Anti-CD20 (rituximab) treatment improves atopic eczema

BACKGROUND: Atopic eczema (AE) is a chronic inflammatory skin disorder characterized by eczematous skin lesions, pruritus, and typical histopathologic features. OBJECTIVE: We asked whether depletion of B cells by monoclonal anti-CD20 antibody therapy (rituximab) would improve severe AE. METHODS: Six patients (4 women and 2 men) with severe AE received 2 intravenous applications of rituximab, each 1000 mg, 2 weeks apart. To evaluate the efficacy of rituximab, we monitored clinical parameters (eczema area and severity index, pruritus), total and allergen-specific IgE levels, skin histology, and inflammatory cells and cytokine expression in the skin and peripheral blood before and after therapy. RESULTS: All patients showed an improvement of their skin symptoms within 4 to 8 weeks. The eczema area and severity index significantly decreased (before therapy, 29.4 +/- 4.3; week 8, 8.4 +/- 3.6; P < .001). Histologic alterations such as spongiosis, acanthosis, and dermal infiltrate, including T and B cell numbers, also dramatically improved. However, whereas blood B cells were below detectable levels as a consequence of rituximab administration, skin B cells were reduced by approximately 50% only. Expression of IL-5 and IL-13 was reduced after therapy. Moreover, whereas allergen-specific IgE levels were not altered, we observed a slight reduction in total IgE concentrations in blood. CONCLUSIONS: B cells play a major role in AE pathogenesis. Treatment with an anti-CD20 antibody leads to an impressive improvement of AE in patients with severe disease.

Evidence that anti-muscarinic antibodies in Sjögren's syndrome recognise both M3R and M1R

Inhibitory anti-muscarinic receptor type 3 (M3R) antibodies may contribute to the pathogenesis of Sjögren's syndrome (SS), and putative anti-M3R blocking antibodies in intravenous immunoglobulin (IVIg) have been suggested as a rationale for treatment with IVIg. We investigated the presence of subtype-specific anti-MR autoantibodies in healthy donor and SS sera using MR-transfected whole-cell binding assays as well as M1R and M3R peptide ELISAs. Control antibodies against the second extracellular loop of the M3R, a suggested target epitope, were induced in rabbits and found to be cross-reactive on the peptides M3R and M1R. The rabbit antibodies had neither an agonistic nor an antagonistic effect on M3R-dependent ERK1/2 signalling. Only one primary SS (out of 5 primary SS, 2 secondary SS and 5 control sera) reacted strongly with M3R transfected cells. The same SS serum also reacted strongly with M1R and M2R transfectants, as well as M1R and two different M3R peptides. Strong binding to M1R and low-level activities against M3R peptides were observed both in SS and control sera. IVIg showed a strong reactivity against all three peptides, especially M1R. Our results indicate that certain SS individuals may have antibodies against M1R, M2R and M3R. Our results also suggest that neither the linear M3R peptide nor M3R transfectants represent suitable tools for discrimination of pathogenic from natural autoantibodies in SS.

Long-term maternal imprinting of the specific B cell repertoire by maternal antibodies

Maternal antibodies protect newborns whilst they are immunologically immature. This study shows that maternal antibodies can also shape the B cell repertoire of the offspring long after the maternal antibodies themselves become undetectable. V(H)DJ(H) gene-targeted (VI10) mice expressing a heavy chain specific for vesicular stomatitis virus (VSV) produce a 20-fold increased spontaneous titer of VSV-neutralizing antibodies. When transferred from mother to offspring, these antibodies prevented accumulation of Ag-specific transitional type 2 and marginal zone B cells with an activated phenotype and favored selection to the B cell follicles. This effect was B cell-intrinsic and lasted up to adulthood. The pups nursed by mothers producing specific antibodies developed higher endogenous antibody titers of this specificity which perpetuated the effects of specific B cell selection into the mature follicular compartment, presumably by blocking auto-Ag-dependent development of transitional type 2 B cells in the spleen. This repertoire change was functional, as following infection of adult mice with VSV, those pups that had received specific maternal antibodies as neonates had increased pre-immune titers and mounted strong early IgG neutralizing antibodies.

Partial overlap of anti-mycobacterial, and anti-Saccharomyces cerevisiae mannan antibodies in Crohn's disease

AIM: To test whether humoral immune reaction against mycobacteria may play a role in anti-Saccharomyces cerevisiae antibodies (ASCA) generation in Crohn's disease (CD) and/or whether it correlates with clinical subtypes. METHODS: The dominant ASCA epitope was detected by Galanthus nivalis lectin (GNL)-binding assay. ASCA and IgG against mycobacterial lysates (M avium, M smegmatis, M chelonae, M bovis BCG, M avium ssp. paratuberculosis (MAP)] or purified lipoarabinomannans (LAM) were detected by ELISA. ASCA and anti-mycobacterial antibodies were affinity purified to assess cross-reactivities. Anti-mycobacterial IgG were induced by BCG-infection of mice. RESULTS: GNL bound to different extents to mycobacterial lysates, abundantly to purified mannose-capped (Man) LAM from M tuberculosis, but not to uncapped LAM from M smegmatis. Fifteen to 45% of CD patients but only 0%-6% of controls were seropositive against different mycobacterial antigens. Anti-mycobacterial IgG correlated with ASCA (r = 0.37-0.64; P = 0.003-P < 0.001). ASCA-positivity and deficiency for mannan-binding lectin synergistically associated with anti-mycobacterial IgG. In some patients, anti-mycobacterial antibodies represent cross-reactive ASCA. Vice-versa, the predominant fraction of ASCA did not cross-react with mycobacteria. Finally, fistulizing disease associated with antibodies against M avium, M smegmatis and MAP (P = 0.024, 0.004 and 0.045, respectively). CONCLUSION: Similar to ASCA, seroreactivity against mycobacteria may define CD patients with complicated disease and a predisposition for immune responses against ubiquitous antigens. While in some patients anti-mycobacterial antibodies strongly cross-react with yeast mannan; these cross-reactive antibodies only represent a minor fraction of total ASCA. Thus, mycobacterial infection unlikely plays a role in ASCA induction.

Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody

BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.

6th International Immunoglobulin Symposium: poster presentations

The posters presented at the 6th International Immunoglobulin Symposium covered a wide range of fields and included both basic science and clinical research. From the abstracts accepted for poster presentation, 12 abstracts were selected for oral presentations in three parallel sessions on immunodeficiencies, autoimmunity and basic research. The immunodeficiency presentations dealt with novel, rare class-switch recombination (CSR) deficiencies, attenuation of adverse events following IVIg treatment, association of immunoglobulin (Ig)G trough levels and protection against acute infection in patients with X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVID), and the reduction of class-switched memory B cells in patients with specific antibody deficiency (SAD). The impact of intravenous immunoglobulin on fetal alloimmune thrombocytopenia, pregnancy and postpartum-related relapses in multiple sclerosis and refractory myositis, as well as experiences with subcutaneous immunoglobulin in patients with multi-focal motor neuropathy, were the topics presented in the autoimmunity session. The interaction of dendritic cell (DC)-SIGN and alpha2,6-sialylated IgG Fc and its impact on human DCs, the enrichment of sialylated IgG in plasma-derived IgG, as wells as prion surveillance and monitoring of anti-measles titres in immunoglobulin products, were covered in the basic science session. In summary, the presentations illustrated the breadth of immunoglobulin therapy usage and highlighted the progress that is being made in diverse areas of basic and clinical research, extending our understanding of the mechanisms of immunoglobulin action and contributing to improved patient care.

Comparison of drug retention rates and causes of drug discontinuation between anti-tumor necrosis factor agents in rheumatoid arthritis

OBJECTIVE: Tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of severe rheumatoid arthritis (RA), yet drug discontinuation is common. The aim of this study was to compare treatment retention rates and specific causes of anti-TNF discontinuation in a population-based RA cohort. METHODS: All patients treated with etanercept, infliximab, or adalimumab within the Swiss Clinical Quality Management RA cohort between 1997 and 2006 were included in the study. Causes of treatment discontinuation were broadly categorized as adverse events (AEs) or nontoxic causes, and further subdivided into specific categories. Specific causes of treatment interruption were analyzed using a Cox proportional hazards model and adjusted for potential confounders. RESULTS: A total of 2,364 anti-TNF treatment courses met the inclusion criteria. Treatment discontinuation was reported 803 times: 309 with etanercept, 249 with infliximab, and 245 with adalimumab. Drug inefficacy represented the largest single cause of treatment discontinuation (55.8% of cases). The median time of receiving anti-TNF therapy was 37 months, but discontinuation rates differed between the 3 anti-TNF agents (P < 0.001), with shorter retention rates for infliximab (hazard ratio [HR] 1.24, 99% confidence interval [99% CI] 1.01-1.51). The specific causes of treatment discontinuation revealed an increased risk of AEs with infliximab (HR 1.4, 99% CI 1.003-1.96), mostly due to an increased risk of infusion or allergic reactions (HR 2.11, 99% CI 1.23-3.62). Other discontinuation causes were equally distributed between the anti-TNF agents. CONCLUSION: In this population, infliximab was associated with higher overall discontinuation rates compared with etanercept and adalimumab, which is mainly due to an increased risk of infusion or allergic reactions.

Immunoglobulin M-enriched intravenous immunoglobulin inhibits classical pathway complement activation, but not bactericidal activity of human serum

Acute or even hyperacute humoral graft rejection, mediated by classical pathway complement activation, occurs in allo- and xenotransplantation due to preformed anti-graft antibodies. Intravenous immunoglobulin (IVIg) preparations can prevent complement-mediated tissue injury and delay hyperacute xenograft rejection. It is known that IgM-enriched IVIg (IVIgM) has a higher capacity to block complement than IVIgG. Different IVIgs were therefore tested for specificity of complement inhibition and effect on anti-bacterial activity of human serum. IVIgM-I (Pentaglobin), 12% IgM), IVIgM-II (IgM-fraction of IVIgM-I, 60% IgM), and three different IVIgG (all >95% IgG) were used. The known complement inhibitor dextran sulfate was used as control. Hemolytic assays were performed to analyze pathway-specificity of complement inhibition. Effects of IVIg on complement deposition on pig cells and Escherichia coli were assessed by flow cytometry and cytotoxicity as well as bactericidal assays. Complement inhibition by IVIgM was specific for the classical pathway, with IC50 values of 0.8 mg/ml for IVIgM-II and 1.7 mg/ml for IVIgM-I in the CH50 assay. Only minimal inhibition of the lectin pathway was seen with IVIgM-II (IC50 15.5 mg/ml); no alternative pathway inhibition was observed. IVIgG did not inhibit complement in any hemolytic assay. Classical pathway complement inhibition by IVIgM was confirmed in an in vitro xenotransplantation model with PK15 cells. In contrast, IVIgM did not inhibit (mainly alternative pathway mediated) killing of E. coli by human serum. In conclusion, IgM-enriched IVIg is a specific inhibitor of the classical complement pathway, leaving the alternative pathway intact, which is an important natural anti-bacterial defense, especially for immunosuppressed patients.

Autoantibodies directed against bactericidal/permeability-increasing protein in patients with cystic fibrosis: association with microbial respiratory tract colonization

BACKGROUND: Cystic fibrosis (CF) is associated with the appearance of serum autoantibodies directed against bactericidal/permeability-increasing protein (BPI). OBJECTIVES: To determine the age-specific seroprevalence rates of anti-BPI-IgG and IgA in a population of patients with CF and to correlate anti-BPI antibody concentrations with microbial respiratory tract colonization and pulmonary function variables at the time of serum sampling and 6 years thereafter. METHODS: Determination of BPI antibodies of the IgG and IgA isotypes using a commercial enzyme-linked immunosorbent assay in sera of a CF serum bank of 1992; correlation of anti-BPI antibody concentrations with age, clinical score, pulmonary function variables in 1992 and 1998, total serum immunoglobulin isotype concentrations and respiratory tract colonization with Pseudomonas aeruginosa and Aspergillus spp. RESULTS: Seventy-one patients (age in 1992, 14.1 +/- 7.5 years) were studied. Reactivities for anti-BPI-IgG and IgA were found in 28 (39%) and 26 (37%) patients, respectively. The seroprevalence of anti-BPI-IgA, but not IgG, increased significantly with age. P. aeruginosa colonization was associated with elevated concentrations of anti-BPI-IgG (P = 0.003) and IgA (P = 0.037). There were significant negative correlations between pulmonary function variables (vital capacity, forced expiratory volume in 1 s) in 1992 and 1998, respectively, and concentrations of anti-BPI-IgG or IgA in a multiple regression analysis. Anti-BPI-IgG, but not IgA, remained significantly associated with P. aeruginosa colonization (P = 0.006) and with reduced vital capacity (P = 0.01) in 1998 after correction for total serum isotype concentration. CONCLUSIONS: Anti-BPI-IgG are strongly associated with concurrent P. aeruginosa colonization and with long term restrictive pulmonary function abnormalities.

Intravenous immunoglobulins--understanding properties and mechanisms

High-dose intravenous immunoglobulin (IVIg) preparations are used currently for the treatment of autoimmune or inflammatory diseases. Despite numerous studies demonstrating efficacy, the precise mode of action of IVIg remains unclear. Paradoxically, IgG can exert both pro- and anti-inflammatory activities, depending on its concentration. The proinflammatory activity of low-dose IVIg requires complement activation or binding of the Fc fragment of IgG to IgG-specific receptors (FcgammaR) on innate immune effector cells. In contrast, when administered in high concentrations, IVIg has anti-inflammatory properties. How this anti-inflammatory effect is mediated has not yet been elucidated fully, and several mutually non-exclusive mechanisms have been proposed. This paper represents the proceedings of a session entitled 'IVIg--Understanding properties and mechanisms' at the 6th International Immunoglobulin Symposium that was held in Interlaken on 26-28 March 2009. The presentations addressed how IgG may affect the cellular compartment, evidence for IVIg-mediated scavenging of complement fragments, the role of the dimeric fraction of IVIg, the anti-inflammatory properties of the minor fraction of sialylated IgG molecules, and the genetic organization and variation in FcgammaRs. These findings demonstrate the considerable progress that has been made in understanding the mechanisms of action of IVIgs, and may influence future perspectives in the field of Ig therapy.

JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

Kinetics of maternally acquired anti-hepatitis A antibodies: prediction of waning based on maternal or cord blood antibody levels

BACKGROUND Timing is critical for efficient hepatitis A vaccination in high endemic areas as high levels of maternal IgG antibodies against the hepatitis A virus (HAV) present in the first year of life may impede the vaccine response. OBJECTIVES To describe the kinetics of the decline of anti-HAV maternal antibodies, and to estimate the time of complete loss of maternal antibodies in infants in León, Nicaragua, a region in which almost all mothers are anti-HAV seropositive. METHODS We collected cord blood samples from 99 healthy newborns together with 49 corresponding maternal blood samples, as well as further blood samples at 2 and 7 months of age. Anti-HAV IgG antibody levels were measured by enzyme immunoassay (EIA). We predicted the time when antibodies would fall below 10 mIU/ml, the presumed lowest level of seroprotection. RESULTS Seroprevalence was 100% at birth (GMC 8392 mIU/ml); maternal and cord blood antibody concentrations were similar. The maternal antibody levels of the infants decreased exponentially with age and the half-life of the maternal antibody was estimated to be 40 days. The relationship between the antibody concentration at birth and time until full waning was described as: critical age (months)=3.355+1.969 × log(10)(Ab-level at birth). The survival model estimated that loss of passive immunity will have occurred in 95% of infants by the age of 13.2 months. CONCLUSIONS Complete waning of maternal anti-HAV antibodies may take until early in the second year of life. The here-derived formula relating maternal or cord blood antibody concentrations to the age at which passive immunity is lost may be used to determine the optimal age of childhood HAV vaccination.

Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Short communication: differential immunoglobulin transfer during mastitis challenge by pathogen-specific components

Mastitis induced by Escherichia coli is often characterized by severe clinical signs, indicating a more powerful combat of the immune system against the pathogen compared with Staphylococcus aureus infections, which are often represented by chronic and subclinical diseases. The aim of this study was to test the major pathogenic component lipopolysaccharide (LPS) from E. coli and lipoteichoic acid (LTA) from Staph. aureus for their effects on blood-milk barrier integrity and the related transfer of immunoglobulins and lactate from blood into milk. A similar somatic cell count (SCC) increase was achieved by intramammary challenge of 1 quarter of 5 cows with 20 µg of LTA, and 8 cows with 0.2 µg of LPS (maximum log SCC/mL: 7). Milk IgG(1) concentrations increased in LPS- but not in LTA-challenged quarters. Milk IgG(2) concentrations were increased in treated quarters at 3h after LPS, and 6h after LTA challenge. Higher maximum levels of IgG(2) were reached in milk of LPS-treated quarters (173 ± 58 μg/mL) than of LTA-challenged quarters (62 ± 13 μg/mL). Immunoglobulin G(1) and IgG(2) levels did not change in control quarters. l-Lactate concentrations in milk increased 4h after LPS and 5h after LTA challenge and reached higher maximum levels in LPS- (221 ± 48 mg/L) than in LTA-treated quarters (77 ± 18 mg/L). In conclusion, a mammary inflammation on a quantitatively similar level based on SCC increase achieves a more efficient transfer of blood components such as IgG(2) via the blood-milk barrier if induced by LPS from E. coli than by LTA from Staph. aureus. This pathogen-specific difference may play an important role in the cure rate of the respective intramammary infection, which is usually lower in Staph. aureus- than in E. coli-induced mastitis.

Anti-Americanism in Europe: Theoretical Mechanisms and Empirical Evidence

One of the most popular explanations for post-9/11 anti-Americanism argues that resentment against America and Americans is mainly a function of the US government’s unpopular actions. The present article challenges this interpretation: first, it argues that neither the vitality of the resentment in times when the United States had no influence in the respective parts of the world nor its recent radical manifestations are accounted for in a political reductionist framework. In fact, specific traditions of anti-Americanism have an influence on the negative attitudes observed today, as a comparison between Britain, France, Germany, and Poland reveals. Second, this article suggests an alternative theoretical approach. Anti-Americanism can be explained by two basic mechanisms: it functions as a strategy to project denied and disliked self-concepts onto an external object, and it offers an interpretation frame for complex social processes that allows to reduce cognitive dissonance. Multivariate analyses based on empirical data collected in the Pew surveys of 2002 and 2007 show the fruitfulness of our theoretical approach.