune system of the neonate. These observations suggest that placental cells may have a primary role in fetal defense.     

Immature T lymphocytes in human cord blood identified by monoclonal antibodies: a model for the study of the differentiation pathway of T cells in humans

The reactivity of human cord blood lymphocytes was assessed against a panel of monoclonal antibodies (MoAb). The mean proportion of OKT3+ cells (pan-T) was significantly lower in cord blood (52 +/- 13.8%; mean +/- SD) compared with that of adult blood (75 +/- 8.9%) and paralleled well with the E-rosette-forming capacity (50 +/- 16.3%). Both the proportions of OKT4+ cells (helper/inducer phenotype) and of OKT8+ cells (suppressor/cytotoxic phenotype) were significantly reduced in cord blood (43 +/- 11.8% vs 50.3 +/- 7.4% and 20 +/- 10.3% vs 25.6 +/- 6.0%, respectively), while the overall OKT4/OKT8 ratio was increased compared with adult blood (2.87 +/- 1.83 vs 2.04 +/- 0.61). Unlike adult blood, in 30 of the 35 samples of cord blood an overlap was observed between the total proportion of OKT4+ and OKT8+ cells (65 +/- 15.2%) and that of OKT3+ cells (52 +/- 14.3%). Although small numbers of cells coexpressing both antigens were occasionally found, double-staining analysis showed that the overlap in cord blood was mostly due to an expanded proportion of OKT3 (Leu-4)-/OKT8 (Leu-2)+ cells. Relevant proportions of OKT6+ (common thymocyte antigen) and OKT10+ (thymocytes, activated T cells, precursor cells) cells were found in cord blood as opposed to adult blood (10.8 +/- 8.6% vs 0.6 +/- 0.6% and 67 +/- 18.0% vs 8 +/- 2.1%, respectively), while terminal deoxynucleotidyl transferase-positive cells were observed only in two samples of cord blood. A small proportion of T cells (E-rosette+) reacted with the MoAb OKIa1 (HLA-DR). Finally, the proportion of cord blood cells recognized by the MoAb Leu-7 (HNK-1 clone) was almost negligible compared with adult blood (2.8 +/- 2.4% vs 15 +/- 7.5%). These data confirm the immaturity and heterogeneity of cord blood lymphocytes and demonstrate the presence at birth of circulating lymphocytes which express a surface phenotype reminiscent of that found in the late stages of intrathymic differentiation and in some human T-cell leukemias. Human cord blood may thus represent a suitable model for the study of the differentiation pathway of normal and pathological T-cells in humans.     

Interleukin-2- and mitogen-activated NK-like killer cells from highly purified human peripheral blood T cell (CD3+ N901-) cultures

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.     

A superior analysis of coenzyme Q10 in blood of humans, rabbits and rats for research

The quantitative analysis of coenzyme Q10 (CoQ10) in samples of whole human blood has been refined to allow a 2- to 3-fold increase in the number of analyses per day, and reduction of cost to approximately 15% of the previous cost. The method is simple yet maintains reliability. The standard error was 0.2% (n = 6). The variation in blood levels of CoQ10 for human subjects for each of three months was approximately 5% in comparison with the control value (n = 5). For 30 human males, of 18-50 years (26 +/- 6) in age, and for 30 human females, of 18-50 years (26 +/- 9), the mean blood level of CoQ10 was 0.71 +/- 0.13 microgram/ml and 0.70 +/- 0.18 microgram/ml respectively. The mean blood levels of CoQ10 of rabbits (n = 28) was 0.29 +/- 0.07 micrograms/ml, and that for rats (n = 29) was 0.23 +/- 0.03 micrograms/ml.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of thyrotropin-releasing hormone on immune functions of peripheral blood mononuclear cells

The tripeptide thyrotropin-releasing hormone (TRH) works as a hypothalamic hormone, but is found also outside the brain in intrinsic nerve fibers of the gastrointestinal tract. There is evidence that TRH modulates the activity of immunocompetent cells, although there are only very few data on TRH-mediated immune effector functions. Since we could recently show that TRH inhibits monocyte activities we were also interested in other possible TRH modulated immune functions. Peripheral blood mononuclear cells (PBMC) from ten healthy subjects were cultured for 7 days and pulsed with 0.125 and 0.250 microgram/ml Pokeweed mitogen (PWM). 10(-12) to 10(-6) M TRH was added simultaneously with PWM. Lymphocyte proliferation [(3H]thymidine incorporation), interferon-gamma (IFN-gamma) activity (RIA) and immunoglobulin activities (IgG, IgM, IgA; ELISA) were determined in the supernatants. We could demonstrate a TRH-dependent decrease in PWM-pulsed IgG activity with significant (alpha = 0.05) values at 10(-8) and 10(-10) M (-29 +/- 6%/-16 +/- 3% for PWM 0.125 microgram/ml and -17 +/- 9%/-11 +/- 9% for PWM 0.250 microgram/ml). This inhibitory effect could be abolished by an anti-TRH antiserum. There was no TRH effect on IgM and IgA activities, IFN-gamma activity and lymphocyte proliferation compared with the PWM stimulated values alone. The described TRH effect on the polyclonal IgG response by PBMC gives further evidence for a functional link between the immune system and the endocrine system, although its underlying mechanism is not yet clear.     

Comparative studies on in vitro reactivity of fresh and cryopreserved pig lymphocytes.

The effect of cryopreservation on in vitro reactivity of pig lymphocytes was studied. Peripheral blood mononuclear cells (PBMC) were frozen by controlled-rate freezing and stored in liquid nitrogen (LN2) between 4 and 36 days. Following thawing 74.7 +/- 2.6% of cells were recovered of which 94.5 +/- 0.9% were viable as determined by trypan blue exclusion. Functional parameters measured included the concentration of free intracellular Ca2+ ([Ca2+]i) in resting and mitogen-stimulated PBMC, mitogen and alloantigen-induced blastogenesis, as well as cell-mediated cytotoxicity. Irrespective of storage time and cell donor, [Ca2+]i in frozen-thawed PBMC (67.7 +/- 4.3 nM) was significantly lower (P less than 0.001) when compared to fresh cells (96.2 +/- 4.5 nM). In addition, cryopreserved PBMC only weakly responded with an increase of [Ca2+]i after stimulation by various concentrations of phytohemagglutinin (PHA). Following activation by PHA (2 micrograms/ml) for 4 days fresh lymphocytes (84,047 +/- 5475 cpm) incorporated significantly more (P less than 0.005) [3H]thymidine than frozen PBMC (66,001 +/- 4117 cpm). A similar difference in proliferation rates (P less than 0.05) between fresh (10,046 +/- 1915 cpm) and frozen-thawed PBMC (5852 +/- 1304 cpm) was observed in one-way mixed lymphocyte cultures (MLC), while the spontaneous incorporation of radiolabel was unchanged in frozen stored cells. By using MLC-derived cytotoxic effector cells (E) and [3H]thymidine-labeled concanavalin A blasts as targets (T), cryopreserved PBMC displayed a severe deficiency of cytotoxic effector functions at all tested E:T ratios. These results indicate that pig PBMC are very sensitive to LN2 storage although some immunological functions are more affected by cryopreservation than others.     

Proliferation patterns of peripheral blood lymphocytes in CLL patients: cytophotometric and microfluorimetric study

Peripheral blood lymphocytes of 19 patients with CLL, 9 patient with LS and 10 healthy donors were studied by Feulgen cytophotometry, 3HTdR autoradiography, A0 microfluorimetry and PHA stimulated cultures. In CLL the bulk of cells are in G0 (80.6 +/- 3.7%) the rest are in G1 (16.3 +/- 3.6%) and S + G2 (3.0 +/- 1.0%). Thymidine LI values were two orders lower (0.098 +/- 0.04). In five cases combined autoradiographic and cytophotometric study on the same cells revealed 6-14% of cells arrested in S. In peripheral blood of LS patients G0 cells also predominate, and only in 3 cases cytophotometry revealed hyperdiploid (S + G2) cells. In normal lymphocytes 1.5 hrs after PHA stimulation A0 binding increases on the average by 80% compared to unstimulated cultures and remains at this level during 12 hrs. CLL and LS cells behave nearly the same with the only difference: the 80% increase is observed only after 3-4.5 hrs in culture. G0----G1 flow rate in case of normal lymphocytes is higher than for neoplastic cells but both are recruited into cell cycle during all the period in culture. G1----S transition is delayed in case of LS lymphocytes and strongly inhibited in CLL lymphocyte cultures compared to normal cells. The possible mechanisms of these features are discussed.     

Response of nasal blood flow to neurohormones as measured by laser-Doppler velocimetry

Laser-Doppler velocimetry (LDV) has been adapted to measure nasal blood flow (NBF) in the mucosa of human volunteers. Resting NBF was 42.4 +/- 2.1 ml X 100 g-1 X min-1 in 19 nonatopic subjects and 37.9 +/- 1.7 ml X 100 g-1 X min-1 in 24 atopic subjects. Topical saline, but not water, reduced ipsilateral NBF by 15.4 +/- 6.6% (n = 22) without affecting contralateral NBF. Administration of 60 microgram of oxymetazoline reduced NBF by 26.5% (n = 28), whereas 120 microgram resulted in a 54.3% reduction. Phenylephrine produced a dose-related reduction in NBF with an ID50 (dose producing 50% reduction) of 1,456 microgram. Methacholine (0.006 to 12 mg) had no significant effect on NBF when studied alone or after oxymetazoline pretreatment. Therefore, LDV can be employed to monitor NBF, which has been found to be sensitive to alpha-adrenergic, but not cholinergic, stimulation.     

The effect of estrogen on the incorporation of 3H-thymidine by PHA-stimulated human peripheral blood lymphocytes

The effect of estrogen on the incorporation of tritiated thymidine by phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes was evaluated in 15 adult males. Varying concentrations of diethylstilbestrol diphosphate (DEP-S) were added to peripheral blood lymphocytes with and without PHA to study the effects of estrogen on blastogenesis. Maximum suppression of blastogenesis occurred after the addition of 500 mcg/ml culture of DEP-S. The absence and presence of DEP-S 500 mcg/ml culture resulted in a 52% reduction in lymphocytic reactivity (p.002). It was concluded that this reduction or suppression of lymphocytic blastogenesis in the presence of estrogen suggests that the palliative effects of estrogenic therapy in treating patients with hormone-dependent tumors may be countered by its adverse effect on the host's immunologic responsiveness to malignancy.     

Response of baboon peripheral blood lymphocytes to phytohemagglutinin: time-course and dose-response relationships.

Optimal conditions for studying phytohemagglutinin(PHA)-induced transformation of baboon peripheral blood lymphocytes were determined. Maximal stimulation, as determined by uptake and incorporation of tritiated thymidine (total cell and trichloroacetic-acid-precipitable radioactivity), occurred at a PHA level of 12.5 microgram in a culture volume of 0.25 ml containing 2 x 10(5) lymphocytes. Optimal stimulation occurred after a total incubation period of 114 h, the last 18 h of which was in the presence of 1muCi of the labeled DNA precursor per culture. While there was considerable variation in the extent of responsiveness of lymphocytes from individual animals, the shape of the dose-response and time-course curves for most mitogen concentrations was generally similar.     

Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes.

Human T-cell leukemia virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). Lymphocytes from patients with ATL or TSP/HAM display abnormal proliferation properties in culture. Here we report that purified, soluble Tax1 protein can be taken up by, and stimulate proliferation of, uninfected human peripheral blood lymphocytes (PBLs) that have been stimulated with phytohemagglutinin (PHA). Tax1 was 40 to 70% as active as interleukin-2 (IL-2) in stimulating proliferation of PBLs. Heat inactivation, chloroform extraction, and immunoprecipitation with antisera specific for Tax1 each abolished the ability of the protein to stimulate lymphocyte proliferation. Tax1 failed to stimulate PBL proliferation in the absence of PHA. After an initial round of cell division, Tax1-treated PBLs exhibited prolonged sensitivity to IL-2-induced proliferation. These results indicate that Tax1 can stimulate lymphocyte proliferation in culture and imply that extracellular Tax1 may be involved in the spontaneous proliferation of TSP/HAM lymphocytes and the IL-2-dependent proliferation of ATL lymphocytes.     

Cell-mediated immunity to influenza virus antigen and mitogen in guinea pigs: measurement with a semimicro protein synthesis assay

A rapid and precise method for the assay of cell-mediated immune response basing on protein synthesis stimulation of mitogen-activated guinea pig lymphocytes is modified in a way that enables the study of virus-immunological problems. When used as a micromethod it has the following advantages over conventional methods: short-term cell culture, need of low quantities of cells and rapid preparation of great numbers of samples for radioactivity measurements. In this study we report the results of comparative experiments on measuring lymphocyte stimulation after addition of PHA and stimulation of sensitized lymphocytes following contact with homologous influenza virus antigen in vitro. The most important reaction parameters are as follows: 5-6 . 10(5) spleen lymphocytes/microculture in microtiter plates, use of Eagles's MEM cell culture medium without leucine, supplemented with HEPES buffer and 10% autologous guinea pig serum; optimum lymphocyte stimulation by addition of 0.5 microliter PHA or 0.1-1.0 microgram virus protein/ml; immuno-stimulation by PHA can be measured in vitro already after 6 h and by influenzavirus antigen already after 24 h.     

Concanavalin A increases glyoxalase enzyme activities in polymorphonuclear leukocytes and lymphocytes

Glyoxalase I converts methylglyoxal and glutathione to S-lactoylglutathione and glyoxalase II converts this compound to D-lactic acid, regenerating glutathione in the process. A recent study from my laboratory has provided evidence that S-lactoylglutathione modulates microtubule assembly in vitro whereas concanavalin A (Con A) has been shown to increase microtubule occurrence in polymorphonuclear leukocytes (PMN). The present report describes the dose-dependent activation by Con A of both glyoxalase I and II in PMN and lymphocytes. In nine experiments with PMN, Con A (100 microgram/ml) increased glyoxalase I and II activities by 19 +/- 8% and 12 +/- 10% (mean +/- S.D.). In 17 experiments with lymphocytes, activation of the two enzymes by 10 microgram/ml Con A was 30 +/- 14% and 28 +/- 8%. Changes occurred after a 1-min incubation with Con A and persisted for at least 60 min. Since both enzyme activities are increased it is not clear if S-lactoylglutathione levels are increased or decreased but presumably they change. The present findings are compatible with the hypothesis that Con A increases microtubule occurrence in PMN by affecting the glyoxalase enzymes. They also represent a newly described early biochemical change caused by Con A in lymphocytes.     

Functional characterization of ecto-5'-nucleotidase-positive and -negative human T lymphocytes

Functional studies were performed on human peripheral blood T lymphocytes stained with goat anti-5'-nucleotidase antibodies and separated into ecto-5'-nucleotidase (ecto-5'-NT)-positive and -negative populations using the FACSTAR fluorescence-activated cell sorter. On the average, ecto-5'-NT+ T cells contained 34 +/- 13% CD4+ and 55 +/- 15% CD8+ cells, whereas ecto-5'-NT-T cells contained 65 +/- 12% CD4+ and 23 +/- 8% CD8+ cells. Staining with anti-5'-NT antibodies did not significantly alter the ability of unseparated T cells to proliferate in response to PHA or PMA, or in a MLR. However, prior incubation with anti-5'-NT antibodies did inhibit the ability of irradiated T cells to provide help for PWM-stimulated Ig synthesis by as much as 55%. In five separate experiments, ecto-5'-NT-T cells demonstrated an equal or better ability to incorporate [3H]TdR after PHA stimulation or in a MLR, as compared with ecto-5'-NT+ T cells. Similarly, ecto-5'-NT- T cells were not diminished in their ability to provide help for autologous B cells in a PWM-driven system. Clearly, the inability of ecto-5'-NT- T cells from patients with a variety of immunodeficiency diseases to function in these assays cannot be explained solely by their lack of ecto-5'-NT activity. In contrast, ecto-5'-NT-positive and -negative T cells showed markedly different dose-response curves for proliferation in response to PMA. Ecto-5'-NT+ T cells responded to lower doses of PMA (1.0 ng/ml) than did ecto-5'-NT- T cells and showed a two- to eight-fold greater rate of [3H]TdR incorporation at 3 to 10 ng of PMA per ml. Ecto-5'-NT+ T cells may have a protein kinase C that is more accessible or more easily activated or may utilize an alternate pathway of activation when stimulated with low concentrations of PMA.     

Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Biochemical properties of conjugates of urokinase-type plasminogen activator with a monoclonal antibody specific for cross-linked fibrin

Equimolar mixtures of recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody (MA-15C5) directed against fragment-D dimer of human cross-linked fibrin were conjugated, using the cross-linking agent N-succinimidyl 3-(2-pyridyldithio)propionate (PySSProSu). The conjugate (rscu-PA/MA-15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment-D dimer with a yield of 42 +/- 15% (mean +/- SD, n = 3), contained an average of 1.2 +/- 0.3 IgG molecules/rscu-PA molecule. On non-reduced SDS/PAGE it migrated as a main band with apparent Mr of 200,000. Specific amidolytic activities expressed/mass of u-PA were less than 250 IU/mg for rscu-PA/MA-15C5 and rscu-PA, 140,000 +/- 13,000 IU/mg and 100,000 +/- 17,000 IU/mg for their plasmin-generated two chain derivatives rtcu-PA/MA-15C5 and rtcu-PA respectively. Specific activities on fibrin plates were 100,000 +/- 24,000 IU/mg and 130,000 +/- 49,000 IU/mg for rscu-PA/MA-15C5 and rtcu-PA/MA-15C5 respectively, as compared to 180,000 +/- 15,000 IU/mg for both rscu-PA and rtcu-PA. Activation of plasminogen with rscu-PA/MA-15C5 (Km = 0.37 +/- 0.16 microM, k2 = 0.0063 +/- 0.0030 s-1 or rtcu-PA/MA-15C5 (Km = 19 +/- 3.0 microM, k2 = 2.0 +/- 0.10 s-1) in purified systems followed Michaelis-Menten kinetics with Km and k2 values comparable to those of rscu-PA and rtcu-PA. In an in vitro system composed of a 125I-fibrin-labeled whole human plasma clot immersed in citrated human plasma, dose- and time-dependent lysis was obtained; 50% lysis in 2 h required 1.4 microgram/ml of rscu-PA or 0.33 microgram/ml of rtcu-PA, but only 0.22 microgram u-PA/ml of rscu-PA/MA-15C5 or 0.15 microgram u-PA/ml of rtcu-PA/MA-15C5. Addition of purified fragment-D dimer reversed the increased fibrinolytic potency of rscu-PA/MA-15C5 in a concentration-dependent way (50% inhibition at 7.2 micrograms fragment-D dimer/ml). Thus, conjugation of u-PA moieties with the fibrin-specific antibody MA-15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4-fold for rscu-PA and 2.2-fold for rtcu-PA.     

In vivo T cell activation, in vitro defective IL-2 secretion, and response to influenza vaccination in elderly women.

IL-2 secretion in response to mitogenic stimulation, assayed in vitro, is significantly reduced in circulating T lymphocytes isolated from healthy old people, but the significance of this abnormality and how it relates to in vivo IL-2 secretion remain unclear. We found that IL-2 secretion in response to PHA plus PMA by peripheral blood T cells isolated from 10 out of 32 (31%) healthy old individuals (mean age 86 yr, range 74-97) was significantly decreased compared with results obtained in 23 younger individuals (mean age 34 yr, range 23-46). This IL-2 secretion defect in vitro was reversible after a 3-day incubation in the absence of activators. The 10 healthy old individuals who had defective IL-2 secretion in vitro also showed increased levels of serum IL-2. T cells from 22 healthy old and 22 young individuals, who had normal IL-2 secretion (geometric mean +/- log of 1 SD: 139 +/- 0.3 U/ml and 212 +/- 0.31 U/ml, respectively) in vitro, showed a remarkable transient T cell defect in IL-2 secretion (15 +/- 0.47 U/ml for the old, 54 +/- 0.28 U/ml for the young) 15 days after influenza vaccination. IL-2 secretion became normal again 30 days after vaccination. The T cell-IL-2 activity, expressed as a T cell-IL-2 activity score (calculated as the logarithm of the serum IL-2 U/ml divided by the logarithm of the IL-2 secretion U/ml, in vitro) was significantly increased in elderly non-responders after influenza vaccination (mean +/- 1 SD: 1.4 +/- 0.51) compared with elderly (0.44 +/- 0.13) and younger responders (0.3 +/- 0.2). Our data suggest that in vitro defective IL-2 secretion is a consequence of T cell activation which seems to occur in a significant proportion of healthy elderly individuals and may be clinically relevant inasmuch as it appears to prevent the normal vaccine-induced antibody response.     

Production of interleukin-1-alpha and interleukin-2 by mononuclear cells in healthy adults in relation to different experimental conditions and to the presence of growth hormone

Several reports support the view that the growth hormone (GH) possesses a number of important immunomodulatory properties. This study was undertaken to determine in vitro the role of the GH on interleukin (IL) production. Cultures of blood peripheral lymphocytes obtained from human normal adults were performed in RPMI medium in the presence or absence of phytohemagglutinin (PHA), heated normal serum (NHS) 1% and GH 12.5-500 microgram/l. After incubation from 15 h to 4 days at 37 degrees C in a humidified atmosphere containing 5% CO2, cells were discarded and the supernatants were tested for their contents of IL-1 alpha and IL-2 measured using the Amersham radioimmunoassay system. The results of these in vitro experiments show that: (1) the bulk cultures from peripheral lymphocytes are suitable to study the IL-1 and IL-2 production; (2) in optimal conditions for IL production (incubation during 48 h in the presence of PHA and NHS) no effect of GH was observed on IL production; (3) in the absence of PHA GH acts at physiological doses (less than 100 ng/ml) by increasing the IL production. This effect of GH was optimized with a short time of incubation (16 h) and in the simplest conditions of cultures, that is to say in the absence of serum and of PHA: thus in the presence of GH 100 ng/ml the IL-1 production increases from 0.53 to 3.86 fmol (tubes) and IL-2 increases from 0.18 to 3.22 fmol (tubes). These differences are significant (p less than 0.001). We conclude that GH acts in vitro on mononuclear cells to induce IL production.