workshop e infections and complement

Workshop E Infections and Complement

Institut fur Immunologie und Serologie, Klinische und experimentelle Immunpathologie, Universitat Heidelberg, FRG

E.1 Functionally important domains of C9 as defined by monoclonal antibodies to C9 inhibiting hemolysis

J. BAUSBACK, R. KONTERMANN, and E. W. RAUTERBERG

Ten monoclonal antibodies (MAB) had been recently raised against human purified C9 in our laboratory, i.e. M3, M6, Ml0, Mll, M15, M22, M37, M38, M41 and M61. The MABs were selected for equally strong reactivity in micro-ELISA. They were now applied to further analyse the different domains of C9 in hemolysis. Epitopes defined by the MABs had been characterized by SDS-PAGE immunoblots (SPI) under reducing and non-reducing conditions using either purified human C9 or its fragments. The latter were obtained by controlled proteolysis using alpha-thrombin (generating the NH,-terminal C9a fragment and the COOH-terminal C9b fragment) or trypsin or by BNPS-skatole cleavage. All MABs revealed a strong binding with monomeric C9 (mC9) and various reactivities with its fragments. Binding characteristics of the MABs were analysed using poly-C9 (pC9): M15 and M37 did not react with pC9 in non-reduced SPIs. Tested under the conditions of a dot immunoblot, M37 was stronger binding to pC9 than to m C9 . Taking all results together, we could localize the epitopes of M61 and M3 to the 10 kD spanning C9a2 fragment, Ml0 and M22 to the 4 kD C9a3 part, Mll to the 8 kD C9a., M6 to an epitope on either C9a3 or C9a. and M38 to C9b •. M37 and M41 could only be associated with C9a: They failed to react with reduced fragments. The antibody M15 could not be attributed to any C9 domain by SPI: It reacted exclusively with non-reduced mC9. IgG of the ten MABs was isolated from ascites by protein G-chromatography. Their inhibiting effects at varying final concentrations (cone) on hemolysis of ChEACl-8 by limiting amounts of C9 (z = 1) was compared. ChEACl-8 had been prepared by incubation of ChEA with C8-defective human serum and purified C8. Commercially available MAB C9-42 was used as positive inhibitory control. Both, C9-42 and M15 revealed a 50 % inhibition at about 1 Itg/ml. Four other MABs, i.e. Mll, M3, Ml0, M61, showed this effect at 10, 12.5,25 and 78 Itg/ml, respectively. M37 was only weakly inhibitory. The other MABs (including M38 against the COOH-terminal C9b.) did not inhibit hemolysis even at a cone. of 100 Itg/ml. Our findings suggest that: 1) the COOH-terminal end of C9 does not penetrate the membrane, that 2) M15 is directed to an epitope necessary for interaction with C8 which is only present on native C9 but shielded in poly-C9, and that 3) different epitopes on distant parts of the C9 chain are obviously relevant for lysis each being relatively restricted in size. The latter is suggested by the fact that only one of the two MABs Ml0 and M22 reacting with the small 4 kD C9a4 fragment adjacent to the thrombin cleavage site is an inhibitor of lysis.

XXth Meeting of the Society of Immunology . 59

Institute of Immunology, University of Marburg, and! Institute of Immunobiology, University of Freiburg, FRG

E.2 Influenza A virus-induced release of tumor necrosis factor-a (TNF-a) from macrophages is enhanced by lipopolysaccharide (LPS), lipid A, and lipopeptides

A. BENDER, M. NAIN, F. HINDER, S. HORCH, W. BESSLER!, and D. GEMSA

Cell wall components of gram-negative bacteria stimulate many macrophage functions. The effects of viruses on macrophage functions have received little attention. In this study, the murine macrophage cell line PU5-lo8 (0.5 X 106/ml) was infected with influenza A/PR8 (100 and 333 HAU/ml). The release of TNF-a was used as a criterion of macrophage reactivity. When incubated for 24 h after infection with influenza A virus or treatment with LPS, lipid A, or the synthetic lipopeptide segments Pam} Cys-Ala-Gly and Pam} Cys-Ser-Gly, moderate amounts of TNF-a were released from macrophages. However, a strong potentiation of TNF-a synthesis was observed in virus-infected macrophages that were simultaneously exposed to low concentrations (10 to 100 ng/ml) of either of the bacterial compounds. Thus, gramnegative bacterial cell wall constituents enhance the responsiveness of virus-infected macrophages as shown by synthesis and release of the monokine TNF-a. Superinduced TNF-a may not only act as a cytotoxic compound but, more importantly, as an activating factor for leukocytes involved in antiviral defense. These results indicate that macrophages are intrinsically able to mount an efficient antimicrobial response when simultaneously exposed to viral and bacterial infections.

Institute for Medical Microbiology, * Institute of Immunology, Johannes-Gutenberg-University, Hochhaus am Augustusplatz, 6500 Mainz, FRG

E.3 Inhibition of interleukin 4-driven T cell proliferation by C3d, a cleavage product of the complement component C3

W. DXUBENER, E. SCHMITI", T. NESBIGALL, T. HORN, E. RODE*, and U. HADDING

In the course of the physiological breakdown of C3, the cleavage fragments C3d and C3c were finally produced. We have generated C3d by cleavage of guinea pig C3b with pancreas kallikrein, and the C3d was purified by the use of an anionic FPLC column. It was recently shown that this C3d is able to suppress the IL 2-driven proliferation of mouse helper and cytotoxic T cells. We examined whether the inhibitory capacity of C3d on T cells was restricted on the IL 2-driven proliferation or if T helper cells of type 2 which use IL 4 as autocrine growth factor could also be influenced by C3d.

We found that C3d is able to inhibit the IL4-driven proliferation of the insulin-specific mouse T cell clone ST2/K9.3/4 in a dose-dependent manner. The suppressive effect was specific for C3d because neither C3, C3b nor C3c had any effect on the proliferative response of ST2/K.9.3/4 cells on IL 2 or IL 4. The amount of C3d induced suppression was similar when ST2/K.9.3./4 cells were stimulated with IL2 or IL4.

We conclude that soluble C3d is a suppressive agent which acts on cytotoxic and both types of mouse T helper cells.

60 . XXth Meeting of the Society of Immunology

Institute of Immunology and Dept. of Internal Medicine, Vniv. of Heidelberg; Gambro Dialysatoren, Hechingen, FRG

E.4 Terminal complement complex (TeC) generation during hemodialysis: a superior index for bioincompatibility of cellulosic and synthetic membranes and their sterilization

R. DEPPISCH, v. SCHMITI, J. BOMMER, E. RITZ, and E. W. RAUTERBERG

Hemodialysis (HD) can be associated with severe adverse reactions. Contact of blood with dialysis membranes induces the release of potent inflammatory mediators from cells or serum progenitors. Complement (C) activation is thought to play an important role, although C3a and C5a generation is a parameter for bioincompatibility of controversially discussed value. Other investigators claimed that hypersensitivity against ethylene oxide (ETO) might be of more relevance. Recent studies pointed to a multifunctional role of TCC, the pore forming C5b-9 complex, i.e. to serve as an inducer of eicosanoid or cytokine release from a variety of target cells. In the present study, we evaluated TCC levels during HD comparing three different types of membranes and two types of sterilization. Five long-term dialysis patients were subjected under comparable conditions to HD sequentially six times with each type of dialyzer, i.e. cuprophane (CV) steam sterilized (CV-S), CV sterilized with ETO (CV-E), hemophan (H) steam sterilized (H-S), H sterilized by ETO (H-E) and polysulphone sterilized by ETO (PS-E). EDTA-plasma samples withdrawn during HD from the arterial inlet and the venous outlet were subjected to TCC-measurements by a specific micro-ELISA based on monoclonal antibodies against TCC neoantigens. First contact of blood induced with all membranes a sudden increase of TCC concentrations in venous outlets during the first 30 min of HD. The TCC peak values ranked in the following order: CV-E (505 Vlml) > H-E (57 VI ml) > PS-E (36 Vlml). The same order was seen in each individual patient. Generation of TCC by the dialyzers was faster than its removal from circulation as reflected by raised arterial concentrations at the end of HD. Interestingly, both CV-S and H-S membranes at all points of the kinetic measurements induced lower TCC generation than their ETO-sterilized counterparts. It is tempting to speculate that the higher frequency of adverse reactions with ETOsterilized membranes may in part be due to a TCC dependent effect. PS-E generating significant amounts of TCC in our study had been earlier claimed to be a C non-activating membrane. Our findings, thus, indicate that TCC is a bioincompatibility index which better discriminates between different membranes than the anaphylatoxins C3a or C5a.

1 Institute of Immunology, Klinikum Steglitz, FV Berlin, 2 Landesinstitut fur Tropenmedizin, Berlin

E.5 Interleukin 2 receptor in patients with AIDS and with localized and systemic parasitic diseases

T. DIAMANTSTEIN\ H. FELDMEIER2, K. ZWINGERBERGER2, G. HARMS2, and o. JOSIMOVICALASEVId

Serum levels of cell-free IL 2 receptor were elevated above normal in serum of patients with helminthic and protozoal infections as well as in AIDS patients. Whereas HIV-positive subjects and patients with a helminthic infection limited to the intestinal tract (ascariasis,


trichuriasis) have a similar level of IL 2R as healthy controls, patients with s. stercoraJis, a parasite with repetitive larval passages in host tissue, showed a significant increase of IL 2R (p < 0.01). Patients chronically infected with liver flukes or with s. mansoni also showed elevated serum IL 2R level (4-5fold than normal). The mean concentration of IL 2R in patients with hepatosplenic schistosomiasis was almost twice as high as in those with the disease limited to the intestinal tract. The parasitic infections associated with activation of B cells and functionally disturbed T cells, as visceral leishmaniasis and malaria show elevated serum IL 2R level which may be the consequence of an exuberant expression and release of the IL 2R by B lymphocytes. Similar to AIDS patients HIV -positive subjects with high toxoplasmose titer showed elevated levels of soluble IL 2R. This finally suggests that elevated soluble IL 2R in AIDS is mainly due to activation of B lymphocytes by polyclonal stimulation of the cells by opportunist infections. In conclusion, we have demonstrated that the elevated serum IL 2R level in systemic parasitic infections is releated to distinct clinical conditions of the disease.

Institute of Immunology and Genetics, German Cancer Research Center, D-6900 Heidelberg, FRG

E.6 Requirement for cysteine by T lymphocytes and release of cysteine by macrophages

H.-P. ECK, S. ROTH, H. GMUNDER, and W. DROGE

Patients with the acquired immunodeficiency syndrome (AIDS) have, on the average, markedly elevated plasma concentrations of glutamate (i.e. 2 X 1O-4M) and reduced concentrations of methionine and cystine (BioI. Chem. Hoppe-Seyler 369: 143 (1988». The importance of cyst( e )ine for lymphocyte functions was already suggested by studies from many laboratories which showed that various lymphocyte functions including mitogenic responses of both T cells and B cells and the production of antibody in lymphocyte cultures is strongly augmented by the administration of (unphysiologically) high concentrations of cystine or cysteine. Using cell culture conditions with approximately physiological amino acid concentrations, we now show that the proliferative activity and intracellular cysteine concentrations of mitogenically stimulated T lymphocytes and of a T cell clone are determined decisively by the extracellular concentration of cysteine. Changes of the extracellular cystine or methionine concentrations, in contrast, were found to have little or no effect. Macrophages were found to consume cystine and to release cysteine. Elevated extracellular concentrations of glutamate (2 X 10-4M) reduce the capacity of macrophages to accumulate cysteine in the extracellular space. Mitogenically stimulated T cells contain higher levels of intracellular cysteine if cultured together with macrophages, but this effect is strongly inhibited in cultures with elevated concentrations of glutamate. The observed effects may be relevant for the pathogenetic mechanism of the acquired immunodeficiency syndrome.


lInstitut fur Hygiene, 2Univ.-Klinik f. Neurologie, 3 Inst. f. Med. Chemie u. Biochem., 4 Ludwig-Boltzmann-Institut f. AIDS-Forschung, Universitat Innsbruck, Fritz-PreglStraBe 3, 6010 Innsbruck, Austria

E.7 HIV antigen and neopterin in immune stimulation of patients in Tanzania

P. HENGSTERl, E. SCHMUTZHARD2, D. FUCHs3, B. SOLDERl, G. BITTERLICHt, C. LARCHERt, G. REIBNEGGER2, H. WACHTER2 . ., and M. P. DIERICHl.4

Progression of human immunodeficiency virus (HIV) in T cells depends on the cells' activation. This state of T cell activation can be specifically and quantitatively detected by determination of neopterin which is secreted by macrophages upon stimulation with interferon y. We have investigated HIV-antigen and neopterin in serum and urine in 253 Tanzanians. Two Tanzanians with antigen were detected, one without antibodies, one with antibodies and symptoms of AIDS. In addition, 8 individuals had antibodies but no detectable antigen. All these persons had elevated, up to very high, neopterin levels (963-2312), mean 1739 !lmoVl. The one with a detectable amount of antigen had 1744 !lIDo1/1 neopterin. Furthermore, antibody positive individuals without symptoms and with negative antigen test, also had elevated levels (26)-1565), mean 1097 !lmoi/l. Among the sero-negative population in Tanzania, 80 % had neopterin values with urine neopterin from 56--8101 !lIDol/l (mean 390 !lIDoVI) and serum neopterin 4.>-123.8 nmoVI (mean 14.7 nmoVI) while among unpaid voluntary Austrian blood donor populations neopterin is positive in only 1.59 %. In spite of undetected HIV antigen, this high amount of neopterin, indicating high levels of T cell activation and high levels of HIV progression, might explain why in Tanzania of 10 seropositives 6 have clinically overt AIDS, while in Austria (2500:160) of 10 seropositives, 0.6 have the disease. It seems very adequate to determine neopterin for measuring stimulation as a necessary condition for virus propagation and therefore as a prognostic marker.

Supported by Land Tirol and L.-Boltzmann-Gesellsch.

Institute for Hygiene, Innsbruck, Austria

E.8 The synthesis and expression of CRl on RAJI cells is enhanced by a factor in human serum

M. Hosp, G. TOPAR", J. MOST, T. F. SCHULZ, A. L. PETZER, and M. P. DIERICH

The growth of RAJ! cells in serum-free cultures can be stimulated by monoclonal antibodies to CR2, the receptor for C3d and the Epstein-Barr virus. These effects cannot be observed if trace amounts of serum are present in the culture medium. Comparing the numbers of CR2 expressed on the cell surfaces, we found significantly less receptors on cells grown under serum-free conditions than on cells from serum-containing media, as assessed by immunofluorescence analysis. In ELISA experiments, we could show that not only the surface molecules but also the intracellular pool of CR2 is increased after addition of serum to the culture medium and decreased after withdrawal of serum. These observations were further confirmed by Northern blot studies. A marked increase in CR2-messenger RNA expression was demonstrated in cells from serum-containing cultures. Thus, the number of CR2 on the


cell surface, the intracellular pool and the transcription of this receptor, are at least partly controlled by one or more factors in human serum. Preliminary results indicate that the main activity is contained in a serum fraction with a molecular weight of more than 70,000 dalton.

* Part of the M.D. thesis.

Supported by FWF P 6054.

i Institute for Hygiene, University of Innsbruck, Austria, 2 Basel Institute of Immunology, Basel, Switzerland, and 3 Behringwerke Marburg, FRG

E.9 HSV Type 1 interaction with human complement-binding specifity of a viral pathogenicity factor

H. P. HUEMER\ M. BROKER3, J. LAMBRIS2, and M. P. DIERICH i

In order to localize peptide structures on HSV -1 glycoprotein C (gC-l) which bind to human complement component C3b, we have raised monoclonal antibodies to gC-l and mapped them on recombinant gC expressed as a ~-galactosidase fusion protein in E. coli (AMANN et al., Gene 32, 203-215). In contrast to Glorioso's antibodies which are' directed against four carbohydrate containing antigenic sites and all inhibit rosette formation betWeen HSV type 1 infected cells and EAC3b (FRIEDMAN et al.,J. Virol. 60, 470-475), we could detect a carbohydrate independent antigenic site which is not involved in C3b-gC interaction. Based on the finding that anti-gC antibodies which inhibited C3b binding did not recognize recombinant gC we have more evidence for the importance of glycosilation in this interaction. Looking for the gC binding site on the C3 molecule, we used polyvalent rabbit sera against C3 fragments in rosette inhibition assay. Only antisera raised against fragments of the a-chain inhibited rosette formation. Since antisera directed against epitopes of the N-terminus and some directed against the C-terminal portion of the a-chain acted inhibitory, it can be assumed that it is more likely a conformational than a single epitope on C3b which is responsible for binding to HSV glycoprotein C.

This work was supported by a grant of the Austrian FWF (P6054).

Institut fUr Immunologie, GSF, Landwehrstr. 61, Miinchen, FRG

E.10 Cell-bound monoclonal anti-Thy-1 antibodies exhibit differential affinity for macromolecular C1 complex versus free C1q

U. KUMMER, s. THIERFELDER, and J. MYSLIWIETZ

Experiments with cell-bound monoclonal antibodies (anti-Thy-l mAb of rat and mouse origin) have indicated that only certain isotypes (mouse IgG2a and rat IgG2b mAb) exhibit a high intrinsic affinity for the Clq subunit of the first complement component. However, the use of native serum (under physiological conditions approximately 70 % of the Clq subunit is calculated to be associated with the C1r2s2 tetramer in a calcium-dependent complex) in uptake experiments together with a detection system consisting of HRP-conjugated anti-Clq, antiClr and anti-CIs reagents revealed that even mAb of the rat IgG2a isotype and the IgM class (mouse and rat) generated stable associations between intact Cl via the Clq recognition unit on the cell surface. In contrast, the strength of the interaction between these members of anti-


Thy-1 mAb and the C1q recognition unit, free of C1r2s2 (purified C1q or EDTA-treated serum), was below a certain «threshold affinity», so that C1q is readily dissociated from the activator during multiple wash-off steps of the assay. We think that the increased affinity of intact Clover subunit C1q for cell-bound mAb (and presumably for all specific C1-binder) may be found in the molecular structure of C1 itself. In macromolecule C1, the C1rzsz tetramer is located in the centre of C1q and wrapped around the «stems» between the globular heads and the central bundle. Compared with the C1q subunit, free of tetramer, the molecular arrangement of C1 might be responsible for a reduced mobility of the globular heads of the recognition unit. One would expect that a change in mobility of the binding region (C1q heads) in intact C1 could enhance its binding affinity due to a longer binding period to the activator.

Institut rur Rechtsmedizin, Universitat Mainz, FRG, * Universitatskinderklinik, Wiirzburg, FRG

E.11 Virus neutralisation: functional differences between C4A and C4B

H.-J. KYTZIA, J. CHRISPEELS, T. KAUFMANN, C. RITINER, and H. W. KRETH"

Subacute sclerosing panencephalitis (SSPE) and several other slow-virus or autoimmune diseases are strongly associated with deficiency of human complement component C4A. To assess the physiological role of C4 in the early phases of virus infections, a homologous model assay system to determine fluid phase virus neutralization was developed. It employed genetically C4-deficient human serum as source of antibody and complement components, highly purified C4 isotypes, and wild-type mumps virus as target. Remaining virus infectivity was indicated by cytopathogenic effects on green monkey kidney (VERO) cell monolayer cultures. The results were: 1) Human C4-deficient serum was almost inactive as compared to normal human serum. Its very low residual activity was almost entirely due to neutralizing antibody. Purified C4 had no detectable influence upon virus infectivity. 2) Addition of physiological amounts of purified C4 restored the neutralizing activity of human C4-deficient serum. 3) In this respect, C4A samples (n = 3; C4A3, C4A4) tested were by one order of magnitude more active than C4B samples (n = 4; C4B1). This finding is contrary to the threefold higher hemolytic activity of C4B. These data lend further support to our hypothesis that, due to functional differences between C4 isotypes and allotypes, partial C4 deficiency plays a causal role in the pathogenesis of several virus associated diseases.

Supported by Deutsche Forschungsgemeinschaft, SFB 3111D6.

Institut fiir Immunbiologie der Universitat, D-7800 Freiburg, Institut fiir Organische Chemie der Universitat, D-7400 Tiibingen, FRG

E.12 Immunogenicity of synthetic oligopeptides corresponding to epitopes of Friends murine leukemia virus glycoprotein covalently linked to lipopeptides

R. LINK, T. BOLTZ, R.-P. HUMMEL, W. TRaGER, G. JUNG, P. KOCH, and W. G. BESSLER

Epitopes of Friends murine leukemia virus (FMuL V) envelope glycoprotein were predicted by our program HYCON using the parameters hydrophilicity, hydropathy, acrophilicity,


flexibility, alpha-, beta-, turn- and coil-conformation probabilities. Five peptides corresponding to these epitopes were synthesized using the Fmoc/tBu approach and covalently coupled to the B lymphocyte mitogen Pam3Cys-Ser (Pam3Cys-(S-2,3-bis(palmitoyloxy)(2RS)-propyl)-N-palmitoyl-(R)-Cys-Ser) which constitutes the N-terminal part of the lipoprotein from the outer membrane of Escherichia coli. By means of isotype-specific enzymelinked immunoassays, we determined the magnitude of antibody production and the isotype pattern following immunization with the lipopeptide-antigen-conjugates in STU mice as well as in Balb/c mice. The results demonstrate that these conjugates induce specific immunoglobulin production in vivo without the addition of any further adjuvant.

Supported by a grant from BMFT/BGA.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050, Australia

E.13 Evidence for T cell recognition of a purified lipophosphoglycan from Leishmania major in mice

H. MOLL, G. F. MITCHELL, E. HANDMAN, and M. J. MCCONVILLE

Human isolates of the intramacrophage protozoan parasite Leishmania major can infect mice and produce a range of cutaneous disease patterns, similar to the situation in man, depending on the strain of inbred mouse. On the surface of L. major promastigotes, a lipophosphoglycan (LPG) can be detected that serves as a receptor for macrophage recognition and establishment of intracellular residency. Previous studies in our laboratory have shown that the purified LPG, given with killed Corynebacterium parvum as an adjuvant, can vaccinate genetically susceptible mice against cutaneous leishmaniasis. In order to elucidate the immunological mechanisms leading to protection, we have studied both the T and B cell reactivity to L. major LPG that had been isolated from promastigotes. Both genetically resistant and genetically susceptible mice infected with L. major promastigotes were able to develop specific delayed type hypersensitivity (DTH) reactivity to L. major LPG. This evidence suggests that the purified LPG molecule can activate antigen-specific T cells. Studies using genetically susceptible mice vaccinated with L. major LPG in liposomes and C. parvum revealed that the frequency of L. major-reactive T cells in these mice was only slightly higher than in mice treated with adjuvant alone. However, the serum of LPG-vaccinated mice had significantly increased titers of LPG-specific IgG antibodies. The data show that the purified L. major LPG has the capacity to induce both T and B cell responses. Potential mechanisms leading to the development of protective immunity to cutaneous leishmaniasis will be discussed.

WHO Immunology Research and Training Centre, Institute of Biochemistry, University of Lausanne, Chemin des Boveresses, CH -1066 Epalinges, Switzerland

E.14 Analysis of Leishmania-specific T cell clones in vitro and in vivo

I. MULLER and J. A. LOUIS

Leishmaniases are infectious diseases of protozoan origin, caused by obligate intracellular parasites of the genus Leishmania. Acquired immunity against Leishmania major, the etiologic agent of cutaneous leishmaniasis, depends on the interaction of specific T lymphocytes and parasitized mononuclear phagocytes. In the murine model of experimentally induced cutane-


ous leishmaniasis parasite-specific L3T4+ T lymphocytes are required for the immunological control of cutaneous leishmaniasis. However, specific L3T4+ T cells are not only important in the resolution of L. major induced lesions, they also playa role in susceptibility to infection and are thereby detrimental for the host. This capacity of parasite-specific L3T4+ T cells to exacerbate the course of infection has been demonstrated by adoptive transfer experiments in our laboratory. Because of the central role of L3T4+ T cells in exacerbation or protection, we established T cell lines and clones from highly susceptible, infected BALB/c mice. Two of the derived T cell clones proliferated in vitro upon stimulation with syngeneic accessory cells and live L. major promastigotes as antigen and secreted IL 2. There was no response detectable when preparations of killed L. major promastigotes were used as antigen. Adoptive transfer of these cells to naive syngeneic BALB/c mice resulted in a clear reduction of lesion development. Even three months post infection the lesions of T cell recipients were 70-80 % smaller than in the control group. Enumeration of viable L. major parasites present in the lesions confirmed these findings.

Supported by the Swiss Natl. Fdt, WHO and DFG.

! Department of Pediatrics, University of Diisseldorf, FRG, 2 Kinderspital der Universitat Ziirich, Switzerland, 3 Istituto di Patologia Generale, Universita di Trieste, Italy

E.15 Evidence for the association between the polymorphic form «A» of the C8a-y-subunit and the deficiency of the C8~-subunit in humans

W. NURNBERGER!, H. PIETSCH!, R. SEGER2, L. RONCELLI3, F. TEDESCOl, and V. WAHN!

A sodiumdodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblot system was developed that allowed the demonstration of the C8a-y and the C8~ subunits, and also the differentiation of the C8a-y polymorphic forms (AB, A, and B) directly from serum samples. AB shows a triple band (termed C8a-y1, C8a-y2, and C8a-y3), A shows the C8a-y2 and C8ay3, and B shows the C8a-y1 and C8a-y3 bands. Thus, A lacks the C8a-y1, which has the highest molecular weight on SDS-PAGE. Sera from 12 patients with C8~ deficiency, from 10 of their parents, and from 68 normal healthy individuals (Diisseldorf area) were analyzed. The patient group represents about 30 % of the C8~-deficient patients described in the literature.

In the normals, the main polymorphic forms were found in similar distribution as described (1). In contrast, all the C8~-deficient patients possessed form A, and their parents had a significantly higher frequency of the form A:

Normal controls C8~-deficient patients Parents of C8~-deficient patients

* p < 0.01, and ** p < 0.05 (chi-quadrat-test)

C8 - polymorphic form AB A B

34/68 0112 3/10

23/68 12112* 7/10'f*

11/68 0/12 0/10

Possible interpretations of these findings should consider the genetic control of the C8 subunits, and also the process by which the C8 molecule is synthesized, assembled and secreted. We favour the thesis, that form A arises in the absence of C8~ or in the presence of low levels of C8~ by conversion of C8a-y1 to C8a-y2 or C8a-y3.

1. RITTNER, C., HARGESHEIMER, W., STRADTMANN, B., BERTRAMS,J., BAUR, M. P., PETERSEN, B. H. 1986. Am. J. Mun. Gen. 38: 482.


Institute for Hygiene and Boltzmann Institute for AIDS-Research, Innsbruck, Austria

E.16 Recombinant peptides derived from the envelope of HIV -2 in the serodiagnosis of HIV -2 infections

W. OBERHUBER, T. F. SCHULZ,j. M. HOFBAUER, P. HENGSTER, C. LARCHER, H. WACHTER, and M. P. DIERICH

We produced recombinant envelope-derived peptides of HIV-2, corresponding to amino acids 555-761 (expression plasmid pHIV-2/1), 393-773 (pHIV-2/2), 5-392 (pHIV-2/3) and 393-554 (pHIV-2/4) by expressing several restriction fragments of the HIV-2 env-gene in the bacterial expression vector pEX-3. These recombinant envelope peptides were then used as antigens on Western blots and ELISA to detect antibodies to HIV-2 in human sera. Of 18 HIV-2 positive sera available to us, 17 reacted strongly with those recombinant peptides which were derived from, or extended into, the transmembrane protein of HIV -2 (pHIV -2/1, pHIV-2/2). In contrast, recombinant peptides derived from the external envelope glycoprotein (pHIV-2/3, pHIV-2/4) were only weakly recognized by two sera. In contrast to what we expected on the basis of published findings, that the envelopes of HIV-1 and HIV-2 are antigenically distinct, we did observe some cross-reactivity of some human sera containing antibodies to HIV-1 with the HIV-2 peptides derived from the transmembrane protein of HIV-2. In spite of this cross-reactivity, a serological distinction can be made between antisera to HIV -1 and HIV -2 by simultaneous testing in ELISA on recombinant peptides derived from the transmembrane protein of HIV-1 and HIV-2.

Supported by: FWF P 6054 and Ludwig-Boltzmann-Gesellschaft.

Max-Planck-Institut fur Immunbiologie, Freiburg, ! Universitats-Hautklinik, Heidelberg; 2 Ernst-Rodenwaldt-Institut FbVI, Mainz, FRG

E.17 T cell response in mice against Borellia burgdorfcri

U. E. SCHAIBLE, M. D. KRAMER!, K. JUSTUS2, C. MUSETEANU, and M. SIMON

«Lyme Borreliosis» is a multisystemic bacterial infection in man and other mammals caused by the tick transmitted spirochete Borrelia burgdorferi (B.b.). The severe clinical manifestations such as arthritis, encephalomyelitis, myocarditis and acrodermatitis are thought to be controlled by reactions secondary to the specific immune response. In order to test this assumption, we have established a B.b. infection in the mouse system. Mice of genetically distinct inbred strains were tested for their T cell response in vivo in the delayed type hypersensitivity (DTH) assay. After inoculation of mice with intact or inactivated B.b. DTH could be elicited with soluble B.b. antigen 10 d later. All mouse strains tested so far except BALB/c showed a considerable DTH comparable in B10 congenic strains of different H-2 haplotype. In addition, no differences were observed in B6 mice of either sex. No DTH was observed using either other species of spirochetes (Leptospira interrogans, Treponema phagedenis) or unrelated bacteria (Mycobacterium tuberculosis) for elicitation or immunization, which indicates, that the DTH is specific for B.b. Antigen specific T cells from spleen and lymph nodes were isolated from B.b. immunized mice and were expanded in vitro in the presence of optimal concentrations of soluble B.b. protein. Histological examinations of mice experimentally infected with B.b. revealed pathological events similar to those seen in humans


including perivascular infiltrations in heart, liver, kidney and brain and enlarged follicles containing multinucleated giant cells in spleen. The data suggest the mouse to be a suitable model to investigate the involvement of B.b. specific T cell response in the pathogenesis of «Lyme disease».

Abteilung Angewandte Immunologie, Institut f. Radiologie und Pathophysiologie, DKFZ, Heidelberg, FRG, and Department of Immunology, Madurai Kamaraj University, Madurai, India

E.18 Mycobacterium leprae-induced modulation of CD2 promotes immunologic unresponsiveness in lepromatous leprosy

B. SCHRAVEN, v. MUTHUKARRUPPAN, and S. C. MEUER

Despite normal numbers of circulating T lymphocytes, peripheral blood cells of patients with bacterial index positive lepromatous leprosy have a strongly reduced capacity to form rosettes with sheep red blood cells. This phenomenon is due to diminished or absent CD2 expression and associated with reduced functional T cell responses to mitogens and antigens. Moreover, T cell activation as induced by monoclonal antibodies directed at CD2 (<<alternative pathway of T cell activation») is inhibited. Importantly, a strongly suppressed T cell proliferation following antigen receptor triggering via T3-Ti can be observed as well. CD2 modulation results from an interaction between as yet unknown components of M. leprae and CD2. This is concluded from experiments in which analogous changes as observed in vivo could be induced following incubation of peripheral blood mononuclear cells from healthy individuals with preparations of M. leprae but not M. tuberculosis. Thus, a selective loss of CD2 occurs. Such a treatment is not cytotoxic and does not affect surface expression of other known T cell differentiation antigens such as CD3, CD4, CD8. In addition, incubation of T cells with M. leprae in vitro results in reduced responses to triggering of both major T cell activation pathways by monoclonal antibodies as well as reduced proliferation following mitogeninduced activation. These findings provide new insight into the pathophysiology of lepromatous leprosy and may explain reasons for the overall immunologic unresponsiveness observed in this disease.

Supported by a scientific exchange program between DFG and INSA.

Max-Planck-Institut fur Immunbiologie, Stubeweg 51, D-7800 Freiburg, FRG

E.19 Recognition of malarial solid phase antigens by murine immune T cells

S.]. SLADE, S. S. GILLARD, and]. LANGHORNE

Certain malarial proteins have already been isolated, characterized and implicated as potential vaccine components; however, their efficacy as T cell immunogens has not been studied extensively. We are currently investigating T cell recognition and function in antimalarial immunity. Solid-phase T cell proliferation assays are being developed following electrophoretic separation of Plasmodium chabaudi proteins to screen relevant antigens. Initial


screening is performed using immune T cells rather than T cell clones in order to obtain a greater selection window of T cell reactivity. Our preliminary results indicate that primed and immune T cells can proliferate in response to extracts of the blood stages adsorbed onto nitrocellulose. The ability to present antigens in this way allow us to analyse proteins which have been extracted and purified in the presence of detergents. Other methods of presentation of antigens to murine T cells are under investigation to optimise the screening assays. It is important to elucidate antimalarial immunogens which provoke a good protective response; accordingly, all proteins separated and/or isolated are simultaneously examined in in vivo protection assays.

lInstitut fur Hygiene and 2 Ludwig-Boltzmann-Institut fur AIDS-Forschung, Innsbruck, Austria, and lPaul-Ehrlich-Institut, Frankfurt, FRG

E.20 HIV and HIV -infected cells activate the complement system

B. M. SOLDER!, T. F. SCHULZt, P. HENGSTERl, C. LARCHERl, G. BITIERLICHt, A. EIGENTLERt, J. LOWER3, R. KURTH3, H. WACHTER2, and M. P. DIERICH1

Herein, we report that HIV as well as HIV-infected cells trigger the complement system. HIV -infected H9 cells were incubated in normal human serum and deposition of C3 fragments on the surface of these cells was demonstrated using an anti human C3d antibody in an immunofluorescence assay. Membrane fluorescence was also detected on cells incubated in serum in the presence of 20 mmol EGTA and 5 mmol MgCl2 or C4 deficient serum but not in heat-inactivated serum or nortnal serum in the presence of 10 mmol EDTA, suggesting that HIV-infected cells trigger the complement system via the alternative pathway. Using a complement fixation technique, we could show that also purified HIV directly activates the complement system. This however seems to occur via the classical pathway, as both purified HIV as well as recombinant gp160 only led to complement activation after incubation in nortnal human serum but not in the presence of 10 mmol EDTA or 20 mmol EGTA and 5 mmol MgCl2 or C4 deficient serum. Deposition of C3 fragments is not followed by lysis of HIV infected cells but is capable of mediating immune adherence to human erythrocytes. These findings suggest that HIV as well as HIV-infected cells trigger the complement system via the classical and alternative pathway, respectively, and that coating of HIV with C3 fragments might playa role in infectivity of cells bearing complement receptors.

This work was supported by L.B.G. and by a grant of the Gertnan B.M.F.T.

Institute for Clinical Microbiology, University Erlangen, Wasserturtnstr. 3, D-8520 Erlangen, FRG

E.21 Stage-specific antigens of Leishmania major identified by monoclonal antibodies

W. SOLBACH, M. LOHOFF, C. BOGDAN, and M. ROLLINGHOFF

Two monoclonal antibodies (mab SN3/8, mab H19) to Leishmania major (L. major) have been prepared by fusion of NS1 myeloma cells with spleen cells from BALB/c mice


chronically infected with parasites. Mab SN3/8 specifically bound to surface structures of the promastigote and amastigote form of the parasite as well as to parasitic excretion products. In contrast, mab H19 recognized antigens exclusively distributed on the surface of amastigotes. Parasite-infected splenic macrophages from chronically infected mice, but not from normal animals displayed cellular surface antigens that were recognized by both antibodies. The distinct visual appearance of immunofluorescence staining suggested that mab H19 recognized L. major specific epitopes different from those detected by mab SN3/8. This was confirmed by Western blot analysis of parasite lysates separated by SDS-PAGE which revealed that mab H19 recognized a 11 kd protein, whereas mab SN3/8 stained a non-protein structure, which presumably is similar to the lipophosphoglycan described by HANDMAN et al. (1). Functional in vivo studies showed that BALB/c mice treated with mab SN3/8 prior to infection with virulent L. major or infected with parasites preincubated with mab SN3/8 developed a significantly more severe disease than control animals. The antibodies thus allow to clearly identify stage specific L. major antigens and offer the possibility for further functional studies in vivo.

1. HANDMAN, E., and G. F. MITCHELL. 1985. Proc. Natl. Acad. Sci. USA 82: 5910-5914.

This work was supported by the Johannes-und-Frieda-Marohn-Stiftung.

Institute of Virus Research and * Institute of Immunology, German Cancer Research Center, Heidelberg, FRG

E.22 Replication of herpes simplex virus in the B cell line J OK-l

K. THIELE, G. MOLDENHAUER*, and H. KIRCHNER

For the past few years, our laboratory has been attracted by the possibility to study replication of herpes simplex virus (HSV) in the cellular component of the immune system. Therefore, we investigated HSV replication in monocytes, B cells and T cells of peripheral blood. Here, we want to present our recent studies on a persistent infection of JOK-l, a human leukemic cell line, which was isolated from a patient with hairy cell leukemia. Originally, JOK-l was characterized as a hairy cell line, but it could be shown that it is in fact a human lymphoblastoid B cell line. Persistent infection by HSV was maintained for 10 months (time of this communication). The titer of infectious virus peaked after 5 days of infection and remained at this level all the time. The proportion of cells producing infectious viruses increased up to 20 % by 72 h post infection. Although cytopathic effects were observed, cells were not completely lysed by the virus. Immunofluorescence studies revealed that HSVpositive cells ranged from seventeen up to fifty percent in different infected cultures. Usually, there was no detectable difference in cell growth of the infected cultures as compared to uninfected cells. Studies at the DNA level revealed, high amounts of virus genomes in the cells, yet the level of viral DNA varied from day to day, which probably is due to cell differentiation in the culture. To obtain further evidence that differentiation events in the cell culture are responsible for HSV-replication we cloned infected JOK-l cells and investigated viral titers and content of viral genomes in the clones. None of the clones obtained harbored viral DNA, so that probably cells will be infected by HSV only at a certain stage of differentiation. Following infection cells will be lysed by the virus. Analysis of cells stained with several B cell differentiation markers revealed differences between mock- and HSV-infected JOK-l cells, which we are going to investigate in more detail.


Institute of Immunology, University of Heidelberg, FRG

E.23 C5 activation is not necessarily associated with the cleavage of C5a-chain

U. TRAUGOlT, U. ROTHER, G. KAISER, and M. KIRSCHFINK

In the absence of both the classical and the alternative complement pathway, convertase C5 and C6 can be transformed to lytic activity by a variety of physicochemical means. It has been shown that C56 hemolytic activity (C56f) is generated by freezing and thawing a mixture of purified components C5 and C6 and that this proceeds via an intermediate C56 which itself is not hemolytic active. Whereas C5-activation by its natural enzymes leads to the cleavage of C5a-chain to form C5a and C5b, no C5a could be detected when C5 and C6 were activated by freezing and thawing. Native C5, C5b6 (purified from zymosan-activated serum), C56f and its hemolytically inactive intermediate C56 were compared in Western blot analysis regarding integrity of C5a-chain. C5a-chain remained uncleaved in the protein complexes C56f and C56 at 116 kD molecular weight like in native C5. As expected the a-chain in C5b6 was converted to a' at 110 kD. These results are in contrast to the accepted proposition that cleavage of C5achain is a prerequisite for C5 activation.

Div. of Immunol., Med. Inst. of Environmental Hygiene at University of Dusseldorf, Auf'm Hennekamp 50, D-4000 Dusseldorf, FRG

E.24 Oral treatment of mice with the drug D-Pencillamine (D-Pen) haptenates their peritoneal macrophages and primes their T cells

S. VOGELER and E. GLEICHMANN

D-Pen induces a variety of adverse immunological side-effects, the pathogenic mechanism of which are unknown. One possibility is that D-Pen spontaneously haptenates lymphoid cells and/or macrophages so that T cells react against them in a GVH-like fashion. Consistent with this hypothesis Lyt 1 +2- Th cells from mice injected with D-Pen s.c., or with D-Penhaptenated spleen cells i.v. could specifically be restimulated, if D-Pen was presented to them on spleen cells preincubated with the drug for 20 h (1). Here, we demonstrate that oral administration of D-Pen is also able to sensitize. BALB/c mice received 5-7 mg of D-Pen per day in the drinking water for 1-12 weeks. Peritoneal macrophages and cells from Peyer's patches from such mice were used as stimulator cells without further incubation with D-Pen in vitro. They were combined in vitro with responder cells taken from the lymph nodes of animals that had been primed with D-Pen by s.c. injection of the drug at the base of the tail. A significant proliferation of the responder cells was observed. In another experiment, stimulator cells again consisted of peritoneal macrophages obtained from orally treated mice. They were cocultured with lymph node cells obtained from the same mice. Again, a significant proliferation of responder cells was observed.

1. NAGATA, N., U. HURTENBACH, E. GLEICHMANN. 1986. Specific sensitization of Lyt-l+r T cells to spleen cells modified by the drug D-penicillamine or a stereoisomer. J. Immunol. 136: 136.


Max-Planck-Institut f. expo Medizin, Gottingen, FRG

E.25 Some properties of on radical-treated human C5

W. VOGT, I. VON ZABERN, and R. NOLTE

It has been previously communicated that treatment with hydroxyl radical generating systems (H20 2 ; xanthine oxidase, iron-catalyzed) convert human C5 to a C5b-like, quasi activated though uncleaved new species (C5(H20 2)) (VOGT et aI., Immunol. Letters 14: 209 (1987)). This product is capable of binding C6, and the resulting complex causes reactive lysis of non-sensitized red cells in cooperation with complement components C7-C9. We have now found that formation of the active C5(H20 2)6 complex is a rather slow process, taking about one hr at 37"C for the complex to become fully active. If incubated alone at 37°C, C5(H20 2)

«decays», i.e. it looses the ability to react with C6 and late components. At O°C, C5(H20 2)

remains functionally active for weeks. The active species comprises the whole, uncleaved C5 molecule; it is not cleaved by the C3/C5 convertase, CVFBb, nor is it utilized in immune haemolysis assays, indicating that C42, too, does not split C5(H20 2) into C5a and C5b. The reason for the failure is probably lack of recognition by the complement enzymes: C5(H20 2)

has lost the binding site for surface-fixed C3b (and probably for CVF); such binding is, however, a prerequisite for the proper conformation of C5 to be recognized and cleaved by the complement convertases (VOGT et aI., Immunology 34: 29 (1978)). In contrast, C5(H20 2) is more readily attacked by plasma kallikrein and plasmin than native C5. The cleavage pattern differs from that of the complement convertases. While C5(H20 2) itself is not chemotactic, it generates a chemotactic product upon cleavage by plasma kallikrein, which may, however, not be C5a but a larger fragment. The reactions described may provide a mechanism for the generation and/or enhancement of chemotactic activity in tissues by-passing the participation of the early complement reaction sequence.

Institute for Clinical Microbiology, University Erlangen-Niirnberg, Erlangen, FRG

E.26 The outer membrane of Yersinia enterocolitica as inhibitor of the alternative complement pathway: regulatory role of antibodies

E. WACHTER and V. BRADE

Alternative complement pathway (ACP) activation is considered an important nonspecific defence mechanism in the early stage of bacterial infection. However, several pathogenic bacteria such as Streptococcus pneumoniae, group B Streptococcus type III or Haemophilus influenzae were already described to be poor activators of the ACP. Our recent experiments with Yersinia enterocoJitica 0:3 (Y. ent.) in guinea pig serum revealed resistance to ACP activation as indicated by survival in EGTA-Mg-serum or C4-deficient-serum and by low grade ACP-C3-inactivation. Influence of lipopolysaccharide (LPS) variation and outer membrane proteins (OMP) on the ACP activation were studied according to growth temperature (22°C or 37"C) and plasmid content (p+/p-). Low temperature greatly stimulates synthesis of LPS side chains, whereas plasmid-coded OMPs are expressed only at 37°C. Independent on LPS side chain length and OMP expression all bacteria showed low grade C3 inactivation via ACP. In addition, neuraminidase treatment of Y. ent. was also ineffective with respect to ACP activation. After treatment of Y. ent. with polyclonal rabbit antiserum consumption of C3 via ACP was greatly enhanced. Monoclonal antibodies (mab) specific for LPS of Y. ent. showed


an identical effect on ACP activation. In contrast, mab specific for OMP was ineffective. The positive role of anti-LPS mab was apparent with all bacteria independent of LPS side chain length and plasmid expression. Anti-LPS(mab)-mediated ACP activation included all terminal complement components up to C9. In spite of ACP activation, killing of antibody-coated Y. ent. occurred with bacteria grown at 22°C, but not at 37 DC. Resistance of Y. ent. grown at 37°C was independent of plasmid content. The molecular basis for this temperature-dependent resistance is unknown.

In summary our experiments demonstrate an inhibitory role of the outer membrane on ACP activation by Y. ent. Only specific anti-LPS antibody can overcome this inhibition. A regulatory role for OMP on ACP activation or killing of Y. ent. was not demonstrable.

Supported by DFG Br 446/8.

Institut fiir Blutgerinnungswesen und Transfusionsmedizin der Universitat Diisseldorf, Moorenstr. 5,4000 Diisseldorf 1, FRG

E.27 Relative assessment of cellular immune reactivity by comparative determination of free IL 2R, TNF alpha, IFN gamma, neopterin levels in the sera of ARC/AIDS patients under autovaccination treatment

P. WERNET, G. KOGLER, G. BRINGMANN-VASEN, A. HOLDER, B. M. E. KUNTZ,]. W. SCHEJA, H. LEHNERT, and H. TH. BRUSTER

In longitudinal measurements of free IL 3 receptors, TNF alpha, IFN gamma, neopterin levels in the sera of 55 ARC/AIDS patients under treatment with an autovaccination protocol different groups of patients with very high levels of IL 2R could be defined. The simultaneous presence of elevated TNF alpha and IFN gamma values in the sera of certain of these patients appears to correlate with no detectable HIV antigen (p24). This may serve as a prognostically good sign for these patients. On the other hand, high neopterin levels inversely correlated with low absolute numbers of CD 4 + cells and with recurrent opportunistic infections. Further correlations of these serological parameters signaling cell-mediated changes in immunity together with the systematic assessment of HIV -, CMV - and EBV -serology may allow a better understanding of immunopathological processes in ARC/AIDS patients with no, or certain, therapeutic regimens.

Institut fiir Immunbiologie der Universitat, D-7800 Freiburg, Institut fiir Organische Chemie der Universitat, D-7400 Tiibingen, FRG

E.28 Detection of HIV -1 and HIV -2 infections using lipopeptide conjugates as antigens in enzyme immunoassays

F. WIEDEMANN, T. BOLTZ, R.-P. HUMMEL, W. TRaGER, G. ]UNG, K. SCHAUERTE, and W. G. BESSLER

For the establishment of HIV -1 and HIV -2 peptide immunoassays, we synthesized peptides derived from the transmembrane glycoprotein of HIV-1 and the analogous region of HIV-2 using the FmocltBu strategy (1). We coupled the N-termini of the peptides (HIV-1 peptide:


LGLWGCSGKLIC; HIV-2 peptide: NSWGCAFRQVq to the synthetic lipopeptide Pamr Cys-(S-2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl(R)-Cys-Ser) in order to improve the attachment of the peptides to the microtiter plates via hydrophobic interactions. 117 of 121 HIV-1 infected patients and 0 of 142 controls reacted with PamjCys-Ser- HIV-1(598-609) cyclic disulfide. The four nonreactive patients showed similar weak reactions in a commercially available ELISA. 5 out of 5 HIV -2 infected patients and 0 of 48 controls were positive with PamjCys-Ser- HIV-2(593-603) cyclic disulfide. Similar computer predicted structures can be found in the amino acid sequences of HIV-1, HIV-2, Simian immunodeficiency virus (SIV), and Friends murine leukemia virus (FMuL V). It may be that these particular peptide segments containing a disulfide bridge constitute immunodominant epitopes despite the variability of the amino acid sequences.

1. GNANN, J. W., et al. 1987. Science 237: 1346.

Supported by BGA.

Inst. for Immunology, Univ. of Heidelberg, Progen Biotechnik, 6900 Heidelberg, and RobertKoch-Inst., BGA, 1000 Berlin 65, FRG

E.29 The anaphylatoxin C3a bears at its C-terminus a neoantigenic determinant with diagnostic potential: analysis with a synthetic octapeptide C3a (69-76)

G. ZILOW, W. NASER, A. FRIEDLEIN, A. BADER, H. SCHAFER, and R. BURGER

Activation of the complement component C3 by proteolytic cleavage leads to the generation of the anaphylatoxin C3a. This potent mediator contributes to inflammation and has immunoregulatory activity. C3a seems to be involved, together with C5a, in the pathogenesis of the adult respiratory distress syndrome (ARDS) observed e.g. in poly trauma or sepsis patients. The determination of C3a in plasma or bronchoalveolar lavage fluid of ARDSpatients provides one of the few diagnostic parameters with prognostic potential. The biologically active site of the C3a resides in its C-terminal amino acids (73-77). The Cterminus of C3a is exposed only after proteolytic cleavage and might therefore represent a neoantigenic determinant. We obtained a monoclonal antibody (mAb) H453 to this molecular site of C3a and identified it as neoantigenic determinant with diagnostic potential. H453 was obtained after immunization with a synthetic octapeptide (OP) coupled to a carrier protein (KLH). The OP corresponded the amino acids 69-76 of C3a, i.e. the C-terminus of C3adesArg present in body fluids. The mAb reacted in ELISA with C3a and with KLH-OP but not with uncleaved C3 or with unsubstituted KLH. Free OP efficiently blocked the binding of H453 whereas binding of another mAb to C3a (H13) remained unaffected. H453 reacted strongly in immunoblotting analysis with purified C3a or with C3a in zymosan-activated serum, but failed to react with uncleaved C3. Obviously, the corresponding determinant is masked as a linear sequence in the native C3 molecule and is exposed only after generation of C3a. The mAb permitted the establishment of a novel C3a ELISA using an indirect sandwich system. The assay had a sensitivity in the nanogram range (1-5 ng/ml). The presence of high amounts of C3 did not interfere with C3a-determination in patient plasma or in normal plasma reconstituted with purified C3a-desArg. Therefore, removal of C3a from the sample by precipitation was not required, in contrast to previously described assays applying radiolabelled C3a-desArg. This new assay should facilitate quantitation of C3a in samples from patients at risk for ARDS.

workshop e infections and complement

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