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OBJECTIVE: Measurement of inflammatory mediators is an important tool to assess inflammation. We have, therefore, conducted a survey within the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study to evaluate the inter-relationship between soluble CTLA-4 (sCTLA-4) and other inflammatory markers. MATERIALS AND METHODS: This is a population-based study, designed to quantify the circulating serum levels of sCTLA-4 and other inflammatory markers such as CRP and pro-inflammatory cytokines and chemokines by in-house ELISA, Immuno-turbidimetry and multiplex ELISA, respectively. A total of 1016 Swedish Caucasians aged 70 years old were recruited. The statistical analysis was performed by ANOVA. RESULTS: The levels of sCTLA-4 were directly related to the levels of pro-inflammatory cytokines such as IL-6, IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma and chemokines such as IL-8. However, the levels of sCTLA-4 were inversely related to the levels of MCP-1. Also, we could not demonstrate any relation between the levels of sCTLA-4 and CRP or soluble adhesion molecules. CONCLUSIONS: Circulating sCTLA-4 could be used as a biomarker for inflammation, potentially reflecting dysregulated T lymphocytes.

Genetic association of programmed cell death-1 (PDCD1) has been implicated in several autoimmune inflammatory disorders. Hence, in this study, our main objective is to evaluate the association of PDCD1 gene to Wegener's granulomatosis (WG). We, thus, analyzed three single nucleotide polymorphisms (SNPs) in PDCD1 gene among WG patients and controls. Further, we quantified circulating serum levels of soluble (s) PD-1 in patients and controls. The methodologies used were ABI Taqman allelic discrimination and restriction fragment length polymorphism for genotyping and in-house ELISA for quantifying sPD-1. Statistical relevance was analyzed by Fischer's exact test. As a result, reduced AA homozygote for SNP in intron-1 was observed, among the patients. However, no association was demonstrated after Bonferroni correction. Also, no differences in genotype and allele frequency were elucidated for SNPs in intron-4 and exon-5. Moreover, we could not demonstrate circulating sPD-1. In conclusion, we show no association of selected SNPs in PDCD1 gene with WG.

Sundewall A-C, Lefvert AK, Olsson R. (Department of Clinical Chemistry, Huddinge University Hospital, Huddinge, and Department of Medicine II, Sahlgrenska University Hospital, Gothenburg, Sweden.) Anti-acetylcholine receptor antibodies in primary biliary cirrhosis. Low concentrations of acetylcholine receptor antibodies were found in 16 out of 17 patients with primary biliary cirrhosis. Seven patients were treated or had been treated with penicillamine. Ten untreated patients had antibody levels corresponding to those found in the treated group. Our data support the presence of receptor antibodies of both IgG and IgM class.

In order to investigate the potential involvement of PTPN22 R620W in the pathogenesis of myasthenia gravis (MG), we performed a case-control study including 409 Swedish MG patients and 1557 normal controls. The W620 variant was significantly overrepresented in patients (odds ratio, 1.52; 95% confidence interval, 1.21-1.90; p=0.00027). Incubation of patient (n=100) derived PBMC cells with the autoantigen, the acetylcholine receptor, resulted in a significantly higher number of cells producing anti-AChR antibodies and IL-2 in W620 carriers, suggesting that PTPN22 W620 may be a loss-of-function variant in MG.

Objectives – To measure clinical and immunological parameters in a patient with myasthenia gravis (MG) treated with antibodies against CD25 (basiliximab, Simulect®). Patient and methods – Injections of basiliximab were given repeatedly together with cyclosporin A and corticosteroids for 9 months to a patient with severe MG. Her muscle function score was monitored and the immunological parameters were followed using ELISA, flow cytometry and radioimmunoassay. Results – The patient improved moderately and corticosteroid treatment could be withdrawn. The percentage of activated CD4+ T cells decreased during treatment, while that of ‘naive’ T cells increased. The serum levels of sCD28, sCD152, sCD80, sCD86 and IL-10 decreased. The treatment was stopped due to repeated infections. Conclusion – Treatment with basiliximab appears to be suitable only for severely ill patients who do not respond to conventional treatments. However, careful monitoring of side effects is necessary.

Objectives. The molecular mechanisms underlying the regulation of the CD152 (CTLA-4) gene are largely unknown. Two single nucleotide polymorphisms (SNPs) located in the promoter region are suspected to contribute to the pathogenesis of myasthenia gravis (MG) through regulation of gene expression. Setting, Subjects and Design. One hundred and sixty-five unrelated Swedish-Caucasian patients with MG (103 females and 62 males, age 17 to 92 years) and 148 ethnically matched healthy individuals were studied. Gene typing of two SNPs (T/C−1772 and A/G−1661) and quantification of soluble CD152 were performed in the patients. Besides the association studies, the function of these two SNPs is characterized. Results. We present new genetic associations of two SNPs in the CD152 gene with human MG. These SNPs located in the promoter region are involved in transcriptional binding activity for Nuclear Factor I (NF-1) and c/EBPbeta, as demonstrated using chromatin immunoprecipitation and electromobility shift assay. MG patients with the T/C−1772 polymorphism have elevated levels of sCD152 in sera. Conclusions. The two SNPs in the promoter region are associated with MG and might cause abnormal alternative splicing and affect the expression of CD152, thereby contributing to the pathogenesis of MG.

Background: Myasthenia gravis (MG) is an autoimmune disorder of neuromuscular transmission. The majority of patients show abnormal thymic histology. Setting: The study was performed at the Myasthenia Gravis Centre, Karolinska University Hospital, and at the Immunological Research Laboratory, Centre for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden. Patients and methods: Information was collected retrospectively from 1956 and prospectively from 1975 on clinical data, concomitant diseases, concentration of serum acetylcholine receptor antibodies (AChR-abs), immunosuppressive treatment (IS) and response to it, in 537 patients of whom 326 were thymectomised. Follow-up time was 1.5–50 years. Results: Age at onset of MG increased from a median age of 24 years before 1975 to a median age of 61 years after 2000. Thymoma was found in 65, hyperplasia (HPL) in 185 and a normal thymus in 76 patients. The trans-sternal surgical approach for thymectomy was used in 255 patients (78%). In five patients with thymoma, MG appeared after thymectomy. Of 537 patients, 466 (87%) had circulating AChR-abs. IS was given to 300 (56%) patients, mostly those with thymoma (85%). In total, 441 patients (82%) showed an improvement. One-third of patients with HPL, a quarter of those with thymoma, one-fifth of those with a normal thymus and one-seventh of those not operated on went into remission. Conclusion: The prognosis for the majority of patients with MG is favourable, irrespective of thymic histology. The cause may be the use of immunomodulating therapy.

Objectives – To measure clinical and immunological parameters in a patient with myasthenia gravis (MG) treated with antibodies against tumour necrosis factor-alpha (infliximab, Remicade®). Patient and Methods – A patient with severe MG received repeated injections of infliximab. His muscle function score was monitored and the immunological parameters were followed using enzyme-linked immunosorbent assay, flow cytometry and radioimmunoassay. Results – The patient improved in muscle fatigability tests and the levels of antibodies against the acetylcholine receptor decreased during treatment. The activation marker human leucocyte antigen-DR on CD4+ T cells also decreased. Conclusion – Treatment with infliximab might be beneficial for patients with severe MG but demands careful monitoring of possible serious side-effects.

Objective. The T cell co-stimulatory factors CD28 and CTLA-4 and their ligands CD80 and CD86 occur as receptors on T cells and antigen-presenting cells and also in soluble forms in the circulation. We determined the levels of soluble co-stimulatory molecules in patients with abdominal aortic aneurysm (AAA) and normal individuals. We further correlated these soluble co-stimulatory molecules to other clinical parameters of importance such as age of the patient, presence of hypertension, size of the aneurysm and levels of matrix metalloproteinases-9 and C-reactive protein. Design, setting, subjects. This case-control study was designed to quantify the circulating levels of soluble co-stimulatory molecules by an in-house enzyme linked immunosorbent assay. A total of 314 subjects participated in the study including 100 patients and 214 normal controls. The statistical analysis was performed by Mann–Whitney test and Spearman's correlation rank test. Results. Our results show increased plasma levels of sCD28, sCD86 (P = 0.0001) and decreased plasma levels of sCTLA-4 (P = 0.0018) in the patients compared with normal individuals. The levels of these factors were not related to the age of the patient, size of aneurysm or levels of C-reactive protein in plasma. There was, however, a significant inverse relationship between the concentrations of sCTLA-4 and sCD80 with matrix metalloproteinase-9. Conclusions. We suggest that soluble co-stimulatory molecules serve as biomarkers for the estimation of immune activation in AAA patients.

Objectives – To describe two patients with myasthenia gravis (MG) and prolactinoma and analyze the associations between MG and prolactin (PRL) levels. Design – Two case reports and a case–control study of PRL levels in 192 patients with MG. Participants – The Immunological Research Laboratory, Center for Molecular Medicine, Department of Medicine and the Department of Neurology, Karolinska Institutet, Stockholm, Sweden; St Petersburg Medical Academy for Postgraduate Studies, and St Petersburg State Medical Pediatric Academy, Russia. Results – Two women with MG and thymic hyperplasia accompanied by prolactinomas are described. The levels of plasma PRL were raised in 101 women with MG, but not in 91 men. There was an association between high PRL levels and high levels of autoantibodies against the acetylcholine receptor. Conclusions – There is an association of MG with raised levels of PRL in women. PRL has stimulating effects on immune activation and the increased levels might thus be implied in the pathophysiology of MG.

Background Altered stress response is characteristic for subjects with abnormal aggressive and antisocial behavior, but the underlying biological mechanisms are unclear. We hypothesized that autoantibodies (autoAbs) directed against several stress-related neurohormones may exist in aggressive subjects. Methods Using enzyme-linked immunosorbent assay, we studied whether autoAbs directed against corticotropin (ACTH), α-melanocyte-stimulating hormone (α-MSH), oxytocin, and vasopressin are present in serum of male subjects with conduct disorder and prisoners with history of violence. Healthy blood donors served as control subjects. Results Both conduct disorder and prisoners groups displayed strongly increased levels of ACTH-reactive immunoglobulin G (IgG) and immunoglobulin M (IgM) autoAbs compared with control subjects. Levels of oxytocin-reactive IgM autoAbs were slightly increased in both groups of aggressive subjects, whereas levels of vasopressin-reactive IgG and IgM autoAbs were lower only in conduct disorder. No differences in the levels of α-MSH-reactive autoAbs were found between aggressive and control subjects. Conclusions High levels of ACTH-reactive autoAbs as well as altered levels of oxytocin- and vasopressin-reactive autoAbs found in aggressive subjects may interfere with the neuroendocrine mechanisms of stress and motivated behavior. Our data suggest a new biological mechanism of human aggressive behavior that involves autoAbs directed against several stress-related neurohormones.

The aim of the Malmö International Brother Study (MIBS) is to evaluate host genetic factors associated with the development of inhibitory antibodies in patients with hemophilia. Factor VIII gene mutations and genetic polymorphisms of the IL1beta, IL4, and IL10 genes, known to influence antibody production in autoimmune diseases, were analyzed in 164 patients (124 with severe, 26 with moderate, and 14 with mild disease) in 78 unrelated families with hemophilia A. Seventy-seven (47%) patients in 54 families had a history of inhibitors (57 high responding, 20 low responding). Inversions were found in 36 families (75 patients). There was no association between the development of inhibitor and the IL1beta Taq I RFLP alleles in exon 5 or the –590 C/T single nucleotide polymorphism (SNP) in the promoter region of IL4. There was, however, a strong association between an allele with 134 bp in one of the CA repeat microsatellites, IL10G, located in the promoter region of the IL10 gene, and the development of inhibitor (odds ratio [OR], 4.4; 95% confidence interval [95% CI], 2.1-9.5; P < .001). The association was consistent in the subgroup of families with severe hemophilia and inversions. IL10 is the first gene located outside the causative factor VIII gene mutation to be associated with inhibitor development.

Dendritic cells (DC), as initiators and orchestrators of immune responses, control both naive and primed T cell responses. Depending on their maturation stage, DC promote immunity or tolerance. Here we investigated (1) the phenotype and cytokine secretion patterns of IL-10-modulated immature DC (IL-10-DC) and lipopolysaccharide (LPS)-driven mature DC (LPS-DC) in comparison with unmodulated immature DC (imDC) and (2) the effects of IL-10-DC, and of LPS-DC, vs. imDC on autologous T cell responses in patients with myasthenia gravis (MG) compared with healthy controls (HC). All three types of DC derived from MG significantly increased the levels of CD4+CD25+ T cells and of their subfraction expressing CD69, when compared to DC derived from HC. IL-10-DC induced production of IL-10 and IL-4 by T cells from MG patients, but only IL-10 production from HC. LPS-DC activated autologous T cells as reflected by augmented CD25, CD69 and CTLA-4 expression on CD4+ T cells, without differences between MG and HC. This was associated with increased production of both Th1 (IFN-gamma) and Th2 (IL-10 and IL-4) cytokines by T cells. These results indicate that DC-induced activation of autologous T cells is more pronounced in MG than in HC. In addition, DC-induced T cell responses in MG vs. HC are more Th2-prone.

Cytolytic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in the down-regulation of antigen-activated immune responses. The aberrant CTLA-4 expression is characterized by low surface and intracellular levels of CTLA-4 protein, impaired up-regulation of CTLA-4 in T cells in response to ConA stimulation and high levels of soluble CTLA-4 (sCTLA-4) in serum. The serum levels of sCTLA-4 are positively correlated with the serum concentration of antibodies against the acetylcholine receptor. The (AT)n polymorphism in the 3′-untranslated region contributes to decreased mRNA stability and, hence, to reduced expression of CTLA-4.

Objectives. To analyse the association of autoantibodies against cardiolipin (CL) and oxidized low density lipoproteins [copper-oxidized low density lipoprotein (oxLDL), malondialdehyde-modified LDL (MDA-LDL)] with rheumatoid arthritis (RA) and cardiovascular complications. Methods. One hundred and twenty-one patients with RA were consecutively included. Autoantibodies were determined by ELISA. Healthy individuals from the same region were used as controls. Results. Levels of IgG, IgM and IgA antibodies against MDA-LDL and CL, as well as IgG and IgA antibodies against oxLDL were increased in the patients (P

Ninety-five percent of patients with palmoplantar pustulosis are smokers at onset of the disease. The aim of this study was to determine whether these patients have serum antibodies to nicotinic acetylcholine receptors (nAChR ab) and if their sera induce a specific immunofluorescence in normal palmar skin. Sera from 45 patients with palmoplantar pustulosis and 23 patients with chronic hand eczema were analysed for muscle nAChR ab, and immunofluorescence was performed on healthy palmar skin. Forty-two percent of the patients with palmoplantar pustulosis but none of the eczema patients had raised levels of nAChR ab. Immunofluorescence showed staining on endothelial cells in the papillary dermis in 47% of all sera from patients with palmoplantar pustulosis and in those with nAChR ab in 68%. On palmar skin from smokers there was also a staining of the sweat duct. Sera from patients with chronic hand eczema were negative. Our findings indicate that palmoplantar pustulosis is an autoimmune disease, possibly induced by smoking.

The cytotoxic T lymphocyte associated protein 4 (CTLA-4) gene (Ctla-4) is a candidate gene for autoimmune disease. We here report results of two single nucleotide polymorphisms (SNPs) in the Ctla-4, a +49 A/G SNP in CDS1 and a C/T promoter SNP at position -318. There were no differences in these two SNPs between patients and healthy individuals. The frequency of allele G and genotype G/G at position +49 in CDS1 was increased in patients with thymoma when compared with patients with normal and hyperplastic thymic histopathology. Patients with the G/G genotype had signs of immune activation manifested as higher levels of serum IL-1beta and higher percentage of CD28(+) T lymphocytes. There was a strong linkage between the 86bp allele in the 3'-UTR and the A(+49) allele in CDS1. Our results suggest that the SNP at position +49 in CDS1 might be associated with the manifestations of MG.

Cytotoxic T-lymphocyte-associated antigen (CTLA-4) is an important downregulator of T-cell activation. In order to analyze the expression and regulation of CTLA-4 on human peripheral T cells, CTLA-4 mRNA and protein expression were determined using analysis by reverse transcription–polymerase chain reaction (RT–PCR) and FACs, respectively. Intracellular CTLA-4 was constitutively expressed in unstimulated CD4+ and CD8+ T cells. Interleukin (IL)-2 induced a dose-dependent increase of both intracellular and surface expression of CTLA-4 (CD152). Most of the CD4+ and CD8+ cells expressing CTLA-4 also expressed CD25. Interferon (IFN)-γ induced the upregulation of CTLA-4 expression via antigen-presenting cells (APC) activation. The CTLA-4delTM mRNA (550 bp) had a shorter half-life than the full length CTLA-4 mRNA and the expression was downregulated upon activation of the cells by treatment with IL-2. Given an inhibitory role of CTLA-4 and CD4+ CD25+ T cells in immune responses, the present findings suggest that IL-2-induced immunosuppression may result from its stimulatory effect of the CTLA-4 expression.

Human autoimmune myasthenia gravis (MG) is associated with the IL-1beta TaqI RFLP allele 2. Individuals positive for this allele have high levels of inducible IL-1beta in their peripheral blood. Here, we have characterized MG induction and the immune response elicited by Torpedo acetylcholine receptor (AChR) immunization in wild-type and IL-1beta deficient (-/-) mice. Compared with wild-type mice, IL-1beta-/- mice were relatively resistant to induction of clinical experimental autoimmune myasthenia gravis (EAMG). Draining lymph node cells from IL-1beta-/- mice showed poor proliferative capacity upon AChR stimulation in vitro. Both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) cytokine responses were reduced and levels of serum anti-AChR antibodies decreased in IL-1beta-/- mice compared to wild-type mice. Taken together, these results reveal a critical role for IL-1beta in the induction of MG in mice, and support a role for IL-1beta in the pathogenesis of MG in man.

Circulating immune complexes (CIC) are frequently found in postinfarction patients. The constituents of these CIC are mostly unknown.The objective of the current study was to assess whether CIC containing alimentary proteins and antibodies against these proteins are implicated in precocious myocardial infarction (MI).Seventy-six survivors (67 men and 9 women, mean age 39 years) of a first MI before the age of 45 years were enrolled in this study. Two control groups were included. One group consisted of age-matched, randomly selected, population-based healthy individuals, 79 men and 11 women, without features of coronary heart disease. An additional control group was used only for the determination of serum antibodies against some of the alimentary proteins and consisted of 139 healthy blood donors, 95 men and 44 women, with a mean age of 42 years. Sucrose density gradient centrifugation, gel filtration and precipitation by polyethylene glycol were used for the isolation of CIC, and enzyme-linked immunosorbent assay (ELISA) was used to measure the immunoglobulin levels and specific antibodies against alimentary proteins in both sera and isolated CIC. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis and Western blotting were used to determine alimentary proteins in the CIC.Alimentary antigens/antibodies were present in immune complex form in seven out of 14 (50%) postinfarction patients who had persistent high concentrations of CIC, the latter constituting 18% of the entire group. Antibodies of the IgG isotype predominated. A rise in CIC, signs of activation of the classical complement pathway, and a rise in plasma concentrations of von Willebrand factor antigen (vWFAg) were evident within 1 week in four patients subjected to a 2-week elimination diet followed by a single challenge with cow's milk.This study suggests that dietary proteins occasionally give rise to persistent CIC, which may predispose to MI at a young age.

Abstract —Atherosclerosis is an inflammatory disease, and the involvement of immune mechanisms in disease progression is increasingly recognized. Immunization with oxidized low density lipoprotein (LDL) decreases atherosclerosis in several animal models. To explore humoral and cellular immune reactions involved in this protection, we immunized apolipoprotein E knockout mice with either homologous plaque homogenates or homologous malondialdehyde (MDA)-LDL. Immunization with both these antigen preparations reduced lesion development. The plaques contained immunogen(s) sharing epitopes on MDA-LDL, MDA–very low density lipoprotein, and oxidized cardiolipin. This shows that a T-cell–dependent antibody response was associated with protection against atherosclerosis. The protection was associated with specific T-cell–dependent elevation of IgG antibodies against MDA-LDL and oxidized phospholipids, and the increased titers of IgG antibodies were correlated with decreased lesion formation and lower serum cholesterol levels.

Background —Circulating immune complexes (CICs) and autoantibodies against oxidatively modified LDLs (oxLDLs) and cardiolipin occur in patients with atherosclerosis and myocardial infarction (MI). The ability of such CICs and antibodies to predict myocardial infarction (MI) was investigated in a prospective nested case-control study in which healthy 50-year-old men were followed for 20 years. Methods and Results —Two hundred fifty-seven men were included in the study, and 119 developed MI (39 died) between 50 and 70 years of age. One hundred thirty-eight randomly chosen men who did not develop MI up to 70 years of age served as controls. The prevalence of elevated levels of CICs and the concentration of CICs in men who developed MI were higher than in those who remained healthy. The concentration of CICs at age 50 was associated with a marked increased risk for MI, and this risk was independent of other conventionally recognized risk factors. There was a positive correlation between the levels of CIC and IgG antibodies to cardiolipin in men who developed MI. The level of IgG antibodies and the prevalence of elevated IgG and IgM antibodies to cardiolipin were higher in those who developed MI and had CICs than in those without CICs. Among men homozygous for C4 null alleles, those who developed MI had higher concentrations of CICs than did those who remained healthy. Conclusions —This prospective study shows that CICs alone or in combination with autoantibodies against cardiolipin in healthy males at 50 years of age predict subsequent MI between the age of and 70 years.

Background and Purpose —Anticardiolipin antibodies (aCL) have been proposed to be an independent risk factor for stroke. To test this hypothesis, a nested case-control study was performed to compare aCL with the other known risk factors for stroke. Methods —Within the framework of the World Health Organization Multinational Moni toring of Trends and Determinants in Ca rdiovascular Disease (MONICA) project and the Västerbotten Intervention Program (VIP) health survey, 44 725 men and women were enrolled and followed up from January 1, 1985, through August 31, 1996. Individuals free from cardiovascular events were followed up, and 123 developed stroke (on average, 34.1 months after blood sampling; 21 cerebral hemorrhage and 102 cerebral infarction); they were compared with 241 age- and sex-matched control subjects from the same population. ELISA was used for the analysis of IgG, IgM, and IgA aCL. Results —IgM-aCL were present in 11.4% of patients (14/123) who developed stroke and in 4.1% of individuals (10/241) who remained healthy ( P =0.013, OR 2.97, 95% CI 1.28 to 6.89). The OR for the levels of IgM-aCL was 1.34 ( P =0.01, 95% CI 1.07 to 1.68) without adjustment for other risk factors and 1.24 when adjusted for hypertension, diabetes mellitus, cigarette smoking, and use of smokeless tobacco ( P =0.077, 95% CI 0.98 to 1.56). There was no difference between patients and controls for the prevalence or level of IgG-aCL and IgA-aCL and also no difference between patients with cerebral hemorrhage and cerebral infarction for the prevalence of all 3 isotypes of aCL. Conclusions —We conclude that aCL are associated with future stroke but do not constitute an independent risk factor.

CD28 is required to promote T cell proliferation and cytokine production, while the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) functions as a negative modulator for T cell activation. We previously reported that alleles with longer PCR products (designated as allele xx) in an (AT)n polymorphism in Ctla-4 are associated with myasthenia gravis with thymoma, while the shortest allele, 86, is negatively associated with the disease. Here, we demonstrate that serum IL-2 sRα increases parallel to the length of (AT)n in Ctla-4 . Periphereal blood mononuclear cells (PBMC) from patients with Ctla-4 xx/xx contained higher activity of telomerase than patients bearing Ctla-4 86/86. Blockade of CTLA-4 increased the telomerase activity in PBMC stimulated by acetylcholine receptor in vitro. There was a positive correlation between the expression of CD28 and CTLA-4 on anti-CD3 activated PBMC, suggesting a balance between CD28 and CTLA-4. Cells from patients with Ctla-4 xx/xx had the highest level of T cell proliferative responses upon the addition of anti-CD28 antibodies to the anti-CD3 containing culture system while cells from patients with Ctla-4 86/xx had an intermediate and cells from patients with Ctla-4 86/86 the lowest increase. The current results point to the (AT)n in Ctla-4 as a myasthenia gravis facilitating mutation under certain permissive environments by influencing the T cell reactivity via the CD28 pathway.

Human monoclonal antibodies against cardiolipin, native and oxidatively modified LDL and against apoB100 were obtained by Epstein-Barr virus transformation of peripheral blood mononuclear cells followed by cloning. The antibodies were used to analyse the cross-reactivity between monoclonals with different specificities and to study the repertoire of antibodies present in sera from patients. Monoclonal antibodies selected for binding to cardiolipin were the least cross-reactive, whereas antibodies against different modifications of LDL frequently also reacted with other modifications. Inhibition studies showed that serum antibodies against cardiolipin and against oxidatively modified LDL were to a different extent inhibited by the different monoclonal antibodies and thus markedly heterogeneous.

The classical risk factors, hypercholesterolemia, smoking, hypertension and diabetes, explain only a part of the epidemiological features of atherosclerotic coronary heart disease. Investigations in the past few years have shown involvement of immunological mechanisms in atherosclerosis. Circulating immune complexes accelerate atherosclerosis both in experimental animal models and in humans. The fourth component of complement (C4) plays an important role in the solubilisation and elimination of immune complexes. C4 consists of two allotypes, C4A and C4B. An earlier report showed an association between C4B null alleles (C4B*Q0) and myocardial infarction and to infarction related mortality. In the present investigation, C4A*Q0 and C4B*Q0 were studied in two population samples. The first (Group I) was a cross sectional study of 100 consecutive males with myocardial infarction before the age of 45 years and 164 population based healthy controls, age and sex matched. The second (Group II) was a nested case control study in which a cohort of 50 year-old males were followed for 20 years for development of myocardial infarction between 50-60 and 60-70 years, and the results compared with those who did not develop MI. We observed no association of homozygous and/or heterozygous C4A*Q0 or C4B*Q0 with myocardial infarction occurring in the age groups45, 50-60 and 60-70 years or with the infarction related mortality (P0.05). The prevalence/frequency of C4A*Q0 and C4B*Q0 was not related to the age at which MI occurred. The prevalence of C4A*Q0 was not affected by age. We thus conclude that partial deficiency of C4 does not appear to be a major risk factor for myocardial infarction.

Interleukin-10 (IL-10) is an important pleiotropic cytokine with both anti-inflammatory and B lymphocyte stimulating functions. The expression of IL-10 is tightly controlled. A bi-allelic polymorphism and 2 CA repeat microsatellites located in the promoter region of IL-10 gene were analysed in Swedish myasthenia gravis (MG) patients and ethnically matched healthy individuals. The prevalence of a 'high secretor' phenotype of IL-10 (IL-10 1 G/G) was higher in IL-1beta TaqI polymorphism allele 2 positive healthy individuals ('high secretor' phenotype for IL-1beta) than in healthy individuals negative for this allele. No such balance was found in MG patients. In one of the microsatellites, IL10.R, allele 112 was associated with patients having normal thymic histology. No relation of IL10.R allele 112 to proinflammatory cytokine gene polymorphisms and serum IgG, IgM and autoantibodies against nicotinic acetylcholine receptor (nAchR-Ab) was found. In another microsatellite, IL10.G, allele 134 was associated with patients having higher level of nAchR-Ab in their circulation. Our results demonstrated novel genetic markers within IL-10 gene indicating different mechanisms for IL-10 involved in the disease.

Circulating immune complexes can lead to vascular inflammation and premature atherosclerosis and the fourth component of complement, C4, plays an important role in the removal of immune complexes. The objective of this study was to analyze the relation between circulating immune complexes and C4 null alleles in patients operated on for peripheral vascular disease before the age of 50. The prevalence of circulating immune complexes and null alleles of C4 (C4Q0) was determined in 62 patients with peripheral atherosclerosis requiring surgery before 50 years of age and in a matched control group. C4A and C4B null alleles (C4A*Q0, C4B*Q0) were determined by electrophoresis of plasma, followed by immunofixation. C4A and C4B concentrations were measured by ELISA. Circulating immune complexes were determined by sucrose density gradient centrifugation and gel filtration. There was no difference in the distribution of C4Q0 between patients and controls. The patients had higher prevalences and levels of circulating immune complexes. This was correlated with the presence of C4Q0, especially C4A*Q0. There was an inverse correlation of concentration of circulating immune complexes with C4A levels and with ratio of C4A/B levels. Thus, a significant proportion of patients with premature peripheral atherosclerosis had circulating immune complexes and C4A*Q0 enhanced the propensity to immune complex formation. This might represent one mechanism for vascular damage in this patient group.

T lymphocytes are implicated in the pathogenesis of systemic vasculitis such as Wegener’s granulomatosis (WG) and polyarteritis nodosa (PAN). In the present study, we have characterized in detail the T-cell receptor (TCR) of peripheral blood T cells from eight vasculitis patients of known HLA class II genotypes. We used flow cytometry to outline the exact TCR V gene expression, complementarity determining region 3 (CDR3) fragment analysis to estimate the degree of clonality and cDNA sequencing to define the exact TCR  or β chain sequences. The TCR CDR3 region interacts with antigenic peptides presented by HLA molecules, and it is normally immensely diverse. It was therefore of particular interest to identify a common dominating TCR BV8-F/L-G-G-A/Q-G-J2S3 β chain sequence in the CD4+T cells of four unrelated vasculitis patients. Furthermore, this BV8-associated CDR3 motif was linked to the HLA-DRB1*0401 allele, as well as to active disease and/or an established BV8+ CD4+ T-cell expansion. In contrast, age- and HLA-matched patients with rheumatoid arthritis did not harbor the described BV8 motif. These results strongly suggest that BV8+ CD4+ T cells with the described CDR3 motif recognize a specific antigen presented by DR4 molecules, indicating the existence of a common vasculitis-associated antigen.

The aim of this study was to analyze associations between myasthenia gravis (MG) and polymorphisms in the tumor necrosis factor (TNF) region in 79 Swedish patients and 155 unrelated controls. The frequency of the TNFa2 allele of a microsatellite located 3.5 kb upstream of the lymphotoxin alpha (LT- α ) gene in the TNF region was found to be increased in overall MG patients compared to controls. The frequency of the short 5.5 kb fragment (TNFB*1) of a bi-allelic Nco I RFLP polymorphism located at the first intron of the LT- α gene was increased in patients with an early onset of disease compared to patients with a later onset.

Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) plays a pivotal role in downregulating both the cellular and the humoral response by suppressing ongoing responses of activated T cells. Our earlier study showed that genetic variations in interleukin-1 genes confer susceptibility to myasthenia gravis, especially in patients having the lowest risk from major histocompatibility complex genes. Here we describe an association of Ctla-4 gene to the disease with thymoma and a higher prevalence of CTLA-4 gene polymorphism allele 104 in patients positive for IL-1beta TaqI allele 2, an IL-1beta 'high secretor' phenotype. There was no association in patients with hyperplasia and normal thymic histology. These results further advocate that MG is a polygenetic disease and suggest that co-stimulators such as CTLA-4 and CD28 might have an important role in the pathogenesis of the disease.

Interleukin 1 (IL-1)beta, TaqI restriction fragment length polymorphism (RFLP) in exon 5 and IL-1 receptor antagonist (IL-1Ra) polymorphism, variable numbers of an 86-bp tandem repeat (VNTR), were analysed in 107 patients with myasthenia gravis (MG) and 82 ethnically matched healthy control (HC) individuals. Positive association was found with IL-1beta TaqI RFLP allele 2 carriage in MG (OR = 2.007), while allele 1 was negatively associated with MG (OR = 0.498). When homozygous individuals for allele 2 were considered, the association was stronger (OR = 4.630), indicating a dose effect of allele 2. Analysis of IL-1beta TaqI RFLP in relation to HLA-B8 demonstrated that the allelic association was more pronounced in patients without HLA-B8 (OR = 2.813). There was no difference in IL-1Ra VNTR allelic distribution in MG patients compared with HC. However, MG patients who were noncarriers of IL-Ra allele 2 had a significantly higher percentage of IL-1beta TaqI RFLP allele 2 carriage (OR = 3.085), while there was no such difference in IL-1Ra allele 2 carriers. Our results demonstrate a new genetic marker in MG, which exerts its maximum effect in patients with the lowest MHC-associated susceptibility. We propose a possible pathogenetic role of IL-1beta and a possible intrinsic dyregulation of IL-1 in MG.

Patients with Wegener's granulomatosis have a high prevalence of expanded populations of CD4+ and CD8+ T cells bearing different alpha/beta T cell receptors. To elucidate the role of these populations, we studied the phenotypic and functional characteristics of 13 expanded T cell populations in four patients for a period of 35-51 months. The expanded populations generally showed a persistently high expression of the activation markers HLA-DR and CD25. This expression was independent of the activity of the disease. The expanded populations also expressed CD45RO and/or CD45RA and most of them expressed CD57 but not CD28. Analysis of intracellular presence and secretion of IFN-gamma, IL-2, and IL-4 showed that most of the expanded cell populations contained and/or secreted more of these cytokines than the nonexpanded populations, with an especially high expression/secretion of IFN-gamma and IL-2. The expanded populations showed little proliferative response to Con A and OKT3. The proliferative response of the cells was partly restored after preincubation in medium alone. Some of the expanded populations were associated with disease activity, thus suggesting a link between expanded T cells and the disease. The activated status of the expanded populations and the tendency for certain populations to correlate in magnitude with disease activity suggest their involvement in the disease process. The relative stability of these cell populations indicates that the stimulus driving them is persistent, in agreement with the chronicity of the disease.

Beta2-adrenergic receptors (beta2AR) are present on both lymphocytes and skeletal muscle cells. Antibodies and T cells that react with these receptors are present in patients with myasthenia gravis (MG). Immune reactivity against the beta2AR may thus modify both the immune and the muscle functions in MG. In this study, we analysed the density and affinity of beta2AR on peripheral blood mononuclear cells using a radioligand binding assay. The density (Bmax) of the receptor on cells from patients with MG was significantly lower than that on cells from patients with other neurological disorders and healthy individuals. The affinity (Kd) of the receptor and the concentration of the second messenger, cAMP, in the cells did not differ between the groups. Serum antibodies against beta2AR were demonstrated in 22% of 27 MG patients vs. 0% of 26 healthy controls. Incubation of cells with serum or purified IgG containing antibodies against the beta2AR resulted in a decline in ligand binding of the receptor in samples from three out of five patients. Thus, this study suggests that a downregulation of the beta2AR may occur in MG. This downregulation might be of importance in the patho-genesis of the disease and its symptoms.

Eleven patients with myasthenia gravis were followed for three years after thymectomy. Acetylcholine receptor-specific T-cell stimulation was found in 8/11 patients before operation as compared to 2/11 three years after thymectomy. Changes of T-cell antireceptor-reactivity were commonly paralleled by changes in disease severity. The numbers of cells secreting IL-2 upon stimulation with human acetylcholine receptor correlated with those secreting IFN-γ. T-cell reactivity against a monoclonal acetylcholine receptor antibody did not decrease after thymectomy. Such reactivity could reflect a beneficial immune response counteracting anti-receptor reactivity. The frequency of autoantibody-secreting cells remained unchanged, while the serum concentration of acetylcholine receptor antibodies started to decrease one year after thymectomy. All examined thymus-cell suspensions contained autoreactive T- and B-lymphocytes. There was a preferential enrichment of autoreactive lymphocytes in the thymus in a few patients with recent onset of disease

SUMMARY Takayasu's arteritis (TA) is a chronic large vessel vasculitis with a predilection for the aortic arch and its branches. T lymphocytes may be important in the pathogenesis, as they have been found to infiltrate the vascular lesions. To elucidate further the role of T cells in the disease, we studied circulating CD4+ and CD8+ T cells, expression of the activation marker (HLA-DR), marker for naive (CD45RA) and primed (CD45RO) cells and the different variable α/β (AV/BV) gene segments on them. The TCR AV/BV repertoire was studied using a panel of 15 T cell receptor (TCR) V-specific MoAbs by flow cytometry in 18 patients and 23 age- and sex-matched controls. Patients had a higher percentage of AV12S1 (P

The principal symptoms of myasthenia gravis (MG), muscle weakness and fatigue due to impaired neuromuscular transmission, are caused by autoantibodies to the muscle nicotinic acetylcholine receptor (AChR). The mechanisms underlying the autoimmune response, however, appear to be initiated by activation of specific HLA class II–restricted CD4+ T lymphocytes. Thus, central to elucidating the causation of MG is determining how T cells are recruited to contribute to misguided immunological assaults on the major autoantigenic target, AChR. By combining a polymerase chain reaction (PCR)–based strategy and Southern blot technique, we have analyzed the frequency of expression of 22 individual T cell receptor (TCR) Vβ gene subfamilies in CD4+ and CD8+ peripheral blood T cell subsets derived from eight MG patients and seven healthy controls. The quantification of relative usage of individual TCR Jβ gene segments was performed by hybridization of PCR-amplified products (specifically Vβ1-Cβ) with a complete panel of 32P-5′-end-labeled Jβ-specific oligonucleotide probes, followed by scanning analysis of autoradiographs. Comparisons of data obtained from Vβ analyses of T cells from MG patients with those from healthy individuals established that MG patients significantly overexpressed Vβ1, Vβ13.2, Vβ17, and Vβ20 gene family members within both CD4+ and CD8+ T cell sub-populations. Moreover, analysis of the relative utilization of individual TCR Jβ gene segments in Vβ1+/CD4+ and Vβ1+/CD8+ T lymphocytes revealed distribution patterns in patients indistinguishable from those recorded in the corresponding cell subsets derived from controls. T lymphocytes from MG patients displayed a biased overexpression of four TCR Vβ gene segments: Vβ1, Vβ13.2, Vβ17, and Vβ20. The relative frequencies of association of individual Vβ1 (Dβ)Jβ combinations revealed that Jβ gene usage in the Vβ1-overrepresented T cell subsets had normal distribution patterns. It can thus be deduced that Jβ gene segment products appear not to have a selective effect on the process leading to overexpression of Vβ1 exons in MG patients. Hence, our observations suggest a possible role for superantigen(s) in the T cell activation in MG patients.

SUMMARY An important and yet unresolved question concerns the mode of T cell recognition of idiotypic epitopes on immunoglobulin molecules in humans. Results from murine and human studies show that some idiotype-specific T cells recognize conformational epitopes on immunoglobulin, and such T cells are not MHC-restricted. In the present study T cell stimulation induced by idiotypic determinants on the autologous monoclonal IgG (M-components) from patients with monoclonal gammopathies was studied. In parallel, T cell stimulation in response to a conventional antigen, purified protein derivative, was also examined. It is shown that, as with conventional antigen, idiotype-induced T cell stimulation requires the presence of antigen-presenting cells (APC; monocytes and/or B cells), and is MHC class II (DR)-restricted. B cells, but not monocytes, can present idiotypic determinants to T cells at very low antigen concentrations, while monocytes do so only when antigen is present at high concentrations. Antigen processing and presentation is abrogated by treatment of APC with chloroquine. In conclusion, our study demonstrates that human idiotype-specific T cells recognize processed idiotypic determinants presented by MHC class II (HLA-DR) molecules on APC, and that B cells require about 1000-fold less antigen than monocytes.

Oxidized low density lipoproteins (oxLDL) stimulated human blood T cells to a specific cytotoxic response which autologous PHA activated blood mononuclear cells which had been pre-incubated with oxLDL. The cytotoxic reaction was exerted by CD8+ enriched cells but not by CD4+ enriched cells. Allogeneic target cells from HLA mismatched donors were not damaged and the reaction was consistently inhibited by monoclonal antibody against HLA class I. Antigen-specific cytotoxic T cells might contribute to lysis of cells in the atherosclerotic plaque that have taken up oxLDL and thus participate in the inflammatory process associated with atherosclerosis.

The majority of patients with myasthenia gravis were shown to have T and B cells specific for a beta-bungarotoxin binding protein, presynaptic membrane receptor (PsmR). Such autoreactive T cells may be subdivided into different subsets according to the pattern of cytokine production. In this study the authors examined the subpopulation of the T cells by analysing their IFN-gamma and/or IL-4 secretion pattern. T cell response to human muscle acetylcholine receptor (AChR) was examined in parallel. PsmR-stimulated IFN-gamma secretion was found in 60%, and IL-4 secretion in 48% of the patients. Cells stimulated to secrete both IFN-gamma and IL-4 or IFN-gamma only were the most common patterns. Treatment of the cells with a mouse anti-human HLA-DR antibody abolished the secretion of cytokines. There was a positive correlation between the numbers of PsmR-reactive and AChR-reactive T cells. In conclusion, the results show that PsmR-stimulated T cells secreted IFN-gamma and/or IL-4. This T cell response is MHC class II restricted. Thus, this study indicates that both Th1/Th2 or Th0 subsets of the T cells are involved in the autoimmune response in the disease.

Objectives. To investigate the prevalence of anti-cardiolipin and antiendothelial cell antibodies in patients operated on for atherosclerotic peripheral vascular disease before 50 years of age. The hypothesis to be tested was whether antibodies associated with an immune/inflammatory damage to the vascular wall were associated also with early atherosclerosis. Design. A case-control study. Setting. Departments of surgery and an immunological research laboratory, and routine laboratories of two tertiary referral hospitals. Subjects. All patients operated for atherosclerotic peripheral vascular disease before 50 years of age. Sixty-two patients (33 males, 29 females), and 67 age- and sex-matched controls participated. The diagnosis of atherosclerosis was made on the basis of the clinical presentation and angiographic visualization of the diseased vasculature. Main outcome measures. Subjects were compared for the prevalence of anticardiolipin and antiendothelial cell antibodies, altered serum lipoprotein levels, smoking, diabetes, hypertension, and signs of inflammation. Results. Antibodies against endothelial cells and cardiolipin were found in 12.9 and 14.5% of the patients, respectively, which was higher than observed in the control group (P < 0.05). Sixty per cent of patients with antibodies were females. Conventional risk factors were more often noted in the patient group. However, patients with antibodies against endothelial cell and/or cardiolipin had a lower prevalence of hyperlipidaemia/dyslipidaemia when compared to patients without these antibodies (P < 0.05). Conclusions. Antibodies against endothelial cells and cardiolipin are present in a subset of patients with clinical and angiographic diagnosis of severe premature atherosclerotic peripheral vascular disease. The lower occurrence of hyperlipidaemia/dyslipidaemia in patients with autoantibodies, in comparison to patients without the antibodies, suggests that these antibodies have a role in vascular damage.

The presence of idiotype-reactive T-cell subsets and their relation to the tumor load were analyzed in 9 patients with monoclonal gammopathy of undetermined significance (MGUS), in 12 patients with multiple myeloma (MM) clinical stage I, and in 9 patients with MM stage II/III. An enzyme-linked immunospot assay was used to identify interferon-gamma (IFN-gamma)-, interleukin-2 (IL-2)-, or IL-4-secreting T cells after stimulation by F(ab')2 fragments of monoclonal IgG. The response to autologous IgG was significantly higher than that induced by isotypic monoclonal IgG. Comparable results were obtained in a proliferation assay (3H-thymidine incorporation). A total of 8 of 9 patients with MGUS, 7 of 12 patients with MM stage I, and 3 of 9 with MM stage II/III had T cells secreting IFN-gamma and/or IL-2 (T helper [Th1] type-1 cells), whereas cells secreting both Th1 and Th2 or Th0 types of cytokines were more frequent in patients with MM, particularly in those with MM stage II/III. The number and frequency of Th1-type cells were significantly higher in MGUS patients as compared with those of MM stage II/III. The results indicate that idiotype-reactive T cells of the Th1 and Th2 or Th0 subsets were present in MGs and might provide indirect evidence that idiotype-reactive Th1-type cells may have a regulatory impact on the human tumor B cells.

SUMMARY Wegener's granulomatosis (WG) and polyarteritis nodosa (PAN) are systemic necrotizing vasculitides of unknown etiology. These disorders run a fatal course if untreated. T lymphocytes are implicated in the pathogenesis of WG, since they have been found to infiltrate affected organs, and sIL-2R correlates with disease activity. To elucidate further the role of T cells in necrotizing vasculitis, we have used a panel of 12 TCR V-specific MoAbs to investigate the number of cells expressing certain Vα and Vβ gene segments in the CD4+ and CD8+ subsets of altogether 11 patients with WG or PAN. In the group of patients, we found abnormal expansions of T cells using particular TCR Vα or β gene products. These T cell expansions were more numerous, of a dramatically higher magnitude, and frequently more often found in the CD4 subset, compared with T cell expansions identified in healthy individuals. In long-term studies of the T cell expansions for up to 18 months, a heterogeneous pattern was revealed, with no obvious correlation to clinical features such as disease activity or treatment. Studies of TCR V gene usage in this group of patients may help in understanding the pathogenesis of necrotizing vasculitis, and in the identification of unknown antigens, and may open the possibility to a highly selective immunotherapy by targeting disease-mediating T cells.

Abstract One hundred patients who had survived a myocardial infarction before the age of 45 years and 90 age- and sex-matched healthy individuals were investigated for circulating immune complexes (CICs) and the presence of complement C4 null alleles (C4Q0). CICs were found in increased concentrations in 20% of patients and 6.7% of control subjects. Patients and control subjects had the same prevalence of C4Q0. CICs were present in all patients and in 36% of the control subjects homozygous for C4Q0. Patients and control subjects heterozygous for C4Q0 had CICs in 71% and 0%, respectively. The high prevalence and a high concentration of CICs were particularly associated with C4A*Q0. Patients homozygous for C4A*Q0 had concentrations of LDL that were lower than found in other patients. The increased concentration of CICs associated with genetic deficiencies of the complement factor C4 might thus be an additional etiological factor for the development of chronic vascular damage and premature myocardial infarction.

Idiotype-specific T cells were characterized in patients with multiple myeloma stage I by analysing idiotype-induced DNA synthesis (3H-thymidine incorporation), IL-2 and IFN-gamma production at the single cell level (ELISPOT) (in vitro tests) and delayed type hypersensitivity (DTH) skin reaction (in vivo test). In seven out of eight patients at least one of the four tests was positive. In five patients three or more tests were positive. One patient was negative in all four tests. Six patients had both IL-2 and IFN-gamma-secreting cells and three of them also a DTH response. Furthermore, those three patients with a proliferative response also had IL-2 and IFN-gamma-secreting cells induced by the idiotype. The data indicate that part of the idiotype-specific T cell fraction belongs to the CD4 Th1 cell population. Whether CD8-specific T cells also were present could not be ruled out. The present study provides further support for the existence of idiotype-specific T cells in multiple myeloma. Such cells might be an important target for an immune-mediated therapeutic approach.

Agalactosyl IgG (Gal(0) is a glycoform lacking terminal galactose from the oligosaccharides situated on the Fc. The percentage of circulating IgG that is Gal(0) is increased in a a number of autoimmune diseases, and in certain chronic infections associated with autoantibody production. However it is not known whether this represents decreased galactosylation of all IgG, or an increase in the relative concentration of a subset of agalactosyl antibodies of specificity relevant to the disease process. Since there is currently no way to separate agalactosyl from galactosylated IgG, we devised an assay for the relative degree of galactosylation of antibody to tetanus toxoid (TT), an antigen irrelevant to the diseases studied, and compared this value with the %Gal(0) of the whole circulating IgG. In rheumatoid arthritis (RA) and tuberculosis (TB), a raised %Gal(0) in serum IgG was reflected in a parallel rise in the extent to which antibody to TT was agalactosyl. In SLE a rise in %Gal(0) was seen in the presence of very little rise in agalactosyl anti-TT, and in myasthenia gravis (MG), where serum %Gal(0) is normal, an abnormally low percentage of the anti-TT was agalactosyl. These results imply that in RA and TB a systemic influence is downregulating the galactosylation even of irrelevant IgG. However in SLE and MG antibodies of specificities not studied here must be responsible for the %Gal(0) found in serum. It remains to be seen whether these are the autoantibodies involved in the disease process.

The pharmacokinetics of recombinant human granulocyte-macrophage colony- stimulating factor (rhGM-CSF), induction of anti-GM-CSF antibodies, and clinical effects related to the induction of the antibodies were analyzed in patients with metastatic colorectal carcinoma (CRC) who were not on chemotherapy (n = 20, nonimmunocompromised patients). rhGM- CSF (250 micrograms/m2/d; Escherichia coli-derived) was administered subcutaneously for 10 days every month for 4 months. Eight patients with multiple myeloma (MM) on intensive chemotherapy followed by rhGM- CSF treatment were also included (immunocompromised patients). After a single injection of GM-CSF at the first cycle in CRC patients, the maximum calculated concentration (Cmax) was 5.24 +/- 0.56 ng/mL; the half life (T1/2) was 2.91 +/- 0.8 hours; and the area under the concentration curve (AUC) was 30.86 +/- 6.03 hours x ng/mL (mean +/- SE). No anti-GM-CSF antibodies were detected. During the subsequent cycles, 95% of the CRC patients developed anti-GM-CSF IgG antibodies, which significantly altered the pharmacokinetics of rhGM-CSF at the third and fourth cycles with decreased Cmax (2.87 +/- 0.57 ng/mL; P < .05), T1/2 (1.57 +/- 0.2 hours; P < .05), and AUC (14.90 +/- 4.10 hours x ng/mL; P < .005). The presence of anti-GM-CSF antibodies significantly reduced the GM-CSF-induced enhancement of granulocytes, and there was a clear tendency for a decreased increment of monocytes. Antibodies diminished systemic side effects of rhGM-CSF. Only 1 of 8 MM patients showed a very low anti-GM-CSF antibody titer after GM-CSF therapy, as shown by enzyme-linked immunosorbent assay and Western blot. Therefore, in nonimmunocompromised patients, exogenous nonglycosylated GM-CSF induced an anti-GM-CSF IgG antibody response in practically all patients, which seemed to be of clinical significance. In immunocompromised patients, virtually no significant antibody response was shown.

The prevalence of peripheral blood B cells secreting antibodies reacting with the F(ab')2 fragment of monoclonal IgG was studied in five patients with multiple myeloma (MM), nine patients with monoclonal gammopathy of undetermined significance (MGUS) and six healthy controls. An enzyme-linked immunospot assay allowed direct visualization of antibody producing B cells. All patients had B cells producing antibodies to autologous or allogeneic monoclonal IgG. Autoreactive cells were found more frequently than alloreactive cells in seven out of nine patients with MGUS and three out of four patients with MM. The same frequency of alloreactive cells in the patient groups was detected in healthy individuals. These findings show the existence of B cells producing anti-idiotypic antibodies which could be a part of an idiotypic network in monoclonal gammopathies.