Peter Willeke

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 One patient developed pneumonia 2 weeks after treatment and was withdrawn.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.

Interleukin 1beta and tumour necrosis factor alpha secreting cells are increased in the peripheral blood of patients with primary Sjögren's syndrome.

Objective: To study systemic alterations of cytokine secreting peripheral blood mononuclear cells (PBMC) in primary Sjögren’s syndrome (pSS) and their relation to common clinical and immunological manifestations of this disease. Methods: PBMC spontaneously secreting tumour necrosis factor α (TNFα), interleukin 1β (IL1β), and interleukin 6 (IL6) were assessed by enzyme linked immunospot (ELISPOT) analysis in a cohort of 31 patients with pSS fulfilling the modified European classification criteria. Nineteen healthy volunteers served as controls. ELISPOT results were correlated with glandular and extraglandular manifestations and autoantibody titres—that is, rheumatoid factor (RF) isotypes, anti-Ro/SS-A, anti-La/SS-B as determined by an enzyme linked immunosorbent assay (ELISA) technique. Results: The number of TNFα and IL1β secreting cells was significantly higher in patients with pSS than in controls. No differences were detected in the number of IL6 secreting PBMC. Patients with recurrent parotid swelling (RPS) had a significantly increased number of IL1β secreting PBMC. Moreover, the number of IL1β secreting PBMC correlated with the disease duration (rs=0.479; p<0.01) and with the concentration of IgM RF (rs=0.63; p<0.01) and IgG RF (rs=0.42; p<0.05). Other autoantibodies did not correlate with cytokine secreting PBMC. Conclusion: The increased systemic secretion of IL1β and TNFα in patients with pSS points to a pathogenic impact of these cytokines in this autoimmune disease. In particular the correlation of IL1β secreting PBMC with RPS and RF production indicates that IL1β is a crucial regulator in the development of local and systemic disease manifestations.

Extended haplotype analysis reveals an association of TNF polymorphisms with susceptibility to systemic lupus erythematosus beyond HLA‐DR3

Objective: To study the relative contribution of tumour necrosis factor (TNF) and HLA‐DRB1 polymorphisms to the genetic susceptibility to systemic lupus erythematosus (SLE) via an extended haplotype analysis. Methods: We performed an association study in 205 unrelated German Caucasian patients with SLE fulfilling the 1997 revised American College of Rheumatology (ACR) criteria. Healthy age‐, ethnically‐ and sex‐matched individuals (n = 157) served as controls. HLA‐DRB1 typing was performed by a sequence‐specific oligonucleotide hybridisation assay. Two TNF single nucleotide polymorphisms (SNPs) and two multiallelic microsatellites were analysed by mutagenically separated polymerase chain reaction (PCR) or fragment length analysis, respectively. Extended haplotypes were reconstructed with the PHASE software. Results: Alleles for all polymorphic loci studied and the most frequent haplotypes showed a significantly different distribution between SLE patients and controls. The alleles HLA‐DR2, DR3, TNFd1, TNF2, TNFB*1, and TNFa2, designated as risk alleles, and the extended haplotypes DR3‐TNFd1‐TNF2‐TNFB*1‐TNFa2 and DR2‐TNFd3‐TNF1‐TNFB*2‐TNFa11 prevailed in SLE patients. TNF risk alleles were strongly positively linked with HLA‐DR3 and negatively linked with HLA‐DR2. Thus, in HLA‐DR3 haplotypes individual effects of TNF polymorphisms could not be resolved. By contrast, HLA‐DR2 showed an association with SLE independently of TNF risk alleles, while the risk increased further when they were present. In haplotypes lacking HLA‐DR2 and DR3, the alleles TNFd1 and TNF2 contributed independently to SLE susceptibility. Conclusion: Extended haplotype analysis revealed HLA‐DR3 independent associations of TNF polymorphisms with susceptibility to SLE. Haplotypes that have been shown to be associated with different TNF‐α production capacity may prevail in different disease subgroups.

Etanercept tolerance in a patient with previous infliximab-induced hepatitis

Dear Editor, We read with great interest the report by Harada et al. [1], who described the first case of autoimmune hepatitis exacerbated by etanercept administration in a patient with rheumatoid arthritis (RA). Hepatitis is a rare complication of treatment with tumor necrosis factor (TNF)-α antagonists. Several cases associated with infliximab therapy have been described [2]. To further contribute to the understanding of the mechanisms that lead to hepatitis induction by both TNF-a antagonists, we would like to present our observation in a RA patient who developed hepatitis during infliximab therapy and was subsequently treated with etanercept without recurrence. A 41-year-old woman with a history of rheumatoid factornegative, erosive RA since 1999 presented to our outpatient clinic in 2001. Since sulfasalazine, methotrexate, and a combination of methotrexate with leflunomide were not effective and the patient had progressive disease, infliximab was started (3 mg/kg body weight), in addition to methotrexate (15 mg/ week subcutaneously), with folic acid supplementation and prednisolone (10 mg/day). She received repeated infliximab infusions after 2 and 6 weeks, and then every 6 to 8 weeks. As the patient improved, prednisolone was gradually tapered to 5 mg/day. She had normal liver enzymes. In 2004, increased levels of alanine aminotransferase (ALT, 1061 U/l), aspartate aminotransferase (AST, 2019 U/l), and alkaline phosphatase (244 U/l) were detected. Consequently, all medications were withdrawn, except prednisolone. Laboratory testing showed an increase in the antinuclear antibody (ANA) titer previously present at 1/160 to 1/640 and positive anti-smooth muscle antibodies (ASMA). Tests for antibodies against doublestranded DNA, mitochondria, liver–kidney microsomes, and soluble liver antigen/liver pancreas antigen, as well as for hepatitis A, B, and C and cytomegalovirus, were negative. Liver biopsy revealed lymphoplasmacytic inflammatory cell infiltration predominating the portal areas, necrosis of hepatocytes, and mild periportal fibrosis. Prednisolone was started (initial dose of 1 mg kg day), and within 4 weeks, normalization of ALT (17 U/l) and AST (29 U/l) was observed. When prednisolone was reduced to 12.5 mg/day after 2 months, the patient developed a severe flare of her joint symptoms. Since treatment options were limited at that time, etanercept was started (2×25 mgweekly). Joint symptoms improved and prednisolone was tapered to 5 mg/day. During a follow-up period of more than 3 years on etanercept, significantly increased ALT and AST values have not recurred, while low titers of ANA and ASMA remained positive. Likewise, Goldenberg and Jorizzo [3] have treated a patient with pyoderma gangrenosum and autoimmune hepatitis with etanercept and reported no adverse effects. Our observation of etanercept tolerance in a patient who had previous liver disease with features of autoimmune hepatitis during infliximab therapy suggests that both TNF antagonists might exert different effects on mechanisms that result in liver inflammation. Etanercept might be tolerated in patients at risk of exacerbation of autoimmune hepatitis. However, it remains to be clarified whether non-TNF-α biologicals might be safer still for these patients.

Interferon-γ Is Increased in Patients with Primary Sjogren's Syndrome and Raynaud's Phenomenon

OBJECTIVES To determine the prevalence of Raynauds phenomenon (RP) in patients with primary Sjogrens 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.

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 Sjögrens 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-γ and TNF-α) 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 Sjögrens 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.

Emerging Treatment Strategies and Potential Therapeutic Targets in Primary Sjogrens Syndrome

Primary Sjögrens syndrome (pSS) is a common autoimmune disease which can lead to considerable complications and diminished quality of life. Recent insights into disease mechanisms and the advent of biological agents have provided new options for the treatment of pSS. In particular, B cell targeted intervention has shown promising results. In this review, we focus on emerging treatment strategies and therapeutic targets beyond B cells. Interference with proinflammatory cytokines and mechanisms that link innate and adaptive immunity offers new options in the treatment of pSS. Approaches directed against interleukin (IL)-1beta, Toll-like receptors and the inflammasome are emerging. Targeting IL-12, IL-18, the IL-23/IL-17 system, macrophage migration inhibitory factor and chemokines might be considered. The inhibition of apoptosis of glandular cells, the promotion of cell regeneration and organ-specific stem cell transplantation are potential strategies directed at preserving and restoring functional exocrine tissue. The recognition of patients who benefit most from a particular strategy might help to design more efficient therapeutic approaches. Since efficacy of many agents depends on the presence of residual functional glandular tissue, future studies should focus on patients with recent onset of pSS.

The role of interleukin‐10 promoter polymorphisms in primary Sjögren's syndrome

Objective: To determine the impact of a broad spectrum of different polymorphisms within the interleukin‐10 (IL‐10) promoter gene on disease susceptibility to primary Sjögrens syndrome (pSS), clinical manifestations, and autoantibody production. Methods: We genotyped 111 unrelated German Caucasian patients with pSS and 145 healthy controls for the single nucleotide polymorphisms (SNPs) at positions −2849, −2776, −2769, −2763, −1349, −1082, −851, −819, −657, and −592 and for the microsatellites IL10.R and IL10.G. Allele and haplotype distributions were compared between patients and controls and between subgroups of patients with different clinical and laboratory findings. Results: We found no significant differences in the allele or haplotype frequencies between pSS patients and healthy controls. After Bonferroni correction we found a significant association of the ACC haplotype (at the −1082, −819, and −592 loci) with immunoglobulin (Ig)A antibodies to anti‐α‐fodrin. Conclusion: Overall we found no associations of IL‐10 promoter polymorphisms with the susceptibility to pSS in our cohort. The finding that the production of IgA anti‐α‐fodrin antibodies is associated with polymorphisms within the IL‐10 promoter region suggests a genetic contribution to the generation of these antibodies.

Platelet counts as a biomarker in ANCA-associated vasculitis

Objectives: To determine whether platelet (PLT) counts might serve as a biomarker to distinguish between active anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and remission and also between active disease and systemic infection. Method: PLTs were analysed before treatment in patients with AAV in the active state and in remission. PLTs were also analysed in AAV patients with acute infections. The results were correlated with clinical manifestations, the Birmingham Vasculitis Activity Score version 3 [BVAS(v.3)], and other laboratory findings [i.e. C-reactive protein (CRP), leucocytes, differential count, procalcitonin (PCT)]. Diagnostic accuracy was calculated with a receiver operating characteristic (ROC) curve. Results: PLT counts were significantly increased in 98 patients with AAV during the active disease state [median: 405 PLTs/nL; interquartile range (IQR) 288–504] compared to patients in remission (246 PLT/nL; IQR 214–289) (p < 0.001). We found a correlation of PLT counts in active disease with the BVAS(v.3) (r = 0.582, p < 0.001). In AAV patients with systemic infections (n = 37), PLT counts exhibited significantly lower values (226 PLT/nL; IQR 163–273) compared to patients with active disease (p < 0.001). In the ROC curve analysis, the area under the curve (AUC) of PLTs was significantly larger when distinguishing active disease from systemic infection (AUC 0.868) compared to leucocytes (AUC 0.590), CRP (AUC 0.522), or procalcitonin (AUC 0.515) (p < 0.001). Conclusions: PLT counts were found to correlate with disease activity in AAV and thus may be used to represent immunological activity. In addition, PLT counts serve as a marker that can distinguish acute infection from active disease.

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.