Workshop R: Innovations - Methods and Therapeutics

WORKSHOPR Innovations _. Methods ond Therapeutics

University of Leipzig, Biosciences, Dept. of Immunobiology, Leipzig, Germany

R.l «MECH» - a novel type of immunostimulatory active chemical substance

K. DROSSLER and S. LEISTNER

MECH is the code word of [3(2-mercaptoethyl)quinazoline-2,4(IH,3H)-dione], a novel immunostimulatory active substance that has been proven effective in a variety of animal models. This low molecular weight (222,2) chemical compound was synthesized in our laboratory (see EP 454064 AI). It is an odorless, crystalline substance, has a chiefly lipophilic nature and is only slightly soluble in water. When given by the oral route it is quickly absorbed from the gastro-intestinal tract leading to high serum concentrations already one hour after ingestion. In an acute toxicity test female rats (Shoe: WIST) were given single doses of 3000 mg/kg or 6000 mg/kg MECH orally. Neither mortality nor any symptom of poisoning could be recognized. The immunostimulatory activity of MECH has been investigated in mice and guinea pigs. When CBA mice were immunized i.p. on day 0 with sheep red blood cells and given a dosis of 2 mg MECH/kg on days -2, -1, 0, +1 and +2 the values of IgM-type plaque-forming spleen cells (IgM-PFCs) at peak response on day +4 were significantly increased in respect to controls. Under similar conditions, the numbers of IgG-PFCs monitored on day +6 were up to 4 times higher than in controls. The delayed-type hypersensitivity reaction recorded by footpad swelling test was slightly increased but strongly prolonged; the rejection of allogeneic skin grafts was not influenced. In guinea pig MECH was found to increase the contact sensitivity to 2,4-dinitrofluorobenzene and the specific T -lymphocyte proliferation in vivo. Under in vitro culture conditions MECH augmented the LPS-induced IL-l synthesis from murine and human macrophages in a dose-response manner and simultaneously, the prostaglandin-E2 production was strongly decreased.

University of Leipzig, Biosciences, Dept. of Tmmunobiology, Leipzig, Germany

R.2 Influence of the immunostimulatory active substance MECH on the release of macrophage derived immunomodulating mediators

H. GARN and K. DROSSLER

The newly synthesized compound MECH [3-(2-Mercaptoethyl)-chinazolin-2,4-(IH,3H)-dione] is known to cause immunostimulating effects in vivo when using mice and guinea pigs. Some of the results of the in vivo experiments indicate that macrophages

24th Meeting of the Society of Immunology . 233

may play a role in the immunomodulating action of the substance. Therefore, we investigated the in vitro effects of MECH on the release of different factors by murine peritoneal exudate macrophages and human monocytes. We couldn't see any effect when the substance was added at concentrations ranging from 10-4 to 10-8 M to macrophage monolayers even after incubation times up to seven days. In contrast to these results, when MECH was introduced together with lipopolysaccharide (LPS) as a natural stimulant, striking effects were observed. Under these conditions, the application of MECH resulted in a dose-dependent increase of interleukin-l (IL-l) in the culture supernatants as compared to that found in LPS-stimulated controls. Simultaneously, the amount of prostaglandin E2 (PGE2) released by the LPS-stimulated macrophages was significantly decreased. The release of IL-6, tumour necrosis factor-a (TNF-a) and leukotriene B4 (LTB4) was only slightly influenced. All of the described activities were observed using murine macrophages or human monocytes equally. These results are strong indications for our hypothesis that the in vivo immunostimulating effects of MECH are at least partially due to the effects of the substance on macrophage functions.

I Institute for Medical Immunology, HU Berlin, Charite, 2Institute for Immunology, FU Berlin, Klinikum Steglitz, and 3Schering AG, Berlin, Germany

R.3 Characterization of a neutralizing scFv directed against human TNF-a in vitro and in vivo

G. GROTZ!, C. RIESEl, T. MICHELI, E. OELSSNER!, M. IRMLER3, U. MARX!, T. DIAMANTSTEIN2, H.-D. VOLK l, and R. VON BAEHR!

We constructed an scFv from the variable domains of a neutralizing mAb directed against human TNF-a because of its therapeutical interest. Periplasmatic expression in E. coli resulted only in a low yield of purified protein. Therefore cytoplasmatic expression leading to inclusion bodies was preferred. Using a refolding procedure we got a ten-fold higher yield of purified protein. This material was used for in vitro and in vivo characterization. The scFv has a similar affinity as the Fab-fragment but a ten-fold lower avidity compared to the parental mAb. We showed in immunochemical studies with recombinant receptors that the binding of rhu TNF-a to both receptors could be competed with the scFv. This could be reflected in a neutralizing capacity similar to the Fab-fragment in an in vitro assay using the cell line L929. Furthermore we will present first results of in vivo studies of the scFv using a test to neutralize lethality of rhu TNF-a in C3H/HeJ-mice.

234 . 24th Meeting of the Society of Immunology

Institute of Medical Microbiology and Hygiene, Technical University of Munich, Munich, Germany

R.4 Differential action of the immunosuppressive agent leflunomide on T cells in vivo and in vitro: Inhibition of proliferation yet preservation of TCR-triggered T cell function

R. LANG, H. WAGNER, and K. HEEG

The recent reports that HWA 486 (leflunomide) prevents allograft rejection in rats raises the question about the substance's mode of action on T cells. We have analyzed here the effects of leflunomide on T cell reactivity in vivo and in vitro in the murine system. Leflunomide strongly inhibited T cell proliferation following stimulation with lectins, monoclonal anti-TCR antibodies, and alloantigens. However, lymphokine production (IL-2) and expression of IL-2 receptor (IL-2R) was sustained upon stimulation. Similar to this, cytolytic effector function of all ore active CTL was not impaired by leflunomide. Preliminary results of cell cycle analyses suggest that leflunomide blocks the cell cycle in Gl or early S-phase. To analyze the effects of leflunomide on T cells in vivo we injected mice with the superantigen SEB which activates all V~8-expressing T cells. As in control mice high serum levels of IL-2 could be measured, in addition V~8+ T lymphocytes expressed IL-2R. Moreover, early apoptosis « 24 h) of SEB reactive T cells was not impaired by leflunomide. In contrast, SEB-induced clonal in vivo expansion (day 2 after injection) was completely blocked by leflunomide. Hence, leflunomide primarily inhibits T cell proliferation yet does not interfere with the triggering of a variety of T cell functions. This might have important consequences for the use of leflunomide as an immunosuppressive agent.

I Institute Molecular Pharmacology, Medical School Hannover, Germany; 2Institute Clinical Pharmacology, Guangzhou College of Traditional Medicine, China

R.S Effects of the alkaloid sinomenine on mediator release, cytokine synthesis, and proliferation of immunocompetent cells

LIANG LJU1,2,j. RIESEl, K. RESCH I ,and V. KAEVER1

The alkaloid sinomenine (extracted from Sinomenium acutum) is used in the traditional chinese medicine for treatment of chronic inflammatory diseases such as rheumatoid arthritis. In vivo anti-inflammatory and analgesic actions have been well demonstrated by chinese researchers. We have investigated in vitro effects of this drug in several cellular systems. Our results show that sinomenine could significantly inhibit leukotriene C4 and prostaglandin E2 synthesis in mouse peritoneal macrophages or the mouse macrophagelike cell line H4-7 after short-time stimulation with zymosan or calcium ionophore. In H4-7 cells production of nitric oxide (measured after 48 hrs) and tumor necrosis factor (determined after 20 h) induced by interferon-y/lipopolysaccharide was reduced by sinomenine, too. This drug also exhibited a marked inhibition on the proliferation of murine spleen cells and human peripheral mononuclear cells either activated by mitogens or by two-way mixed lymphocyte reaction. Although half-maximal concentrations


needed for the described in vitro inhibitory effects seem rather high (10-4-10-3 M) different viability assays gave no evidence for a direct cytotoxic action of sinomenine in our experiments.

Abteilung Immunologie und Transfusionsmedizin, Medizinische Hochschule Hannover, Hannover, Germany

R.6 CTLp-frequencies in monozygotic twins detected by limiting dilution analysis

}. LOTZ, D. PEEST, R. LEO, R. JACOBS, M. STOLL, A. KEMPER, H. LINK, H. DIEDRICH, W. STANGEL, and H. DEICHER

Limiting dilution analysis of cytotoxic T -lymphocyte precursors (CTLp) has been proposed to be a useful tool for donor selection in allogenic bone-marrow transplantation. We present a culture-system in which irradiated cells of party A served as stimulators for proliferation of party B cells. 50000 irradiated stimulator cells were cocultured with viable responder cells diluted in 8 steps from 50000 to 3125 cells/well. 30 wells for each dilution were set up. Generated CTLs were measured in a 51Cr-release assay after 7 days incubation. Frequencies of allogen-specific CTLp were calculated by least-square approximation. Measured CTLp frequencies run from 1 in 4000 to less than 1 in 300000 peripheral blood lymphocytes of HLA matched bone-marrow donor! recipient pairs. With this system we were able to detect CTLp-frequencies in 3 out of 5 pairs of twins all proven to be monozygotic by HLA analysis as well as lymphocyte- and erythrocyte surface markers. Control mixed-lymphocyte cultures were all unreactive in these monozygotic twins. Based on these results we propose that histocompatibility antigens are not solely determined by genetic inheritance but might also be influenced by environmental or mutagenic factors.

Supported by Dieter Schlag Stiftung.

GSF-Institut fur Immunologie, Munchen, Germany

R.7 T cell depleting effector functions of a chimeric anti-Thy-1.2 antibody with deletion of the CH 1 domain

R. MOCIKAT, P. LANG,}. MYSLIWIETZ, and S. THIERFELDER

To generate T cell depleting chimeric antibodies (Ab) specific for the murine Thy-1.2 antigen (Ag) we substituted the immunoglobulin heavy chain constant (CH) region by the human IgG 1 sequence by homologous recombination in the hybridoma cell line. With a frequency of 0.4 x 10-7 we obtained clones which have undergone an illegitimate recombination event leading to deletion of at least a part of the CHI gene segment. eDNA sequencing revealed splicing of the VH to the hinge domain, but all cysteines involved in interchain bonding were present. Nevertheless, as a consequence of the altered conformation the S-S bridges between H and It chains cannot be formed. The It chains are non-


covalently associated to a H chain dimer and can dissociate from it. This unusual behavior has not been observed with the hitherto known CHI-deficient Ab whose antigenic specificities are not defined or which have no L chains at all, as in a subgroup of H chain disease. The known specificity and the well-defined biological effects of our immunosuppressive parent Ab allowed us to assess the significance of the labile It chain pairing as to Ag binding and effector functions. Using a novel FACS method we quantitated the equilibrium of associated and dissociated form and showed that the average number of It chains per Ab molecule is 1.3. However, Ag binding turned out to be possible only for those molecules which have two It chains. For those Ab the avidity is unaffected. In vivo testing revealed a reduced T cell depleting efficiency as compared to an intact mouse/human chimeric IgGl Ab. The graft-versus-host reaction after bone marrow transplantation could not be prevented. We showed that this is due to the fact that only a part of the Ab can bind to the target cells and not to altered effector functions. For example the fixation of Clq was not reduced by the abnormal conformation.

Institute of Biophysics, School of Medicine, University of Leipzig, Leipzig, Germany

R.B Determination of the hydrogen peroxide concentration during phagocytic activity with the luminescent system luminol/hypochlorites

S. MUELLER and J. ARNHOLD

The generation of reactive oxygen species by polymorphonuclear leukocytes (PMN) and macrophages is considered to play an important part in antimicrobic and anticancerous activity and also in tissue injury. In this context, hydrogen peroxide acts as a central oxygen metabolite which leads additionally to more reactive oxygen species in various enzymatical and nonenzymatical steps. The determination of the hydrogen peroxide concentration during phagocytic activity still remains insufficiently solved until now. Our studies on the chemiluminescent mechanism of luminol by hypochlorite have offered a new possibility of determining hydrogen peroxide. According to these investigations and those of other authors luminol is oxidized by hypochlorite to a diazaquinone in a first step. The diazaquinone leads in a specific reaction with hydrogen peroxide to a strong chemiluminescence. On the basis of this mechanism a luminescent assay was developed in order to detect hydrogen peroxide up to 10-9 M in a time of less than 2 sec. Hence, it is possible to determine hydrogen peroxide even if the concentration changes in time like during phagocytic processes. Subsequently the concentration-time curve of hydrogen peroxide in the presence of phagocyting PMN was measured. In addition, the effect of superoxide dismutase, catalase and sodium azide was investigated. Our results reveal that this luminescent method provides a specific and sensitive method to determine hydrogen peroxide in the extracellular compartment of phagocytic cells. According to this, the hydrogen peroxide concentration rises up to 10-3 M during the phagocytic process. Finally, the application of the assay is discussed in order to characterize the functional state of PMN in respect to inflammatory diseases and the influence of drugs and cell regulators.


Baxter Deutschland GmbH, UnterschleiBheim, Germany

R.9 Extracorporeal elimination of human immunoglobulin with IgG-Therasorb

J. MOLLER-DERLlCH, A. DU MOULIN, and R. SPAETHE

Immunoapheresis as extracorporeal technique to eliminate predefined components from the patient's plasma has been proven to be a safe and highly specific method. The therapeutic procedure was first introduced by STOFFEL et al. (1). IgG-Therasorb columns contain a highly specific immobilized antibody raised against IgG heavy chains and kappa and lambda light chains. In an in vitro immunoapheresis model, which mimics clinical conditions, kinetics of IgG, IgM and IgA reduction was investigated. A preclinical study with normal human plasma and 17 sera from highly sensitized dialysis patients and patients suffering from autoimmune diseases showed that on average 93 % of human IgG and 76-80 % both of IgM and IgA were eliminated. Analysis of IgG subclasses revealed an equal reduction of IgG 1-4. By eliminating human immunoglobulin removal of harmful antibodies like cytotoxic anti HLA antibodies or autoantibodies could be demonstrated. Main fields of clinical application for IgG-Therasorb columns will be kidney transplantation and autoimmune diseases.

1. W. STOFFEL, H. BORBERG, V. GREVE (1981). Lancet II, p. 1005-1007.

Humboldt University Berlin, Charite, Institute of Medical Immunology and 'Practice of Medicine (Haematology/Oncology) Berlin, Germany

R.10 Human primary lymphoid tissue culture in the new hybrid bioreactor system T ecnomouse

A. NAGEL, H. MATTHES', U. MARX, and R. VON BAEHR

The hybrid bioreactor system Tecnomouse is an accepted tool for in vitro production of monoclonal antibodies. Because of an optimal combination of supply of nutrients via hollow fibres and supply of oxygen via silicon membranes directly to the cell growth chamber, this system seems to be well equipped for the cultivation of exacting human tissue culture also.

To analyze the properties of the bioreactor system human bone marrow and thymic tissue were cultivated simultaneously in different bioreactor units. Tissue could be kept viable over several weeks. The extracapillary (EC) cell culture chamber was analyzed after fermentation procedures. We found that the periodic harvests taken from this chamber during fermentation did not reflect the real situation within the bioreactor in terms of viability and total cell counts. High concentrations of IL-6, IL-8 were generated in both cultures. Predominantly CD-13+ and CD-1S+ cells were found at the end of the bone marrow culture. Possible applications of the Tecnomouse bioreactor in the field of in vitro modulation of human lymphoid organs are discussed.


Klinik fur Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, Hannover, Germany

R.ll Immunosuppression with an anti-lL2 monoclonal antibody: evaluation of the immunosuppressive mechanism in liver allografted patients

B. NASH AN, H.J. SCHLITT, K. WONIGEIT, and R. PICHLMAYR

Monoclonal antibodies (mAb) are used in experimental and clinical settings to improve immunosuppression. The mechanism of action including the therapeutic effect and the induction of side effects is various and seems to relate to the interaction with the target side as well as the isotype of the mAb. We report here on the results of an ongoing clinical study (randomized phase 3 trial) in liver allografted patients which are treated either with a mAb (BT 563, murine IgG 1) that binds to the a chain of the IL2 receptor or ATG. 20 patients received the mAb for 12 days at a dosage of 10 mg/d. Lymphocyte subpopulations (CD3, CD4, CD8, CD45RA, CD25 and BT 563 coated cells) were measured by flow cytometry for 4 weeks. Levels of soluble IL-2 receptor were evaluated by an ELISA test. The presence or absence of FC-receptors for mouse IgG 1 in these patients was analyzed by stimulating PBMC by the CD3 mAb WT31 (IgGl). BT 563 sera levels were measured by ELISA and flow cytometry. Results: 1. No reduction of lymphocytes was observed during therapy and no effect in lymphocyte subpopulations was noted. 2. During therapy no cells carrying CD25, but coated cells were seen. 3. No major alteration in soluble IL-2 receptor concentrations was measured during therapy. 4. In vitro tests of high and low responsiveness of IgG 1 demonstrated that 25 % were of the high responder type caused by Fc receptor polymorphism. 5. Levels of free, binding BT 563 were 1 to 4 ~g/ml during therapy. 6. No episode of rejection was observed postoperatively. Conclusion: According to the above data BT 563 is coating and thus blocking lymphocytes. There was no evidence of depletion or modulation. This assumption is supported by the observation of stable levels of soluble IL-2 receptor in patient sera as well as high levels of free binding mAb. The in vitro stimulation of PBMC via the Fe-receptor has no clinical relevance as there was no difference between high and low responders in the clinical outcome, lymphocyte subpopulations or soluble IL-2 receptor levels. Thus the immunosuppressive action of BT 563 is most likely the effect of a functional blockade of IL-2 receptors giving the opportunity of selective immunosuppressIOn.

Humboldt University Berlin, Charite, Institute of Medical Immunology, Berlin, Germany

R.12 Endotoxin in hybridoma technology - production of monoclonal antibodies for in vivo use

P. PREIKSCHAT, V. VON BAEHR, U. MARX, and R. VON BAEHR

A number of monoclonal antibodies with various potential in vivo applications are currently subject to preclinical analyses. Several of them have been licensed for clinical use. As most of these antibodies are produced by hybridoma technology endotoxin contaminations have become major problem.


We investigated the influence of different cell culture techniques and methods of down stream processing on the level of endotoxin in final antibody preparations. Samples from ascites fluids, suspension culture supernatants and harvests derived from high cell density bioreactor- cultivation were analyzed.

Furthermore, different purification procedures (e.g. ultrafiltration, affinity chromatography over protein-A and -G, ion exchange chromatography) were compared in regard to the reduction of endotoxin contaminations.

The advantages of high cell density bioreactors for production of monoclonal antibodies for in vivo use were demonstrated. The efficiency of different purification procedures was checked. To verify the results we obtained with the routinely used LALtest (Chromogenix) we performed several variants of endotoxin tests in parallel.

Boehringer Mannheim GmbH, Biochemical Research Center, Penzberg, Germany

R.13 A non-radioactive immunoassay for the measurement of lymphocyte proliferation

E. RUSSMANN, C. DOPPLER, and M. HINZPETER

Assays for the assessment of cell proliferation have become a key technology in cellular immunology and a number of short-term assays have been developed, including dye exclusion techniques, diverse colorimetric assays and tritiated thymidine uptake. At present the incorporation of 3H-thymidine into DNA is widely used and accepted as a measure of DNA synthesis and cell proliferation. But this assay is laborious and hampered by the involvement of radioactive isotopes, specialized equipment and radioactive waste. To avoid toxic hazards like scintillation fluids and radioactive nucleotide precursors non-radioactive assays for cell proliferation have been developed and evaluated in many laboratories. However, in contrast to the 3H-thymidine incorporation assay which specifically measures DNA-synthesis all other methods measure total cell number or the number of viable cells. Therefore the use of colorimetric cell proliferation assays is limited. They can not be used for the study of lymphocyte proliferation where a high percentage of non proliferating but viable cells result in a high background which will falsify the assay results. The use of 5-bromo-2' -deoxyuridine (BrdU) instead of 3H_ thymidine offers a novel non-radioactive alternative to the radioactive assay. This technique is based on the incorporation of BrdU into DNA followed by its detection with anti-BrdU specific monoclonal antibody. Like thymidine, the pyrimidine analogue BrdU is incorporated into the DNA when cells enter the S-phase. Therefore, the BrdU incorporation assay analyses cell proliferation only, whereas colorimetric, cell metabolic assays would give no or misleading results. To evaluate the performance of the BrdU ELISA the sensitivity of the BrdU-ELISA was compared to that of the 3H-thymidine assay. The comparison was done with human peripheral blood lymphocytes stimulated with PHA or anti-CD3 and with murine thymocytes stimulated with IL-l and IL-2. In all assay systems tested so far we obtained comparable dose response curves using the different indicators. In conclusion, we found the BrdU-ELISA to be an attractive alternative compared to the 3H-thymidine assay. It neither requires radioactive isotopes, special protective precautions or expensive equipment nor does a radioactive waste problem exist. The complete assay from the onset of the microculture up to data analysis can be performed in the same microtiter plate. Together with on-line computer processing (data collection, calculation and report generation) this technique allows the fast throughput of large numbers of samples.


Humboldt University Berlin, Charite, Institute of Medical Immunology, Berlin, Germany

R.14 In vitro cultures of human PBLs and macrophages using a miniaturized perfusion bioreactor

M. SCHLArKE, S. KOCH, C. LIEBENTHAL, M. SEIFERT, W. D. DOcKE, and U. MARX

In vitro cultures of human PBL's and macrophages are a substantial part of studying immune mechanisms. Supply of cells with nutrients and O 2 is limited in usual cell culture techniques (flasks, plates, roller bottles, spinners etc.). Because of the impossibility of culturing this cells in a three-dimensional space, a wide range of immunological phenomena can't be analyzed in these in vitro systems (e.g. adherence, chemotaxis in spleen and thymus). To improve supply with nutrients and oxygen, a new miniaturized hollow fibre bioreactor was used for in vitro culture of mononuclear cells from the peripheral blood of healthy donors. Previous studies indicate, that the system can provide high densities of tumor cells with necessary nutrients over several weeks. A serum-free cell culture was performed in the bioreactor system. Over a period of more than 5 weeks, viable cells could be detected in the bioreactor harvests. An activation of Tcells and macrophages could be shown using FACS-analysis. Regarding adherence, deficiency of the used perfusion membrane could be proved. For this reason, a set of various hollow fibre membranes was tested in parallel, respecting their properties to enhance cell growth and adherence. Possible applications of this bioreactor system in the fields of in vitro immunization and culture of stimulated or transformed immune cells are discussed.

Institut fur Immunologie, Munchen, Germany

R.1S Changed pattern of recurrences of colorectal cancer patients after adjuvant treatment with murine monoclonal antibody 17-1 A

E. SCHNEIDER-GADICKE, G. SCHLIMOK, R. RAAB, W. SCHMIEGEL, S. SAID, K. HOFFKEN, and G. RIETH MOLLER

In a prospective clinical trial, 185 patients with Dukes C colorectal cancer were randomized to be either treated with surgery followed by 900 mg of m17-1A or by surgery alone (control group). Analysis of the presence and pattern of recurrence was performed on 166 eligible patients. Since smaller tumor burden, assumed to be more accessible for antibodies and cytotoxic effector cells, is present at distant sites at the beginning of adjuvant therapy, the distinction between local recurrence and distant metastasis was of particular interest. After a median clinical follow-up of 5 years, 46 treated patients were found to be tumor-free as compared to 28 patients in the control group. Furthermore, an excess of distant metastases as first event was observed among patients in the control arm. For patients treated with m17-lA, recurrences in liver and lung numbered 14 and 6 respectively. These incidence rates were exceeded in the control patients with 22 and 11 events, respectively. When plotted according to Kaplan-Meier estimates, a clear treatment effect could be demonstrated for the group treated with monoclonal antibody 17 -lA (p = 0.002). No corresponding reduction of local recurrence


was found among patients receiving m17-lA. In conclusion, these observations support the hypothesis of the original protocol that monoclonal antibodies are most effective in eradicating micrometastatic foci.

lInstitut fur Klinische Biochemie and 2Klinik fur Hautkrankheiten der U niversitat Bonn, Bonn, Germany

R.16 CD4-ELlSA: a method for determining CD4+ -T -lymphocytes and a possible benefit for quality assurance programs

L. SOMMER 1, R. BAuER2, and A. VON RUECKER 1

The determination of absolute CD4-T -lymphocyte counts by flow cytometry has various drawbacks. Most important is the fact that it requires results from three laboratory methods: the white blood cell count (WBC), the differential count to determine the percentage of lymphocytes and immunophenotyping to detect the CD4 positive T -lymphocytes. A recent external quality assessment trial in Germany (1) showed that the sum of errors is significant. The coefficient of variation (CV) in the case of CD4-T-Iymphocytes was 20--24%; the maximum and minimum values varied by approx. 270 %. On the basis of such findings, it seems doubtful whether reliable clinical care can be given to e.g. AIDS patients. A CD4-ELISA which measures CD4 surface molecules in suspension after lysing of cells would be helpful to avoid the cited drawbacks providing CD4 surface molecules are expressed in a constant manner on CD4-T-cells and the amount of CD4 surface molecules on other cells (e.g. monocytes) can be ignored. This point is controversial. Our preliminary results show that the overall correlation between flow cytometric CD4-T-Iymphocyte counts and CD4-ELISA (TRAxTM) enumeration data is satisfactory. Coefficients of correlation ranged from 0.77 to 0.94 depending on the flow cytometer and monoclonal antibodies used in comparison. Low CD4-T -lymphocyte counts « 500 [tl) observed in AIDS patients were associated with a decrease of specific CD4-immunofluorescence per cell in flow cytometric data. This effect was partly compensated for by the relative increasing amount of CD4dim-

monocytes in CD4-ELISA results, as evaluation of flow cytometric data shows. Since true reference methods for cell surface molecules are still lacking, we think the use of a CD4-ELISA kit to monitor AIDS patients is comparable to flow cytometric methods, but no less problematic. A combination of both techniques may help procedure more reliable CD4-cell data and assist in quality assurance programs.

1. A. VON RUECKER et al.: Klin. Lab. 1993 (in press).


Forschungsinstitut Borstel, Borstel; 1 Dept. of Urology, Medical University of Lubeck, Lubeck, Germany

R.17 Inhibition of interleukin (IL)-2 and interferon (IFN)-y release and bacillus Calmette-Guerin (BCG)-induced cytotoxicity by pentoxifylline (POF)

A. THANHAUSER, N. REILING, A. BOHLE1, C. SCHLUTER, M. ERNST, H.-D. FLAD, and

A.J. ULMER

Pentoxifylline (POF) has been reported to be an effective drug in inhibiting tumor necrosis factor-a (TNF-a) response during either endotoxemia or OKT-3 therapy. However, the production of IL-l and IL-6 is not affected or even increased by POF. Furthermore, POF reduced serum-IL-2 after administration of OKT-3 or lipopolysaccharide in mice. To gain further insight into the influence of POF on human immunocompetent cells we investigated the effect of POF on BCG- or phytohemagglutinin (PHA)-induced DNA synthesis and lymphokine production. Furthermore, we focussed our interest on the generation of lymphokine-activated killer (LAK) cells and BCG-activated killer (BAK) cells in the presence of POF. The results show that DNA synthesis of PBMC stimulated with either BCG or PHA was inhibited by POF. Interestingly, we could demonstrate that the addition of POF led to a decrease not only of the monokine TNF-a, but also of the lymphokines IL-2 and IFN-y. Whereas BCGinduced IL-2 and IFN-y release is inhibited at the mRNA level by POF, PHA-induced mRNA expression of these lymphokines is not affected by POF. Thus, the existence of a posttranscriptional regulation of PHA-induced lymphokine release by POF can be assumed. The observed inhibition of lymphokine release is correlated with an inhibitory effect of POF on BCG-induced cytotoxicity against bladder tumor cells, while IL-2 generated LAK cells are not affected by POF.

Supported in part by BMFT, No. 01K18827 and Institut Merieux, Leimen, Germany.

University of Leipzig, Biosciences, Dept. of Immunobiology, Leipzig, Germany

R.1S A monoclonal antibody-based ELISA-system for quantification of MECH in body compartments and cell-lines

U. WAGNER, K. OHLSEN, and K. DROSSLER

A competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detecting even small amounts of the newly described immunostimulant [3-(2-Mercaptoethyl)-chinazolin-2,4-(lH,3H)-dione] (MECH). Hapten-carrier conjugates (MECHHGG) were prepared and used for immunization of Balb/c mice as a first step of monoclonal antibody (mAb) production. The generated mabs were characterized in respect to their specificity and affinity. One of them, designated 3AS/Fll (isotype IgG2a, k), reacted specifically with the quinazoline ring system and partially with the -CH2Smoiety in the side chain at position of MECH. Moreover, this mAb also recognizes naturally occurring metabolites of MECH. To establish an ELISA-system for quantification of MECH in body fluids, tissue extracts and cell-lines, 3AS/Fll was biotinylated.


Antibody binding to solid phase-immobilized MECH -carrier conjugates was determined by using a streptavidin biotinylated horseradish peroxidase complex. The minimal detectable concentration of soluble MECH in phosphate-buffered saline (0.05 % Tween 20) was 0.4 ng/m!. Orally applicated MECH was found in serum already 10 minutes later and peaked 2 hours after ingestion. The substance and/or their metabolites could also be detected in cerebrospinal fluid, different cell-lines, spleen and kidney. The highest amounts were measured in the liver.

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Workshop R: Innovations - Methods and Therapeutics

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