Methods for the in vitro determination of an individual disposition towards TH1- or TH2-reactivity by the application of appropriate stimulatory antigens.

In this study we performed several methods for the determination of cytokines (RT-PCR for the demonstration of cytokine mRNA and flow cytometry for the analysis of intracellular cytokines) and compared them with a recently established test system stimulating peripheral blood mononuclear cells (PBMC) with TH1- and TH2-relevant recall antigens and analysing type 1 and type 2 cytokines by ELISA. Aim of the study was therefore to evaluate the reliability of TH1/TH2 cytokine profiles in two individuals with different types of an allergic/atopic disposition: one of them showed a strong TH1/type 1-mediated tuberculin-reaction (subject A), the other (subject B) revealed elevated IgE-levels and eosinophil counts (TH2/type 2-mediated). PBMC were incubated with the type 1-antigen purified protein derivative (PPD) and the type 2-antigen tetanus-toxoid (TT) for seven days. From the comparison of ELISA with RT-PCR and flow cytometry-analysis it became evident that all three methods allowed the definition of subject A as a 'type 1-responder'. Subject B showed a pure type 2-response in the ELISA method; PCR and flow cytometry analysis revealed the simultaneous production of type 1- and type 2-cytokines resulting in a mixed type 1/type 2-profile. Active immunization of subject A with TT at the end of the observation period of 12 months resulted in a transient shift from type 1- to a mixed type 1/type 2-profile (simultaneous PPD-induced IFN-gamma- and TT-induced IL-5 production). From this pilot study based on clear cut clinical criteria concerning either a humoral or cellular immunological reactivity towards allergens/antigens it is suggested that the determination of type 1/type 2-cytokines by ELISA in supernatants of PBMC stimulated with type 1/type 2-relevant antigens is a useful approach for a better classification of 'type1-' or 'type 2-responder'.

Analysis of immunoregulatory T-helper cell subsets in patients with multiple sclerosis: relapsing-progressive course correlates with enhanced T H1, relapsing-remitting course with enhanced T H0 reactivity.

In this study, we analysed the recall antigen-induced cytokine production by peripheral blood mononuclear cells (PBMC) from 31 patients with multiple sclerosis (MS) with a relapsing-remitting (rr) and a relapsing-progressive (rp) course and from 40 healthy controls. Cells were stimulated with purified protein derivative (PPD; type 1 response) and tetanus toxoid (TT; type 2 response). Cytokines were determined in the supernatants by ELISA. One of the interesting findings was that healthy controls showed more frequently an IL-5 production after incubation with TT than MS-patients (68% vs.37%; p<0.01), while the type 1 reactivity was only slightly enhanced in MS patients as compared to the controls. However, within the MS patients, there was a significant difference in the incidence of the type 1 reactivity comparing patients with an rp and an rr course (60% vs. 24%; p<0.05). Furthermore, the frequency of a type 0 profile (simultaneous PPD-induced IFN-gamma and TT-induced IL-5 production) was fourfold higher in rr than in the rp patients (43% vs. 10%, p<0.05). In vitro analysis of cytokine profiles in MS could therefore be an interesting approach to evaluate the prognosis of MS (rr vs. rp) already at the beginning of the disease. Thus, it seems that the presence of a type 0 profile is a valid indicator for a favorable course, while a type 1 profile is rather associated with rp MS.

[Autoimmune hepatitis and overlap syndrome: diagnosis]. / Autoimmunhepatitis und Overlap-Syndrom: Diagnostik.

Autoimmune hepatitis (AIH) is a rare autoimmune disease (incidence about 5% among all chronic liver disorders) that reflects a loss of tolerance to normal hepatic proteins. AIH is characterized by female preponderance, hypergammaglobulinemia, extrahepatic syndromes and a good response to immunosuppressive treatment. AIH may be subdivided into two or three subtypes. AIH type 1 is characterized by antinuclear autoantibodies (ANA) and/or smooth muscle antibodies (SMA). SMA are actin-specific, can occur without ANA and their presence relates strongly to AIH. AIH type 2 is defined by the presence of anti-liver-kidney microsomal antibodies (LKM-1). Patients with AIH type 2 are typically younger at the time of disease onset, exhibit higher inflammatory activity, suffer more frequent relapses under immunosuppressive treatment and are more likely to progress to cirrhosis. AIH type 3 is characterized by autoantibodies against the soluble liver antigen (SLA) and liver-pancreas antigen (LP), but ANA/SMA are frequently present and, therefore, some authors consider this autoantibody manifestation as belonging to AIH type 1. Antineutrophil cytoplasmic antibodies (ANCA) recognize cytoplasmic or nuclear components of neutrophilic granulocytes and are detected with high prevalence in patients with autoimmune liver diseases. They are associated with AIH type 1 but not with AIH type 2. However, 40-70% of patients with primary sclerosing cholangitis (PSC) also produce these autoantibodies. Autoimmune cholangitis is an idiopathic disorder with mixed hepatocellular and cholestatic findings that typically has antinuclear antibodies (ANA). It may be considered as an atypical form of primary biliary cirrhosis. It has been recognized that some forms of AIH may also occur with variable incidence and severity especially in patients with primary biliary cirrhosis (overlap AIH/PBC) or primary sclerosing cholangitis (AIH/PSC). On the basis of clinical, biochemical, serological, histological and radiological criteria a clear distinction between these conditions can be readily made in the majority of cases. An association of AIH-typical autoantibodies (anti-LKM-1, anti-SLA/LP) in association with antimitochondrial autoantibodies (AMA) almost confirm the overlap syndrome AIH/PBC. In PSC patients expressing typical ERCP findings and suffering from inflammatory bowel disease (IBD), the diagnosis of an overlap syndrome between PSC/AIH can be readily made in the presence of ANCA and AIH relevant autoantibodies. Apart from this kind of overlap syndrome involving different types of autoimmune disorders within the liver AIH can be also associated with other organspecific autoimmune disorders as documented in the autoimmune polyglandular syndrome type 1 (APS-1). In this disease homozygosity for a defect in a single gene (AIRE) leads to a broad spectrum of organ specific autoimmune diseases.

In vivo-induction of antibodies to mistletoe lectin-1 and viscotoxin by exposure to aqueous mistletoe extracts: a randomised double-blinded placebo controlled phase I study in healthy individuals.

BACKGROUND: Several studies have been performed in tumour patients to analyse the immunological response to mistletoe extracts. Considering the fact that these extracts are given subcutaneously in most instances, the kind of application resembles a typical immunization schedule. We therefore wanted to see how those extracts act on immunocompetent cells of healthy individuals hoping that this kind of provocation test may give new informations about a more specific application of these extracts in certain diseases. SUBJECTS/METHODS: 47 healthy individuals were exposed for twelve weeks either to Iscador Quercus special (IQ) known to be rich in mistletoe lectin (ML)-1 (n = 16), to Iscador Pini (IP) being poor in ML-1 but enriched in viscotoxins (n = 15), or to placebo (physiological saline) (n = 16) in a randomised, double-blinded placebo-controlled study. Humoral immunoreactivity was analysed by measuring antibodies towards the two compounds ML-1 and viscotoxin VA2 (VA2). Sera were collected in intervals of four weeks up to week 12 and again three months after last exposure. RESULTS: None of the subjects had antibodies to ML-1 or VA2 before exposure. In week 12, anti-ML-1 antibodies of the IgG-type were found in all 16 IQ-treated individuals but only 6 of the 15 probands exposed to IP. In contrast, anti-VA2 IgG-antibodies could be detected in all individuals of both groups. The antibodies were preferentially of the IgG1 and IgG3 type while antibodies of the IgA and IgM type were produced only in a few probands. Antibodies of the IgE-type occurred only in the IQ-exposed individuals and were directed against ML-1 but not VA2. None of the probands receiving placebo developed antibodies to ML-1 or VA2. Severe side effects were not observed in any of the probands. CONCLUSIONS: These data obtained in healthy individuals clearly indicate that IQ and IP-extracts can induce antigen-specific humoral responses. They may, therefore, provide, a solid basic for the evaluation of the humoral immune response in disease states.

L-tryptophan contaminant 'peak E' induces the release of IL-5 and IL-10 by peripheral blood mononuclear cells from patients with functional somatic syndromes.

In 1989, the development of eosinophilia myalgia syndrome (EMS) was observed in some patients after the intake of l-tryptophan containing several contaminants, including 1,1'-ethylidenebis[l-tryptophan] ('peak E'). Since l-tryptophan has been taken particularly by individuals suffering from functional somatic syndromes (FSS), such as fibromyalgia syndrome (FMS), we put forward the hypothesis that EMS may have developed preferentially in patients with FSS as an allergic reaction towards the contaminant peak E. We therefore studied the immunological reactivity towards l-tryptophan and peak E in these individuals (n = 12) and compared these data with those obtained in 12 healthy controls and 12 patients with other chronic disorders. Peripheral blood mononuclear cells (PBMC) were cultured for 7 days with pure l-tryptophan and peak E. Supernatant fluids were collected at day 7. The type 2 cytokines IL-4, IL-5 and IL-10, and the type 1 cytokines IL-2 and IFN-gamma, were determined by a double sandwich ELISA. PBMC from seven of the 12 FSS patients, but only three of the 24 controls, produced cytokines after incubation with peak E (P < 0.05). Interestingly, six of the seven FSS patients reacting with peak E produced IL-5 and/or IL-10. In contrast, PBMC from only one patient with other chronic disorders and one healthy control secreted type 2 cytokines in response to peak E. The observed heightened type 2 reactivity towards the more immunogenic contaminant 1,1'-ethylidenebis[l-tryptophan] in FSS patients may therefore be taken as an additional argument for our concept that EMS may have developed as a kind of drug-induced allergic disease.

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects.

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.

Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.

The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.

Induction of T helper cell type 1 response and elimination of HBeAg during treatment with IL-12 in a patient with therapy-refractory chronic hepatitis B.

This study reports the increase of immunoregulatory T helper cell type 1 response and elimination of HBV-DNA during IL-12 therapy in a patient with chronic hepatitis B virus infection who had not responded to three previous interferon-alpha therapies and one treatment with Famciclovir over a period of 6 years. The patient received IL-12 at a dose of 0.5 microgram/kg bodyweight weekly. Peripheral blood mononuclear cells were isolated before and during IL-12 application and incubated for 7 days with specific type 1 (purified protein derivative) and type 2 (tetanus-toxoid) TH cell antigens as well as with a macrophage/monocyte activating antigen (Bacille Calmette-Guérin). In the supernatants cytokines were determined by a double-sandwich ELISA. After 8 weeks HBV-DNA became negative and HBeAg seroconversion to anti-HBeAg occurred. Immunologically the loss of viremia was accompanied by a strong increase of the purified protein derivative-induced production of the type 1 cytokine interferon-gamma (1219 pg/mL before, 13,138 pg/mL after IL-12 therapy). Furthermore, Bacille Calmette-Guérin-induced secretion of the macrophage/monocyte-associated cytokines IL-1, tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor was higher at the end of therapy. This case indicates that IL-12 enhances type 1 T helper cell activity which may be a predisposition for elimination of HBeAg and successful treatment of hepatitis B.

Sequential occurrence of primary sclerosing cholangitis and autoimmune hepatitis type III in a patient with ulcerative colitis: a follow up study over 14 years.

In 1983, a female patient born in 1963 presented with symptoms of ulcerative colitis and typical clinical and histological signs of primary sclerosing cholangitis (PSC). At this time only pANCA were positive while other marker antibodies for autoimmune liver disorders could not be detected. In summer 1987 the clinical picture changed and was replaced by laboratory and histological signs typical of autoimmune hepatitis (AIH). Thus, IgG levels increased considerably and cholestatic enzymes became normal. For the first time, anti-liver-pancreas antibodies (LP), a diagnostic marker for AIH type III could be detected. In the following years several relapses occurred also induced by repeated discontinuation of immunosuppressive therapy. Symptoms of colitis persisted but signs of cholestasis remained absent for the following ten years. In 1997, colitis exacerbated again and colectomy had to be performed together with liver transplantation. Surprisingly, histology of the explanted liver now showed the typical features of PSC stage III/IV while the significant criteria for AIH were now lacking. Thus, progression to cirrhosis was, probably, mainly induced by the biliary destructive and fibrotic process although biochemical and serological data were clearly indicative of an autoimmune, i.e. AIH-related manifestation.

A longitudinal study of autoantibodies against central nervous system tissue and gangliosides in connective tissue diseases.

Our objective was to investigate longitudinally, antibodies against central nervous tissue (anti-CNS) derived from bovine brain and gangliosides GM 1, GD1a, GD1b, and GT1b in 91 patients with connective tissue diseases (systemic lupus erythematosus, n = 38; mixed connective tissue disease, n = 16; primary Sjogren's syndrome, n = 7; progressive systemic sclerosis, n= 13; polymyositis/dermatomyositis, n=4; overlap syndrome, n = 5; undifferentiated connective tissue disease, n = 8). Anti-CNS and anti-ganglioside antibodies, measured by enzyme-linked immunosorbent assay, were found in 73% and 63% of patients, respectively. Anti-CNS positive sera were also reactive in Western blotting in 74% of cases and recognized up to 14 different polypeptides from 29 to 130 kDa. Anti-CNS and anti-ganglioside antibodies reflected only in a limited extent the disease activity. In 27 of 58 patients, anti-CNS antibodies remained positive independently of disease activity and antibody levels did not correlate with the phases of exacerbations. A total of 36 of 60 anti-CNS-positive patients, in contrast to two of 22 anti-CNS-negative patients, had major neuropsychiatric manifestations (P < 0.001). Anti-ganglioside antibodies were not significantly associated with neuropsychiatric manifestations. In conclusion, our longitudinal data suggest that anti-CNS antibodies may be an important marker for the diagnosis of cerebral involvement in connective tissue diseases, but the pathogenic role of these autoantibodies remains to be determined.

Immunomodulatory properties of rumalon, a glycosaminoglycan peptide complex, in patients with osteoarthritis: activation of T helper cell type 2 cytokines and antigen-specific IgG4 antigen-specific igG4 antibodies.

OBJECTIVE: To assess the immunogenic properties of the glycosaminoglycan peptide complex Rumalon, an aqueous extract of bovine cartilage and bone marrow frequently used in patients with osteoarthritis (OA). METHODS: Sera from 31 patients with OA who had received several series of Rumalon injections (Group 1, n = 17: before therapy and after one injection series; Group 2, n = 6: after 2-3 injection series; Group 3, n = 4: after 4-8 injection series; Group 4, n = 4: after 9-18 injection series) were tested by ELISA for antibodies against Rumalon and its components as well as by a double sandwich ELISA for type 1 [interferon-gamma, interleukin 2 (IL-2)] and type 2 cytokines (IL-4, IL-5, IL-10, IL-13). RESULTS: After the first injection series antibodies to Rumalon were induced in 7 of the 17 patients that were all negative before therapy. The antibodies were preferentially of the IgG4 type. IgG4 levels were increased during therapy (ELISA optical density x 1000 in Group 1: 73.9 +/- 209.5; Group 4: 1354.5 +/- 307.6), and in Group 4 all patients had developed these antibodies. Upon analysis of cytokine levels, there was a significant increase in IL-5 (Group 1 before therapy 407.4 +/- 257.1 pg/ml, Groups 3 and 4: 1409.4 +/- 963.1 pg/ml; p < 0.001) and to a lesser extent of IL-10 during therapy (Group 1 before therapy 950.2 +/- 867.8 pg/ml, Groups 3 and 4: 2817.8 +/- 3127.3 pg/ml; p < 0.05), while type 1 cytokines were not affected. CONCLUSION: Rumalon appears to have immunomodulatory properties and preferentially stimulates IgG4 antibodies via the activation of type 2 cytokines in vivo. Whether these phenomena can be correlated with the postulated therapeutic effect of Rumalon in patients with OA remains to be seen, but pain relief via release of endorphins by Th2 cells could be one explanation.

Acute liver failure due to enalapril.

This report presents a 46-year-old man who was treated for hypertension with the angiotensin-converting-enzyme (ACE) inhibitor enalapril. After 3 years of continuous treatment he presented with jaundice and progressive liver failure that continued despite withdrawal of the medication. The patient was taking no other medication. All known causes of acute liver failure could be excluded indicating a drug-induced liver damage after long-term treatment with enalapril. Analysis of liver biopsies revealed a pathomorphological pattern comparable to than observed in severe halothane hepatitis. Serological studies including T-cell stimulation with enalapril and a broad spectrum of tests for autoimmunity including autoantibodies against calreticulin, the major Ca2+ and Zn2+ binding protein of the endoplasmic reticulum and suggested to be involved in the pathogenesis of halothane hepatitis were negative. Thus, the mechanism of enalapril-induced liver injury remains obscure. Liver failure progressed and finally led to orthotopic liver transplantation. To our knowledge, this is the longest duration of chronic treatment with an ACE inhibitor before liver failure occurred. In addition, liver failure progressed despite withdrawal of the medication. It is concluded that even after long-term treatment with an ACE inhibitor liver failure may be induced. Therefore, regular monitoring of liver enzymes should be considered.

Overlapping but distinct specificities of anti-liver-kidney microsome antibodies in autoimmune hepatitis type II and hepatitis C revealed by recombinant native CYP2D6 and novel peptide epitopes.

Anti-liver-kidney microsome antibodies (anti-LKM) occur in autoimmune hepatitis (AIH) type II and in a subset of patients with hepatitis C. Anti-LKM1 in AIH are directed against cytochrome P4502D6 (CYP2D6), but conflicting data exist concerning the specificity of anti-LKM in hepatitis C. The aim of this study was to evaluate binding specificities of anti-LKM antibodies in both diseases using novel test antigens as well as their inhibitory capacity on CYP2D6 enzyme activity. Sera from 22 patients with AIH type II and 17 patients with hepatitis C being anti-LKM-positive in the immunofluorescence test were investigated for binding to native recombinant CYP2D6 and liver microsomes by ELISA and immunoblotting, and to synthetic peptides covering the region 254-339 (254-273, 257-269, 270-294, 291-310, 307-324, 321-339, 373-389) as well as the novel peptide 196-218 by ELISA. Furthermore, all sera were tested for inhibition of CYP2D6-dependent bufuralol 1'-hydroxylase activity. Twenty of the 22 AIH type II sera (91%) and nine of the 17 hepatitis C sera (53%) were positive for CYP2D6 by ELISA and/or immunoblotting. The previously described major peptide epitope comprising CYP2D6 amino acids 257-269 was recognized by 16 of the 22 AIH sera but by only one hepatitis C serum. A further epitope, 196-218, could be defined for the first time as another immunodominant epitope for AIH because it was recognized by 15 of the 22 AIH (68%) but only three of the 17 hepatitis C sera (18%). With the exception of the peptide 254-273, the other peptides showed no significant reactivity. Analysing the inhibitory properties of anti-LKM antibodies it emerged that 95% of AIH sera and 88% of hepatitis C sera inhibited enzyme function. These data indicate that anti-LKM antibodies in AIH and hepatitis C react with CYP2D6, as shown by their inhibitory activity, and that besides the known epitope 257-269 a further immunodominant epitope exists on CYP2D6 which is recognized by sera from patients with AIH II but hardly by sera from patients with hepatitis C.

High prevalence of antibodies to calreticulin of the IgA class in primary biliary cirrhosis: a possible role of gut-derived bacterial antigens in its aetiology?

BACKGROUND: In a preliminary study we showed that antibodies to the endoplasmic reticulum protein calreticulin (CR) occur in primary biliary cirrhosis (PBC) and autoimmune hepatitis type 1 (AIH). Since anti-CR antibodies have also been found in patients with infectious diseases, we investigated their prevalence and immunoglobulin classes in patients with various hepatic and intestinal diseases, hoping to get some information on a possible relationship between an infectious trigger and the induction of a certain class of anti-CR antibodies. METHODS: Sera were tested for anti-CR antibodies of the IgA, IgG, and IgM class by Western blotting, using CR isolated from human liver: in autoimmune liver diseases (primary biliary cirrhosis (PBC) (n = 86) and autoimmune hepatitis (AIH) type 1 (n = 57)), alcoholic liver cirrhosis (ALC) (n = 32), viral liver infections (acute hepatitis A (n = 8), acute hepatitis B (n = 20), and chronic hepatitis C (n = 28)), and intestinal diseases (Crohn disease (CD) (n = 30), acute yersiniosis (n = 26)). Sera from 100 healthy individuals served as negative controls. RESULTS: The most prominent finding was the high prevalence of anti-CR antibodies of the IgA class and the similarity in the anti-CR antibody class pattern in PBC (IgA, 62%; IgG, 43%; IgM, 55%) and yersiniosis (IgA, 62%; IgG, 39%; IgM, 42%). Class IgA anti-CR antibodies also occurred frequently in ALC (IgA, 44%; IgG, 41%; IgM, 19%). In contrast, in AIH anti-CR antibodies were predominantly of class IgG (IgA, 28%; IgG, 60%; IgM, 33%). In hepatitis A anti-CR antibodies were absent. In the other diseases they had a low prevalence and were mostly of class IgG (acute hepatitis B: IgA, 0%; IgG, 15%; IgM, 0%; chronic hepatitis C: IgA, 7%; IgG, 21%; IgM, 0%; CD: IgA, 13%; IgG, 20%; IgM, 13%). Of the healthy individuals 7% had anti-CR antibodies exclusively of class IgG. CONCLUSIONS: The high prevalence of anti-CR antibodies of class IgA in patients with PBC and yersiniosis as well as in alcoholic liver disease reflects a reactivity of the gut-associated immune system and could imply that a still undefined gut-derived bacterial (?) agent may trigger PBC.

Characterisation of immunological reactivity of patients with adverse effects during therapy with an aqueous mistletoe extract.

Drugs, which are effective are also bond to exert adverse effects. This is also true for mistletoe extracts. Extracts from European mistletoe (Viscum album, VAL) belong to the complementary therapeutic regimens and are used for adjuvant cancer therapy. This study was performed to characterise immunological reactivity of patients with adverse effects during treatment with an aqueous VAL extract (Helixor). VAL-stimulated proliferation and cytokine release of peripheral blood mononuclear cells (PBMC) and anti-mistletoe lectin (ML)-1 antibody production were investigated. 34 patients with proven adverse effects due to VAL therapy (group 1) and 9 patients with unproven relation (group 2) were studied and compared to 14 tumour patients treated with VAL for more than 2 years without side effects (TTP). VAL-stimulated proliferation of PBMC of group 1 was significantly enhanced as compared to group 2 patients and TTP. PBMC from patients with local manifestations proliferated significantly stronger than those from patients with systemic symptoms. Anti-ML-1 antibodies of the IgE type were produced in patients with proven adverse effects but not in patients without adverse effects. Production of Th1 and Th2 specific cytokines varied considerably, indicating that different mechanisms were involved in the induction of adverse effects. In conclusion, our study provide evidence that adverse effects towards VAL (Helixor) are seldom and are dominated by an application site reaction suggesting the involvement of delayed type hypersensitivity (DTH) reactions.