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Mitogens are able to stimulate T cells in human peripheral blood lymphocyte cultures to form thermostable rosettes with sheep erythrocytes (TSER). Previously we characterized TSER-forming cells as BL/T2-, DR- and 4F2-positive, e.g. activated T lymphocytes. In this study the evidence is presented that mitogen-induced TSER-forming cells are the responding, proliferating lymphocytes. We studied the sequential activation of human lymphocytes by various mitogens. PHA-induced TSER-forming cells could not been further activated neither by PWM nor by Con A. DNA synthesis rate in Con A-preactivated lymphocytes was enhanced by PHA but not by PWM. PWM-induced TSER-forming cells, however, were further activated as well by PHA as by Con A. These data suggest the heterogeneity of the pathways by which different mitogens activate human immune cells. PWM- and PHA-induced TSER-forming cells could support B lymphocyte differentiation, when PWM was added to the cultures, whereas Con A-preactivated cells failed, indicating different functional activities induced by mitogenic lectins.

The Pokeweed mitogen (PWM)-inducing in vitro synthesis of human IgG and IgM was determined by enzyme immunoassay (EIA) in culture supernatants of PWM-stimulated peripheral blood lymphocytes (PBL) from 83 healthy donors ranging from 24-46 years. Seventy five per cent were characterized as normal responders (200-2000 ng/ml IgG and IgM), 10% classified as high responders (2000-3000 ng/ml), and another 10% had a very high response (more than 5000 ng/ml of both IgG and IgM). One of 83 donors was shown to be a PWM non-responder. In 83 unstimulated cultures from the same donors 660 +/- 75 ng/ml IgG and 60 +/- 10 ng/ml IgM were measured after 8 days of incubation. Ten donors exhibited similar dose dependence of PWM-induced Ig synthesis but differed in time kinetics (optimum between 8 and 12 days of culture). Regression analysis of IgG and IgM production in 83 PWM-stimulated lymphocyte cultures from different healthy donors showed a correlation coefficient of r = 0.627. Intracellular Ig in PWM-stimulated cells was quantified by EIA technique after cell lysis in time kinetic experiments. Reproducibility and accuracy of the test system are described in detail. An excess of monocytes of more than 20% in the culture caused an inhibition of the Ig synthesis (calculated on constant numbers of B cells). Monocytes were further immobilized in FCS-coated culture plates and pulsed with PWM for 2 h. Adding unstimulated, nonadherent cells to the plates after removing the mitogen resulted in an Ig production comparable to that with the classical method.

Human lymphocytes derived from peripheral blood, the spleen and lymph nodes were fused to the HAT-sensitive heteromyeloma cell line CB-F7. The Ig-producing initial cell lines were selected, and the supernatants were further analyzed for specific antigen binding (ELISA). IgM-antibodies were found which reacted with self- and non-self antigens of different molecular origin (nucleotides, proteins, carbohydrate structures). These antibodies were called multireactive (multispecific). The multispecific IgM-producing human hybridomas occurred with higher frequencies in the spleen (6.9% of IgM-producers) cell fusions than in experiments where peripheral blood-derived lymphocytes were fused (2.7%). There were no hybridomas producing multireactive antibodies detected in fusion material from lymph nodes. The greatest number of multireactive IgM was seen when PBL from SLE or anti-HIV-positive patients were hybridized to CB-F7 cells. Representative cell lines were cloned and recloned. The multireactivity of the IgM produced by really monoclonal cells, however, was preserved.

Lectin-activated human peripheral blood lymphocytes were able to form thermostable rosettes with sheep red blood cells (SRBC). Capability to form thermostable rosettes (TSER) after lectin treatment was comparable in time kinetics and dose dependence to cell size distribution changes. Enhancement of TSER formation by human lymphocytes exposed to PHA and Con A, however, occurred before 3H-Thymidine incorporation could been registered. Lectin treatment of lymphocytes from 20 healthy donors for 48 hrs induced 49 +/- 4% (PHA), 33 +/- 5% (Con A), and 20 +/- 2% (PWM) thermostable rosette forming cells, whereas in unstimulated cultures were found 4 +/- 1%. Furthermore, thermostable rosette forming cells were enriched by gradient centrifugation and characterized as activated lymphocytes of T-origin by SE rosetting and binding studies with various monoclonal antibodies. It was shown that preparation of TSER forming cells after lectin activation makes it possible to enrich the fraction of lectin-stimulated T lymphocytes.

In 28 healthy adults aged 19 to 52 years, normal values of the TG lymphocytes amount to 17.8 +/- 2.3% and 253.1 +/- 96.1 per microliters respectively and the non TG/TG ratio to 4.72 +/- 0.82. When compared with normal controls, patients on dialysis had a disturbed non TG/TG ratio (4.72 +/- 0.82 vs. 6.00 +/- 1.94, p less than 0.05). During the first posttransplant month the total TG cell count was significantly reduced, but the relative TG cell count did not significantly differ from the normal as well as praeoperative TG cell count. In connection with rejection crises we could not observe any changes in the TG subset. Beside this general dynamics we observed two different kinds of changes of the non TG/TG ratio in the individual posttransplant course. Either the non TG/TG ratio were lower than praetransplant or higher. In 9 out of 9 cases the lowering of the non TG/TG ratio (that means a relative or absolute increase of Fc-IgG receptor bearing T cells) were connected with a good graft function. In 4 out of 6 cases a postoperative increase of the non TG/TG ratio were connected with an early graft failure. A change of T subsets for the benefit of TG cells which includes suppressor cells seems to be favourable with regard to the graft survival. Therefore, we think the determination of the prae- and posttransplant non TG/TG ratio is of some prognostic value.

In a group of 23 patients with systemic lupus erythematosus by means of monoclonal antibodies differentiation markers were investigated on peripheral blood T-lymphocytes. Individual specific differences in the percentage of T1-, T3-, T4- and T8-positive cells were found, indicating various possibilities of immunological disorders in this disease. Furthermore, in comparison with healthy donors, an increased number of T4-, T8-double stained cells was detected in the peripheral blood of patients with systemic lupus erythematosus. Normally, this cell phenotype can be detected in the thymus only. Therefore, it becomes necessary to perform parallel and simultaneous determinations, respectively, of several T cell markers in order to gain a more exact insight into the disturbances of the immune system and to exclude incorrect interpretations of the phenotypical distribution of T cell markers.

The mutations in the genes determining the levels of extracellular enzymes production were transferred into Bacillus subtilis 168 strain by genetic transformation technique. The method used has permitted registering the transfer of pleiotropic genes. Seven amylase producers were detected among 126 his+ transformants screened, as well as five metalprotease producers among 246 gly+ transformants. Cotransfer of pap and hpr mutant genes linked with his+ or gly+ genes might result in finding the producers among the mentioned prototrophic transformants. Selection of linked markers in transformation, presented in the paper, is discussed to be a useful technique for obtaining producer strains for industrial production.

During the last years monoclonal antibodies have become an important constituent of the clinical diagnostic systems for the detection of various humoral and cellular antigens. By means of monoclonal antibodies new knowledge could be observed in the typing of leukaemias and in the functional characterization of immune cells. Furthermore, possibilities of therapeutical use of monoclonal antibodies and present problems concerning this matter are discussed. The development of human monoclonal antibodies will be helpful in the solution of some of these problems.

The autologous mixed lymphocyte culture (AMLC) in humans has been reviewed. Normal human T cells are stimulated to proliferate in vitro when co-cultured with mitomycin-C-treated or X-irradiated autologous non-T-cells. This reactivity, termed autologous mixed lymphocyte reaction, is thought to represent a self-recognitive mechanism that might be important in regulating the cellular interactions involved in the generation of normal immune response. The nature of the stimulator cell remains a matter of controversy, with evidence suggesting that monocytes, B cells, or dendritic cells may be the major stimulatory populations. In addition, activated T cells expressing HLA-D/DR antigens are also capable of stimulating autologus T cells in AMLC. It was shown that treatment of stimulator cells with anti-HLA-DR antibodies resulted in a significant decrease in their stimulatory capacity in AMLC. These observations suggest that HLA-D/DR antigens, the human counterpart of murine Ia antigens coded for by the I-E subregion of the H-2 complex, expressed on non-T cells or activated T cells participate in the stimulation of AMLC. However, recent findings would suggest that HLA-DS, the human equivalent of murine I-A, is more important than HLA-DR in inducing proliferation in the AMLC. The nature of the T cell subpopulations stimulated by autologous non-T cells has not been clearly elucidated. Evidence has been presented to suggest that either helper T cells or suppressor T cells, as well as cytotoxic cells may be activated. Recent evidence involving the use of monoclonal antibodies that identify mutually exclusive T cell subsets suggests only T cells with the T4+ (helper/inducer) phenotype are triggered directly, but in the presence of such cells, T8+ (suppressor/cytotoxic) cells may also be activated. The T cells which respond in AMLC are a subset of the Con A-responsive T cell population and are separate from allogeneic MLC-responding T cells. Furthermore, the responding cells are T cells forming rosettes with autologous erythrocytes and have surface and functional characteristic of post-thymic precursors. Recent studies indicate that in AMLC between T and non-T cells or T and activated T cells phenotypically distinct subpopulations of T lymphocytes respond by proliferation and express distinct immunoregulatory function. Thus, it seems that distinct species of Ia antigens expressed on activated T cells as opposed to those expressed on non-T cells could participate in the triggering process for the stimulation of functionally distinct human T cell subpopulations.(ABSTRACT TRUNCATED AT 400 WORDS)

Human lymphocytes were derived from the spleen of a 15-year-old female patient after partial splenectomy. High cell yield and percentage of human B-lymphocytes (over 30%) gave useful conditions for their fusion to mouse myeloma cells (P3 X63/Ag8/653). Fusion efficiency (cultures with growth) was 96%. From 288 initially seeded cultures 176 showed IgM and only one IgG production as determined by enzyme immunoassays. Twenty primary cell lines were cloned with differing results and 17 heterohybridoma cell clones with high IgM production rates could be established for more than 6 months in vitro. The present results show the possibility to produce human monoclonal antibodies in heterohybrids derived from human spleen lymphocytes.

The ELISA is the established screening technique for the detection of antibodies directed against HIV. The first generation assays, mostly based on the sandwich principle, employed purified virus from cell culture and gave both false-positive and false-negative results. Sandwich-type assays preferentially detect IgG antibodies, require a high serum dilution and are two-step procedures. In order to detect an immune response as early as possible after infection anti-HIV antibodies of the IgM class should also be measured. To this end a competitive ELISA has been developed using a solid phase-adsorbed recombinant HIV envelope protein and an enzyme-labelled human monoclonal antibody. This detects both IgM and IgG antibodies, the results are available within 1 h and a serum predilution is not necessary.

A solid phase enzyme immunoassay using methylated bovine serum albumin (BSA)-precoated and DNA-coated microtiter plates was developed for the detection of IgG and IgM anti-DNA autoantibodies in the supernatants of lymphocyte cultures and hybridomas. In patients with systemic lupus erythematosus (SLE) the significantly raised IgG anti-DNA antibody synthesis indicates that preactivated anti-DNA clones circulate in the peripheral blood. This was associated with the detection of anti-DNA antibodies in the sera. The screening of 53 supernatants from human x mouse hybridomas showed an antibody to denatured DNA in 16 supernatants.

Histamine (10(-4) M) suppressed the PHA-induced activation of lymphocytes in vitro. This was shown by inhibition of the expression of DR- and 4F2-antigens, of the blast transformation, DNA- and RNA-synthesis, and the formation of thermostable sheep erythrocyte rosettes. Histamine affect these phenomenons only when added to the culture during the first hours. Already 6 hrs after addition of PHA, it exerted no significant suppression. The target of the inhibiting effect of histamine is the G0-G1-transition of the lymphocytes. Cells that already had entered the G1-phase were no more susceptible to histamine.

Counting and separation of auto-rosette-forming lymphocytes is a critical procedure since autologous rosettes are very unstable. It was demonstrated that human serum albumin efficiently stabilizes autologous rosettes, and makes it possible to distinguish at least three T-cell subsets by their different affinities for autologous erythrocytes. About 1% of T-cells have a high affinity for autologous erythrocytes and about 30% intermediate affinity. With a careful procedure, it is possible to detect a further subpopulation with very low affinity for erythrocytes. The sum of these three subsets was about 75% of the T-cells in the peripheral blood.

For mass cultivation of group B streptococci (B-III and B-variant streptococci), a fermenter dialysis culture technique is described and compared to conventional shaking culture and fermenter batch culture techniques. The influence of two kinds of cultivation media on the bacterial yield is demonstrated. The growth rate of the bacteria and the yield of the microbes is higher in modified POPE medium than that observed with Nährbouillon I. The type-specific polysaccharide of B-III-streptococci prepared by phenol-water extraction followed by gel-chromatography can be used as a screening antigen for the production of monoclonal antibodies against B-III-streptococci.

The influence of steroids on lymphocyte responses was tested in vitro. The inhibition of early events of lymphocyte stimulation and cell proliferation after PHA stimulation was estimated. We found, however, different individual responses of immune cells of various donors to the preparations dexamethasone, prednisolone, hydrocortisone or methylprednisolone in both in vitro models tested. Steroid treatment of lymphocyte cultures resulted in both inhibition and enhancement of PWM-driven Ig synthesis in cultures derived from different test persons. In experiments with lymphocytes from patients with acute systemic lupus erythematosus we found a dramatically enhanced PWM-stimulated IgG synthesis which is disturbed in cultures of these patients. The individuality of steroid sensitivity of lymphocyte cultures of healthy test persons and the abnormal reactivity of SLE lymphocytes were discussed in relation to therapeutical applications.

The aim of this study was to develop an optimal technique for cryopreservation of newly formed human and mouse hybridoma cells immediately after fusion. It was shown that human hybridoma cells could be frozen most successfully at a cooling rate of 5 K/min whereas mouse hybridomas at 1 K/min. The percentage of FCS in cryopreservation media (30 or 90%) had no influence on the recovery of both types of hybridomas. For testing the efficiency of freezing, the fusion rate (number of growing and Ig producing hybridomas), the ratio of IgM:IgG-producing initial hybridoma lines and yields of specific wells were analysed. Comparable results were registered when optimally cryopreserved and non-frozen material was studied. Optimization of the cryopreservation of newly formed hybridomas may be of methodological importance, especially when a time-consuming screening for antigenic specificities must be carried out.

The exact chemical characterization of the prepared Escherichia coli K 1 capsule polysaccharide is necessary and a prerequisite for using this antigen as a screening antigen in the production of monoclonal anti-K 1-antibodies. The K 1-antigen, prepared by phenol-water-extraction, was analyzed by protein, RNA, and neuraminic acid determination. An addition, the antigen was subjected to elementary analysis, infrared- and 13C-NMR-spectroscopy, and gelchromatography. After testing the serological specificity, the K 1-antigen was identified as a high molecular polyneuraminic acid.

Activated T cells in the peripheral blood of patients with systemic lupus erythematosus (SLE) were determined using monoclonal antibodies against activation antigens. Elevated percentages of HLA-DR+ T cells were found in association with active disease. In contrast, we observed an increase in IL-2 receptor-bearing T cells in only six out of 16 patients with active disease. In vitro assays, like spontaneous proliferation, response to IL-2, production of IL-2, and immunoglobulin synthesis have shown that the different patterns of activation antigens are related to different functional stages of T-cell activation. The possible therapeutic consequences are discussed.

Dipeptidyl peptidase IV (DP IV), an ectoenzyme in the cell membrane of human T lymphocytes, is an important constituent in the process of lymphocyte activation and proliferation. In the presence of specific inhibitors and antibodies against DP IV several in vitro functions of activated lymphocytes were found to be impaired. These include mitogen induced DNA synthesis, immunoglobulin secretion and production of interferon-gamma. The functional importance of DP IV during activation of lymphocytes is underlined by an increase in cellular DP IV activity upon mitogenic stimulation.

The aim of this work was to optimize the antigen-specific prestimulation of human lymphocytes for further hybridization to produce human monoclonal antibodies. In vitro stimulation of human peripheral blood lymphocytes with purified Tetanus Toxoid (TT) resulted both in an increased amount of secreted specific antibodies and in an enhancement of the portion of anti-TT antibodies in the level of total immunoglobulin secretion. Both IgG and IgM antibodies were found. Donors who had not been boostered in vivo with TT for more than 10 years also showed an antibody response to the antigen in vitro. Five days after in vivo boostering we found in lymphocyte cultures derived from 3 donors an enhanced antibody synthesis in the response to TT and also an increased portion of specific antibodies in the spontaneously secreted immunoglobulin in unstimulated control cultures.

Antibodies coupled to horseradish peroxidase (HRP) were purified by gel filtration and by combination of affinity chromatography and gel filtration. Binding kinetics to solid phase bound antigens were studied of the antibody-enzyme conjugates in the presence and in the absence of unlabelled IgG. The detection limit in enzyme immunoassay was found to be lowered by factor 16 after elimination of unlabelled IgG from the conjugate.

Activation events induced by lectins in human lymphocyte cultures differed not only in time kinetics of their appearance but were also in a different manner inhibited by hydroxyurea (HU). 4F2 and Tac expression, thermostable sheep erythrocyte rosette formation, cell volume distribution changes and the enhancement of the purine metabolic rate [( 3H]adenine incorporation) induced by PHA, Con A or PWM were not influenced by HU treatment, suggesting G1-phase dependency of these markers. The decrease in the mitogen-induced marker expression after 48 h of incubation with HU can be explained by the unability of activated cells to process the cell cycle, to divide and to become newly positive cells. Mitogen-induced RNA synthesis was partially, DNA synthesis, PWM-induced Ig synthesis and lectin-mediated HLA class II antigen expression were totally inhibited by HU. It holds especially for the DR antigen that its appearance in a polyclonal model of lymphocyte activation occurs in a later (postmitotic G1) phase of the cell cycle. The inhibitory effect of HU on late activation parameters could be removed after washing the cells. Mitogen-activated, HU-treated PBL cultures after removing HU continued cell cycle progression without requirement for further addition of lectin.

An enzyme immunoassay for the quantification of human Cu/Zn SOD in serum, urine and erythrocytes was developed applying monoclonal and polyclonal antibodies. The one-step assay is completed within 30 min and enables the detection of 0.3 microgram Cu/Zn SOD per litre. A Cu/Zn SOD concentration of 46 +/- 21.5 micrograms/l and of 1 +/- 0.6 micrograms/mmol creatinine was determined in the serum and the urine, respectively, of healthy individuals. A content of 15 +/- 1.7 ng Cu/Zn SOD was found in 10(6) erythrocytes. Patients with Down's syndrome exhibited a 3.8-fold, a 2-fold and a 1.6-fold higher concentration of Cu/Zn SOD in their serum, urine and erythrocytes.

The kinetics of HLA-DR antigen expression by human peripheral blood monocytes during cultivation in vitro was studied. Immediately after separation by glass adherence, about 60% of monocytes expressed DR antigens as judged by indirect fluorescence staining with a monoclonal antibody (BL-DR/1). Monocytes carefully depleted of lymphocytes, gradually lost their DR antigens during cultivation in the absence of exogenous interferon-gamma (IFN-gamma). However, addition of a few lymphocytes to the adherent cells prevented the decrease of DR antigen expression. Furthermore, it was shown that doses as low as 1 IU/ml of IFN-gamma are sufficient to induce DR antigen expression by cultured monocytes. Experiments with cyclosporin A suggest that lymphocytes contaminating the monocyte preparations can produce spontaneously sufficient amounts of IFN-gamma for maintenance of the DR antigens on monocytes.

The influence of C-reactive protein (CRP) on early and late parameters of mitogen-induced lymphocyte activation in vitro has been tested. Early activation markers (increase of cell size and formation of thermostable SE rosettes) and the lymphocyte proliferation (3H-thymidine incorporation) were changed by CRP (10-50 micrograms/ml), whereas PWM-induced stimulation was inhibited and PHA-induced activation was enhanced. Production of human immunoglobulins in vitro in culture from 12 healthy donors was influenced by CRP in a different manner. Modulation of mitogenic responses of lymphocytes by CRP occurs in an early stage of the cell cycle. Responsible for this phenomenon can be soluble factors, which were removed from the culture by washing the cells after 24 hrs. We discuss here the unspecific modulatory role of CRP in various immune-mediated diseases.