DQ Molecules Research Articles

Characterisation of the HLA-DQ alpha eplet 52SK targeting the DQA1*01 alleles family.

Eplet 52SK is unique in the HLA eplet registry as targeting the whole family of DQA1*01 alleles. It is proposed as an antibody-verified eplet but has not been validated enough to deserve this label. Especially, confusion can occur with reactivity targeting the 52PQ eplet which is present on the DQB1*05 and DQB1*06 alleles families, as DQ molecule stability imposes DQA1*01 to selectively associate with these DQ-β families only. Using two Luminex single antigen (LSA) assays from two vendors, beads bearing DR-α/DQ6 heterodimers, a special build LSA panel of additional DQ beads, and an adsorption/elution strategy relying on cells from deceased donors or recombinant cells solely expressing one DQ antigen, we definitely established the antibody-verified status of eplet 52SK using patients' sera reacting only against the DQ5 and DQ6 beads of the One Lambda LSA panel in routine patients' follow up. We also show that reactivity against this eplet is not a rare event among anti-DQ1 immunisation. This study further strengthens the importance of considering the DQA1 locus in immunological studies of HLA and in organ allocation strategies.

Calcium-dependent HLA-DQ epitope revealed by EDTA mediated inhibition of antibody reactions in the Luminex single antigen bead assay.

Complement mediated interference with the detection of antibodies targeting HLA is a known limitation of the single antigen bead (SAB) Luminex assay. Ethylenediaminetetraacetic acid (EDTA) is currently the serum treatment of choice in most histocompatibility laboratories to block complement activation by chelating calcium. The purpose of this study was to investigate a serum with an antibody reactivity to HLA-DQ6, 7, 8 and 9 molecules, in the Luminex SAB assay, that was inhibited by treatment with EDTA. Serum was from a 55-year-old highly sensitised female renal transplant candidate that contained, among others, antibodies to an epitope containing the 74EL eplet, shared by HLA-DQ6, DQ7, DQ8 and DQ9 molecules. Serum samples were treated with EDTA, dithiothreitol (DTT), or heat prior to testing by SAB assay. EDTA-treated serum was also tested after the addition of calcium chloride (CaCl2). HLA-DQ-specific antibodies were isolated by adsorption/elution method using three informative donor cells and were tested in the absence or presence of EDTA. The antibody reactivity against HLA-DQ6, DQ7, DQ8 and DQ9 in the SAB assay was significantly inhibited by treating serum and eluates with EDTA and was restored by addition of CaCl2. The study represents the first description of a calcium-dependent epitope in HLA molecules. The relevance of this finding is that the treatment of sera with EDTA could lead to false-negative reactions in the SAB assay, which may compromise virtual crossmatching.

Immunological markers of rheumatoid arthritis in the Moroccan population

Aim: few studies exist on HLA-DRB1 alleles and their association with RA in North Africa. We aimed to provide an evaluation of the distribution of the HLA-DRB1* and -DQB1* genes in Moroccan patients with early RA. Additionally, we sought to analyze the relationship between HLA molecules and the production of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP) in these patients. Methods: 65 patients with rheumatoid arthritis benefited from HLA class II typing (DRB1* and DQB1*) and testing for anti-CCP and RF antibodies. The frequency of HLA alleles was compared to that of 180 healthy controls. Logistic regression analysis was used to investigate the association of HLA-DR and -DQ molecules in the different subgroups according to anti-CCP and RF status. Results: The frequency of HLA-DRB1*04 and *11 alleles is increased in patients compared to controls (OR = 1.9; 95% CI (1.06-3.30), P=0.029; OR=2, 95% CI (1-3.83, P=0.047, respectively). 56 patients (86.1%) were RF positive, and 67.7% were anti-CCP positive. A significant increase in the frequency of DRB1*04 alleles was also noted in seropositive patients. Conclusion: Our results suggest the predisposition of the HLA-DRB1*04 allele and the seropositive status of patients with RA. This could be useful in predicting the evolution of RA and establishing a diagnosis in some patients at an early stage of disease use.

Changes upon the gluten-free diet of HLA-DQ2 and TRAFD1 gene expression in peripheral blood of celiac disease patients

BackgroundCeliac disease (CD) is a chronic immuno-mediated enteropathy caused by dietary gluten in genetically susceptible individuals carrying HLA (Human Leukocytes Antigen) genes that encode for DQ2.5 and DQ8 molecules. TRAFD1 (TRAF-type zinc finger domain 1) is a gene recently found associated with CD and defined as a master regulator of IFNγ signalling and of MHC class I antigen processing/presentation. There is no specific drug therapy and the only effective treatment is the gluten-free diet (GFD). The great majority of celiac patients when compliant with GFD have a complete remission of symptoms and recovery of gut mucosa architecture and function. Until now, very few studies have investigated molecular differences occurring in CD patients upon the GFD therapy. MethodsWe looked at the expression of both HLA DQ2.5 and TRAFD1 risk genes in adult patients with acute CD at the time of and in treated patients on GFD. Specifically, we measured by qPCR the HLA-DQ2.5 and TRAFD1 mRNAs on peripheral blood mononuclear cells (PBMC) from the two groups of patients. ResultsWhen we compared the HLA-DQ mRNA expression, we didn't find significant variation between the two groups of patients, thus indicating that GFD patients have the same capability to present gliadin antigens to cognate T cells as patients with active disease. Conversely, TRAFD1 was more expressed in PBMC from treated CD subjects. Notably, TRAFD1 transcripts significantly increased in the patients analyzed longitudinally during the GFD, indicating a role in the downregulation of gluten-induced inflammatory pathways. ConclusionOur study demonstrated that HLA-DQ2.5 and TRAFD1 molecules are two important mediators of anti-gluten immune response and inflammatory process.

HLA DQ protein changes the cell surface distribution pattern of HLA proteins as monitored by Förster resonance energy transfer and high-resolution electron microscopy.

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II-deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6-BLS-1, and were able to detect the effect of the appearance of HLA-DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS-1 and JY human B-cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA-DQ6-transfected tDQ6-BLS-1 cells compared with the parent BLS-1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA-DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6-BLS-1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response.

HLA-DR3 mediated CD4 T cell response against GAD65 in type 1 diabetes patients.

We planned this study to identify diabetogenic glutamic acid decarboxylase (GAD65) peptides possibly responsible for human leucocyte antigen (HLA)-DR3/DQ2-mediated activation of GAD65-specific CD4 T cells in type 1 diabetes (T1D). Top 30 GAD65 peptides, found to strongly bind in silico with HLA-DR3/DQ2 molecules, were selected and grouped into four pools. The peptides were used to stimulate CD4 T cells of study subjects in 16-h peripheral blood mononuclear cell culture. CD4 T cells' stimulation in terms of interferon-gamma (IFN-γ), interleukin (IL)-17, tumor necrosis factor-alpha (TNF-α), and IL-10 expression was analyzed using flow cytometry. Although all four GAD65 peptide pools (PP1-4) resulted in significantly higher expression of IFN-γ by CD4 T cells (p = .003, p < .0001, p = .026, and p = .002, respectively), only pool 2 showed significant increase in IL-17 expression (p < .0001) in T1D patients vs healthy controls. Interpeptide group comparison for immunogenicity revealed significantly higher IFN-γ and IL-17 expressions and significantly lower IL-10 expression for PP2 compared to other groups (p < .0001, p = .02, and p = .04, respectively) in patients but not in controls. Further, group 2 peptides resulted in significant increase in CD4 T cells' expression of IFN-γ and IL-17 (p = .002 for both) and significant decrease in IL-10 (p = .04) in HLA-DRB1*03-DQA1*05-DQB1*02+ patients vs HLA-DRB1*03-DQA1*05-DQB1*02+ controls. The CD4 T cells' expression of IL-17 was significantly higher (p = .03) in recently diagnosed vs long-standing HLA-DRB1*03-DQA1*05-DQB1*02+ T1D patients. GAD65 peptides, particularly those belonging to PP2, induced CD4 T cells to express IFN-γ and IL-17 cytokines in T1D patients, suggesting that group 2 peptides possibly presented by HLA-DR3 molecule to CD4 T cells shift immune balance toward inflammatory phenotype in patients.

Association Between Type 1 Diabetes Mellitus and Celiac Disease: Autoimmune Disorders With a Shared Genetic Background

Type 1 diabetes mellitus (T1DM) and celiac disease (CD) are one of the most recognized related autoimmune disorders as they share a common genetic background that has been found in the HLA genotype, more specifically DQ2 and DQ8 molecules. Studies have shown that environmental factors as early or late exposure to cereals in the first months of life or the acquired viral infections have been implicated in the risk of development of autoantigens. CD, in most cases, is asymptomatic; therefore, it goes underdiagnosed. As a result, it has been linked to late consequences as decreased growth, delayed puberty, and anemia. Also, CD has been considered an independent risk factor for nephropathy and retinopathy. Therefore, in T1DM patients, as high-risk individuals, a CD screening has been recommended, especially to analyze their joint management. A gluten-free diet has been studied and linked to possible benefits in glycemic control or decreasing the hypoglycemic episodes in T1DM and preventing in CD the late bowel mucosal damage as gluten has been well documented as the primary trigger of these autoimmune responses. This article has reviewed the concurrent occurrence of T1DM and CD regarding the pathogenesis, clinical overlaps, screening, and management options.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells

PurposeLimbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. MethodsHuman corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. ResultsLMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. ConclusionThese findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.

Celiac disease: Understandings in diagnostic, nutritional, and medicinal aspects

Celiac disease (CD) is characterized by clinical polymorphism, with classic, asymptomatic or oligosymptomatic, and extra-intestinal forms, which may lead to diagnostic delay and exposure to serious complications. CD is a multidisciplinary health concern involving general medicine, pediatric, and adult gastroenterology, among other disciplines. Immunology and pathology laboratories have a fundamental role in diagnosing and monitoring CD. The diagnosis consists of serological testing based on IgA anti-transglutaminase (TG2) antibodies combined with IgA quantification to rule out IgA deficiency, a potential misleading factor of CD diagnosis. Positive TG2 serology should be corroborated by anti-endomysium antibody testing before considering an intestinal biopsy. Owing to multiple differential diagnoses, celiac disease cannot be confirmed based on serological positivity alone, nor on isolated villous atrophy. In children with classical signs or even when asymptomatic, with high levels of CD-linked markers and positive HLA DQ2 and/or DQ8 molecules, the current trend is to confirm the diagnosis on basis of the non-systematic use of the biopsy, which remains obligatory in adults. The main challenge in managing CD is the implementation and compliance with a gluten-free diet (GFD). This explains the key role of the dietitian and the active participation of patients and their families throughout the disease-management process. The presence of the gluten in several forms of medicine requires the sensitization of physicians when prescribing, and particularly when dispensing gluten-containing formulations by pharmacists. This underlines the importance of the contribution of the pharmacist in the care of patients with CD within the framework of close collaboration with physicians and nutritionists.

Structure of the HLA-DR-DQ-genotype in children with coeliac disease

Introduction. Coeliac disease is a genetically determined disease associated with the HLA-DQ2 and DQ8 genes. According to European data, these alleles are detected in more than 95% of patients. The results of earlier studies in various regions of the Russian Federation and the constituent republics have shown a lower incidence of HLA-DQ2/DQ8 in children with coeliac disease (60–80%). These data served as a basis for discussion about regional genetic features and the potential for disease development in a significant proportion of patients without DQ2/DQ8 alleles.Methods. Genetic screening was conducted on 275 children with coeliac disease. According to the results of the study, the patients were assigned to genetic risk groups.Results. HLA-DQ2/DQ8-alleles were detected in 274 children (99.6%), with 86.9% of patients having DQ2 molecule in genotype and 12.7% having DQ8 molecule. Among 239 DQ2-positive patients, 33.5% were DQ2 homozygous. The remaining DQ2-positive patients had the following HLA-DR-DQ allele distribution: DR3-DQ2/DR5-DQ7 – 8.7%, DR7-DQ2/DR5-DQ7 – 13.4%, DR3-DQ2/ DRx-DQx – 22.2%, DR7-DQ2/DRx-DQx – 2.2%. In five children the incomplete DQ2 molecule was determined, represented by DQA1*0501 allele, while in four patients DQA1*501 was associated with DQB1*0301 allele forming DR5-DQ7 molecule. We found high and significant risk alleles in 29.1% and 22.2% of cases, while moderate and low risk alleles were found in 27.3% and 21.4% of cases respectively.Conclusions: In Russian children with coeliac disease the prevalence of HLA-DQ2/DQ8 alleles corresponds to European data. Low detection frequency of HLA-DQ2/DQ8 molecules in Russian coeliac patients, which was reported earlier, may be due to peculiarities of the approach to the disease diagnostics, which do not correspond to modern Russian and international recommendations.

A19 NOVEL HLA-DQ2 TRANSGENIC MICE DEVELOP GLUTEN-IMMUNOPATHOLOGY FOLLOWING GLUTEN SENSITIZATION

Abstract Background Celiac disease (CeD), an autoimmune and chronic inflammatory enteropathy triggered by the ingestion of gluten, is associated with HLA-DQ2 (~90%) and, to a lesser extent, HLA-DQ8. We previously characterized a humanized mouse model of gluten sensitivity that expresses the HLA-DQ8 allele, and that develops mild innate and adaptive immune pathways related to DQ8+ CeD patients. Gluten peptides recognized in the context of DQ2 differ from those bound to DQ8 molecules and homozygous carriage of HLA-DQ2 provides higher risk for CeD development. Thus, characterization of a transgenic HLA-DQ2 model would allow for the investigation of disease pathways that affect the majority of celiac patients. Aims Our aim was to determine gluten-immune responses and small intestinal pathology in C57BL/6 mice humanized with the HLA DR3-DQ2 gene. Methods To break oral tolerance to gluten, DR3-DQ2 mice were orally sensitized with gliadin (5mg/mL) and cholera toxin (CT; 2.5mg/mL) before being challenged with gluten (10mg/mL) for three weeks. Non-sensitized DR3-DQ2 controls mice received CT and a sham challenge. To determine the effects of long-term exposure to gluten, gliadin sensitized DR3-DQ2 mice were placed on a gluten-containing diet for twelve weeks. Controls were given CT and remained on a gluten-free diet. Pathology was evaluated by CD3+ intraepithelial lymphocytes (IELs) counts; villus-crypt (V/C) ratios. Gluten-induced immune responses were evaluated by anti-tissue transglutaminase-2 (tTG) and anti-gliadin antibodies, inflammatory gene expression by Nanostring Technology, and CD4+ T cell proliferation using flow cytometry. Intestinal permeability was measured in vitro by Ussing Chamber. Results DR3-DQ2 mice that were sensitized and challenged with gluten for three weeks had higher IEL counts, lower V/C ratios, higher anti-gliadin and tTG antibodies, which are used in the serological diagnosis of the disease. Gluten sensitized mice also has higher expression of pro-inflammatory genes, and an induction of gluten specific T cells, but no changes in permeability compared to control mice. DR3-DQ2 mice given a gluten-containing diet for 12 weeks also had higher IEL counts, lower V/C ratios, and higher anti-gliadin and tTG antibodies, but had no changes in permeability compared to controls. Conclusions These results indicate that mice humanized with the HLA DR3-DQ2 gene show innate and gluten-specific immune responses following sensitization. Thus, mice expressing HLA-DQ2 represents a novel model of gluten sensitivity that can be used to investigate celiac disease pathways related to gluten peptide repertoire that binds to DQ2 MHC class II, encoded by the HLA gene expressed by the majority of celiac patients. Supported by CIHR to EFV and OGS to AVC Funding Agencies CIHROGS

A molecular basis for the T cell response in HLA-DQ2.2 mediated celiac disease

The highly homologous human leukocyte antigen (HLA)-DQ2 molecules, HLA-DQ2.5 and HLA-DQ2.2, are implicated in the pathogenesis of celiac disease (CeD) by presenting gluten peptides to CD4+ T cells. However, while HLA-DQ2.5 is strongly associated with disease, HLA-DQ2.2 is not, and the molecular basis underpinning this differential disease association is unresolved. We here provide structural evidence for how the single polymorphic residue (HLA-DQ2.5-Tyr22α and HLA-DQ2.2-Phe22α) accounts for HLA-DQ2.2 additionally requiring gluten epitopes possessing a serine at the P3 position of the peptide. In marked contrast to the biased T cell receptor (TCR) usage associated with HLA-DQ2.5-mediated CeD, we demonstrate with extensive single-cell sequencing that a diverse TCR repertoire enables recognition of the immunodominant HLA-DQ2.2-glut-L1 epitope. The crystal structure of two CeD patient-derived TCR in complex with HLA-DQ2.2 and DQ2.2-glut-L1 (PFSEQEQPV) revealed a docking strategy, and associated interatomic contacts, which was notably distinct from the structures of the TCR:HLA-DQ2.5:gliadin epitope complexes. Accordingly, while the molecular surfaces of the antigen-binding clefts of HLA-DQ2.5 and HLA-DQ2.2 are very similar, differences in the nature of the peptides presented translates to differences in responding T cell repertoires and the nature of engagement of the respective antigen-presenting molecules, which ultimately is associated with differing disease penetrance.

Graphene covalently functionalized with 2,6-diaminoanthraquinone (DQ) as a high performance electrode material for supercapacitors

2,6-Diaminoaquinone molecules were covalently modified onto the surface of GO via a nucleophilic displacement reaction between the epoxy groups on the surface of GO and the –NH2 groups of DQ molecules in the presence of ammonia to form a composite material.

A pilot study of preproinsulin peptides reactivity in Chinese patients with type 1 diabetes.

BackgroundThe aim of our study is to investigate whether preproinsulin (PPI) could trigger a proinflammatory CD4+ T cell response in Chinese patients with type 1 diabetes (T1D).MethodsPeripheral blood mononuclear cells were stimulated by a pool of 13 PPI peptides. Additional five PPI peptides previously proved to be antigenic in other cohorts of patients with T1D were also used. PPI reactive T cell responses were measured by interferon (IFN)‐γ ELISPOT assay.ResultsFifty‐one Chinese patients with T1D were enrolled in this study and 72.34% of them were positive for at least one islet autoantibody. The stimulation index (SI) value of IFN‐γ response to PPI peptide pool or peptides with dominant epitopes was below 3 in patients when SI≥3 was used as the positive cut‐off value. Two peptides (B9‐23 and C19‐A3) restricted to DQ8 or DR4 molecule failed to induce positive IFN‐γ response in patients with high‐risk HLA‐DQ8 or HLA‐DR4/DR9 alleles. RNA‐seq analysis of PPI specific CD4+ T cell lines further showed that most of the IFN‐γ associated genes remained unchanged.ConclusionsThis is the first report of CD4+ T cell epitope mapping of PPI in Chinese T1D. The lack of positive IFN‐γ response to PPI peptides indicates that PPI might not be the principal antigenic candidate for autoreactive CD4+ T cells in Chinese T1D. Therefore, the efficacy of PPI‐based immunotherapies in attenuating proinflammatory CD4+ T cell response requires further investigation.

Association of HLA-DRB1 genotype with younger age onset and elder age onset rheumatoid arthritis in Japanese populations.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by joint destructions and human leukocyte antigen (HLA)-DRB1 is an important genetic risk factor for RA and influences the phenotype of RA. The clinical features of elder age onset RA (EORA) were known to be different from those of younger age onset RA (YORA). Previous studies reported the different association pattern of DRB1 alleles with YORA or EORA. The associations of DRB1 genotype with these RA subsets remained almost unknown. We investigated the genotype association of DRB1 with YORA or EORA in Japanese populations.HLA genotyping was performed in Japanese RA patients and the association of allele or genotype carrier frequencies were analyzed.The genotype frequency of DRB104:05/DRB104:06 (P = .0204, OR 7.69, 95%CI 1.39-42.72), DRB104:05/DRB112:01 (P = .0050, OR 5.53, 95%CI 1.71-17.88), and DRB104:05/DRB115:01 (P = .0124, OR 3.34, 95%CI 1.39-8.02) in YORA was higher than EORA. However, the frequencies of DRB101:01/DRB104:05 in YORA was tended to be lower than EORA (P = .0784, OR 0.14, 95%CI 0.01-2.42). The gene dosage effect of the shared epitope alleles was detected in EORA, but not in YORA. Trans-complementing DQ heterodimer molecules, formed by DQA1 and DQB1 of the haplotypes with and without shared epitope alleles, might explain the higher genotype frequencies of "shared epitope /not shared epitope". Linear regression analyses showed the primary role of DQB104:01 allele for the age at onset of RA.This is the first report for the associations of DRB1 genotype with YORA or EORA in the Japanese population and the differential distribution of the genotypes was noted between these RA subsets. The involvement of DQ molecules for the age at onset of RA was suggested.

Two-Photon Fluorescent Nanoprobe for Glutathione Sensing and Imaging in Living Cells and Zebrafish Using a Semiconducting Polymer Dots Hybrid with Dopamine and β-Cyclodextrin.

Fabrication of hybrid semiconducting polymer dots (Pdots) endowed with special applications in biosensing and bioimaging in living systems has recently received considerable attention. In this study, novel two-photon fluorescent hybrid Pdots, DA-CD@Pdots, were first prepared by poly(styrene-co-maleic anhydride) (PSMA) being grafted with β-cyclodextrin (β-CD) and poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadiazole)] (PFBT) through a nanoprecipitation method, followed by covalent attachment with dopamine (DA) by using an EDC-catalyzed carboxylamine coupling reaction. The DA molecules anchored on the surface of the Pdots were further oxidized to form their quinone-like structures (DQ) and act as good electron acceptors to magnifyingly quench the fluorescence of Pdots by intraparticle photoinduced electron transfer (PET) and the "molecular-wire effect". The finally achieved hybrid DQ-CD@Pdots display enhanced colloidal stability, higher resistibility to environmental effects, and lower biological toxicity. In the presence of glutathione (GSH), DQ molecules on the surface of Pdots are reduced into catechol molecules and result in the inhibition of PET and restoration of the fluorescence of the Pdots. On the basis of the above demonstrations, the hybrid DQ-CD@Pdots are used as fluorescent probes for "turn-on" detection of GSH in the range from 0.01 to 3.0 μM with the detection limit of 2.7 nM. The prepared DQ-CD@Pdots probe is also applied to the GSH detection and imaging in living systems including human cervical carcinoma HeLa cells and living zebrafish with satisfactory results.

HLA and microtubule-associated protein tau H1 haplotype associations in anti-IgLON5 disease.

ObjectivesWe investigated the associations with HLA and microtubule-associated protein tau (MAPT) H1 haplotype in anti-IgLON5 disease, a recently identified disorder characterized by gait instability, brainstem dysfunction, and a prominent sleep disorder in association with IgLON5 antibodies and pathologic findings of a novel neuronal-specific tauopathy.MethodsWe compared the HLA alleles and MAPT H1/H1 genotype of 35 patients with anti-IgLON5 with healthy controls. The on-line server tool NetMHCIIpan 3.1 was used to predict the IgLON5 peptide binding to HLA Class II molecules.ResultsThe HLA-DRB1*10:01-DQB1*05:01 haplotype was overrepresented in patients with anti-IgLON5 disease (OR = 54.5; 95% CI: 22.2–133.9, p < 0.0001). In addition, HLA-DQA was genotyped in 27 patients, and 25 (92.6%) of them had DQ molecules composed by DQA1*01 and DQB1*05 chains compared with 148/542 (27.3%) controls (OR = 43.9; 95% CI: 10.4–185.5, p < 0.0001). Patients DRB1*10:01 positive developed more frequently sleep or bulbar symptoms than those carrying other HLA alleles (70.0% vs 26.7%; p = 0.011). Prediction algorithms identified 2 IgLON5 peptides (1 located in the signal sequence) that showed strong binding to HLA-DRB1*10:01 and other HLA-DRB1, but not to HLA-DQA and HLA-DQB molecules. The MAPT H1/H1 homozygous genotype was present in 20/24 (83.3%) anti-IgLON5 Caucasian patients compared with 54/116 (46.5%) healthy controls (p = 0.0007).ConclusionsThe robust association of anti-IgLON5 disease with distinct HLA Class II molecules supports a primary autoimmune origin. The significant association of MAPT H1 haplotype also suggests that an underlying neurodegenerative process could be involved in anti-IgLON5 disease.

HLA-DQA1 and HLA-DQB1 Alleles, Conferring Susceptibility to Celiac Disease and Type 1 Diabetes, are More Expressed Than Non-Predisposing Alleles and are Coordinately Regulated.

HLA DQA1*05 and DQB1*02 alleles encoding the DQ2.5 molecule and HLA DQA1*03 and DQB1*03 alleles encoding DQ8 molecules are strongly associated with celiac disease (CD) and type 1 diabetes (T1D), two common autoimmune diseases (AD). We previously demonstrated that DQ2.5 genes showed a higher expression with respect to non-CD associated alleles in heterozygous DQ2.5 positive (HLA DR1/DR3) antigen presenting cells (APC) of CD patients. This differential expression affected the level of the encoded DQ2.5 molecules on the APC surface and established the strength of gluten-specific CD4+ T cells response. Here, we expanded the expression analysis of risk alleles in patients affected by T1D or by T1D and CD comorbidity. In agreement with previous findings, we found that DQ2.5 and DQ8 risk alleles are more expressed than non-associated alleles also in T1D patients and favor the self-antigen presentation. To investigate the mechanism causing the high expression of risk alleles, we focused on HLA DQA1*05 and DQB1*02 alleles and, by ectopic expression of a single mRNA, we modified the quantitative equilibrium among the two transcripts. After transfection of DR7/DR14 B-LCL with HLA-DQA1*05 cDNA, we observed an overexpression of the endogenous DQB1*02 allele. The DQ2.5 heterodimer synthesized was functional and able to present gluten antigens to cognate CD4+ T cells. Our results indicated that the high expression of alpha and beta transcripts, encoding for the DQ2.5 heterodimeric molecules, was strictly coordinated by a mechanism acting at a transcriptional level. These findings suggested that, in addition to the predisposing HLA-DQ genotype, also the expression of risk alleles contributed to the establishment of autoimmunity.

Characterization of Magnitude and Antigen Specificity of HLA-DP, DQ, and DRB3/4/5 Restricted DENV-Specific CD4+ T Cell Responses.

Background: Dengue Virus (DENV) associated disease is a major public health problem. Assessment of HLA class II restricted DENV-specific responses is relevant for immunopathology and definition of correlates of protection. While previous studies characterized responses restricted by the HLA-DRB1 locus, the responses associated with other class II loci have not been characterized to date. Accordingly, we mapped HLA-DP, DQ, and DRB3/4/5 restricted DENV-specific CD4 T cell epitopes in PBMCs derived from the DENV endemic region Sri Lanka.Methods: We studied 12 DP, DQ, and DRB3/4/5 alleles that are commonly expressed and provide worldwide coverage >82% for each of the loci analyzed and >99% when combined. CD4+ T cells purified by negative selection were stimulated with pools of HLA-predicted binders for 2 weeks with autologous APC. Epitope reactive T cells were enumerated using IFNγ ELISPOT assay. This strategy was previously applied to identify DRB1 restricted epitopes. In parallel, membrane expression levels of HLA-DR, DP, and DQ proteins was assessed using flow cytometry.Results: Epitopes were identified for all DP, DQ, and DRB3/4/5 allelic variants albeit with magnitudes significantly lower than the ones previously observed for the DRB1 locus. This was in line with lower membrane expression of HLA-DP and DQ molecules on the PBMCs tested, as compared to HLA-DR. Significant differences between loci were observed in antigen immunodominance. Capsid responses were dominant for DRB1/3/4/5 and DP alleles but negligible for the DQ alleles. NS3 responses were dominant in the case of DRB1/3/4/5 and DQ but absent in the case of DP. NS1 responses were prominent in the case of the DP alleles, but negligible in the case of DR and DQ. In terms of epitope specificity, repertoire was largely overlapping between DRB1 and DRB3/4/5, while DP and DQ loci recognized largely distinct epitope sets.Conclusion: The HLA-DP, DQ, and DRB3/4/5 loci mediate DENV-CD4 specific immune responses of lower magnitude as compared to HLA-DRB1, consistent with their lower levels of expression. The responses are associated with distinct and characteristic patterns of immunodominance, and variable epitope overlap across loci.

Relevance of HLA-DQB1*02 Allele in the Genetic Predisposition of Children with Celiac Disease: Additional Cues from a Meta-Analysis

Background and Objectives: Celiac disease (CD) is a multifactorial immune-mediated disorder, triggered by the ingestion of gluten in genetically-predisposed subjects carrying MHC-DQ2 and -DQ8 heterodimers, which are encoded by four HLA-DQ allelic variants, overall. This meta-analysis aims at providing further epidemiological support to the predominant relevance of one specific allele, namely HLA-DQB1*02, in the predisposition and genetic risk of CD. Materials and Methods: We performed a search of MEDLINE/PubMed, Embase, Web of Science, and Scopus, retrieving all publications (case–control study, cross-sectional, and retrospective cohort study) on the association between HLA class II polymorphisms and first-degree relatives (FDRs) of children with CD. After a critical reading of the articles, two investigators independently performed data extraction according to the following inclusion criteria: HLA class II genes, any DQ and DR molecules, and CD diagnosed following the current clinical guidelines. A third participant was consulted for discussion to reach an agreement concerning discrepancies. Results: Our search strategy selected 14 studies as being eligible for inclusion, and those were submitted for data extraction and analysis. These studies were published between 1999 and 2016 and, collectively, enrolled 3063 FDRs. Positive and negative likelihood ratios (LR+ and LR−, respectively) for CD diagnosis, according to the presence of the HLA-DQ genotype coding a complete MHC-DQ2 and/or MHC-DQ8 molecules, were 1.449 (CI 1.279–1.642) and 0.187 (CI 0.096–0.362), respectively. If only the isolated presence of HLA-DQB1*02 allele is considered, the pooled estimation of LR+ was 1.659 (CI 1.302–2.155) and, importantly, the LR− still showed a very good discriminatory power of 0.195 (CI 0.068–0.558). Conclusions: Through our differential meta-analysis, comparing the presence of the genotype coding the full MHC-DQ2 and/or DQ8 molecules with the isolated presence of HLA-DQB1*02 allelic variant, we found that the LR− of the latter analysis maintained the same value. This observation, along with previous evidences, might be useful to consider potential cost-effective widened screening strategies for CD in children.