Respiratory complex I regulates dendritic cell maturation in explant model of human tumor immune microenvironment.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Manually pressurized droplet digital PCR chip for rapid SARS-CoV-2 diagnostics.

Droplet digital PCR (ddPCR) is a technique in which PCR reaction is divided into thousands of nanoliter-sized droplets and has proven to be a great tool in virus diagnostics. Compared to the gold standard system quantitative real-time PCR (RT-qPCR), ddPCR functions particularly well when dealing with samples with low template counts, such as viral concentration. This feature makes the technique suitable for early detection of the virus. In this study, a novel portable PDMS ddPCR chip is introduced. The chip functions without external pumps using manual pressurization with a multichannel pipet. The created droplets are monodispersed and form a monolayer on the chip's collection chamber, from where they can be effortlessly imaged. Droplets were analyzed and counted using artificial intelligence. The use of the manually pressurized chip was demonstrated for a SARS-CoV-2 assay, which takes advantage of isothermal strand invasion-based amplification (SIBA) technology, allowing quick and accurate, even point-of-care analysis of the sample. The results demonstrate that SIBA assays can be divided into nanoliter-sized droplets and used as quantitative assays, giving an approximation of the samples' viral count.

Reduced binding of apoE4 to complement factor H promotes amyloid-ß oligomerization and neuroinflammation.

The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late-onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform-specific binding of apoE to FH alters Aß1-42-mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aß1-42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement-resistant oligomers with apoE/Aß1-42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aß1-42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aß plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.

Repurposing host-guest chemistry to sequester virulence and eradicate biofilms in multidrug resistant Pseudomonas aeruginosa and Acinetobacter baumannii.

The limited diversity in targets of available antibiotic therapies has put tremendous pressure on the treatment of bacterial pathogens, where numerous resistance mechanisms that counteract their function are becoming increasingly prevalent. Here, we utilize an unconventional anti-virulence screen of host-guest interacting macrocycles, and identify a water-soluble synthetic macrocycle, Pillar[5]arene, that is non-bactericidal/bacteriostatic and has a mechanism of action that involves binding to both homoserine lactones and lipopolysaccharides, key virulence factors in Gram-negative pathogens. Pillar[5]arene is active against Top Priority carbapenem- and third/fourth-generation cephalosporin-resistant Pseudomonas aeruginosa and Acinetobacter baumannii, suppressing toxins and biofilms and increasing the penetration and efficacy of standard-of-care antibiotics in combined administrations. The binding of homoserine lactones and lipopolysaccharides also sequesters their direct effects as toxins on eukaryotic membranes, neutralizing key tools that promote bacterial colonization and impede immune defenses, both in vitro and in vivo. Pillar[5]arene evades both existing antibiotic resistance mechanisms, as well as the build-up of rapid tolerance/resistance. The versatility of macrocyclic host-guest chemistry provides ample strategies for tailored targeting of virulence in a wide range of Gram-negative infectious diseases.

Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

HLA allele-specific expression: Methods, disease associations, and relevance in hematopoietic stem cell transplantation.

Varying HLA allele-specific expression levels are associated with human diseases, such as graft versus host disease (GvHD) in hematopoietic stem cell transplantation (HSCT), cytotoxic T cell response and viral load in HIV infection, and the risk of Crohn's disease. Only recently, RNA-based next generation sequencing (NGS) methodologies with accompanying bioinformatics tools have emerged to quantify HLA allele-specific expression replacing the quantitative PCR (qPCR) -based methods. These novel NGS approaches enable the systematic analysis of the HLA allele-specific expression changes between individuals and between normal and disease phenotypes. Additionally, analyzing HLA allele-specific expression and allele-specific expression loss provide important information for predicting efficacies of novel immune cell therapies. Here, we review available RNA sequencing-based approaches and computational tools for NGS to quantify HLA allele-specific expression. Moreover, we explore recent studies reporting disease associations with differential HLA expression. Finally, we discuss the role of allele-specific expression in HSCT and how considering the expression quantification in recipient-donor matching could improve the outcome of HSCT.

Validation of the X-ray microtomography in the assessment of duodenal morphometry and surface area in celiac disease.

Background: Duodenal histology remains the diagnostic reference standard in celiac disease. However, traditional methods have suboptimal sensitivity and reproducibility for early mucosal changes and research purposes. We validated a recently introduced micro-CT imaging method for an accurate digital evaluation of duodenal histomorphometry and mucosal surface areas. Methods: Endoscopic biopsies from 58 individuals were utilized for the micro-CT imaging, selecting histological changes ranging from normal to severely damaged mucosa. The imaging protocol was optimized for practicability and resolution. The Bland-Altman method was applied to test intra- and interobserver variations in the blinded measurements. Results: The 3D micro-CT reconstructions enabled easy and precise digital cutting with optimal orientation and computer-assisted measurement of the surface area. Intraobserver analysis of morphological measurements showed a mean difference of 0.011 with limits of agreement (LA) from -0.397 to 0.375 and a standard deviation (SD) of 0.197. The corresponding figures for interobserver analysis were 0.080, from -0.719 to 0.537 and 0.320, respectively. The intraclass correlation coefficients (ICC) for the intraobserver and interobserver variations were 0.981 and 0.954, respectively. Intraobserver surface area analysis yielded a mean difference of 0.010, LA from -0.764 to 0.785 and an SD of 0.395, and an interobserver analysis mean difference of 0.028, LA from -0.642 to 0.698 and SD of 0.342. The respective ICCs for the intra- and interobserver variations were 0.963 and 0.972. Conclusions: Micro-CT showed excellent accuracy and reproducibility in the evaluation of mucosal morphometry and surface areas. The improved sensitivity for histological changes is a powerful tool for the diagnosis of celiac disease and for clinical and pharmacological studies.

Higher serum infliximab concentrations during induction predict short-term endoscopic response in patients with inflammatory bowel disease.

OBJECTIVE: Measuring of serum infliximab (IFX) induction concentrations might reduce primary non-response rates in inflammatory bowel diseases (IBD), but optimal target concentrations are unclear. We investigated whether IFX induction concentrations predict short-term endoscopic response at week 12 or treatment persistence at week 52. METHODS: Sixty-nine IBD patients (Crohn's disease, n=24; ulcerative colitis, n=45) received standard IFX induction of 5 mg/kg bodyweight at weeks 0, 2, and 6. Responders continued maintenance therapy and underwent follow-up until week 52 or treatment discontinuation. We measured IFX concentrations at weeks 2, 6, and 12, and evaluated treatment response around week 12 with endoscopy or with clinical scores and fecal calprotectin. Using the receiver operating characteristic analysis, we determined optimal IFX concentration thresholds associated with treatment response. We further compared IFX induction concentrations between patients persisting on IFX at week 52 and patients discontinuing treatment due to insufficient response. RESULTS: Responders (74%, 51 out of 69 patients) had significantly higher median IFX concentrations than non-responders at weeks 6 (25.06 vs. 19.68 µg/ml; P = 0.04) and 12 (18.03 vs. 10.02 µg/ml; P = 0.03), but not at week 2 (33.12 vs. 34.20 µg/ml; P = 0.97). Optimal IFX concentration thresholds for induction response were 21.33 and 5.13 µg/ml at weeks 6 and 12, respectively. Fifty-three patients continued IFX maintenance therapy until week 52. Induction concentrations failed to predict persistence on IFX therapy at week 52. CONCLUSION: Higher IFX induction concentrations predict endoscopic short-term response. However, induction concentrations failed to predict long-term persistence on IFX treatment.

Prevalence, Cell Tropism, and Clinical Impact of Human Parvovirus Persistence in Adenomatous, Cancerous, Inflamed, and Healthy Intestinal Mucosa.

Parvoviruses are single-stranded DNA viruses, infecting many animals from insects to humans. Human parvovirus B19 (B19V) causes erythema infectiosum, arthropathy, anemia, and fetal death, and human bocavirus (HBoV) 1 causes respiratory tract infections, while HBoV2-4 are enteric. Parvoviral genomes can persist in diverse non-permissive tissues after acute infection, but the host-cell tropism and the impact of their tissue persistence are poorly studied. We searched for parvoviral DNA in a total of 427 intestinal biopsy specimens, as paired disease-affected and healthy mucosa, obtained from 130 patients with malignancy, ulcerative colitis (UC), or adenomas, and in similar intestinal segments from 55 healthy subjects. Only three (1.6%) individuals exhibited intestinal HBoV DNA (one each of HBoV1, 2, and 3). Conversely, B19V DNA persisted frequently in the intestine, with 50, 47, 31, and 27% detection rates in the patients with malignancy, UC, or adenomas, and in the healthy subjects, respectively. Intra-individually, B19V DNA persisted significantly more often in the healthy intestinal segments than in the inflamed colons of UC patients. The highest loads of B19V DNA were seen in the ileum and colon specimens of two healthy individuals. With dual-RNAscope in situ hybridization and immunohistochemistry assays, we located the B19V persistence sites of these intestines in mucosal B cells of lymphoid follicles and vascular endothelial cells. Viral messenger RNA transcription remained, however, undetected. RNA sequencing (RNA-seq) identified 272 differentially expressed cellular genes between B19V DNA-positive and -negative healthy ileum biopsy specimens. Pathway enrichment analysis revealed that B19V persistence activated the intestinal cell viability and inhibited apoptosis. Lifelong B19V DNA persistence thus modulates host gene expression, which may lead to clinical outcomes.

Coeliac disease re-screening among once seronegative at-risk relatives: A long-term follow-up study.

BACKGROUND: Serological screening of the relatives of coeliac disease patients is widely endorsed. However, the need for and the optimal timing of possible re-testing of once seronegative at-risk individuals for coeliac disease remain unclear. OBJECTIVE: We investigated this issue by inviting a large cohort of previously screening-negative relatives of patients with coeliac disease to participate in a follow-up study. METHODS: Altogether 599 relatives of coeliac disease index patients not diagnosed with coeliac disease in a screening study carried out in 2006-2010 were asked about possible later diagnosis or re-tested with coeliac disease autoantibodies in 2017-2021. Besides incidence, the possible impact of various patient-related clinical factors and HLA haplotype on the later diagnosis or screening positivity was examined. RESULTS: Fifteen (2.5%) relatives were either diagnosed with a coeliac disease (n = 8) during the follow-up period or were found to be screening-positive in the re-testing (n = 7), giving a combined annual incidence of 221/100,000 person-years in all relatives and 336/100,000 among those carrying coeliac disease-associated HLA DQ2/DQ8. The new cases more often carried the high-risk (DQ2.5/2.5 or DQ2.5/2.2; 35.7% vs. 7.4%, respectively, p < 0.001) HLA and were younger at initial screening (23.3 vs. 40.5 years, p = 0.028) and - in spite of a negative screening outcome - had higher median transglutaminase antibody level in the first study than those not affected. There were no significant differences between the affected and non-affected relatives in other demographic data, degree of kinship with the index, current symptoms or frequency of chronic co-morbidities. CONCLUSION: The incidence rate for later coeliac disease diagnosis or new seropositivity in relatives who had been tested once was 221/100,000 person-years in all and 336/100,000 among those carrying at-risk HLA genetics after ∼10 years of follow-up. HLA-typing could help to target a subgroup of relatives who would benefit most from re-testing.

Role of HLA-DQ typing and antitissue transglutaminase antibody titres in diagnosing coeliac disease among Sudanese children with type 1 diabetes mellitus.

OBJECTIVE: The aim of the study was to determine the prevalence of coeliac disease (CD) and to recognise Human leukocyte antigen (HLA)-associated hereditary susceptibility to Sudanese CD patients with type 1 diabetes mellitus (DM1). DESIGN: Antitissue transglutaminase IgA (anti-TG IgA) was measured in the serum of 373 children affected with DM1 aged 1-19-year old and in 100 serum samples from non-diabetic control children. Histological examination was performed in 19 children seropositive for anti-TG IgA (17 DMI and 2 controls). Additionally, PCR-based analysis of Major histocompatibility complex, class II, DQ beta 1 (HLA-DQB1) genotyping was implemented in three study population groups as follows: group 1 (n=25) (+ve DM1 and +ve CD), group 2 (n=63) (-ve DM1 and +ve CD) and control group 3 (n=2) (+ve CD). RESULTS: Twenty-six Sudanese children with DM1 out of 373 (6.97%) were seropositive for anti-TG IgA. Duodenal biopsy revealed Marsh 2 and 3 in 13 out of 17 (76.47%) seropositive anti-TG IgA patients with DM1. Significant association (p<0.05) was detected between the level of anti-TG IgA autoantibodies (IU/mL) and Marsh stage. HLA DQ2 and DQ8 were found in 88% (22/25) and 8% (2/25) of examined patients with CD with DM1, respectively. CONCLUSIONS: Anti-TG IgA titre of greater than 10 times upper limit of normal (≥10× ULN) can be useful for detecting CD in children with type 1 diabetes without duodenal biopsy. HLA testing in children with DM1 appears to provide little added benefit given the high prevalence (96%) of HLA DQ2/DQ8 in children with DM1.

Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix.

Oxygen tension varies during placental and fetal development. Although hypoxia drives early trophoblast invasion, low placental oxygen levels during pregnancy show association with pregnancy complications including fetal growth restriction and preeclampsia. JEG-3 cells are often used as a trophoblast model. We studied transcriptional changes of JEG-3 cells on a uterine leiomyoma derived matrix Myogel. This might be the closest condition to the real uterine environment that we can get for an in vitro model. We observed that culturing JEG-3 cells on the leiomyoma matrix leads to strong stimulation of ribosomal pathways, energy metabolism, and ATP production. Furthermore, Myogel improved JEG-3 cell adherence in comparison to tissue culture treated plastic. We also included PDMS microchip hypoxia creation, and observed changes in oxidative phosphorylation, oxygen related genes and several hypoxia genes. Our study highlights the effects of Myogel matrix on growing JEG-3 cells, especially on mitochondria, energy metabolism, and protein synthesis.

Dissecting the contribution of single nucleotide polymorphisms in CCR9 and CCL25 genomic regions to the celiac disease phenotype.

PURPOSE AND OBJECTIVES: Given their role in homing immune cells to the intestine, CC motif chemokine receptor 9 (CCR9) and its specific ligand CC motif chemokine ligand 25 (CCL25) are interesting candidate genes for celiac disease. These genes are located in regions previously shown to be associated with or linked to celiac disease, but no investigations on their association with various celiac disease phenotypes have so far been conducted. Here we studied such associations of both genotyped and imputed single nucleotide polymorphisms (SNPs) with either regulatory function or exonic location of the CCR9 and CCL25 loci. RESULTS: Exploiting a carefully phenotyped cohort of 625 celiac disease patients and 1817 non-celiac controls, we identified that multiple SNPs with predicted regulatory function (RegulomeDB score ≤3a and/or eQTL effect) located between 100 kB upstream and downstream of CCR9 and CCL25 are associated with celiac disease and/or selected phenotypes. Of the genotyped SNPs in the CCR9 loci, rs213360 with an eQTL effect on CCR9 expression in blood was associated with celiac disease and all investigated phenotypes except high HLA risk. Rs1545985 with an eQTL on CCR9 expression and rs7652331 and rs12493471, both with RegulomeDB score ≤3a, were all associated with gastrointestinal symptoms and malabsorption and the latter additionally with anemia. The genotyped CCL25 SNPs rs952444 and rs882951, with RegulomeDB scores 1d and 1f respectively and eQTL effect on CCL25 expression in small intestine, were associated with gastrointestinal symptoms and malabsorption. The CCL25 SNP rs2303165 identified in sequencing followed by imputation was associated with partial villous atrophy. However, the association did not pass the permutation based multiple testing correction (PEMP2 > 0.05). CONCLUSIONS: We conclude that SNPs in the region of CCR9 and CCL25 with predicted functional effect or exonic localization likely contribute only modestly to various celiac disease phenotypes.

Presence of high-risk HLA genotype is the most important individual risk factor for coeliac disease among at-risk relatives.

BACKGROUND: Family screening has been advocated as a means to reduce the major underdiagnosis of coeliac disease. However, the precise risk of the disease in relatives and the impact of patient- and relative-related individual factors remain obscure. AIMS: To investigate the individual risk of coeliac disease among patients' relatives. METHODS: Altogether 2943 relatives of 624 index patients were assessed for the presence of previous coeliac disease diagnosis, or were screened for the disease. Coeliac disease-associated human leucocyte antigen (HLA) genotype was determined from all participants. The association between individual factors and new screening positivity was assessed by logistic regression. RESULTS: There were 229 previously diagnosed non-index relatives with coeliac disease and 2714 non-affected (2067 first-degree, 647 more distant) relatives. Of these 2714 relatives, 129 (4.8%) were screening-positive (first-degree 5.1%, second-degree 3.6%, more distant 3.5%). The combined prevalence of the previously diagnosed and now detected cases in relatives was 12.2% (6.3% clinically detected, 5.9% screen-detected). In univariate analysis, age <18 years at diagnosis (odds ratio 1.60, 95% CI 1.04-2.45) in index, and age 41-60 years (1.73, 1.10-2.73), being a sibling (1.65, 1.06-2.59) and having the high-risk genotype (3.22, 2.01-5.15 DQ2.5/2.5 or DQ2.5/2.2 vs other risk alleles) in relatives were associated with screening positivity. Only high-risk HLA remained significant (2.94, 1.80-4.78) in multivariable analysis. CONCLUSIONS: Unrecognised coeliac disease was common among at-risk relatives even in a country with an active case-finding policy, and also in relatives more distant than first-degree. The presence of a high-risk genotype was the most important predictor for screening positivity. ClinicalTrials.gov identifier NCT03136731.

Targeted RNA-Based Oxford Nanopore Sequencing for Typing 12 Classical HLA Genes.

Identification of human leukocyte antigen (HLA) alleles from next-generation sequencing (NGS) data is challenging because of the high polymorphism and mosaic nature of HLA genes. Owing to the complex nature of HLA genes and consequent challenges in allele assignment, Oxford Nanopore Technologies' (ONT) single-molecule sequencing technology has been of great interest due to its fitness for sequencing long reads. In addition to the read length, ONT's advantages are its portability and possibility for a rapid real-time sequencing, which enables a simultaneous data analysis. Here, we describe a targeted RNA-based method for HLA typing using ONT sequencing and SeqNext-HLA SeqPilot software (JSI Medical Systems GmbH). Twelve classical HLA genes were enriched from cDNA of 50 individuals, barcoded, pooled, and sequenced in 10 MinION R9.4 SpotON flow cell runs producing over 30,000 reads per sample. Using barcoded 2D reads, SeqPilot assigned HLA alleles to two-field typing resolution or higher with the average read depth of 1750x. Sequence analysis resulted in 99-100% accuracy at low-resolution level (one-field) and in 74-100% accuracy at high-resolution level (two-field) with the expected alleles. There are still some limitations with ONT RNA sequencing, such as noisy reads, homopolymer errors, and the lack of robust algorithms, which interfere with confident allele assignment. These issues need to be inspected carefully in the future to improve the allele call rates. Nevertheless, here we show that sequencing of multiplexed cDNA amplicon libraries on ONT MinION can produce accurate high-resolution typing results of 12 classical HLA loci. For HLA research, ONT RNA sequencing is a promising method due to its capability to sequence full-length HLA transcripts. In addition to HLA genotyping, the technique could also be applied for simultaneous expression analysis.

Clustering based approach for population level identification of condition-associated T-cell receptor ß-chain CDR3 sequences.

BACKGROUND: Deep immune receptor sequencing, RepSeq, provides unprecedented opportunities for identifying and studying condition-associated T-cell clonotypes, represented by T-cell receptor (TCR) CDR3 sequences. However, due to the immense diversity of the immune repertoire, identification of condition relevant TCR CDR3s from total repertoires has mostly been limited to either "public" CDR3 sequences or to comparisons of CDR3 frequencies observed in a single individual. A methodology for the identification of condition-associated TCR CDR3s by direct population level comparison of RepSeq samples is currently lacking. RESULTS: We present a method for direct population level comparison of RepSeq samples using immune repertoire sub-units (or sub-repertoires) that are shared across individuals. The method first performs unsupervised clustering of CDR3s within each sample. It then finds matching clusters across samples, called immune sub-repertoires, and performs statistical differential abundance testing at the level of the identified sub-repertoires. It finally ranks CDR3s in differentially abundant sub-repertoires for relevance to the condition. We applied the method on total TCR CDR3ß RepSeq datasets of celiac disease patients, as well as on public datasets of yellow fever vaccination. The method successfully identified celiac disease associated CDR3ß sequences, as evidenced by considerable agreement of TRBV-gene and positional amino acid usage patterns in the detected CDR3ß sequences with previously known CDR3ßs specific to gluten in celiac disease. It also successfully recovered significantly high numbers of previously known CDR3ß sequences relevant to each condition than would be expected by chance. CONCLUSION: We conclude that immune sub-repertoires of similar immuno-genomic features shared across unrelated individuals can serve as viable units of immune repertoire comparison, serving as proxy for identification of condition-associated CDR3s.

HLA RNA Sequencing With Unique Molecular Identifiers Reveals High Allele-Specific Variability in mRNA Expression.

The HLA gene complex is the most important single genetic factor in susceptibility to most diseases with autoimmune or autoinflammatory origin and in transplantation matching. Most studies have focused on the vast allelic variation in these genes; only a few studies have explored differences in the expression levels of HLA alleles. In this study, we quantified mRNA expression levels of HLA class I and II genes from peripheral blood samples of 50 healthy individuals. The gene- and allele-specific mRNA expression was assessed using unique molecular identifiers, which enabled PCR bias removal and calculation of the number of original mRNA transcripts. We identified differences in mRNA expression between different HLA genes and alleles. Our results suggest that HLA alleles are differentially expressed and these differences in expression levels are quantifiable using RNA sequencing technology. Our method provides novel insights into HLA research, and it can be applied to quantify expression differences of HLA alleles in various tissues and to evaluate the role of this type of variation in transplantation matching and susceptibility to autoimmune diseases.

Independent and cumulative coeliac disease-susceptibility loci are associated with distinct disease phenotypes.

The phenotype of coeliac disease varies considerably for incompletely understood reasons. We investigated whether established coeliac disease susceptibility variants (SNPs) are individually or cumulatively associated with distinct phenotypes. We also tested whether a polygenic risk score (PRS) based on genome-wide associated (GWA) data could explain the phenotypic variation. The phenotypic association of 39 non-HLA coeliac disease SNPs was tested in 625 thoroughly phenotyped coeliac disease patients and 1817 controls. To assess their cumulative effects a weighted genetic risk score (wGRS39) was built, and stratified by tertiles. In our PRS model in cases, we took the summary statistics from the largest GWA study in coeliac disease and tested their association at eight P value thresholds (PT) with phenotypes. Altogether ten SNPs were associated with distinct phenotypes after correction for multiple testing (PEMP2 ≤ 0.05). The TLR7/TLR8 locus was associated with disease onset before and the SH2B3/ATXN2, ITGA4/UBE2E3 and IL2/IL21 loci after 7 years of age. The latter three loci were associated with a more severe small bowel mucosal damage and SH2B3/ATXN2 with type 1 diabetes. Patients at the highest wGRS39 tertiles had OR > 1.62 for having coeliac disease-related symptoms during childhood, a more severe small bowel mucosal damage, malabsorption and anaemia. PRS was associated only with dermatitis herpetiformis (PT = 0.2, PEMP2 = 0.02). Independent coeliac disease-susceptibility loci are associated with distinct phenotypes, suggesting that genetic factors play a role in determining the disease presentation. Moreover, the increased number of coeliac disease susceptibility SNPs might predispose to a more severe disease course.

Differences Between Familial and Sporadic Celiac Disease.

BACKGROUND: It is not known if genetic background, characteristics at diagnosis, physical and psychological well-being, and adherence to a gluten-free diet are comparable between patients with familial or sporadic celiac disease. These issues were investigated in a follow-up study. METHODS: Altogether 1064 patients were analyzed for celiac disease-associated serology, predisposing HLA-DQ, and non-HLA genotypes. Medical data were collected from patient records and supplementary interviews. Current symptoms and quality of life were further evaluated with the Gastrointestinal Symptom Rating Scale (GSRS), the Psychological General Well-Being questionnaire (PGWB), and Short Form 36 (SF-36) questionnaires. RESULTS: Familial and sporadic groups differed (P < 0.001) in the reason for diagnosis and clinical presentation at diagnosis, familial patients being more often screen-detected (26% vs. 2%, P < 0.001) and having less often gastrointestinal (49% vs. 69%) and severe symptoms (47% vs. 65%). The groups were comparable in terms of histological damage, frequency of malabsorption, comorbidities, childhood diagnoses, and short-term treatment response. At the time of the study, familial cases reported fewer symptoms (21% vs. 30%, P = 0.004) and lower prevalence of all (78% vs. 86%, P = 0.007), neurological (10% vs. 15%, P = 0.013), and dermatological (9% vs. 17%, P = 0.001) comorbidities. Dietary adherence and GSRS scores were comparable, but familial cases had better quality of life according to PGWB and SF-36. High-risk genotype HLA-DQ2.5/DQ2.5 was more frequent among familial cases, and four non-HLA SNPs were associated with familial celiac disease. CONCLUSIONS: Despite the greater proportion of high-risk genotypes, familial cases had milder symptoms at presentation than did sporadic cases. Worse experience of symptoms and poorer quality of life in sporadic disease indicate a need for intensified support.

Nonbiopsy Approach for Celiac Disease Is Accurate When Using Exact Duodenal Histomorphometry: Prospective Study in 2 Countries.

GOALS: To test the accuracy of serology-based criteria for diagnosing celiac disease utilizing quantitative histomorphometry. BACKGROUND: The revised European pediatric guidelines allow noninvasive celiac disease diagnosis for a subgroup of children. However, in some of the studies on this issue, the positive predictive value (PPV) of serology has remained suboptimal, possibly because of challenges of histopathology as the reference standard. STUDY: Prospectively enrolled children with transglutaminase 2 antibodies (TGA) above the upper limit of normal (ULN) underwent blood sampling and duodenal biopsy in Finland and Romania. Those with TGA ≥10× ULN, positive endomysium antibodies (EmA), and disease-associated genetics were considered to fulfill triple criteria for celiac disease. Initial histopathologic analysis was conducted using grouped classification, whereupon centralized morphometry was performed. RESULTS: Altogether 88 (54%) children were triple positive. In local evaluation, 99% of triple-positive children and 73% of children with TGA <10× ULN had celiac disease. These figures increased to 100% and 85% after more precise morphometric analysis. Triple-positive children had more anemia and higher median EmA and liver enzyme values than those with TGA<10× ULN; the groups were comparable in other clinical features and laboratory parameters. CONCLUSIONS: When applied as recommended, the nonbiopsy strategy had already yielded excellent PPV regardless of the site of diagnosis or clinical presentation in the local analysis. PPV further increased to 100% with standardized duodenal morphometry.