DARC shuttles inflammatory chemokines across the blood-brain barrier during autoimmune central nervous system inflammation

The Duffy antigen/receptor for chemokines, DARC, belongs to the family of atypical heptahelical chemokine receptors that do not couple to G proteins and therefore fail to transmit conventional intracellular signals. Here we show that during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, the expression of DARC is upregulated at the blood-brain barrier. These findings are corroborated by the presence of a significantly increased number of subcortical white matter microvessels staining positive for DARC in human multiple sclerosis brains as compared to control tissue. Using an in vitro blood-brain barrier model we demonstrated that endothelial DARC mediates the abluminal to luminal transport of inflammatory chemokines across the blood-brain barrier. An involvement of DARC in experimental autoimmune encephalomyelitis pathogenesis was confirmed by the observed ameliorated experimental autoimmune encephalomyelitis in Darc(-/-) C57BL/6 and SJL mice, as compared to wild-type control littermates. Experimental autoimmune encephalomyelitis studies in bone marrow chimeric Darc(-/-) and wild-type mice revealed that increased plasma levels of inflammatory chemokines in experimental autoimmune encephalomyelitis depended on the presence of erythrocyte DARC. However, fully developed experimental autoimmune encephalomyelitis required the expression of endothelial DARC. Taken together, our data show a role for erythrocyte DARC as a chemokine reservoir and that endothelial DARC contributes to the pathogenesis of experimental autoimmune encephalomyelitis by shuttling chemokines across the blood-brain barrier.

Natural anti-FcepsilonRIalpha autoantibodies may interfere with diagnostic tests for autoimmune urticaria.

IgG autoantibodies against the alpha-chain of the high affinity IgE receptor are claimed to play a pathogenetic role in autoimmune urticaria. The best methods for detection of functional autoantibodies are currently the autologous serum skin test and the basophil histamine release assay. A simplified and feasible screening test would facilitate the diagnosis of autoimmune urticaria. Here we offer an explanation for the difficulties in establishing a screening test for autoantibodies directed against the alpha-chain of the high affinity IgE receptor in autoimmune urticaria. Identical autoantibodies in chronic urticaria patients and healthy donors belonging to the natural autoantibody repertoire were found by sequence analysis of anti-alpha-chain autoantibodies isolated by repertoire cloning from antibody libraries. These natural autoantibodies bound to the receptor and triggered histamine release but only if IgE was previously removed from the receptor. Diagnostic assays used for detection of antibodies directed against the IgE receptor may require signal comparison with and without the artificial removal of IgE, immune complexes, and complement in order to avoid false positive or negative results. After IgE removal diagnostic tests will detect natural autoantibodies against the high affinity IgE receptor regardless of whether they are pathogenic or not.

Thyroid dysfunction and anemia in a large population-based study.

OBJECTIVE AND BACKGROUND Anemia and thyroid dysfunction are common and often co-occur. Current guidelines recommend the assessment of thyroid function in the work-up of anemia, although evidence on this association is scarce. PATIENTS AND METHODS In the "European Prospective Investigation of Cancer" (EPIC)-Norfolk population-based cohort, we aimed to examine the prevalence and type of anemia (defined as hemoglobin <13 g/dl for men and <12 g/dl for women) according to different thyroid function groups. RESULTS The mean age of the 8791 participants was 59.4 (SD 9.1) years and 55.2% were women. Thyroid dysfunction was present in 437 (5.0%) and anemia in 517 (5.9%) participants. After excluding 121 participants with three most common causes of anemia (chronic kidney disease, inflammation, iron deficiency), anemia was found in 4.7% of euthyroid participants. Compared with the euthyroid group, the prevalence of anemia was significantly higher in overt hyperthyroidism (14.6%, P < .01), higher with borderline significance in overt hypothyroidism (7.7%, P = .05) and not increased in subclinical thyroid dysfunction (5.0% in subclinical hypothyroidism, 3.3% in subclinical hyperthyroidism). Anemia associated with thyroid dysfunction was mainly normocytic (94.0%), and rarely macrocytic (6.0%). CONCLUSION The prevalence of anemia was higher in overt hyperthyroidism, but not increased in subclinical thyroid dysfunction. Systematic measurement of thyroid-stimulating hormone in anemic patients is likely to be useful only after excluding common causes of anemia.

Sphingosine kinase 2 deficient mice exhibit reduced experimental autoimmune encephalomyelitis: Resistance to FTY720 but not ST-968 treatments

The immunomodulatory drug FTY720 is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that requires activation by sphingosine kinase 2 (SK-2) to induce T cell homing to secondary lymphoid tissue. In this study, we have investigated the role of SK-2 in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. We show that SK-2 deficiency reduced clinical symptoms of EAE. Furthermore, in SK-2-deficient mice, the protective effect of FTY720 on EAE was abolished, while the non-prodrug FTY720-derivative ST-968 was still fully active. Protection was paralleled by reduced numbers of T-lymphocytes in blood and a reduced blood-brain-barrier leakage. This correlated with reduced mRNA expression of ICAM-1, VCAM-1, but enhanced expression of PECAM-1. A similar regulation of permeability and of PECAM-1 was seen in primary cultures of isolated mouse brain vascular endothelial cells and in a human immortalized cell line upon SK-2 knockdown. In summary, these data demonstrated that deletion of SK-2 exerts a protective effect on the pathogenesis of EAE in C57BL/6 mice and that SK-2 is essential for the protective effect of FTY720 but not of ST-968. Thus, ST-968 is a promising novel immunomodulatory compound that may be a valuable alternative to FTY720 under conditions where SK-2 activity is limited.

Protective effect of a germline, IL-17-neutralizing antibody in murine models of autoimmune inflammatory disease.

Monoclonal antibodies (mAbs) inhibiting cytokines have recently emerged as new drug modalities for the treatment of chronic inflammatory diseases. Interleukin-17 (IL-17) is a T-cell-derived central mediator of autoimmunity. Immunization with Qβ-IL-17, a virus-like particle based vaccine, has been shown to produce autoantibodies in mice and was effective in ameliorating disease symptoms in animal models of autoimmunity. To characterize autoantibodies induced by vaccination at the molecular level, we generated mouse mAbs specific for IL-17 and compared them to germline Ig sequences. The variable regions of a selected hypermutated high-affinity anti-IL-17 antibody differed in only three amino acid residues compared to the likely germline progenitor. An antibody, which was backmutated to germline, maintained a surprisingly high affinity (0.5 nM). The ability of the parental hypermutated antibody and the derived germline antibody to block inflammation was subsequently tested in murine models of multiple sclerosis (experimental autoimmune encephalomyelitis), arthritis (collagen-induced arthritis), and psoriasis (imiquimod-induced skin inflammation). Both antibodies were able to delay disease onset and significantly reduced disease severity. Thus, the mouse genome unexpectedly encodes for antibodies with the ability to functionally neutralize IL-17 in vivo.

Screening for anemia in Red Cross blood donor clinics

Accurate screening for anemia at Red Cross blood donor clinics is essential to maintain a safe national blood supply. Despite the importance of identifying anemia correctly by measurement of hemoglobin or hematocrit (hemoglobin/hematocrit) there is no consensus regarding the efficacy of the current two stage screening method which uses the Readacrit$\sp{\rm tm}$ microhematocrit in conjunction with copper sulfate.^ A cross-sectional study was implemented in which hemoglobin/hematocrit was measured, with the present method and four new devices, on 504 prospective blood donors at a Canadian Red Cross permanent blood donor clinic in London, Canada. Concurrently gathered, venous and capillary blood samples were tested by each device and compared to Coulter S IV$\sp{\rm tm}$ determined venous standard readings. Instrument hemoglobin/hematocrit means were statistically calibrated to the standard ones in order to appraise systematic deviations from the standard. Classification analysis was employed to assess concordance between each instrument and the standard when classifying prospective donors as anemic or non-anemic. This was done both when each instrument was used alone (single stage) and when copper sulfate was used as a preliminary screen (two stage) and simulated over a range of anemia prevalences. The Hemoximeter$\sp{\rm tm}$ and Compur M1000$\sp{\rm tm}$ devices had the highest correlations of hemoglobin measurements with the standard ones for both capillary (n.s.) and venous blood (p $<$.05). Analysis of variance (anova) also showed them to be the most accurate (p $<$.05), as did both single and two stage classification analysis, therefore, they are both recommended. There was a smaller difference between instruments for two stage than for single stage screening; therefore instrument choice is less crucial for the former. The present method was adequate for two stage screening as tested but simulations showed that it would discriminate poorly in populations with a higher prevalence of anemia. The Stat-crit and Readacrit, which measure hematocrit, became less accurate at crucial low hematocrit levels. In light of this finding and the introduction of new, effective and easy to use hemoglobin measuring instruments, the continued use of hematocrit as a surrogate for hemoglobin, is not recommended. ^

SEVERITY OF IRON DEFICIENCY ANEMIA AND ITS RELATIONSHIP TO GROWTH AND MORBIDITY IN A POPULATION OF PRE-SCHOOLERS IN RURAL GUATEMALA (IRON DEFICIENCY, MORBIDITY, GROWTH)

The objectives of this study were to determine the nature of the relationship between severity of iron deficiency anemia, response to iron treatment, respiratory and gastrointestinal illness and weight change. Seventy-five pre-school children from rural Guatemala received daily oral iron therapy for an eleven week period, and were classified into one of three groups having different degrees of iron deficiency anemia. Anthropometric and biochemical data were collected prior and after iron treatment; morbidity data were collected throughout the period of treatment. The outcome variables were percentage weight change, percentage of total days ill with any type of symptom, percentage of total days ill with gastrointestinal symptoms, percentage of total days ill with respiratory symptoms, percentage of total days ill with combination syndrome symptoms. Age, sex and socio-economic status, were independent of any of the independent or outcome variables used. On the other hand, the level of hemoglobin covaried with the height of the children, the smallest children were the most severely anemic. The relationships between hemoglobin levels and weight change, frequency of morbidity (gastrointestinal, respiratory and combination syndrome) and total number of days ill with any symptomatology were investigated. No statistical significance was found in these analyses except when contrasting children with normal hemoglobin levels to iron deficient children, where the findings indicated the normal children experienced more gastrointestinal morbidity. The same relationship were again analyzed but including delta hemoglobin as covariate in the analysis, this latter one was found to be significant at 7% when the percentage of days ill from gastrointestinal morbidity was tested against the hemoglobin groups. The relationship found indicates that, all other covariates accounted for, the percentage of days ill from gastrointestinal morbidity will decrease approximately 1% for each 1% increase in delta of hemoglobin. ^

The relationship between obesity, autoimmune disease, and C-reactive protein: A secondary data analysis of the National Health and Nutrition Examination Survey (NHANES) 2009--2010

Autoimmune diseases are a group of inflammatory conditions in which the body's immune system attacks its own cells. There are over 80 diseases classified as autoimmune disorders, affecting up to 23.5 million Americans. Obesity affects 32.3% of the US adult population, and could also be considered an inflammatory condition, as indicated by the presence of chronic low-grade inflammation. C-reactive protein (CRP) is a marker of inflammation, and is associated with both adiposity and autoimmune inflammation. This study sought to determine the cross-sectional association between obesity and autoimmune diseases in a large, nationally representative population derived from NHANES 2009–10 data, and the role CRP might play in this relationship. Overall, the results determined that individuals with autoimmune disease were 2.11 times more likely to report being overweight than individuals without autoimmune disease and that CRP had a mediating affect on the obesity-autoimmune relationship. ^

Microangiopatía trombótica en paciente con síndrome antisintetasa : a propósito de un caso

Las microangiopatías trombóticas se caracterizan por presentar anemia hemolítica microangiopática asociada a plaquetopenia, fallo renal y lesión neurológica. El Síndrome anti-sintetasa es una entidad poco frecuente perteneciente al grupo de las miopatías inflamatorias idiopáticas. El objetivo de esta presentación es comunicar una paciente que desarrolló una púrpura trombocitopénica trombótica en el contexto de un Síndrome anti-sintetasa, asociación inusualmente reportada.

Microarray analysis of autoimmune diseases by machine learning procedures

—Microarray-based global gene expression profiling, with the use of sophisticated statistical algorithms is providing new insights into the pathogenesis of autoimmune diseases. We have applied a novel statistical technique for gene selection based on machine learning approaches to analyze microarray expression data gathered from patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS), two autoimmune diseases of unknown genetic origin that share many common features. The methodology included a combination of three data discretization policies, a consensus gene selection method, and a multivariate correlation measurement. A set of 150 genes was found to discriminate SLE and PAPS patients from healthy individuals. Statistical validations demonstrate the relevance of this gene set from an univariate and multivariate perspective. Moreover, functional characterization of these genes identified an interferon-regulated gene signature, consistent with previous reports. It also revealed the existence of other regulatory pathways, including those regulated by PTEN, TNF, and BCL-2, which are altered in SLE and PAPS. Remarkably, a significant number of these genes carry E2F binding motifs in their promoters, projecting a role for E2F in the regulation of autoimmunity.

Wheat allergy in celiac children

Gluten is the main structural protein complex of wheat with equivalent toxic proteins found in other cereals (rye, barley, and oats) which are responsible for different immunologic responses with different clinical expressions of disease. The spectrum of gluten-related disorders has been classified according to pathogenic, clinical, and epidemiological differences in three main forms: (i) wheat allergy (WA), an IgE-mediated disease; (ii) autoimmune disease, including celiac disease (CD), dermatitis herpetiformis, and gluten ataxia; and (iii) possibly immune-mediated, gluten sensitivity [1]. WA is an immunologic Th2 response with typical manifestations which can vary from dermatological, respiratory, and/or intestinal to anaphylactic reactions. In contrast, CD is an autoimmune disorder, a gliadin-specific T-cell response which is enhanced by the action of intestinal tissue transglutaminase (tTG), with a wide clinical spectrum including symptomatic cases with either intestinal (e.g., chronic diarrhea, weight loss) or extraintestinal features (e.g., anemia, osteoporosis, neurologic disturbances) and silent forms that are occasionally discovered as a result of serological screening [1]. We studied wheat allergy in two children with early diagnosis of CD, who developed immediate allergic symptoms after eating small amounts of wheat flour.

Immunomodulation of experimental autoimmune encephalomyelitis by oral administration of copolymer 1

The activity of copolymer 1 (Cop 1, Copaxone, glatiramer acetate) in suppressing experimental autoimmune encephalomyelitis (EAE) and in the treatment of multiple sclerosis patients when injected parenterally has been extensively demonstrated. In the present study we addressed the question of whether Cop 1 can induce oral tolerance to EAE similar to myelin basic protein (MBP). We now have demonstrated that oral Cop 1 inhibited EAE induction in both rats and mice. Furthermore, oral Cop 1 was more effective than oral MBP in suppressing EAE in rats. The beneficial effect of oral Cop 1 was found to be associated with specific inhibition of the proliferative and Th1 cytokine secretion responses to MBP of spleen cells from Cop 1-fed mice and rats. In all of these assays, oral Cop 1 was more effective than oral MBP. The tolerance induced by Cop 1 could be adoptively transferred with spleen cells from Cop 1-fed animals. Furthermore, Cop 1-specific T cell lines, which inhibit EAE induction in vivo, could be isolated from the above spleen cells. These T cell lines secrete the anti-inflammatory cytokines IL-10 and transforming growth factor type β, but not IL-4, in response to both Cop 1 and MBP. In conclusion, oral Cop 1 has a beneficial effect on the development of EAE that is associated with down-regulation of T cell immune responses to MBP and is mediated by Th2/3 type regulatory cells. These results suggest that oral administration of Cop 1 may modulate multiple sclerosis as well.

Resistance to apoptosis caused by PIG-A gene mutations in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder resulting from mutations in an X-linked gene, PIG-A, that encodes an enzyme required for the first step in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutations result in absent or decreased cell surface expression of all GPI-anchored proteins. Although many of the clinical manifestations (e.g., hemolytic anemia) of the disease can be explained by a deficiency of GPI-anchored complement regulatory proteins such as CD59 and CD55, it is unclear why the PNH clone dominates hematopoiesis and why it is prone to evolve into acute leukemia. We found that PIG-A mutations confer a survival advantage by making cells relatively resistant to apoptotic death. When placed in serum-free medium, granulocytes and affected CD34+ (CD59−) cells from PNH patients survived longer than their normal counterparts. PNH cells were also relatively resistant to apoptosis induced by ionizing irradiation. Replacement of the normal PIG-A gene in PNH cell lines reversed the cellular resistance to apoptosis. Inhibited apoptosis resulting from PIG-A mutations appears to be the principle mechanism by which PNH cells maintain a growth advantage over normal progenitors and could play a role in the propensity of this disease to transform into more aggressive hematologic disorders. These data also suggest that GPI anchors are important in regulating apoptosis.