IL-24 is a common and specific autoantigen of IgE in patients with chronic spontaneous urticaria.

BACKGROUND: The efficacy of omalizumab (anti-IgE) and increased IgE levels in patients with chronic spontaneous urticaria (CSU) suggest autoallergic mechanisms. OBJECTIVE: We sought to identify autoallergic targets of IgE in patients with CSU. METHODS: Serum samples of patients with CSU together with those of patients with idiopathic anaphylaxis and healthy control subjects (7 of each) were screened for IgE autoantibodies by using an array of more than 9000 proteins. Sera of 1062 patients with CSU and 482 healthy control subjects were used in an IgE-anti-IL-24-specific ELISA to investigate the association of IgE-anti-IL-24 and CSU. RESULTS: By using array analyses, more than 200 IgE autoantigens were found in patients with CSU that were not found in control subjects. Of the 31 IgE autoantigens detected in more than 70% of patients, 8 were soluble or membrane bound and expressed in the skin. Of these, only IgE autoantibodies to IL-24 were found in all patients with CSU. In vitro studies showed IL-24 to release histamine from human mast cells sensitized with purified IgE of patients with CSU but not control subjects. By using ELISA, mean ± SD levels of IgE-anti-IL-24 were 0.52 ± 0.24 IU/mL in patients with CSU and 0.27 ± 0.08 IU/mL in control subjects, with 80% of patients with CSU but only 20% of control subjects having levels greater than 0.33 IU/mL (P < .0001). IgE-anti-IL-24 showed acceptable predictive properties for CSU, with a likelihood ratio of 3.9. Clinically, IgE-anti-IL-24 levels showed an association with disease activity, as assessed by the urticaria activity score and with reduced basophil counts. CONCLUSION: Our findings show that patients with CSU frequently exhibit IgE autoantibodies against many autoantigens and that IL-24 is a common, specific, and functional autoantigen of IgE antibodies in patients with CSU.

Autoimmune chronic spontaneous urticaria: What we know and what we do not know.

Chronic spontaneous urticaria (CSU) is a mast cell-driven skin disease characterized by the recurrence of transient wheals, angioedema, or both for more than 6 weeks. Autoimmunity is thought to be one of the most frequent causes of CSU. Type I and II autoimmunity (ie, IgE to autoallergens and IgG autoantibodies to IgE or its receptor, respectively) have been implicated in the etiology and pathogenesis of CSU. We analyzed the relevant literature and assessed the existing evidence in support of a role for type I and II autoimmunity in CSU with the help of Hill's criteria of causality. For each of these criteria (ie, strength of association, consistency, specificity, temporality, biological gradient, plausibility, coherence, experiment, and analogy), we categorized the strength of evidence as "insufficient," "low," "moderate," or "high" and then assigned levels of causality for type I and II autoimmunity in patients with CSU from level 1 (causal relationship) to level 5 (causality not likely). Based on the evidence in support of Hill's criteria, type I autoimmunity in patients with CSU has level 3 causality (causal relationship suggested), and type II autoimmunity has level 2 causality (causal relationship likely). There are still many aspects of the pathologic mechanisms of CSU that need to be resolved, but it is becoming clear that there are at least 2 distinct pathways, type I and type II autoimmunity, that contribute to the pathogenesis of this complex disease.

Sex differences in the drug therapy for oncologic diseases.

There are clear gender-dependent differences in response rates and the probability of side effects in patients treated with chemotherapy. Sex-biased expression levels of metabolic enzymes and transporters in liver and kidney leading to different pharmacokinetics have been described for most common anti-cancer drugs. In women, half-life is often longer, which is associated with improved survival, but also increased toxicity.Some chemotherapy protocols lead to a better response rate in women without increasing toxicity (e.g., cisplatin and irinotecan), while others only increase toxicity, but do not improve response rates in women (e.g., 5-fluorouracil). The increased toxicity often correlates with different pharmacokinetics, but women also show a higher sensitivity to some agents with shorter half-life (e.g., steroids). Organ-specific toxicities like cardiac toxicity after doxorubicin treatment or neurotoxicity associated with ifosfamide are more severe in women due to gender-specific changes in gene expression. Novel therapies like tyrosine kinase inhibitors or monoclonal antibodies show very complex, but clinical significant differences depending on gender. Antibodies often have a longer half-life in women, which is associated with an improved response to therapy.Side effects appear to be highly dependent on different tissue properties, as women have a higher incidence of oral mucositis, but lower rates of gut toxicity. Nausea and vomiting is a greater problem in females during therapy due to the lower activity of anti-emetic drugs. Nausea and vomiting pose a bigger challenge in female patients, as anti-emetic drugs seem to be less effective.

Anti-aquaporin 4 IgG Is Not Associated With Any Clinical Disease Characteristics in Neuromyelitis Optica Spectrum Disorder.

Background: Neuromyelitis optica spectrum disorder (NMOSD) is a clinically defined, inflammatory central nervous system (CNS) disease of unknown cause, associated with humoral autoimmune findings such as anti-aquaporin 4 (AQP4)-IgG. Recent clinical trials showed a benefit of anti-B cell and anti-complement-antibodies in NMOSD, suggesting relevance of anti-AQP4-IgG in disease pathogenesis. Objective: AQP4-IgG in NMOSD is clearly defined, yet up to 40% of the patients are negative for AQP4-IgG. This may indicate that AQP4-IgG is not disease-driving in NMOSD or defines a distinct patient endotype. Methods: We established a biobank of 63 clinically well-characterized NMOSD patients with an extensive annotation of 351 symptoms, patient characteristics, laboratory results and clinical scores. We used phylogenetic clustering, heatmaps, principal component and longitudinal causal interference analyses to test for the relevance of anti-AQP4-IgG. Results: Anti-AQP4-IgG was undetectable in 29 (46%) of the 63 NMOSD patients. Within anti-AQP4-IgG-positive patients, anti-AQP4-IgG titers did not correlate with clinical disease activity. Comparing anti-AQP4-IgG-positive vs. -negative patients did not delineate any clinically defined subgroup. However, anti-AQP4-IgG positive patients had a significantly (p = 0.022) higher rate of additional autoimmune diagnoses. Conclusion: Our results challenge the assumption that anti-AQP4-IgG alone plays a disease-driving role in NMOSD. Anti-AQP4-IgG might represent an epiphenomenon associated with NMOSD, may represent one of several immune mechanisms that collectively contribute to the pathogenesis of this disease or indeed, anti-AQP4-IgG might be the relevant factor in only a subgroup of patients.

On the Lipophilic Nature of Autoreactive IgE in Chronic Spontaneous Urticaria.

Chronic spontaneous urticaria (CSU) is a skin disease related to autoreactive IgE in at least a subgroup of patients. However, the nature of this autoreactive IgE remains poorly characterized. This investigation had three objectives: first, to quantity CSU autoreactive IgE; second, to recognize the patterns of CSU autoreactive IgE compared with healthy control IgE; and third, to investigate the physiochemical nature of CSU autoreactive IgE. Methods: IgE autoreactivity was assessed in sera from 7 CSU and 7 healthy individuals. Autoantigen recognition patterns were assessed using principal component analysis (PCA) and heatmap visualization. Lipophilicity was assessed using NanoOrange reagent. Results: First, although total IgE levels did not differ significantly, the autoreactive proportion of IgE of CSU patients was 62% ± 37%, 1000-fold higher than that of healthy controls 0.03% ± 0.008% (P = 0.0006). Second, CSU autoreactive IgE differed from healthy control IgE by recognizing more and different autoantigens (226 vs. 34; P = 0.01). Third, the median (with 10-90% percentiles) serum level of lipophilic IgE was 39% (38-40%) in 232 CSU patients, 1.4-fold higher than the 28% (26-29%) of 173 healthy controls (P < 0.0001). Furthermore, lipophilicity correlated with autoreactivity (r = 0.8; P < 0.0001), connecting these two observed features. Conclusion: We believe that these novel observations about CSU autoreactive IgE, particularly the finding that it is more lipophilic than that of IgE from healthy individuals, will lead to the development of new diagnostic tests and therapies for autoreactive IgE-mediated diseases.

Immunoglobulin E-Mediated Autoimmunity.

The study of autoimmunity mediated by immunoglobulin E (IgE) autoantibodies, which may be termed autoallergy, is in its infancy. It is now recognized that systemic lupus erythematosus, bullous pemphigoid (BP), and chronic urticaria, both spontaneous and inducible, are most likely to be mediated, at least in part, by IgE autoantibodies. The situation in other conditions, such as autoimmune uveitis, rheumatoid arthritis, hyperthyroid Graves' disease, autoimmune pancreatitis, and even asthma, is far less clear but evidence for autoallergy is accumulating. To be certain of an autoallergic mechanism, it is necessary to identify both IgE autoantibodies and their targets as has been done with the transmembrane protein BP180 and the intracellular protein BP230 in BP and IL-24 in chronic spontaneous urticaria. Also, IgE-targeted therapies, such as anti-IgE, must have been shown to be of benefit to patients as has been done with both of these conditions. This comprehensive review of the literature on IgE-mediated autoallergy focuses on three related questions. What do we know about the prevalence of IgE autoantibodies and their targets in different diseases? What do we know about the relevance of IgE autoantibodies in different diseases? What do we know about the cellular and molecular effects of IgE autoantibodies? In addition to providing answers to these questions, based on a broad review of the literature, we outline the current gaps of knowledge in our understanding of IgE autoantibodies and describe approaches to address them.

Solution structure and backbone dynamics of the Trypanosoma cruzi cysteine protease inhibitor chagasin.

A Trypanosoma cruzi cysteine protease inhibitor, termed chagasin, is the first characterized member of a new family of tight-binding cysteine protease inhibitors identified in several lower eukaryotes and prokaryotes but not present in mammals. In the protozoan parasite T.cruzi, chagasin plays a role in parasite differentiation and in mammalian host cell invasion, due to its ability to modulate the endogenous activity of cruzipain, a lysosomal-like cysteine protease. In the present work, we determined the solution structure of chagasin and studied its backbone dynamics by NMR techniques. Structured as a single immunoglobulin-like domain in solution, chagasin exerts its inhibitory activity on cruzipain through conserved residues placed in three loops in the same side of the structure. One of these three loops, L4, predicted to be of variable length among chagasin homologues, is flexible in solution as determined by measurements of (15)N relaxation. The biological implications of structural homology between chagasin and other members of the immunoglobulin super-family are discussed.

Murine and human mast cell progenitors.

The development of mature mast cells (MCs) from hematopoietic progenitor cells as well as the identification and characterization of committed progenitor cells are a current focus of mast cell research. Most published reports in this area are on the origin and differentiation of MCs in mice. Evidence for the human system, i.e. derived from primary human MCs, is widely lacking. Based on the published data, MCs develop either from a committed progenitor or from a common basophil/mast cell precursor. This review summarizes the current knowledge on MC development and MC differentiation.

Peptide binding at class I major histocompatibility complex scored with linear functions and support vector machines.

We explore two different methods to predict the binding ability of nonapeptides at the class I major histocompatibility complex using a general linear scoring function that defines a separating hyperplane in the feature space of sequences. In absence of suitable data on non-binding nonapeptides we generated sequences randomly from a selected set of proteins from the protein data bank. The parameters of the scoring function were determined by a generalized least square optimization (LSM) and alternatively by the support vector machine (SVM). With the generalized LSM impaired data for learning with a small set of binding peptides and a large set of non-binding peptides can be treated in a balanced way rendering LSM more successful than SVM, while for symmetric data sets SVM has a slight advantage compared to LSM.

A novel method to generate and culture human mast cells: Peripheral CD34+ stem cell-derived mast cells (PSCMCs).

The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 10(8) differentiated human MCs per batch after only three weeks of culture starting with 10(6) total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4-20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1ß or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs.

Detection of circulating tumor-associated antigen depends on the domains recognized by the monoclonal antibodies used: N-terminal trimmed EpCAM-levels are much higher than untrimmed forms.

The measurement of tumor-associated proteins is of high diagnostic value in the follow-up of cancer patients. Most tests ignore that various forms of the protein can exist; especially in epithelial cancers and the soluble receptors they produce. We choose EpCAM as model-antigen to analyze whether tests recognizing different domains of the protein give different results in patients' sera. EpCAM-reactive autoantibodies are present in the sera of patients with colorectal carcinoma, however little is known about the existence and possible relevance of circulating soluble EpCAM protein. Most monoclonal EpCAM-antibodies recognize the first EGF-like repeat and fail to detect N-terminal trimmed protein. We developed a novel ELISA to determine the concentration of serum EpCAM with mAbs recognizing the second EGF-like repeat. In 59 healthy controls, EpCAM concentrations ranged from 232 to 8893ng/ml (mean 1525ng/ml). Levels of EpCAM in 412 patients with adenocarcinoma were somewhat higher with concentrations ranging from 176 to 36,259ng/ml (mean 1971ng/ml). In direct comparison, the untrimmed protein specific ELISA detected lower levels and frequencies as compared to the EGFII-specific ELISA. Only sera with less than 1µg/ml circulating EGFII-EpCAM (66% of the sera) contained EpCAM-specific IgG antibodies. The absence of IgG antibodies in the sera with more than 1µg/ml circulating EpCAM was not due to immune complex formation. Anti-EpCAM IgA and IgM antibodies did not show such a correlation. It will be important to assess whether the presence of high levels of circulating EGFII-EpCAM is associated with side effects in patients given immunotherapy.

Quality of recombinant protein determines the amount of autoreactivity detected against the tumor-associated epithelial cell adhesion molecule antigen: low frequency of antibodies against the natural protein.

The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. (1)H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.

Establishment of early lymphoid organ infrastructure in transplanted tumors mediated by local production of lymphotoxin alpha and in the combined absence of functional B and T cells.

Lymphoid organogenesis is a highly coordinated process involving orchestrated expression of a number of genes. Although the essential role of lymphotoxin alpha (LTalpha) for the normal development of secondary lymphoid organs is well established, it is not clear to which extent it depends upon cooperation with T and B lymphocytes for lymphoid neo-organogenesis. To determine whether LTalpha is sufficient to mediate recruitment of basic elements needed for lymphoid organogenesis, we made use of a LTalpha-transfected cell line as an experimental tool and established tumors in nude and SCID mice. Our data showed that high endothelial venules formed and follicular dendritic cells accumulated and differentiated in response to LTalpha in the absence of lymphocytes. A CD4(+)CD3(-)CD11c(+) cell population that is found in the secondary lymphoid organ was also recruited into tumors expressing LTalpha. Furthermore, in nude mice, B cells migrated in response to LTalpha and formed intratumoral follicles. These B cell follicles were structurally well equipped with follicular dendritic cell networks and high endothelial venules; however, they were not functionally active; e.g., those B cells specific for a surrogate Ag expressed by the tumor were found in the spleen, but not in the tumor. We show that, even in the absence of functional T and B lymphocytes, local expression of LTalpha in transplanted tumors induced typical stromal characteristics of lymphoid tissue, emphasizing that LTalpha is a critically important cytokine for formation of lymphoid organ infrastructure.