Neurocysticercosis (NC) presents a spectrum of clinical manifestations, with two broad clinical entities based on the central nervous system location of the parasite: extraparenchymal (EP-NC) and parenchymal (P-NC). In this work, using quantitative immunoblot methodology, we demonstrate the presence of autoantibodies to brain proteins in CSF from EP-NC, but not P-NC, patients. There was striking correlation between the level of autoantibodies and the levels of the secreted metacestode glycoprotein HP-10, suggesting that the level of stimulation of the autoantibody response may be a function of the number of viable parasites. Nine corresponding proteins autoantigens were provisionally identified by mass spectroscopy.

Forkhead box P3 (FoxP3)(+) regulatory T cells (T-regs) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)-2 receptor alpha chain]. Low-dose IL-2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE T-reg phenotype, we studied its role through developmentally defined regulatory T cell (T-reg) subsets in 45 SLE patients, 103 SLE-unaffected first-degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25-encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to T-reg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4(+)FoxP3(+)CD45RO(-)CD31(+) recent thymic emigrant T-regs. This first component effect influenced the proportions of circulating CD4(+)FoxP3(high)CD45RO(+) activated T-regs. (2) In contrast, patients and unaffected relatives differed sharply in their activated T-reg CD25 state: while relatives as control subjects up-regulated CD25 strongly in these cells during differentiation from naive T-regs, SLE patients specifically failed to do so. This CD25 up-regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated T-regs, but not to their circulating numbers. Both effects were found related to T cell IL-2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early T-regs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up-regulation upon peripheral T-reg activation that is selectively deficient in patients. We expect that T-reg-directed therapies can be monitored more effectively when taking this distinction into account.

A classic T-cell phenotype in systemic lupus erythematosus (SLE) is the downregulation and replacement of the CD3ζ chain that alters T-cell receptor signaling. However, genetic associations with SLE in the human CD247 locus that encodes CD3ζ are not well established and require replication in independent cohorts. Our aim was therefore to examine, localize and validate CD247-SLE association in a large multiethnic population. We typed 44 contiguous CD247 single-nucleotide polymorphisms (SNPs) in 8922 SLE patients and 8077 controls from four ethnically distinct populations. The strongest associations were found in the Asian population (11 SNPs in intron 1, 4.99 × 10(-4) < P < 4.15 × 10(-2)), where we further identified a five-marker haplotype (rs12141731-rs2949655-rs16859085-rs12144621-rs858554; G-G-A-G-A; P(hap) = 2.12 × 10(-5)) that exceeded the most associated single SNP rs858554 (minor allele frequency in controls = 13%; P = 4.99 × 10(-4), odds ratio = 1.32) in significance. Imputation and subsequent association analysis showed evidence of association (P < 0.05) at 27 additional SNPs within intron 1. Cross-ethnic meta-analysis, assuming an additive genetic model adjusted for population proportions, showed five SNPs with significant P-values (1.40 × 10(-3) < P< 3.97 × 10(-2)), with one (rs704848) remaining significant after Bonferroni correction (P(meta) = 2.66 × 10(-2)). Our study independently confirms and extends the association of SLE with CD247, which is shared by various autoimmune disorders and supports a common T-cell-mediated mechanism.

Background Regulatory T cell (Treg) defects are associated with autoimmune diseases [1]. Immunoglobulin E (IgE) is elevated in primary immunodeficiencies associated to Regulatory T cell (Treg) defects [2] and is known to be positively correlated to disease activity in Systemic Lupus Erythematosus (SLE) [3,4]. More specifically, Treg frequency (increased) and CD25 expression (decreased) are dissociated in patients with active SLE [5,6]. Objectives To establish the relationship between circulating Treg frequency/number and phenotype and total IgE/IgE auto-antibodies and test the hypothesis that total IgE could be a marker of a generalized Treg deficit. Methods We first tested a large cross-sectional collection of plasma samples from unselected patients with a diagnosis of SLE according to ACR criteria (n=123) and from healthy controls (n=153). We then started a prospective longitudinal study of SLE patients (n=33, 30 females, mean age 41±10 years). A median of 4 samples were obtained per patient. Clinical characteristics, SLEDAI 2 K as index of disease activity, SELENA-SLEDAI score for definition of flare and sample collection were undertaken. During follow-up 13/33 patients experienced a flare. Samples were analysed by an ELISA “in house” method optimized for total IgE, antigen-specific IgE (anti-DNA, Sm, RNP), IgG anti-DNA and IgG anti-Sm. FACS analysis determined Treg frequency (CD4+CD45RO+Foxp3high) and CD25 MFI expression. The Mann-Whiney Test and Spearman correlation were used with values of p<0.05 considered significant. Results While there was no significant difference in total IgE, IgE anti-DNA was higher in patients (p=0.02) and positively correlated with SLEDAI (p=0.009). In the longitudinal prospective collection there was a positive correlation between IgG anti-DNA (p=0.002) or IgE anti-DNA (p=0.0003) and disease activity. IgE anti-DNA was increased in some patients with high SLEDAI and no IgG anti-DNA. The frequency of active Treg was positively correlated with SLEDAI (p=0.006) and with IgG anti-DNA (p=0.0004) while their CD25 expression was negatively correlated with SLEDAI (p=0.008) and IgG anti-DNA (p=0.002). There was no association between total IgE or IgE anti-DNA and Treg frequency or CD25 expression in either the cross-sectional study or the longitudinal patient collection. Conclusions These results indicate that unlike IgG anti-DNA, IgE anti-DNA is not associated with Treg frequency or CD25 expression, raising questions regarding its affinity and pathogenicity. Still, as previously described, IgE anti-DNA behaved as a biomarker of SLE disease activity. In this study we demonstrate that it may be a useful biomarker in the absence of IgG anti-DNA. References Bennett Nat Genet 2001 27(1):20-1. Ozcan J Allergy Clin Immunol 2008. 122(6):1054-62. Charles Nat Med 2010 16(6):701-7. Dema Plos One 2014 9(2)e90424. Bonelli Ann Rheum Dis 2008 67(5):664-71. Suen Immunology 2009 127(2):196-205. Acknowledgements Jocelyne Demengeot (JD) for grant submission and ongoing support, JD, W Haas and J Lafaille for helpful discussions and R van Vollenhoven for scientific input. Staff at UIC and ICBAS (Porto), HSM, CHLO, Santarém, HFF, HCC and Patients and Patient Association for sample collections. Funding FCT EXPL/DTP-PIC/0644/2012 Disclosure of Interest None declared

ABSTRACT Several immunomodulatory factors are involved in malaria pathogenesis. Among them, heme has been shown to play a role in the pathophysiology of severe malaria in rodents, but its role in human severe malaria remains unclear. Circulating levels of total heme and its main scavenger, hemopexin, along with cytokine/chemokine levels and biological parameters, including hemoglobin and creatinine levels, as well as transaminase activities, were measured in the plasma of 237 Plasmodium falciparum -infected patients living in the state of Odisha, India, where malaria is endemic. All patients were categorized into well-defined groups of mild malaria, cerebral malaria (CM), or severe noncerebral malaria, which included acute renal failure (ARF) and hepatopathy. Our results show a significant increase in total plasma heme levels with malaria severity, especially for CM and malarial ARF. Spearman rank correlation and canonical correlation analyses have shown a correlation between total heme, hemopexin, interleukin-10, tumor necrosis factor alpha, gamma interferon-induced protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1) levels. In addition, canonical correlations revealed that heme, along with IP-10, was associated with the CM pathophysiology, whereas both IP-10 and MCP-1 together with heme discriminated ARF. Altogether, our data indicate that heme, in association with cytokines and chemokines, is involved in the pathophysiology of both CM and ARF but through different mechanisms.

Mechanisms of acquired protection to malaria in asymptomatic Plasmodium falciparum carriers are only partially understood. Among them, the role plays by the self-reactive antibodies has not been clarified yet. In this study, the relationship between repertoires of circulating self-reactive and parasite-specific immunoglobulin G (IgG), their correlation with cytokine levels, and their association with protection against malaria was investigated in asymptomatic Plasmodium falciparum-infected Gabonese children.

Plasmodium falciparum malaria in India is characterized by high rates of severe disease, with multiple organ dysfunction (MOD)—mainly associated with acute renal failure (ARF)—and increased mortality. The objective of this study is to identify cytokine signatures differentiating severe malaria patients with MOD, cerebral malaria (CM), and cerebral malaria with MOD (CM-MOD) in India. We have previously shown that two cytokines clusters differentiated CM from mild malaria in Maharashtra. Hence, we also aimed to determine if these cytokines could discriminate malaria subphenotypes in Odisha.

Background FOXP3+ regulatory T-cells (Tregs) in Systemic Lupus Erythematosus (SLE) are in a functionally deficient state with a characteristic reduction or absence of surface CD25 (the IL-2 receptor alpha chain). Genetic variation in the CD25-encoding IL2RA locus is associated with other autoimmune disorders. Methods We have studied Treg and Treg subset CD25 by flow cytometry and typed 24 SNPs in the IL2RA locus in 47 SLE patients, 108 SLE-unaffected first-degree relatives of SLE patients, and 61 unrelated control subjects. Results In both SLE patients and unaffected relatives, surface CD25 was found strongly reduced not only in activated, but already in circulating CD4+ FOXP3+ CD45RO-CD31 + recent thymic emigrant (RTE) Tregs. In contrast, unaffected relatives clearly differed from SLE patients in properties of activated CD4+ FOXP3highCD45RO + Tregs, which showed a CD25 upregulation versus non-activated CD45RO- Tregs in these relatives similar to control subjects, while not in SLE patients. The distinction of these two components contributing to the previously described SLE-characteristic Treg CD25 reduction was corroborated by our finding that the two components were influenced by polymorphisms in different regions of the IL2RA locus. Furthermore, we found that only RTE Treg CD25, as well as the genetic variants influencing it, were significantly related to numbers and relative frequencies of circulating activated Tregs, whereas CD25 upregulation upon Treg activation was not. Conclusions Our results point to (a) an intrathymic effect present in an extended population carrying SLE susceptibility factors that is responsible for reduced surface CD25 in early Tregs and a subsequently decreased activation capacity. This effect might be compensated in unaffected relatives by (b) CD25 upregulation upon Treg activation, which seemed functionally independent and was selectively deficient in SLE patients. This second component appears of particular interest for therapeutic targeting.

Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus, the mechanism of which remains to be fully understood. Here we investigated whether ivIg affects two established sub-phenotypes of SLE, namely relative oligoclonality of circulating T-cells and reduced activity of CD4 + Foxp3+ regulatory T-cells (Tregs) reflected by lower CD25 surface density.

In human systemic lupus erythematosus (SLE), diverse autoantibodies accumulate over years before disease manifestation. Unaffected relatives of SLE patients frequently share a sustained production of autoantibodies with indiscriminable specificity, usually without ever acquiring the disease. We studied relations of IgG autoantibody profiles and peripheral blood activated regulatory T-cells (aTregs), represented by CD4(+)CD25(bright) T-cells that were regularly 70-90% Foxp3(+). We found consistent positive correlations of broad-range as well as specific SLE-associated IgG with aTreg frequencies within unaffected relatives, but not patients or unrelated controls. Our interpretation: unaffected relatives with shared genetic factors compensated pathogenic effects by aTregs engaged in parallel with the individual autoantibody production. To study this further, we applied a novel analytic approach named coreferentiality that tests the indirect relatedness of parameters in respect to multivariate phenotype data. Results show that independently of their direct correlation, aTreg frequencies and specific SLE-associated IgG were likely functionally related in unaffected relatives: they significantly parallelled each other in their relations to broad-range immunoblot autoantibody profiles. In unaffected relatives, we also found coreferential effects of genetic variation in the loci encoding IL-2 and CD25. A model of CD25 functional genetic effects constructed by coreferentiality maximization suggests that IL-2-CD25 interaction, likely stimulating aTregs in unaffected relatives, had an opposed effect in SLE patients, presumably triggering primarily T-effector cells in this group. Coreferentiality modeling as we do it here could also be useful in other contexts, particularly to explore combined functional genetic effects.

Propose After autologous stem cell transplantation (ASCT) the immunological B cell compartment recovers slowly. Delays on the recovery of B cell function after autologous stem cell transplantation are due to the low lymphocytes count and to their intrinsic dysfunction.Methods We studied the in vivo B cell reconstitution after ASCT examining the independent effect of polyclonal IgG (PolyIg), Fab or Fc fragments infusions in a murine animal model during a period of 12 weeks. These molecules were used in low doses, mimicking the recommended use of IVIg in the case of hypogammaglobulinemia in humans. Flow cytometry analysis and ELISA tests were conducted to monitor the reconstitution of B cells and serum immunoglobulin production. Panama blot and PCA factor 1 analysis were used to study the kinetics of immunoglobulin repertoires reconstitution. Mechanistic studies were also performed using in vitro cell culture.Results During follow-up after ASCT, peripheral B cells expand independently of treatment, correcting the immediate increase in sBAFF (soluble B cell activating factor) induced by previous intense myeloablation. Treatments with Fab and Fc fragments infusions promote significant IgM and IgG production comparing to control. Although the complete recovery of antibody repertoire is only achieved at the end of follow-up after ASCT, there is an earlier and significantly stronger recovery in the treated mice, which is evident at 9 weeks after ASCT. At 30 weeks after ASCT, normal values of antibody repertoire were detected in all individuals. Mechanistic studies show that Fab and Fc fragments promote IgG1 production by indirect pathways.Conclusions The results presented here demonstrate that polyclonal immunoglobulin indirectly improves the function of the reconstituted B cells and their IgG production by means of Fc-mediated effects on bystander cells. These results further stimulate the discussion about the advantages of IVIg therapy during immune reconstitution after human ASCT.

Plasmodium falciparum infection generally induces elevated total plasma levels of immunoglobulins, some of which recognize self- or parasite-specific antigens. To our knowledge, we are the first to report high levels of functional immunoglobulin E (IgE) autoantibodies recognizing brain 14-3-3 protein ε in asymptomatic P. falciparum malaria. 14-3-3 ε protein belongs to a family of proteins that binds to CD81, a member of the tetraspanin superfamily elicited in hepatocyte invasion by sporozoites. Levels of expression of 14-3-3 ε protein were found to be increased in vivo and in vitro during Plasmodium yoelii and P. falciparum intrahepatic development. Collectively, these results indicate that self-reactive IgE is produced during malaria. In addition, the negative correlation between levels of self-reactive IgE to 14-3-3 ε protein and parasitemia in asymptomatic malaria due to P. falciparum supports a role for these IgE molecules in defense mechanisms, probably by interfering with development of liver-stage parasites through the CD81 pathway.

Many multifactorial biologic effects, particularly in the context of complex human diseases, are still poorly understood. At the same time, the systematic acquisition of multivariate data has become increasingly easy. The use of such data to analyze and model complex phenotypes, however, remains a challenge. Here, a new analytic approach is described, termed coreferentiality, together with an appropriate statistical test. Coreferentiality is the indirect relation of two variables of functional interest in respect to whether they parallel each other in their respective relatedness to multivariate reference data, which can be informative for a complex effect or phenotype. It is shown that the power of coreferentiality testing is comparable to multiple regression analysis, sufficient even when reference data are informative only to a relatively small extent of 2.5%, and clearly exceeding the power of simple bivariate correlation testing. Thus, coreferentiality testing uses the increased power of multivariate analysis, however, in order to address a more straightforward interpretable bivariate relatedness. Systematic application of this approach could substantially improve the analysis and modeling of complex phenotypes, particularly in the context of human study where addressing functional hypotheses by direct experimentation is often difficult.

Autoimmune Polyglandular Syndrome Type II (APSII) is characterized by the co-occurrence of clinical insufficiency of at least two endocrine glands. Although, HLA determinants of APSII predisposition have been identified, little attention has been paid to non-HLA genes. Here, we used SNP genotyping in a Sequenom platform and genetic association tests to study a cohort of 60 APSII Tunisian patients presenting highly frequent co-occurrence of Autoimmune Thyroid Disease (AITD) and Type 1 Diabetes (T1D) and lower frequency of Addison’s disease (AD). We tested the high a priori possibility that well-established non-HLA autoimmunity loci were involved in APSII and confirmed five association signals to APSII, namely: (1) two T1D-associated SNPs, in CTLA4 and IL2RA, suggest their involvement in T1D pathogenesis in this cohort; (2) two SNPs in STAT4 and IL15 not previously associated to endocrinopathies, are possibly involved in co-occurrence of organ autoimmunity in APSII, and; (3) one SNP in TNF alpha showed association to APSII irrespective of AD. While this work was performed in a relatively small cohort, these results support the notion that the non-HLA genetic component of APSII include genetic factors specific of particular autoimmune manifestations as well as genetic factors that promote the co-occurrence of multiple autoimmune endocrinopathies.

Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus. Relative oligoclonality of circulating T-cells in SLE has been reported. Also CD4+Foxp3+ regulatory T-cells (Tregs) have a characteristically reduced activity in SLE, reflected by CD25 surface density. Aiming to study the role of Tregs for ivIg therapy, we characterized Tregs and determined TCR spectratypes of four Vb families with reported oligoclonality, in 15 lupus patients (14 with SLE and one with discoid LE) treated with ivIg in cycles of 2-6 consecutive monthly infusions. Among these 15 patients, 11 responded with clinical improvement. Cell counts, cytometry and TCR spectratypes were obtained from peripheral blood at various time points before, during and after ivIg treatment. T-cell oligoclonality was assessed as Vb-familywise repertoire perturbation, calculated for each patient in respect to an individual reference profile averaged over all available time points. For 11/15 patients, average Vb1/Vb2/Vb11/Vb14 repertoires were less perturbed under ivIg treatment than outside ivIg therapy. The four exceptions with relatively increased average perturbation during ivIg therapy included three patients who failed to respond clinically to an ivIg therapy cycle. Patients' Treg CD25 surface density (cytometric MFI) was, other than Treg/CD4+ frequency, clearly reduced when compared to healthy controls, but not obviously influenced by ivIg. However, patients' average Treg CD25 MFI was found negatively correlated with both Vb11 and Vb14 perturbations measured under ivIg therapy, which indicates a role of active Tregs in the therapeutic effect of ivIg.

Background The main processes in the pathogenesis of cerebral malaria caused by Plasmodium falciparum involved sequestration of parasitized red blood cells and immunopathological responses. Among immune factors, IgG autoantibodies to brain antigens are increased in P. falciparum infected patients and correlate with disease severity in African children. Nevertheless, their role in the pathophysiology of cerebral malaria (CM) is not fully defined. We extended our analysis to an Indian population with genetic backgrounds and endemic and environmental status different from Africa to determine if these autoantibodies could be either a biomarker or a risk factor of developing CM.

A comparative analysis of the plasma concentrations of soluble Fas-L (sFas-L) and their correlation with hemoglobin levels and malaria severity was carried out in cohorts of P. falciparum-infected patients from Gabon and India.Young patients from Gabon had plasma levels of sFas-L that increased with disease severity.In contrast, in Indian adults plasmatic sFas-L levels were more elevated in UM and SNCM than in CM.In both Gabonese and Indian subjects, sFas-L concentrations were negatively correlated with haemoglobin rates, which were lower in SNCM than in UM or CM in both populations.We also observed a positive correlation between the level of plasmatic sFas-L with the level of circulating IL-2 receptor in the Indian patients.All these observations suggest a paradoxical role of sFas-L in CM pathogenesis when comparing Gabonese and Indian patients, while a similar role was found to be associated with severe anaemia.