Diagnostic Performance of an Anti-Actin Autoantibody Binding Enzyme Immunodot Blot in Autoimmune Hepatitis Type 1.

BACKGROUND: A serologic hallmark of autoimmune hepatitis (AIH) type 1 are anti-smooth muscle autoantibodies (ASMA) with specificity for filamentous actin (F-actin; AAA (anti-actin antibodies)), traditionally detected by indirect immunofluorescence (IFT) using rat liver, kidney, and stomach tissue sections as substrates. However, IFT is a subjective method requiring an experienced investigator. Therefore, a more objective technique for the detection of AAA may be a helpful diagnostic tool. METHODS: In a retrospective study with cross-sectional design, we evaluated AAA detected by an enzyme immunodot blot (IDB; Liver5 IgG BlueDot, D-tek, Mons, Belgium). Serum samples of patients with AIH type 1 (n = 47) and specified controls (n = 142) were included. For comparison, standard IFT was applied to rat LKS (liver, kidney, stomach) triple tissue sections. RESULTS: IDB readings were done by two independent investigators (92% concordance). The diagnostic sensitivity of the AAA-IDB was 70%, compared to 51% of AAA-IFT (n.s.). The diagnostic specificity of AAA-IDB was significantly lower compared to AAA-IFT (76% vs. 94%; P < 0.0005). Correspondingly, the positive predictive value (49% vs. 75%; P < 0.05) and positive likelihood ratio (2.9 vs. 8.5) differed significantly. Neither prescreening for ANA or ASMA, nor the exclusion of infectious hepatopathies resulted in a significantly better diagnostic performance of the IDB. CONCLUSION: Compared to standard IFT, testing for AAA via IDB did not result in a significantly better diagnostic performance for AIH type 1. A blot with higher antigen binding specificity may be more functional.

Liver involvement in ANCA-associated vasculitis.

The aim of the study was to investigate the incidence, the clinical course and outcome of liver involvement and autoimmune hepatic diseases in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Liver function tests (LFT) (i.e. aspartate and alanine aminotransferase [AST, ALT], gamma-glutamyl transpeptidase [gamma-GT], alkaline phosphatase [ALP] and total bilirubin) were analysed at disease onset in therapy-naïve patients and during remission in patients with granulomatosis with polyangiitis (GPA, n = 67), microscopic polyangiitis (MPA, n = 28) and eosinophilic granulomatosis with polyangiitis (EGPA, n = 14). Results were correlated to the Birmingham Vasculitis Activity Score version 3 (BVAS v.3). Also, serologic tests for other autoimmune hepatic diseases were performed in these patients. During the active state, LFT abnormalities could be detected in 54 AAV patients (49.4 %). ALT, gamma-GT and ALP were significantly higher in GPA patients compared to MPA or EGPA patients at disease onset (p < 0.05). Increased values for gamma-GT in GPA patients correlated with the BVAS (p < 0.01) and were associated with pulmonary involvement, pulmonary-renal syndrome and a longer time to remission. Increased LFT in GPA patients decreased subsequently towards normal levels after initiation of therapy (p < 0.01). No case of severe liver involvement or autoimmune hepatic liver diseases was found in AAV patients. Liver involvement was mainly restricted to GPA patients, is associated with the disease activity and indicates a poorer outcome in patients with GPA. Progressive liver involvement or autoimmune hepatic diseases were not observed.

Interleukin-6 promoter haplotypes are associated with etanercept response in patients with rheumatoid arthritis.

Indices prognosticating anti-tumor necrosis factor (TNF) response in patients with rheumatoid arthritis are a matter of interest. Differential outcome under anti-TNF and anti-interleukin-6 (IL-6) therapy raises the question whether genetic polymorphisms that have previously been linked to IL-6 production are associated with response to anti-TNF therapy. Fifty (50) rheumatoid arthritis (RA) patients were treated with etanercept (median 36 weeks, range 4-52). In terms of the EULAR response criteria, 25 patients responded well, 17 patients moderately and 8 patients not. By direct sequencing, the patients and 91 matched healthy controls were genotyped for the IL-6 promoter SNPs -597G > A (rs1800797), -572G > C (rs1800796) and -174G > C (rs1800795) and for an AnTn microsatellite tract at -373. Alleles and haplotypes were tested for association with disease susceptibility and therapy response. No significant difference was seen in the genotype distribution between patients and healthy controls. Confirming the results of previous studies, we observed a trend of -174G being more frequent in patients with a good or moderate therapy response. Beyond that, carriage of the A9T11 microsatellite allele within the -174G haplotype was associated most closely with a favourable response (relative risk 1.31; 95 % confidence interval 1.02-1.68). A subtle analysis of the IL-6 promoter giving respect to its complex haplotypic structure results in more precise information as to the association of genotypes with the long-term etanercept response. Despite a conclusive hypothesis that a genetically determined IL-6-dominated RA responds less well to anti-TNF, more work has to be done to provide us with reliable information regarding the functional aspects of these genetic polymorphisms.

Putative IL-10 Low Producer Genotypes Are Associated with a Favourable Etanercept Response in Patients with Rheumatoid Arthritis.

Outcome predictors of biologic therapeutic drugs like TNF inhibitors are of interest since side effects like serious infections or malignancy cannot be completely ruled out. Response rates are heterogeneous. The present study addressed the question whether in patients with rheumatoid arthritis (RA) interleukin-10 (IL-10) promoter genotypes with potential relevance for IL-10 production capacity are associated with response to long-term treatment with etanercept. Caucasian RA patients that, according to the EULAR criteria, responded well (n = 25), moderately (n = 17) or not (n = 8) to etanercept therapy (median 36 months, range 4-52), and 160 matched controls were genotyped for the IL-10 promoter SNPs -2849 G>A (rs6703630), -1082 G>A (rs1800896), -819 C>T (rs1800871) and -592 C>A (rs1800872). Haplotypes were reconstructed via mathematic model and tested for associations with disease susceptibility and therapy response. We identified the four predominant haplotypes AGCC, GATA, GGCC, and GACC in almost equal distribution. Patients that responded well carried the putative IL-10 low producer allele -2849 A or the haplotypes AGCC and GATA (RR 2.1 and 4.0, respectively; 95% CI 1.1-4.0 and 1.1-14.8), whereas an unfavourable response was associated with carriage of the putative high producer haplotype GGCC (RR 1.9, 95% CI 1.1-3.3). No significant associations of alleles or haplotypes with disease susceptibility were observed. In RA, a low IL-10 production which is genetically determined rather by haplotypes than by SNPs may favour the response to etanercept treatment. Iatrogenic blockade of TNF may reveal proinflammatory effects of its endogeneous antagonist IL-10. Further studies are needed to correlate these genetic findings to direct cytokine measurements.

Interferon-gamma is increased in patients with primary Sjogren's syndrome and Raynaud's phenomenon.

OBJECTIVES: To determine the prevalence of Raynaud's phenomenon (RP) in patients with primary Sjogren's syndrome (pSS) and to identify clinical and immunological characteristics associated with this manifestation. Since increased interferon-gamma (INF-gamma) has been associated with RP, we also compared the INF-gamma production in pSS patients with or without RP. METHODS: RP was diagnosed if pSS patients presented with characteristic sequence of skin color changes of the digits. In uncertain cases noninvasive vascular tests were performed by ultrasound examination. The secretion of INF-gamma by peripheral blood mononuclear cells was assessed by enzyme-linked immunospot analysis. Further, we examined the expression of different lymphocyte activation markers (CD25, CD45RO, CD69) on CD4+ T-cells by flow cytometric analysis. RESULTS: Thirty-six of 108 patients with pSS had RP. In these patients we found a significantly increased number of INF-gamma-secreting peripheral blood mononuclear cells compared with patients without RP or to healthy controls. Further, in patients with RP a significantly increased percentage of CD25-positive T-helper cells was detectable. In addition we found an association of leukopenia, thyroiditis, and lower C3 levels with RP in pSS patients. CONCLUSIONS: These results suggest a pathogenic role of INF-gamma in pSS patients with RP. Whether the RP is immune-mediated or whether INF-gamma directly causes vasospasm still remains to be elucidated.

Macrophage migration inhibitory factor may contribute to vasculopathy in systemic sclerosis.

Increasing evidence supports the concept of macrophage migration inhibitory factor (MIF) as a central proinflammatory cytokine in autoimmune diseases. To further evaluate its role in systemic sclerosis (SSc), serum levels of MIF were determined by enzyme-linked immunoassay, and correlations to clinical manifestations were analyzed in 43 patients. MIF levels were significantly increased in patients (median, 18.8; range, <0.015-189 ng/ml) in comparison to healthy controls (n=43, 8.0, <0.015-36.5 ng/ml; P<0.0005). MIF values were higher in diffuse than in limited cutaneous SSc (P<0.005). Patients with pulmonary hypertension and recurrent digital ulcers showed higher MIF levels than patients without these manifestations (P<0.005). This association was also observed in limited cutaneous SSc. Sequential studies revealed decreased MIF levels after initiation of immunosuppressive therapy. MIF levels were not significantly different in patients with and without macrovascular disease of the peripheral arteries. The results suggest that MIF might contribute to inflammation and vasculopathy in SSc.

Mycophenolate sodium treatment in patients with primary Sjögren syndrome: a pilot trial.

The aim of this study was to evaluate the efficacy and safety of mycophenolate sodium (MPS) in patients with primary Sjögren syndrome (pSS) refractory to other immunosuppressive agents. Eleven patients with pSS were treated with MPS up to 1,440 mg daily for an observation period of 6 months in this single-center, open-label pilot trial. At baseline, after 3 months, and after 6 months, we examined the clinical status, including glandular function tests, as well as different laboratory parameters associated with pSS. In addition, subjective parameters were determined on the basis of different questionnaires. Treatment with MPS was well tolerated in 8 of 11 patients. Due to vertigo or gastrointestinal discomfort, two patients did not complete the trial. One patient developed pneumonia 2 weeks after treatment and was withdrawn. In the remaining patients, MPS treatment resulted in subjective improvement of ocular dryness on a visual analogue scale and a reduced demand for artificial tear supplementations. However, no significant alterations of objective parameters for dryness of eyes and mouth were observed, although a substantial improvement of glandular functions occurred in two patients with short disease duration. In addition, treatment with MPS resulted in significant reduction of hypergammaglobulinemia and rheumatoid factors as well as an increase of complement levels and white blood cells. MPS promises to be an additional therapeutic option for patients with pSS, at least in those with shorter disease duration. Further investigations about the efficacy and safety of MPS in pSS have to be performed in larger numbers of patients.

Increased serum levels of macrophage migration inhibitory factor in patients with primary Sjögren's syndrome.

The objective of this study was to analyse levels of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) in patients with primary Sjögren's syndrome (pSS) and to examine associations of MIF with clinical, serological and immunological variables. MIF was determined by ELISA in the sera of 76 patients with pSS. Further relevant cytokines (IL-1, IL-6, IL-10, IFN-gamma and TNF-alpha) secreted by peripheral blood mononuclear cells (PBMC) were determined by ELISPOT assay. Lymphocytes and monocytes were examined flow-cytometrically for the expression of activation markers. Results were correlated with clinical and laboratory findings as well as with the HLA-DR genotype. Healthy age- and sex-matched volunteers served as controls. We found that MIF was increased in patients with pSS compared with healthy controls (p < 0.01). In particular, increased levels of MIF were associated with hypergammaglobulinemia. Further, we found a negative correlation of MIF levels with the number of IL-10-secreting PBMC in pSS patients (r = -0.389, p < 0.01). Our data indicate that MIF might participate in the pathogenesis of primary Sjögren's syndrome. MIF may contribute to B-cell hyperactivity indicated by hypergammaglobulinemia. The inverse relationship of IL-10 and MIF suggests that IL-10 works as an antagonist of MIF in pSS.

Clinical utility of the anti-CCP assay: experiences with 700 patients.

Our objective was to determine the frequency of antibodies to cyclic citrullinated peptides (CCPs) in a series of patients with a variety of rheumatic diseases. Seven hundred consecutive serum samples from patients at an outpatient clinic were tested for the presence of rheumatoid factor (RF) and anti-CCP. Clinical diagnosis, radiographic information, and other laboratory data were taken from patients' charts. The sensitivity and specificity of anti-CCP reactivity for the diagnosis of rheumatoid arthritis (RA) were 74.0% and 94.5%, respectively; the corresponding results for RF were 69.7% sensitivity and 81.0% specificity. Highest rates of false-positive RF tests were found in patients with SLE (18.3% vs. 12.7% CCP), Sjögren's syndrome (73.3% vs. 3.3% CCP), and a control group with chronic hepatitis (24.7% vs. 1.3% CCP). The detection of anti-CCP is useful for the diagnosis of RA because of its similar sensitivity but higher specificity compared with RF. Anti-CCP also helps to diagnose other inflammatory and noninflammatory diseases (especially connective tissue diseases) by reducing the rate of false-positive results in comparison with RF.

The mutagenically separated polymerase chain reaction is a rapid and reliable method for genotyping of the tumour necrosis factor-alpha promoter polymorphism (-308 G/A).

BACKGROUND: The tumour necrosis factor-alpha (TNF alpha) promoter polymorphism (-308 G/A) has been shown to be associated with the susceptibility to and/or the severity of diverse diseases such as infections, autoimmunity, and malignancies. We developed a genotyping technique based on the mutagenically separated polymerase chain reaction (MS-PCR) which may be useful in the clinical risk assessment. METHODS: Different length allele-specific primers and an unspecific complementary strand primer were used in a one-tube assay. At least one PCR product was generated in a single reaction obviating the need for an internal control amplification. Introduction of additional base substitutions into the allele-specific primers led to a clear-cut separation between the alleles through the reduction of cross-reactions during amplification. The only post-PCR step required was the separation of allelic PCR products by size upon agarose gel electrophoresis. RESULTS: The allele frequencies in 300 German healthy Caucasians were 0.84 for TNF1 (-308 G) and 0.16 for TNF2 (-308 A) in accordance with published data obtained with the conventional RFLP method. No significant deviation from Hardy-Weinberg equilibrium was observed. The specificity of MS-PCR was confirmed by sequence-based typing. CONCLUSIONS: MS-PCR is a rapid, reliable, and cost-effective technique for genotyping of the TNF alpha promoter polymorphism (-308 G/A).

Differentiation of RNP- and SM-antibody subsets in SLE and MCTD patients by a new ELISA using recombinant antigens.

Connective tissue diseases often have overlapping clinical features and laboratory abnormalities. The distinctiveness of mixed connective tissue disease (MCTD) as an entity is of scientific interest and practical importance. In order to discriminate between MCTD and SLE patients we used a newly developed, commercially not available ELISA with recombinant antigen expressed in Baculovirus infected cells. This ELISA detects antibodies against RNP and Sm in complex as well as the subsets U1-snRNP 68 kDa, RNP-A, RNP-C (RNP), Sm-BB' and SS-D. We analyzed 66 RNP-positive consecutive patients prediagnosed as SLE or MCTD/overlap-syndrome. 45/66 patients were found to be U1-snRNP-68 kDa positive (27 SLE, 18 MCTD), 51/66 RNP-A [36,15] and 44/66 RNP-C [31,13]. 35/66 had antibodies against Sm-BB' (30 SLE, 5 MCTD), 10/66 against Sm-D (all SLE). 28/66 were found to be U1-snRNP-68 kDa and Sm-BB' positive (23 SLE, 5 MCTD), while 8/66 where U1-snRNP-68 kDa and Sm-D positive (all SLE). The combination of antibodies against 68 kDa, Aand C was exclusively observed in 6 MCTD patients, while the combination against 68 kDa, A, C, Sm-BB' and Sm-D was restricted to 8 patients with SLE. The antibody combination to 68 kDa, A, C and Sm-BB' was also found in 11/20 SLE patients with major organ involvement. In SLE and MCTD, determination of subsets of antibodies against Ul-snRNP-68 kDa and Sm-complex allows a differentiation of patient subgroups with more definite diagnoses and potential prognostic impact.

Concomitant reduction of disease activity and IL-10 secreting peripheral blood mononuclear cells during immunoadsorption in patients with active systenic lupus erythematosus.

In patients with systemic lupus erythematosus (SLE) increased secretion of interleukin 10 (IL-10) by peripheral blood mononuclear cells (PBMC) is associated with overproduction of pathogenic autoantibodies. Herein we report the effect of immunoadsorption (IA) on the number of IL-10 secreting PBMC in patients with active SLE. Three courses of IA were performed in 9 patients with active SLE (SLAM 15,9+/-2,9). Each course consisted of 3 treatments on consecutive days. ELISPOT assay using IL-10 specific monoclonal antibodies was used to determine the number of IL- 10 secreting PBMC before and after each treatment. Eleven healthy, age and sex matched volunteers served as controls. Anti-ds-DNA autoantibodies were reduced to 67+/-14% after the treatment period. Before therapy patients showed significantly more spontaneously IL-10 secreting PBMC than controls (p<0.01). After the first course a significant reduction of the IL-10 secreting cells to normal levels was observed which paralleled the development of disease activity (p<0.01). Our results demonstrate a concomitant reduction of disease activity, anti-ds-DNA antibodies and the number of IL-10 secreting PBMC. The production of anti-ds-DNA antibodies and IL-10 are interdependent. Thus we conclude that IA is beneficial in SLE by depletion of these pathogenic antibodies which may lead to a reduced IL-10 secretion in vivo.

Effect of the interleukin-6 promoter polymorphism (-174 G/C) on the incidence and outcome of sepsis.

OBJECTIVE: A biallelic polymorphism within the human interleukin (IL)-6 gene promoter region (-174 G/C) has been shown to affect IL-6 transcription in vitro and IL-6 plasma levels in healthy adults. Because IL-6 is excessively released into the circulation during sepsis and closely correlates with the clinical course, we studied whether this promoter polymorphism has an effect on the incidence and/or outcome of sepsis. DESIGN: Population-based association study in critically ill patients and healthy controls. SETTING: Surgical intensive care unit (ICU) in a German university hospital. PATIENTS: Surgical patients (n = 326) of German Caucasian origin with an ICU stay of at least 3 days admitted between 1997 and 1999 were prospectively enrolled. In a subset of 50 patients, sepsis was diagnosed according to consensus criteria (American College of Chest Physicians 1992). Healthy sex-matched adults of the same ethnic and geographic background served as controls. INTERVENTIONS: Blood sampling. MEASUREMENTS AND MAIN RESULTS: The (-174 G/C) polymorphism was genotyped by an allele-specific polymerase chain reaction. IL-6 plasma levels were determined by enzyme-linked immunosorbent assay. Genotype distribution and allele frequencies did not differ significantly between patients with or without sepsis and healthy controls. In patients who finally succumbed to sepsis, significantly less GG homozygotes were observed compared with survivors (p = .008). Median systemic IL-6 levels in septic patients closely correlated with outcome (p < .0001) but were not associated with the IL-6 promoter genotype. CONCLUSIONS: The IL-6 promoter polymorphism (-174 G/C) does not affect the incidence of sepsis. However, the GG homozygous genotype is significantly associated with an improved survival in sepsis. Because this association is independent from the systemic IL-6 response, we suggest that other genetically linked polymorphisms may be the primary cause.