Identifying the main physical and socioeconomic drivers of illegal landfills in the Canary Islands.

The management of disposed waste in illegal landfills (ILs) is a significant problem in contemporary societies due to respective hazards for the environment and human health. This paper presents a characterisation of ILs on the islands of La Palma (LP) and Gran Canaria (GC) based on multivariable statistical analysis. Inspection of numerous sites on both islands revealed a total of 153 and 286 ILs on LP and GC, respectively. A geospatial database was created composed of different potentially explanatory features of different typology (177): waste type, control and vigilance, socioeconomic, accessibility, distance to elements of interest, visibility and physical. The degree of association between the explanatory features and the occurrence of ILs was analysed with the support of exploratory statistics and the multivariable analysis techniques of principal component analysis (PCA) and binary logistic regression (LR). PCA explained 82.34% and 81.83% of total data variance in LP and GC, respectively, considering 7 and 6 components (Kaiser-Mayer-Olkin; LP: 0.715; GC: 0.711). The LR models for LP and GC had an overall accuracy of 93.5% and 92.5%. In LP and GC, 6 of 23 features and 9 of 21 features were, respectively, selected. The features most associated with the occurrence of ILs were: in LP, building density, distance to agricultural spaces and distance to green zones; in GC, the industrial activity indicator, density of ground use transition to artificial covers, density of greenhouses and distance to communication routes.

Eosinophilia secondary to lenalidomide therapy.

WHAT IS KNOWN AND OBJECTIVE: Limited data are available on eosinophilia as a drug adverse event. We describe a case of eosinophilia from lenalidomide therapy. CASE DESCRIPTION: A 50-year-old woman received lenalidomide, dexamethasone and cyclophosphamide as POEMS syndrome treatment. Eosinophil count rose during lenalidomide treatment and decreased in the periods off treatment. Naranjo nomogram suggested a probable association between the use of lenalidomide and eosinophilia. WHAT IS NEW AND CONCLUSION: Eosinophilia has rarely been described with lenalidomide. This case shows a clear temporal relationship between lenalidomide and eosinophilia.

Statin-associated autoimmune myopathy: A distinct new IFL pattern can increase the rate of HMGCR antibody detection by clinical laboratories.

BACKGROUND AND OBJECTIVE: Statin-associated autoimmune myopathy (SAAM) with anti-HMGCR antibodies has recently been described. Several specific immunoassays are in use to detect HMGCR antibodies. In the course of systematic autoantibody screening we recognized a new distinct IFL staining pattern on rat liver sections that regularly coincided with anti-HMGCR antibodies. In this study we investigated whether this new IFL pattern is specifically associated to statin-associated autoimmune myopathy and corresponds to anti-HMGCR antibodies. PATIENTS AND METHODS: Twenty-three patients positive for anti-HMGCR antibodies (14 diagnosed with SAAM) were investigated for anti-HMGCR antibodies by two ELISA assays and confirmed by immmunoblot. HMGCR associated liver IFL pattern (HALIP) was detected by indirect IFL and the reactivity against HMGCR was confirmed by immunoabsorption using purified human HMGCR antigen. 90 patients with other autoimmune diseases and 45 non-autoimmune statin treated patients were studied as controls. RESULTS: 21 out of 23 (91%) anti-HMGCR positive patients were HALIP positive. The staining was completely and specifically removed by immunoabsorption with human purified HMGCR. None of the control sera from autoimmune patients or non-autoimmune statin treated subjects was positive for HALIP. Statistical concordance between HALIP and anti-HMGCR antibody specific tests was 98.7%, kappa 0.95. CONCLUSIONS: A new and distinct IFL staining pattern (HALIP) is associated to HMGCR associated myopathy. Absorption and concordance studies indicate that the antigen recognized in the liver by HALIP is HMGCR or a closely related protein. Awareness of this new pattern can help to detect HMGCR autoantibodies in statin treated patients tested for autoimmune serology.

Genetics of Graves' Disease: Special Focus on the Role of TSHR Gene.

As most autoimmune diseases, inherited predisposition to Graves' disease (GD) is polygenic with the main contributory genes being located in the HLA region. Also, as in other autoimmune diseases, family linkage, candidate gene association, and GWAS studies have identified an expanding number of predisposing genes (CTLA4, CD40, PTPN22...) and 2 of them, TG and TSHR, are thyroid specific. In spite of this expanding number of associated genes, it has been estimated that all together they account for only a 20% of the heritability of GD. TSHR is of special interest as it codes for the target of TSHR stimulating antibodies (TSAbs), which are unequivocally pathogenic and an exception in autoimmunity by being stimulating rather than neutral, blocking, or cytotoxic. This is surprising because the generation of stimulating TSHR antibodies by immunisation of laboratory animals has been remarkably difficult, suggesting an underlying mechanism that favours stimulating over neutral or blocking anti-TSHR antibodies must be operating in GD patients. Besides, after HLA, TSHR is the gene most tightly associated to GD. The TSHR polymorphisms conferring susceptibility are located in the unusually large intron 1. Two mechanisms have been already put forward to explain its association with GD. According to one, the risk alleles determine an increase in the expression of TSHR mRNA splice variants that code for a soluble form of the receptor. The wider distribution of soluble TSHR would favour its immunogenicity and the development of an autoimmune response to it. It does not explain why it becomes immunogenic, as immunogenicity and distribution are not necessarily connected, nor why the immune response focus to the production of stimulating antibodies. According to the second mechanism proposed, the risk alleles determine a lower TSHR expression in the thymus and this would favour the escape of more TSHR reactive T cells, that is, central tolerance failure. The unexpected finding that thymocytes express TSHR and that TSAbs stimulate them lead to postulate that this would accelerate their egress from the thymus and a less efficient deletion of the TSHR self-reactive T cells. It can be envisaged that these autoreactive T cells may enhance the production of TSHR-Abs in the germinal centres of the thyroid draining lymph nodes, especially of those capable of further stimulating the egress of autoreactive T cells from the thymus. This mechanism, which does not exclude the former, provides and insight of the way in which TSAbs are favoured over neutral or blocking antibodies. Finally this would explain the frequent finding of thymic hyperplasia in GD patients.

HLA-DQ2/DQ8 and HLA-DQB1*02 homozygosity typing by real-time polymerase chain reaction for the assessment of celiac disease genetic risk: evaluation of a Spanish celiac population.

Celiac disease (CD) is a complex autoimmune disorder caused by ingestion of gluten in genetically susceptible individuals. Different genetic risk factors have been identified, but virtually all patients are human leukocyte antigen (HLA)-DQ2 and/or HLA-DQ8 positive. We describe a new, fast, accurate and simple real-time polymerase chain reaction (PCR)-based assay for the genotyping and homozygosity analysis of the CD-related HLA alleles. The assay overcomes the major limitations of protocols currently in use, allowing HLA-DQ2/DQ8 genotyping by using only three real-time PCR reactions. For the appraisal of DQ2 homozygosity, only one more reaction is needed. These reactions are easily automated and suitable for large screening studies in diagnostic procedures, as it is demonstrated by their successful application in our HLA diagnostic laboratory. Finally, we assessed the clinical relevance of this real-time PCR-based assay by studying a cohort of fully characterized patients. As expected, all CD patients had at least one of the CD-associated alleles, and the highest CD risk was indicated by the presence of the HLA-DQ2.5 heterodimer (HLA-DQA1*05-DQB1*02) with HLA-DQB1*02 in homozygosity.

Novel and atypical splicing mutation in a compound heterozygous UNC13D defect presenting in Familial Hemophagocytic Lymphohistiocytosis triggered by EBV infection.

Familial Hemophagocytic Lymphohistiocytosis type 3 (FHL3) is a genetic disorder caused by mutations in UNC13D gene, coding the granule priming factor Munc13-4 that intervenes in NK and T cell cytotoxic function. Here we report the case of a 17-month-old girl with prolonged symptomatic EBV infectious mononucleosis and clinical symptoms of hemophagocytic syndrome. In vitro functional analysis pointed to a degranulation defect. The genetic analysis of UNC13D gene identified initially a heterozygous mutation (c.753+1G>T) in the donor splice-site that resulted in exon 9 skipping (maternal allele). Mutations in other genes were considered, but additional analysis of UNC13D cDNA revealed in the paternal allele a heterozygous transition from G to A (c.2448-13G>A) at the 3' acceptor splice-site in intron 25, generating a new acceptor splice-site that leads to a frameshift and a premature STOP codon. Allele specific amplification of the cDNA confirmed the absence of a functional mRNA from the paternal allele. This case illustrates an atypical compound heterozygous UNC13D mutation affecting the RNA splicing that generates a typical FHL3 phenotype.

Calculated globulin (CG) as a screening test for antibody deficiency.

Calculated globulin (total protein - albumin) is usually tested as part of a liver function test profile in both primary and secondary care and determines the serum globulin concentration, of which immunoglobulins are a major component. The main use hitherto of calculated globulin is to detect paraproteins when the level is high. This study investigated the potential to use low levels of calculated globulin to detect antibody deficiency. Serum samples with calculated globulin cut-off < 18 g/l based on results of a pilot study were collected from nine hospitals in Wales over a 12-month period. Anonymized request information was obtained and the samples tested for immunoglobulin levels, serum electrophoresis and, if appropriate, immunofixation. A method comparison for albumin measurement using bromocresol green and bromocresol purple was undertaken. Eighty-nine per cent (737 of 826) samples had an immunoglobulin (Ig)G level of < 6 g/l using the bromocresol green methodology with a cut-off of < 18 g/l, and 56% (459) had an IgG of < 4 g/l. Patients with both secondary and primary antibody deficiency were discovered and serum electrophoresis and immunofixation showed that 1·2% (10) had previously undetected small paraproteins associated with immune-paresis. Using bromocresol purple, 74% of samples had an IgG of < 6 g/l using a cut-off of < 23 g/l. Screening using calculated globulin with defined cut-off values detects both primary and secondary antibody deficiency and new paraproteins associated with immune-paresis. It is cheap, widely available and under-utilized. Antibody-deficient patients have been discovered using information from calculated globulin values, shortening diagnostic delay and time to treatment with immunoglobulin replacement therapy.

Overexpression of metallothionein I/II: a new feature of thyroid follicular cells in Graves' disease.

CONTEXT: One salient feature of autoimmune thyroid disease is the inappropriate expression of human leukocyte antigen (HLA) class II molecules by thyroid follicular cells. Metallothioneins (MT) are small proteins induced by tissue stress that can contribute to restoring homeostasis of tissue inflammation and have been found to be increased in a transcriptomic analysis of Graves' disease (GD) glands. METHODOLOGY: To assess the role of MT in the pathogenesis of GD, we analyzed MT-I and -II expression and distribution in GD-affected thyroid glands (n = 14) compared with other thyroid diseases (n = 20) and normal thyroid glands (n = 5). Two-color indirect immunofluorescence and semiquantitative morphometry were applied. The relationship between MT and HLA class II expression was analyzed by their degree of colocalization in GD sections, and in vitro induction kinetics and expression of these molecules on the HT93 thyroid cell line were compared by quantitative RT-PCR and flow cytometry using interferon-γ and zinc as stimuli. RESULTS: MT were clearly overexpressed in nine of 14 GD glands. MT expression distribution in GD was almost reciprocal to that of HLA class II. In vitro analysis of MT and HLA class II demonstrated that MT is induced more slowly and at a lower level than HLA. Moreover, the main MT inducer, zinc, reduces interferon-γ-induced class II expression. CONCLUSIONS: These findings show that MT and HLA class II play very different roles in the autoimmune process by affecting the thyroid gland, thereby pointing to the possible role of MT as a marker of cell stress and homeostasis restoration in GD.

Specific T-cell proliferation to myelin peptides in relapsing-remitting multiple sclerosis.

BACKGROUND: The identification of major immunogenic peptides in multiple sclerosis (MS) is of great importance for the development of antigen-specific therapies. Cellular reactivity against a selected mix of seven myelin peptides was evaluated in vitro. The evolution of this reactivity over time and its correlation with clinical variables was also analysed. MATERIAL AND METHODS: Forty-two patients with MS, 15 with other demyelinating diseases and 40 healthy donors (HD) were studied. Cell proliferation was measured by 3[H] thymidine incorporation into samples obtained at 0, 3, 6 and 12months of MS patient follow-up. RESULTS: A positive reaction to the peptide mix was detected in 31 of the 42 patients (74%), 12 of the 40 HD (30%) and 6 of the 15 (40%) patients with other demyelinating diseases. Patients with positive proliferation had greater disability (EDSS score, 3 [1-5.5] vs. 1.0[1-2], P=0.021), higher number of relapses (7±4.1 vs. 3±1.2, P<0.001) and shorter time since the last relapse (9±7.5 vs. 32±12.3months, P=0.036). After 12months of follow-up, cell reactivity was maintained in 33 patients (78%). CONCLUSION: A high percentage of patients exhibit a significant and maintained reactivity to myelin peptides over time. Therefore, this mix may be useful as a source of antigen in the development of protocols aimed at inducing specific tolerance in MS.

Comparative study of clinical grade human tolerogenic dendritic cells.

BACKGROUND: The use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (Good Manufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy. METHODS: Tolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-α, IL-ß and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced. RESULTS: Differences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-γ) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-γ production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells. CONCLUSIONS: Our results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.

Gene expression profiles for the human pancreas and purified islets in type 1 diabetes: new findings at clinical onset and in long-standing diabetes.

Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing beta cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non-classical class I human leucocyte antigen and leucocyte immunoglobulin-like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C-C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long-standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.

Dendritic cells pulsed with antigen-specific apoptotic bodies prevent experimental type 1 diabetes.

Dendritic cells (DCs) are powerful antigen-presenting cells capable of maintaining peripheral tolerance. The possibility to generate tolerogenic DCs opens new therapeutic approaches in the prevention or remission of autoimmunity. There is currently no treatment inducing long-term tolerance and remission in type 1 diabetes (T1D), a disease caused by autoimmunity towards beta cells. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow islet regeneration. Apoptotic cells--a source of autoantigens--are cleared rapidly by macrophages and DCs through an immunologically silent process that contributes to maintaining tolerance. Our aims were to prevent T1D and to evaluate the re-establishment of peripheral tolerance using autologous DCs pulsed in vitro with apoptotic bodies from beta cells. Immature DCs derived from bone marrow of non-obese diabetic (NOD) mice were obtained and pulsed with antigen-specific apoptotic bodies from the beta cell line NIT-1. Those DCs that phagocytosed apoptotic cells diminished the expression of co-stimulatory molecules CD40 and CD86 and reduced secretion of proinflammatory cytokines. Moreover, these cells were resistant to increase the expression of co-stimulatory molecules after lipopolysaccharide activation. The administration of these cells to NOD transgenic mice expressing interferon-beta in their insulin-producing cells, a model of accelerated autoimmune diabetes, decreased diabetes incidence significantly and correlated positively with insulitis reduction. DCs pulsed with apoptotic cells that express disease-associated antigens constitutes a promising strategy to prevent T1D.

Myelin peptides in multiple sclerosis.

The development of specific therapies for organ-specific autoimmune diseases requires the identification of relevant immunogenic epitopes, recognized by both pathogenic T cells and autoantibodies. Here, we review the most relevant studies focused in the identification of peptides in multiple sclerosis (MS) and the distinct T cell reactivity induced in patients compared to controls. Only a few studies reported significant differences in terms of T cell reactivity to them. The current knowledge on this issue, and the diagnostic and therapeutic possibilities opened by the identification of pathogenic MS epitopes are discussed in this paper.

Copy number variation in the CCL4L gene is associated with susceptibility to acute rejection in lung transplantation.

Lung transplantation (LT) has become an accepted therapy for selected patients with advanced lung disease. One of the main limitations to successful LT is rejection of the transplanted organ where chemokines are pivotal mediators. Here, we test the relationship between copy number variation (CNV) in the CCL4L chemokine gene and rejection risk in LT patients (n=161). Patients with no acute rejection showed a significantly lower mean number of CCL4L copies than patients that showed acute rejection (1.66 vs 1.96, P=0.014), with an even greater number of gene copies seen in patients with more than one episode of acute rejection (1.66 vs 2.30, P=0.001). Additionally, patients with > or =2 CCL4L copies had a significantly higher risk of acute rejection compared with patients that had 0-1 CCL4L copies (odds ratio 2.65; 95% confidence interval, 1.33-5.28; P=0.0046). A combined analysis of CCL4L CNV and the rs4796195 CCL4L single nucleotide polymorphism demonstrated that the effect of CCL4L copy number in acute rejection is mainly because of the number of copies of the CCL4L1 allelic variant. This finding constitutes the first report of CNV as a correlate factor in allograft rejection.

Influx of recent thymic emigrants into autoimmune thyroid disease glands in humans.

Autoimmune thyroid diseases (AITD) are considered as prototypic organ-specific autoimmune diseases, yet their underlying aetiology remains poorly understood. Among the various pathophysiological mechanisms considered, a failure of central tolerance has received little attention. Here we present evidence in favour of dysregulated thymic function playing a role in AITD. Flow-cytometric analyses conducted in peripheral blood lymphocytes from 58 AITD patients and 48 age- and-sex-matched controls showed that AITD patients have significantly higher blood levels of CD4(+)CD45RA(+), CD4(+)CD31(+) and CD4/CD8 double-positive T lymphocytes, all markers of recent thymic emigrants (RTE). In addition, the alpha-signal joint T cell receptor excision circles (TRECs) content (a molecular marker of RTEs) was higher in the group of AITD patients older than 35 years than in age-matched controls. This was independent from peripheral T cell expansion as assessed by relative telomere length. Comparisons of TREC levels in peripheral blood lymphocytes and intrathyroidal lymphocytes in paired samples showed higher levels within the thyroid during the initial 30 months of the disease, indicating an influx of RTE into the thyroid during the initial stages of AITD. Additionally, a lack of correlation between TREC levels and forkhead box P3 expression suggests that the intrathyroidal RTE are not natural regulatory T cells. These results uncover a hitherto unknown correlation between altered thymic T cell export, the composition of intrathyroidal T cells and autoimmune pathology.

Population structure in copy number variation and SNPs in the CCL4L chemokine gene.

The recent description of a large amount of copy number variation (CNV) in the human genome has extended the concept of genome diversity. In this study we integrate the analysis of CNV and single nucleotide polymorphisms (SNPs) in the human CCL4L chemokine gene. CCL4L is a nonallelic copy of CCL4/MIP-1beta chemokine and displays a CNV that also includes the CCL3L gene, a nonallelic copy of CCL3/MIP-1alpha. This CNV and two functionally relevant CCL4L SNPs (rs4796195 and rs3744595) have been recently associated to HIV pathology in three independent studies. We have quantified the CCL4L copy number and genotyped both SNPs in samples from HGDP-CEPH Diversity Panel. A strong correlation between CCL4L CNV and one of the SNPs analyzed is found, whereas no significant linkage disequilibrium is found between the two SNPs despite their close distance (647 bp), suggesting a recent appearance of the second SNP when the diversity in the first one and CNV had already been generated. The present study points out that in genes with CNV, it may be a key issue to combine the assessment of gene copy number with the genotyping of relevant SNPs to understand the phenotypic impact of genome variation in the immune response.

Natural killer cells are required for accelerated type 1 diabetes driven by interferon-beta.

The destruction of beta cells by the islet infiltrating lymphocytes causes type 1 diabetes. Transgenic mice models expressing interferon (IFN)-beta in beta cells, in the non-obese diabetic (NOD) strain and in a diabetes-free, major histocompatibility complex-matched, homologous strain, the non-obese resistant (NOR) mice, developed accelerated type 1 diabetes after 3 weeks of age. Our aim was to determine if natural killer (NK) cells could affect the acceleration of the disease. We determined the amount of NK cells in the pancreas, spleen and lymph nodes from NOD rat insulin promoter (RIP)-IFN-beta mice. Pancreatic cytokines were assessed by quantitative real-time polymerase chain reaction and protein arrays. To confirm the relevance of NK cells in the acceleration of autoimmune diabetes this subset was depleted with anti-asialo GM1 antibodies. An increase of intrapancreatic NK cells characterized the accelerated onset of diabetes both in NOD and NOR RIP-IFN-beta transgenic models. Cytokines involved in NK function and migration were found to be hyperexpressed in the pancreas from accelerated diabetic mice. Interestingly, the depletion of NK cells in vivo abolished completely the acceleration of diabetes. NK cells connect innate to adaptive immunity and might play a role in autoimmunity. We report here that NK cells are required critically in the pancreas for accelerated diabetes. This model links inflammation to acceleration of beta cell-specific autoimmunity mediated by NK cells.