Retro-inverso D-peptides as a novel targeted immunotherapy for Type 1 diabetes.

Over the past four decades, the number of people with Type 1 Diabetes (T1D) has increased by 4% per year, making it an important public health challenge. Currently, no curative therapy exists for T1D and the only available treatment is insulin replacement. HLA-DQ8 has been shown to present antigenic islet peptides driving the activation of CD4+ T-cells in T1D patients. Specifically, the insulin peptide InsB:9-23 activates self-reactive CD4+ T-cells, causing pancreatic beta cell destruction. The aim of the current study was to identify retro-inverso-d-amino acid based peptides (RI-D-peptides) that can suppress T-cell activation by blocking the presentation of InsB:9-23 peptide within HLA-DQ8 pocket. We identified a RI-D-peptide (RI-EXT) that inhibited InsB:9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to DQ8 expressed on human B-cells. RI-EXT prevented T-cell activation in a cellular antigen presentation assay containing human DQ8 cells loaded with InsB:9-23 peptide and murine T-cells expressing a human T-cell receptor specific for the InsB:9-23-DQ8 complex. Moreover, RI-EXT blocked T-cell activation by InsB:9-23 in a humanized DQ8 mice both ex vivo and in vivo, as shown by decreased production of IL-2 and IFN-γ and reduced lymphocyte proliferation. Interestingly, RI-EXT also blocked lymphocyte activation and proliferation by InsB:9-23 in PBMCs isolated from recent onset DQ8-T1D patients. In summary, we discovered a RI-D-peptide that blocks InsB:9-23 binding to HLA-DQ8 and its presentation to T-cells in T1D. These findings set the stage for using our approach as a novel therapy for patients with T1D and potentially other autoimmune diseases.

HLA-DQ distribution and risk assessment of celiac disease in a Spanish center

Aims: celiac disease is a multisystem immune-mediated disease triggered by gluten in genetically susceptible individuals. The HLA-DQ2 and/or HLA-DQ8 heterodimers are encoded by the main genetic predisposing factors and their presence is required for the development of the immunological response that leads to the disease. However, the HLA-conferred risk can differ within different countries. The aim of the study was to analyze the risk of Spanish children to develop celiac disease according to their HLA-DQ genotype. Methods: a retrospective observational case-control study was performed using a sample of 475 celiac patients and 628 controls. Results: children carrying the HLA-DQ2.5 had the highest disease risk, especially those with two HLA-DQB1*02 alleles. A similar high risk was observed in HLA-DQ8 homozygous individuals. A risk conferred by HLA-DQ8 in heterozygosity and HLA-DQ2.2 was also found and two patients with celiac disease carried the HLA-DQ7.5 haplotype as the only HLA risk factor. Conclusions: there are four genetic risk categories according to the HLA-DQ genotype. The HLA-DQ7.5 genotype does not confer risk but should not be used to rule out celiac disease when a high suspicion of the disease exists. These findings could be relevant to determine when to perform serological screening in asymptomatic subjects at risk of celiac disease

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Recomendaciones para la elaboración e interpretación de informes genéticos en enfermedad celíaca / Recommendations to report and interpret HLA genetic findings in coeliac disease

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Antibody-defined epitopes on HLA-DQ alleles reacting with antibodies induced during pregnancy and the design of a DQ eplet map.

The concept that HLA antibodies recognize epitopes is leading to new approaches of HLA matching at the epitope level. HLA-DQ plays an important role and many studies have identified structurally defined DQ epitopes specifically recognized by antibodies; they have been recorded in the International HLA Epitope Registry http://www.epregistry.com.br but the list is still incomplete. Pregnancy offers an attractive model to study antibody responses to HLA epitopes. The current analysis was done on 42 DQ-reactive post-pregnancy sera tested in binding assays with a panel of DQ heterodimers. The reactivity of 29 sera corresponded fully to the presence of antibody-verified DQA and DQB epitopes recorded in the Registry. Analysis of the remaining 13 sera led to the identification of additional antibody-defined DQB and DQA epitopes. We have designed the first version of an eplet map for DQ alleles which includes antibody-defined DQA and DQB epitopes and shows sequence positions with polymorphic residues which can be used in HLA epitology studies to identify new antibody-defined DQ epitopes.

Human leukocyte antigen (HLA) typing by DNA sequencing.

DNA sequencing is a powerful technique for identifying allelic variation within the human leukocyte antigen (HLA) genes. Sequencing is usually focused on the most polymorphic exons of the class I (HLA-A, -B, -C) and class II (HLA-DR, -DQ, and -DP) genes. These exons encode the antigen recognition site, the region of the HLA molecule that binds peptides and interacts with the T cell receptor for antigen and natural killer cell immunoglobulin-like receptors (KIR). Sanger sequencing of amplified DNA from each HLA gene from a preparation containing one or two alleles yields a sequence that is used to identify the alleles by comparison with a reference database.

Soluble HLA-DQ2 expressed in S2 cells copurifies with a high affinity insect cell derived protein.

We here describe that soluble HLA-DQ2 (sDQ2) molecules, when expressed in Drosophila melanogaster S2 insect cells without a covalently tethered peptide, associate tightly with the D. melanogaster calcium binding protein DCB-45. The interaction between the proteins is stable in S2 cell culture and during affinity purification, which is done at high salt concentrations and pH 11.5. After affinity purification, the sDQ2/DCB-45 complex exists in substantial quantities next to a small amount of free heterodimeric sDQ2 and large amounts of aggregated sDQ2 free of DCB-45. Motivated by the stable complex formation and our interest in the development of reagents which inhibit HLA-DQ2 peptide binding, we have further characterized the sDQ2/DCB-45 interaction. Several lines of evidence indicate that an N-terminal fragment of DCB-45 is involved in the interaction with the peptide binding groove of sDQ2. Further mapping of this fragment of 54 residues identified a pentadecapeptide with high affinity for sDQ2 which may serve as a lead compound for the design of HLA-DQ2 blockers.

Nasal insulin to prevent type 1 diabetes in children with HLA genotypes and autoantibodies conferring increased risk of disease: a double-blind, randomised controlled trial.

BACKGROUND: In mouse models of diabetes, prophylactic administration of insulin reduced incidence of the disease. We investigated whether administration of nasal insulin decreased the incidence of type 1 diabetes, in children with HLA genotypes and autoantibodies increasing the risk of the disease. METHODS: At three university hospitals in Turku, Oulu, and Tampere (Finland), we analysed cord blood samples of 116 720 consecutively born infants, and 3430 of their siblings, for the HLA-DQB1 susceptibility alleles for type 1 diabetes. 17 397 infants and 1613 siblings had increased genetic risk, of whom 11 225 and 1574, respectively, consented to screening of diabetes-associated autoantibodies at every 3-12 months. In a double-blind trial, we randomly assigned 224 infants and 40 siblings positive for two or more autoantibodies, in consecutive samples, to receive short-acting human insulin (1 unit/kg; n=115 and n=22) or placebo (n=109 and n=18) once a day intranasally. We used a restricted randomisation, stratified by site, with permuted blocks of size two. Primary endpoint was diagnosis of diabetes. Analysis was by intention to treat. The study was terminated early because insulin had no beneficial effect. This study is registered with ClinicalTrials.gov, number NCT00223613. FINDINGS: Median duration of the intervention was 1.8 years (range 0-9.7). Diabetes was diagnosed in 49 index children randomised to receive insulin, and in 47 randomised to placebo (hazard ratio [HR] 1.14; 95% CI 0.73-1.77). 42 and 38 of these children, respectively, continued treatment until diagnosis, with yearly rates of diabetes onset of 16.8% (95% CI 11.7-21.9) and 15.3% (10.5-20.2). Seven siblings were diagnosed with diabetes in the insulin group, versus six in the placebo group (HR 1.93; 0.56-6.77). In all randomised children, diabetes was diagnosed in 56 in the insulin group, and 53 in the placebo group (HR 0.98; 0.67-1.43, p=0.91). INTERPRETATION: In children with HLA-conferred susceptibility to diabetes, administration of nasal insulin, started soon after detection of autoantibodies, could not be shown to prevent or delay type 1 diabetes.

The production and crystallization of the human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 complexed with deamidated gliadin peptides implicated in coeliac disease.

The major histocompatibility complex (MHC) class II molecules HLA-DQ2 and HLA-DQ8 are key risk factors in coeliac disease, as they bind deamidated gluten peptides that are subsequently recognized by CD4+ T cells. Here, the production and crystallization of both HLA-DQ2 and HLA-DQ8 in complex with the deamidated gliadin peptides DQ2 alpha-I (PQPELPYPQ) and DQ8 alpha-I (EGSFQPSQE), respectively, are reported.

A novel HLA-DQB1*06 allele, DQB1*0628, found through routine sequence-based HLA typing and confirmation of DQB1*060302.

In this paper we report the identification of human leukocyte antigen (HLA)-DQB1*0628 and the confirmation of HLA-DQB1*060302.

Estudio coste-efectividad de la aplicación del diagnóstico genético en la valoración de la enfermedad celíaca / Cost-effectiveness study of using genetic diagnosis in evaluation of celiac disease

La enfermedad celíaca (EC) se desarrolla exclusivamente enindividuos genéticamente predispuestos. El objetivo de esteestudio es valorar la utilidad de la determinación del HLADQ2en el despistaje de la EC en población de riesgo. Se hanestudiado un total de 682 individuos: 174 niños celíacos,191 familiares de primer grado de pacientes celíacos, 83niños con diabetes mellitus tipo I, 64 niños con diagnósticodudoso de EC y 164 controles. El HLA-DQ2 positivo lo presentanel 34,7% de los controles, el 96% de la población decelíacos, el 66% de sus familiares de primer grado y el72,2% de los niños diabéticos. El 51,5% de los pacientescon sospecha de enfermedad celíaca y diagnóstico no concluyenteeran HLA-DQ2 positivos y, en este grupo, todos losniños con anticuerpos antiendomisio positivos portaban elHLA-DQ2 aunque en ellos no se confirmara la EC. La caracterizacióngenética de los familiares de los niños celíacos descartala enfermedad en un 34%, no siendo necesario en ellosseguimiento serológico a largo plazo. La elevada prevalenciadel HLA-DQ2 en los niños diabéticos y en pacientes con serologíapositiva de EC y diagnóstico dudoso hace que el estudiogenético sea de escasa utilidad en el despistaje de la EC

Celiac Disease (CD) is developed in only genetically susceptibleindividuals. The aim of this study is to investigatewhether HLA-DQ2 typing is helpful in exclusion of CD inpatients at risk of developing CD. 682 individuals havebeen tested: 174 children with CD, 191 of first degreerelatives of celiac children, 83 children with type 1 diabetes,64 with CD uncertain diagnosis and 164 controls.HLA-DQ2 haplotype was present in 34,7% of controls,96% of celiac children, 66% of relatives, 72,2% of diabeticchildren and 51,5% of children with uncertain diagnosisof CD. In this group, all endomysial antibodies positivepatients with non confirmed CD were HLA-DQ2 positive.HLA-DQ2 typing in first-degree relatives of celiac patientscould eliminate 34% of the population from needing serialautoantibody testing. HLA-DQ2 typing in children with type1 diabetes and children with positive serology and uncertaindiagnosis of CD is not useful because of the high prevalenceof HLA-DQ2 positivity in these study groups

Identification of a novel HLA-DQB1 allele, DQB1*0314, by sequence-based typing in the Korean population.

The polymorphism of HLA class II genes is largely confined to the exon 2 region. Sequence analysis of exon 2 of the DQB1 gene revealed the novel polymorphism in the Korean population. The new DQB1 allele, DQB1*0314, was differed from DQB1*0304 only at codon 46 (GAG-->GGG), corresponding to non-synonymous amino acid change (Glu-->Gly).

Búsqueda activa de casos entre familiares de primer grado de niños celíacos / Case finding study among first degree relatives of coeliac children

Objetivo. Conocer la prevalencia de la enfermedad celíaca entre familiares de primer grado de niños afectos de celiaquía. Estudiar el comportamiento del HLA DQ2 entre los niños y sus familiares. Material y Métodos. Estudio descriptivo transversal de serología de celíaca y de HLA DQ2 entre los familiares de primer grado de niños diagnosticados de enfermedad celíaca en nuestro Área Sanitaria entre 1992 y 2003.Resultados. Se diagnosticaron 54 casos nuevos de enfermedad celíaca en el periodo de estudio (17 varones y 37 mujeres), con una mediana de edad en la biopsia yeyunal de 24 meses. De ellos, el 89 por ciento presentaban clínica digestiva, el 46 por ciento hipocrecimiento/fallo de medro y el 2 por ciento eran asintomáticos. Todos presentaron serología y biopsia yeyunal típica de enfermedad celíaca. El 85 por ciento eran HLA DQ2 positivos. Se pudieron estudiar 44 familias de las 54 iniciales. Se realizó la serología de celíaca en 115 familiares, detectándose cuatro nuevos casos (dos hermanos, una hermana y una madre) (3,5 por ciento de los familiares estudiados). El HLA DQ2 se estudió en 110 familiares, siendo positivo en el 64 por ciento de los mismos (74 por ciento de los padres, 59 por ciento de las madres y 60 por ciento de los hermanos). Los cuatro nuevos casos eran HLA DQ2 positivos. Conclusión. El 3,5 por ciento de los familiares de primer grado de nuestros niños celíacos presentaban marcadores serológicos de celíaca. El HLA DQ2 fue positivo en el 85 por ciento de los niños celíacos y en el 64 por ciento de los familiares estudiados. Los cuatro nuevos casos detectados eran HLA DQ2 positivos (AU)

HLA class II polymorphisms determine responses to bacterial superantigens.

The excessive immunological response triggered by microbial superantigens has been implicated in the etiology of a wide range of human diseases but has been most clearly defined for the staphylococcal and streptococcal toxic shock syndromes. Because MHC class II presentation of superantigens to T cells is not MHC-restricted, the possibility that HLA polymorphisms could influence superantigenicity, and thus clinical susceptibility to the toxicity of individual superantigens, has received little attention. In this study, we demonstrate that binding of streptococcal and staphylococcal superantigens to HLA class II is influenced by allelic differences in class II. For the superantigen streptococcal pyrogenic exotoxin A, class II binding is dependent on DQ alpha-chain polymorphisms such that HLA-DQA1*01 alpha-chains show greater binding than DQA1*03/05 alpha-chains. The functional implications of differential binding on T cell activation were investigated in various experimental systems using human T cells and murine Vbeta8.2 transgenic cells as responders. These studies showed quantitative and qualitative differences resulting from differential HLA-DQ binding. We observed changes in T cell proliferation and cytokine production, and in the Vbeta specific changes in T cell repertoire that have hitherto been regarded as a defining feature of an individual superantigen. Our observations reveal a mechanism for the different outcomes seen following infection by toxigenic bacteria.

A novel HLA-DQB1 allele, DQB1*05022, isolated from the Jing ethnic group in South-west China.

A novel DQB1 allele, DQB1*05022, has been identified from an individual of the Jing ethnic group in South-west China. The sequence was confirmed by cloning and sequencing. The allele differs from DQB1*05021 at codon 47 (TAC to TAT) and from DQB1*05031 at codon 57 (GAC to AGC).

Influence of MHC class II genotype on outcome of infection with hepatitis C virus. The HENCORE group. Hepatitis C European Network for Cooperative Research.

BACKGROUND: The outcome of infection with hepatitis C virus (HCV) varies substantially among individuals. Host genetic factors are likely to give rise to some of this variability. Polymorphisms in the MHC class II loci may influence the outcome of HCV infection; however, reports of MHC class II allele associations have been inconsistent. We aimed to confirm these associations in a cohort of European patients with different clinical outcomes. METHODS: The distribution of MHC class II alleles was compared between patients with self-limiting infection (n=85) and matched patients with persistent infection (n=170); between patients with mild (n=321) and severe (n=321) histological injury; and between patients who responded to interferon (n=96) and those who did not (n=192). The results of these comparisons were confirmed with a second-stage study of self-limiting infection (n=52) versus persistent infection (n=152). FINDINGS: Self-limiting HCV infection was associated with HLA-DRB1*1101 (odds ratio 2.14 [95% CI 1.11-4.12]; p=0.013) and HLA-DQB1*0301 (2.22 [1.24-3.96], p=0.004). Persistent HCV infection was associated with HLA-DRB1*0701 (2.04 [1.03-4.17], p=0.027), and HLA-DRB4*0101 (2.38 [1.29-4.35], p=0.002). These results were confirmed in the second-stage study. No significant associations (p<0.05 after Bonferroni correction) were found between MHC class II alleles and severe histological injury or response to interferon therapy. INTERPRETATION: Specific MHC class II alleles influence susceptibility or resistance to persistent HCV infection.

MICA gene polymorphism and the risk to develop cervical intraepithelial neoplasia.

Cervical intraepithelial neoplasia (CIN) is associated with human papillomaviruses (HPV) and the HLA genes. The MICA (MHC class I chain-related gene A) is expressed by keratinocytes and epithelial cells and interacts with gamma delta T cells. It is therefore possible that MICA might influence the pathogenesis of CIN and cervical cancer through presentation of viral or tumor antigens. To investigate this, we determined the MICA transmembrane allele frequencies in a prospective population-based cohort study from the Västerbotten County in northern Sweden. 74 women developed CIN. 153 control women who remained healthy during follow up were matched for age. Five polymorphic microsatellite alleles of MICA were identified by a polymerase chain reaction-based (PCR) technique using fluorescent-labeled primers. MICA A5 and A5.1 were the most common alleles in this population. None of the alleles of MICA were associated with disease. The frequency of MICA allele A5 was higher among HPV 18 seropositive than HPV 18 seronegative patients but this difference was not significant after the correction of p value. In conclusion, microsatellite allele polymorphism of MICA transmembrane part is not associated with cervical intraepithelial neoplasia.

Jejuna of patients with insulin-dependent diabetes mellitus (IDDM) show signs of immune activation.

The roles of enteric viruses and food antigens as possible triggers in human insulin-dependent diabetes mellitus and the evidence that mucosal-associated homing receptors are important in both human and experimental diabetes prompted us to undertake an immunohistochemical study of intestinal specimens from patients with IDDM. We studied jejunal morphology and immunohistochemistry in 26 patients with IDDM, 13 of whom had the HLA-DQB1*0201 gene and therefore a higher risk of coeliac disease. The findings were compared with those in specimens from age-matched controls. Villous structure and the density of the intraepithelial lymphocytes were normal in every biopsy specimen. The extent of positivity with anti-DR and -DP antibodies in the villous epithelium was significantly greater in the specimens from patients than in those from controls (P = 0.0002 in both comparisons). The crypts were also more positive: for DR P = 0.0001, and for DP P = 0.002. The densities of T cells, CD4+, CD8+, and T cell receptor alpha/beta+ and gamma/delta+ cells in the epithelium and lamina propria were similar in patients and controls, but the patients had significantly more alpha 4/beta 7 integrin+ cells in the lamina propria (P = 0.006). No difference was seen between HLA-DQB1*0201-positive and -negative patients. These findings reflect a stage of inflammation in the structurally normal intestines of patients with IDDM and suggest secretion of inflammatory Th1-type cytokines in the intestine.

HLA-DQB1 locus and the development of atrophic gastritis with Helicobacter pylori infection.

Helicobacter pylori is known to be involved in digestive diseases such as peptic ulcer, atrophic gastritis, and gastric cancer. It is supposed that the incidence of these digestive diseases associated with H. pylori is influenced by the strain diversity of H. pylori, factors involving the host or environment, and the duration of infection. In this study, we directed our attention to HLA, a host factor, and investigated the relation between HLA-DQB1 genotype of H. pylori-infected patients and the development of atrophic gastritis. HLA-DQB1 genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism method on 122 H. pylori-infected patients with atrophic gastritis and 28 uninfected Japanese controls. Infected patients with developed atrophic gastritis were classified as the open type and those with undeveloped atrophic gastritis as the closed type. To estimate the grade of atrophic gastritis reliably, histological and serological evaluations were also undertaken. The allele frequency of DQB1*0401 was significantly higher in the open-type group compared to either the closed-type or the uninfected group. These results suggest that immunogenic factors play an important role in the development of atrophic gastritis in H. pylori-infected patients, and that DQB1*0401 is a useful marker for determining susceptibility to this disease.

Use of complete eluted peptide sequence data from HLA-DR and -DQ molecules to predict T cell epitopes, and the influence of the nonbinding terminal regions of ligands in epitope selection.

In diseases with a strong association with an HLA haplotype, identification of relevant T cell epitopes may allow alteration of the pathologic process. In this report we use a reverse immunogenetic approach to predict possible HLA class II-restricted T cell epitopes by using complete pool sequencing data. Data from HLA-DR2(B1*1501), -DR3(B1*0301), -DQ2(A1*0501, B1*0201), and -DQ8(A1*0301, B1*0302) alleles were used by a computer program that searches a candidate protein to predict ligands with a relatively high probability of being processed and presented. This approach successfully identified both known T cell epitopes and eluted single peptides from the parent protein. Furthermore, the program identified ligands from proteins in which the binding motif of the HLA molecule was unable to do so. When the information from the nonbinding N- and C-terminal regions in the pool sequence was removed, the ability to predict several ligands was markedly reduced, particularly for the HLA-DQ alleles. This suggests a possible role for these regions in determining ligands for HLA class II molecules. Thus, the use of complete eluted peptide sequence data offers a powerful approach to the prediction of HLA-DQ and -DR peptide ligands and T cell epitopes.