Falk Hiepe

International multicenter evaluation of autoantibodies to ribosomal P proteins.

ABSTRACT Autoantibodies to the ribosomal phosphoproteins (Rib-P) are a serological feature of patients with systemic lupus erythematosus (SLE). The reported prevalence of anti-Rib-P antibodies in SLE ranges from 10 to 40%, being higher in Asian patients. The variation in the observed frequency may be related to a number of factors but is dependent in large part on the test system used to detect the autoantibodies. An association of anti-Rib-P with central nervous system involvement and neuropsychiatric manifestations of SLE has been controversial. In the present international multicenter study, we evaluated the clinical accuracy of a new sensitive Rib-P-specific enzyme-linked immunosorbent assay based on recombinant Rib-P polypeptides. The results showed that 21.3% of 947 SLE patients, but only 0.7% of 1,113 control patients, had a positive test result (P < 0.0001). The sensitivity, specificity, positive and negative predictive values, and diagnostic efficiency were determined to be 21.3%, 99.3%, 95.6%, 62.2%, and 65.3%, respectively. When evaluated in the context of participating centers, the prevalence of anti-Rib-P antibodies was found in descending frequency, as follows: China (35%) > Poland (34%) > Japan (28%) > United States (26%) > Germany (Freiburg; 23.3%) > Denmark (20.5%) > Germany (Berlin; 19%) > Mexico (15.7%) > Israel (11.7%) > Brazil (10%) > Canada (8%). The substantial data from this study indicate that the prevalence of anti-Rib-P antibodies may not be restricted to the genetic background of the patients or to the detection system but may depend on regional practice differences and patient selection. We confirm previously reported associations of antiribosomal antibodies with clinical symptoms and serological findings. Remarkably, we found a lower occurrence of serositis in Rib-P-positive lupus patients.

Perturbations of peripheral B lymphocyte homoeostasis in children with systemic lupus erythematosus

Objective: To investigate the distribution of peripheral B cell subpopulations of children with active and inactive systemic lupus erythematosus (SLE) compared with healthy controls. Methods: Peripheral B cell subpopulations of 11 children with SLE (6 with active and 5 with inactive disease) and 14 age matched normal healthy children were analysed. Active disease was diagnosed in children with a flare of SLE, who received treatment by IV cyclophosphamide or IV methylprednisolone pulse to control the disease. Additionally, the peripheral B cells of the children with SLE were compared with those of 13 consecutive patients with adult onset SLE. Results: No major difference was found in the frequency and total number of CD27−/CD19+ naïve B cells and CD27+/CD19+ memory B cells between patients with active and inactive lupus and healthy controls, but there was a significant increase in CD27high expressing plasma blasts in patients with active SLE. These cells coexpress CD38+, HLA-DRdim, surface Iglow and lack the expression of CD20 but are clearly positive for intracellular Ig, indicative of early plasma cells. Most CD138+ cells coexpress CD27high/CD19+. The enhanced frequency of peripheral plasma blasts in children with active SLE is consistent with previous findings in adult patients with SLE, whereas a relative predominance of CD27+ memory B cells was only identified in the adult patients. Conclusions: Profound abnormalities in the distribution of B cell compartments are more pronounced in older patients with SLE, but an enhanced frequency and cell number of peripheral plasma blasts is characteristic of both diseases during active stages. Thus detection of CD27high plasma blasts significantly correlates with active lupus in both children and adults.

Blood dendritic cells in systemic lupus erythematosus exhibit altered activation state and chemokine receptor function

Background Dendritic cells (DCs) have a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Reduced numbers of blood DCs and the accumulation of DCs at inflammatory sites have been observed in SLE. One crucial feature of DCs is their ability to migrate. Objective To analyse the maturation/activation state and the migratory capacity of different DC precursor subsets in SLE to further elucidate their role in autoimmunity. Methods Plasmacytoid DCs (pDCs), myeloid DCs (mDCs) and monocytes from patients with SLE, healthy volunteers and healthy volunteers immunised with tetanus/diphtheria were examined by flow cytometry for expression of subset-specific antigens (BDCA-2, CD11c, CD14, HLA-DR), activation/maturation markers (CD83, CD86, CD40, BLyS) and chemokine receptors (CCR1, CCR5, CCR7, ChemR23). Additionally, migratory capacity to chemokine receptors was investigated in vitro using the chemokines RANTES, CCL19 and chemerin. Results SLE monocytes and mDCs had higher CD86 and B-lymphocyte stimulatory factor (BLyS) expression levels. ChemR23 expression was lower in SLE pDCs and mDCs. Basal and CCL19-specific migration levels were higher in SLE pDCs. Altered DC function in SLE had no correlative changes in chemokine receptor expression, whereas immunisation-induced blood DC migration patterns in healthy donors were accompanied by changes in chemokine receptor expression. Conclusions The phenotypic and migratory disturbances observed in SLE blood DCs could result in altered distribution of DCs in peripheral tissues, contributing to dysregulated immune responses and autoimmunity.

Prolactin enhances the in vitro production of IgG in peripheral blood mononuclear cells from patients with systemic lupus erythematosus but not from healthy controls

OBJECTIVES Recent evidence suggests that prolactin (PRL) plays a part in the pathogenesis of systemic lupus erythematosus (SLE). Because B cell hyperreactivity and autoantibodies are characteristic hallmarks of SLE, this study aimed at assessing the impact of this pituitary hormone on IgG production by stimulating peripheral blood mononuclear cells (PBMC) with PRL. METHODS PBMC from 11 patients with SLE assessed by the ECLAM score and eight healthy controls were incubated with PRL and cultured for seven days. IgG production was measured by enzyme linked immunosorbent assay (ELISA). RESULTS Spontaneous IgG production of SLE PBMC was significantly enhanced compared with that found in healthy controls. After PRL stimulation, the IgG concentrations of supernatants from SLE PBMC were significantly higher than those of unstimulated PBMC (median 394 ng/ml). Of note, the physiological concentration of PRL (20 ng/ml) induced IgG production more effectively (median 1139 ng/ml) than PRL at 100 ng/ml (median 1029 ng/ml). In contrast, preincubation with PRL did not stimulate IgG production in normal PBMC. A significant correlation between PRL induced IgG production and the disease activity (ECLAM) of the patients with SLE was seen. Moreover, the maximum amount of PRL induced IgG depended on the serum PRL concentrations of the patients with SLE. CONCLUSIONS The results suggest that PBMC from patients with SLE have an extraordinarily high susceptibility to PRL, showing the most striking effect at a concentration usually found in vivo. This indicates a potential role for mild hyperprolactinaemia in the pathogenesis of SLE, influencing both IgG production and disease activity.

How to cope with pathogenic long-lived plasma cells in autoimmune diseases

Recently, it has been shown that plasma cells secreting antibodies can be long lived and as such constitute an independent component of immunological memory. They are generated in the context of memory immune reactions and migrate to the bone marrow, where they persist for years and decades. Their survival is dependent on receiving distinct signals provided by cells forming a plasma cell survival niche. They also can migrate to, and survive in, inflamed tissue. In autoimmune diseases long-lived plasma cells secreting autoantibodies provide an as yet unmet therapeutic challenge, because they are resistant to conventional treatments, in particular to immunosuppression and anti-inflammatory drugs. They are eliminated by immunoablation with antithymocyte globulin. This may be the reason why immunoablation followed by reconstitution of the patient’s immune system from haematopoietic stem cells induces long-term remissions in many patients with autoimmune diseases. However, more specific treatments for the elimination of autoreactive, long-lived plasma cells are needed, to avoid the complete temporary immunoincomptence induced by immunoablation, and to decipher the role of long-lived autoreactive plasma cells in human autoimmune diseases in more detail.

Clinical and serological evaluation of a novel CENP-A peptide based ELISA

IntroductionAnti-centromere antibodies (ACA) are useful biomarkers in the diagnosis of systemic sclerosis (SSc). ACA are found in 20 to 40% of SSc patients and, albeit with lower prevalence, in patients with other systemic autoimmune rheumatic diseases. Historically, ACA were detected by indirect immunofluorescence (IIF) on HEp-2 cells and confirmed by immunoassays using recombinant CENP-B. The objective of this study was to evaluate a novel CENP-A peptide ELISA.MethodsSera collected from SSc patients (n = 334) and various other diseases (n = 619) and from healthy controls (n = 175) were tested for anti-CENP-A antibodies by the novel CENP-A enzyme linked immunosorbent assay (ELISA). Furthermore, ACA were determined in the disease cohorts by IIF (ImmunoConcepts, Sacramento, CA, USA), CENP-B ELISA (Dr. Fooke), EliA® CENP (Phadia, Freiburg, Germany) and line-immunoassay (LIA, Mikrogen, Neuried, Germany). Serological and clinical associations of anti-CENP-A with other autoantibodies were conducted in one participating centre. Inhibition experiments with either the CENP-A peptide or recombinant CENP-B were carried out to analyse the specificity of anti-CENP-A and -B antibodies.ResultsThe CENP-A ELISA results were in good agreement with other ACA detection methods. According to the kappa method, the qualitative agreements were: 0.73 (vs. IIF), 0.81 (vs. LIA), 0.86 (vs. CENP-B ELISA) and 0.97 (vs. EliA® CENP). The quantitative comparison between CENP-A and CENP-B ELISA using 265 samples revealed a correlation value of rho = 0.5 (by Spearman equation). The receiver operating characteristic analysis indicated that the discrimination between SSc patients (n = 131) and various controls (n = 134) was significantly better using the CENP-A as compared to CENP-B ELISA (P < 0.0001). Modified Rodnan skin score was significantly lower in the CENP-A negative group compared to the positive patients (P = 0.013). Inhibition experiments revealed no significant cross reactivity of anti-CENP-A and anti-CENP-B antibodies. Statistically relevant differences for gender ratio (P = 0.0103), specific joint involvement (Jaccoud) (P = 0.0006) and anti-phospholipid syndrome (P = 0.0157) between ACA positive SLE patients and the entire SLE cohort were observed.ConclusionsAnti-CENP-A antibodies as determined by peptide ELISA represent a sensitive, specific and independent marker for the detection of ACA and are useful biomarkers for the diagnosis of SSc. Our data suggest that anti-CENP-A antibodies are a more specific biomarker for SSc than antibodies to CENP-B. Furthers studies are required to verify these findings.

Risks of immune system treatments

IN THE POLICY FORUM “CHINA’S ROAD TO SUSTAINABILITY” (2 APRIL, P. 50), J. LIU OVERLOOKS AN important cultural force. China’s worsening environmental conditions have catalyzed a spirit of environmental civilian activism. For example, in 2003, a consortium proposed erecting 13 dams on the Nujiang River. China’s environmental nongovernmental organizations and scholars launched a protest campaign through the Internet and newspapers. The critics argued that as reservoirs behind the dams fi lled up, fl ooding and landslides would imperil habitats. In response, Premier Wen Jiabao suspended the dam project pending an environmental review in 2004 (1). The landmark of environmental civilian activism occurred in Xiamen City in 2007. The local government supported construction of a

Reference standards for the detection of anti-mitochondrial and anti-rods/rings autoantibodies

1.4-billion paraxylene plant near the center of the city. Information about the environmental impact of this project was not made available to the local residents. The people of Xiamen City were outraged when—through cell phone messages and the Internet—they learned of the plant’s environmental risks. A phone text message was circulated among Xiamen citizens in late May calling for a “collective walk” (demonstration). On 1 June 2007, more than 1000 citizens gathered in front of the municipal building to protest. The demonstration forced the local government to cancel the largest industrial project in the history of Xiamen (2). The burgeoning middle class has become the driver of environmental civilian activism. For example, operation of the Likeng trash incinerator in Guangzhou City started in 2005 without any protest, although local farmers worried about health risk (3). In contrast, the proposed Panyu trash incinerator in Guangzhou City in 2009 triggered protests that were led by the middle class (4), who used science-based evidence to openly challenge prevailing notions formulated by the authorities. (In earlier years, standard practice was to obey Beijing-based experts in environmental protection.) In addition, the self-organized middle class forced the local government to open discussion by Internet. By seizing the opportunity for an open discussion, the newly empowered locals took to the streets to protect their environmental rights (4). Recent years have witnessed an impressive growth in environmental protests in China. The number of petitions and mass public protests related to environmental issues has increased by 30% per year in the past few years, although the number of petitions lodged with the Chinese government has dropped (5). The current environmental civilian activism movements have several common characteristics: (i) They are confi ned to one specifi c geographical space. (ii) Their goal is protecting the environment, rather than political rights or commercial interests. (iii) They focus on a specifi c pollutant, rather than general environmental degradation. The local nature of the movement enables the organization of a large number of citizens with little effort in a very short time. Given more open social and political conditions and the increasing size of the middle class in China, environmental civilian activism will certainly be a key driver in China’s transition to sustainability.

In vivo preactivated autoreactive Th cells in healthy individuals

Abstract Background: Anti-mitochondrial antibodies (AMA) are found in >90% of primary biliary cholangitis patients. Anti-rods/rings antibodies (anti-RR) are most commonly associated with interferon-α and ribavirin treatment in hepatitis C patients. Clinical laboratories routinely screen for AMA and anti-RR using indirect immunofluorescence on HEp-2 cells (HEp-2-IFA). Therefore, we sought to establish reference materials for use in AMA and anti-RR testing. Methods: AMA-positive and anti-RR-positive human plasma samples (AMA-REF and RR-REF), identified as potential reference materials based on preliminary data, were further validated by multiple laboratories using HEp-2-IFA, immunoprecipitation (IP), western blotting, IP-western, line immunoassay (LIA), addressable laser bead immunoassay (ALBIA) and enzyme-linked immunosorbent assay (ELISA). Results: AMA-REF showed a strong positive cytoplasmic reticular/AMA staining pattern by HEp-2-IFA to ≥1:1280 dilution and positive signal on rodent kidney/stomach/liver tissue. AMA-REF reacted with E2/E3, E3BP, E1α and E1β subunits of the pyruvate dehydrogenase complex by IP and western blotting and was positive for AMA antigens by LIA, ALBIA and ELISA. RR-REF showed a strong positive rods and rings staining pattern by HEp-2-IFA to ≥1:1280 dilution. RR-REF reacted with inosine monophosphate dehydrogenase by IP, IP-western and ALBIA. RR-REF also produced a nuclear homogenous staining pattern by HEp-2-IFA, immunoprecipitated proteins associated with anti-U1RNP antibody and reacted weakly with histones, nucleosomes, Sm and nRNP/Sm by LIA. Conclusions: AMA-REF and RR-REF are useful reference materials for academic or commercial clinical laboratories to calibrate and establish internal reference standards for immunodiagnostic assays. AMA-REF and RR-REF are now available for free distribution to qualified laboratories through Plasma Services Group.

Peptides of the antigen Sm-D and their use, in particular for the diagnostics of systemic lupus erythematosus (SLE)

The direct analysis of autoantigen-specific Th-cells has been hampered so far by the lack of appropiate methods to directly determine their frequency or functional capabilities. We have applied a set of new techniques to directly identificate and analyze autoantigen-specific T-cells in both affected and healthy people according to their effector functions (e.g. effector cytokine production) after provocation with antigen. n nWe have used these technologies to analyze Th-cells specific for SLE-associated autoantigens, in particular nucleosomes and the ribonucleoprotein La. Suprisingly, in vivo pre-activated autoantigen-specific Th-cells secreting IFNgamma and TNFalpha, could be detected not only in SLE-patients, but also in normal healthy persons, with frequencies ranging from 0.02% to 0.1%. Preactivation of these cells in vivo was confirmed by the fact that they expressed CD45RO but not CD45RA. Some of them had down-regulated expression of CD45RB and CD27. We also detected in healthy donors in vivo preactivated Th-cell specific for the self-antigen alphaB-Cristallin, a small heat shock protein. Up to 0.5% of peripheral Th cells specifically react with IFNgamma secretion upon short term stimulation, a hallmark of a recall response, i.e. in vivo preactivation. n nThe fact that in vivo pre-activated, autoantigen-specific Th-cells can be detected at comparable frequencies and with similar cytokine secretion patterns in blood of normal persons and patients suffering from a disease in which such Th cells are suspected to play a pivotal role, points to mechanisms other than central and peripheral tolerance that control the initiation of those autoimmune reactions.