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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.

BackgroundDuodenal 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.MethodsEndoscopic 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.ResultsThe 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.ConclusionsMicro-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.

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

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.

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.

ObjectiveThe 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).DesignAntitissue 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).ResultsTwenty-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 (pConclusionsAnti-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.

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

Tulehdukselliset suolistosairaudet (IBD) Crohnin tauti (CD) ja haavainen paksusuolentulehdus (UC) ovat maailmanlaajuisesti lisääntyviä sairauksia, joihin liittyy muuntunut suoliston mikrobisto. Infliksimabi (IFX) on TNF-alfan estäjä, jota käytetään IBD:n hoidossa, vaikkakin kolmas osa potilaista voi jäädä ilman hoitovastetta tai menettää sen. Luotettavia testejä hoitovasteen ennustamiseksi ei ole vielä saatavilla. Tutkimuksemme tavoitteena oli selvittää potilaiden ulosteen bakteerit, sienet ja hiivat infliksimabihoidon aikana sekä etsiä hoitovastetta ennustavia mittareita IBD potilailta. Tutkimuksessamme 72 IBD-potilasta (25 CD ja 47 UC) aloitti infliksimabihoidon ja heitä seurattiin vuoden ajan hoidon aloituksesta tai hoidon keskeytykseen saakka. Potilaiden ulostenäytteistä määritettiin sekvensointia hyödyntäen erikseen suoliston bakteerit (16S rRNA geenistä) sekä hiivat ja sienet (ITS1 geenin alueesta). Mikrobiston profiilit määritettiin ennen hoidon aloitusta, 2, 6, 12 viikkoa sekä 1 vuosi hoidon aloituksen jälkeen. IFX hoidon vaste määritettiin kolonoskopialla ja kliinisesti 12 viikon hoidon jälkeen. Tutkimuksessa havaittiin, että potilaiden ulosteen bakteerit sekä sienet ja hiivat erosivat merkittävästi toisistaan infliksimabihoitovasteen mukaan jo ennen hoidon aloitusta. Potilailla, jotka eivät saaneet vastetta hoidosta, oli vähemmän lyhytketjuisia rasvahappoja tuottavia bakteereja erityisesti Clostridia-luokasta sekä enemmän tulehdusta aiheuttavia bakteereja ja sieniä mm. Candida-suvusta. Tämä tulos testattiin bakteerien osalta myös ennustetestillä (ROC), joka erotti toisistaan ne potilaat, jotka saivat hyvän vasteen ja ne, jotka eivät saaneet vastetta (area under the curve > 0.8). Tulosten perusteella ulosteen bakteerit ja hiivat voisivat sopia ennustaviksi mittareiksi IBD-potilaiden infliksimabihoidon vasteen ennustamiseen.

Publisher Copyright: © 2022 The Author(s) 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.

Single cell trapping is demonstrated on superhydrophobic-superhydrophilic patterned microarrays. Superhydrophilic spots that are twice the size of the cells to be trapped are found to be optimal for obtaining single cell trapping. The single cell trapping is based on size exclusivity instead of heavy dilution of the cell suspension and relying on Poisson statistics. The superhydrophobic areas of the array are found to be very resistant toward unwanted adhesion of the cells and thus cleaning steps are not needed after deposition. Based on these properties, 20 mu m superhydrophilic spots are utilized for trapping two types of immune cells, primary peripheral blood mononuclear cells (PBMCs) and THP-1 cells. The cells are trapped from a 10 mu l cell suspension droplet dragged across the surface with different velocities. Single cell trapping efficiencies in the range of 10-30% are shown based on the cell type, the spot type, the seeding velocity and the cell suspension concentration.

Narcolepsy type 1 (NT1) is a chronic neurological disorder having a strong association with HLA-DQB1*0602, thereby suggesting an immunological origin. Increased risk of NT1 has been reported among children or adolescents vaccinated with AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine, Pandemrix. Here we show that pediatric Pandemrix-associated NT1 patients have enhanced T-cell immunity against the viral epitopes, neuraminidase 175–189 (NA175–189) and nucleoprotein 214–228 (NP214–228), but also respond to a NA175–189-mimic, brain self-epitope, protein-O-mannosyltransferase 1 (POMT1675–689). A pathogenic role of influenza virus-specific T-cells and T-cell cross-reactivity in NT1 are supported by the up-regulation of IFN-γ, perforin 1 and granzyme B, and by the converging selection of T-cell receptor TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes, in response to stimulation either with peptide NA175–189 or POMT1675–689. Moreover, anti-POMT1 serum autoantibodies are increased in Pandemrix-vaccinated children or adolescents. These results thus identify POMT1 as a potential autoantigen recognized by T- and B-cells in NT1., Narcolepsy type 1 (NT1) is a severe sleep disorder with strong association to the HLA type DQB1*0602 and increased incidence among children vaccinated with the Influenza A vaccine Pandemrix. Here the authors show that these children develop T and B cell autoimmunity against protein-O-mannosyltransferase 1 via cross-reactivity.

BackgroundDeep 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.ResultsWe 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 CDR3 beta RepSeq datasets of celiac disease patients, as well as on public datasets of yellow fever vaccination. The method successfully identified celiac disease associated CDR3 beta sequences, as evidenced by considerable agreement of TRBV-gene and positional amino acid usage patterns in the detected CDR3 beta sequences with previously known CDR3 beta s specific to gluten in celiac disease. It also successfully recovered significantly high numbers of previously known CDR3 beta sequences relevant to each condition than would be expected by chance.ConclusionWe 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.

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.

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.

The clinical phenotype of celiac disease varies considerably among patients and the dosage of HLA-DQ2.5 alleles has been suggested to be a contributing factor. We investigated whether HLA-DQ2.5 allele dosage is associated with distinct clinical parameters at the time of diagnosis and with patients&rsquo, response to a gluten-free diet. The final cohort included 605 carefully phenotyped non-related Finnish celiac disease patients grouped as having 0, 1 or 2 copies of HLA-DQ2.5. Clinical data at the time of diagnosis and during gluten-free diet were collected systematically from medical records and supplementary interviews. An increasing HLA-DQ2.5 dose effect was detected for celiac disease antibody positivity at diagnosis (p = 0.021) and for the presence of any first-degree relatives with celiac disease (p = 0.011 and p = 0.031, respectively). Instead, DQ2.5-negative patients were suffering most often from classical symptoms at diagnosis (p = 0.007 between HLA groups). In addition, during follow-up they were most often symptomatic despite a gluten-free diet (p = 0.002 between groups). Our results thus suggest that increasing HLA-DQ2.5 dose only has a minor effect on the clinical picture of celiac disease. However, HLA-DQ2.5-negative patients should not be overlooked in clinical practice and particular attention should be paid to this patient group during gluten-free diet.

The pathological mechanisms that lead to the onset and reactivation of celiac disease (CD) remain largely unknown. While gluten free diet (GFD) improves the intestinal damage and associated clinical symptoms in majority of cases, it falls short of providing full recovery. Additionally, late or misdiagnosis is also common as CD presents with a wide range of symptoms. Clear understanding of CD pathogenesis is thus critical to address both diagnostic and treatment concerns. We aimed to study the molecular impact of short gluten exposure in GFD treated CD patients, as well as identify biological pathways that remain altered constitutively in CD regardless of treatment. Using RNAseq profiling of PBMC samples collected from treated CD patients and gluten challenged patient and healthy controls, we explored the peripheral transcriptome in CD patients following a short gluten exposure. Short gluten exposure of just three days was enough to alter the genome-wide PBMC transcriptome of patients. Pathway analysis revealed gluten-induced upregulation of mainly immune response related pathways, both innate and adaptive, in CD patients. We evaluated the perturbation of biological pathways in sample-specific manner. Compared to gluten exposed healthy controls, pathways related to tight junction, olfactory transduction, metabolism of unsaturated fatty acids (such as arachidonic acid), metabolism of amino acids (such as cysteine and glutamate), and microbial infection were constitutively altered in CD patients regardless of treatment, while GFD treatment appears to mostly normalize immune response pathways to “healthy” state. Upstream regulator prediction analysis using differentially expressed genes identified constitutively activated regulators relatively proximal to previously reported CD associated loci, particularly SMARCA4 on 19p13.2 and CSF2 on 5q31. We also found constitutively upregulated genes in CD that are in CD associated genetic loci such as MEF2BNB-MEF2B (BORCS8-MEF2B) on 19p13.11 and CSTB on 21q22.3. RNAseq revealed strong effects of short oral gluten challenge on whole PBMC fraction and constitutively altered pathways in CD PBMC suggesting important factors other than gluten in CD pathogenesis. publishedVersion

Non-biopsy diagnosis of celiac disease is possible in children with anti-transglutaminase 2 antibodies (TGA) &gt, 10&times, the upper limit of normal (ULN) and positive anti-endomysial antibodies (EMA). Similar criteria have been suggested for adults, but evidence with different TGA assays is scarce. We compared the performance of four TGA tests in the diagnosis of celiac disease in cohorts with diverse pre-test probabilities. Serum samples from 836 adults with either clinical suspicion or family risk of celiac disease were tested with four commercial TGA assays, EmA and celiac disease-associated genetics. The diagnosis was set based on duodenal lesion or, in some cases, using special methods. 137 (57%) patients with clinical suspicion and 85 (14%) of those with family risk had celiac disease. Positive predictive value (PPV) for 10&times, ULN was 100% in each TGA test. The first non-diagnostic investigations were encountered with ULN 1.0&times, &ndash, 5.1&times, in the clinical cohort and 1.3&times, 4.9&times, in the family cohort, respectively. Using the assays&rsquo, own cut-offs (1&times, ULN) the PPVs ranged 84&ndash, 100%. Serology-based diagnosis of celiac disease was accurate in adults using different commercial kits and pre-test probabilities using 10&times, ULN. The results also suggest that the ULN threshold for biopsy-omitting approach could be lower.

We have analyzed T cell receptor repertoires in a unique set of thymus samples from a pair of monozygotic twins. While genetics affect the V(D)J rearrangement and generation of junctional sequences, the thymic selections seem largely stochastic and import no detectable inheritable effect at clonal level. Non

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

Isothermal nucleic acid amplification methods can potentially shorten the amount of time required to diagnose influenza. We developed and evaluated a novel isothermal nucleic acid amplification method, RT-SIBA to rapidly detect and differentiate between influenza A and B viruses in a single reaction tube. The performance of the RT-SIBA Influenza assay was compared with two established RT-PCR methods. The sensitivities of the RT-SIBA, RealStar RT-PCR, and CDC RT-PCR assays for the detection of influenza A and B viruses in the clinical specimens were 98.8%, 100%, and 89.3%, respectively. All three assays demonstrated a specificity of 100%. The average time to positive result was significantly shorter with the RT-SIBA Influenza assay (90 min). The method can be run using battery-operated, portable devices with a small footprint and therefore has potential applications in both laboratory and near-patient settings., Highlights • We developed and evaluated novel isothermal nucleic acid amplification method for the rapid detection and differentiation of influenza A and B viruses in human respiratory specimens. • The method displayed high level of sensitivity and specificity and correlated well with those of the reference methods. • The method detected influenza virus within 20 minutes and can potentially be used in near patient or field setting.

Dermatitis herpetiformis (DH) is an extraintestinal manifestation of celiac disease causing an itchy, blistering rash. Granular IgA deposits in the skin are pathognomonic for DH, and the treatment of choice is a lifelong gluten-free diet (GFD). Preliminary evidence suggests that there are patients with DH who redevelop gluten tolerance after adherence to a GFD treatment. To evaluate this, we performed a 12-month gluten challenge with skin and small-bowel mucosal biopsy samples in 19 patients with DH who had adhered to a GFD for a mean of 23 years. Prechallenge biopsy was negative for skin IgA and transglutaminase 3 deposits in 16 patients (84%) and indicated normal villous height-to-crypt depth ratios in the small bowel mucosa in all 19 patients. The gluten challenge caused a relapse of the rash in 15 patients (79%) in a mean of 5.6 months; of these 15 patients, 13 had skin IgA and transglutaminase 3 deposits, and 12 had small-bowel villous atrophy. In addition, three patients without rash or immune deposits in the skin developed villous atrophy, whereas one patient persisted without any signs of relapse. In conclusion, 95% of the patients with DH were unable to tolerate gluten even after long-term adherence to a GFD. Therefore, lifelong GFD treatment remains justified in all patients with DH.

The factors determining the presentation of celiac disease are unclear. We investigated the phenotypic concordance and the distribution of human leukocyte antigen (HLA) risk haplotypes in affected siblings. One hundred sibling pairs were included. Clinical and histological parameters and HLA haplotypes were compared between the first diagnosed indexes and their siblings. The phenotype was categorized into gastrointestinal, extra-intestinal, malabsorption/anemia, and asymptomatic. The phenotype was fully concordant in 21 pairs. The most common concordant phenotype was gastrointestinal (14 pairs). Indexes had more anemia/malabsorption and extra-intestinal symptoms than siblings (45% vs. 20%, p &lt, 0.001 and 33% vs. 12%, p &lt, 0.001, respectively). Twenty siblings and none of the indexes were asymptomatic. The indexes were more often women (81% vs. 63%, p = 0.008). They were also more often seronegative (11% vs. 0%, p = 0.03) and younger (37 vs. 43 year, p &lt, 0.001), and had more severe histopathology (total/subtotal atrophy 79% vs. 58%, p = 0.047) at diagnosis. The indexes and siblings were comparable in other disease features. Pairs with discordant presentation had similar HLA haplotypes more often than the concordant pairs. The phenotype was observed to vary markedly between siblings, with the indexes generally having a more severe presentation. HLA did not explain the differences, suggesting that non-HLA genes and environmental factors play significant roles.

Background: Alpha-mangostin (alpha-MG) is a natural xanthone reported to exhibit rapid bactericidal activity against Gram-positive bacteria, and may therefore have potential clinical application in healthcare sectors. This study sought to identify the impact of alpha-MG on Staphylococcus epidermidis RP62A through integrated advanced omic technologies. Methods: S. epidermidis was challenged with sub-MIC (0.875 mu g/ml) of alpha-MG at various time points and the differential expression pattern of genes/proteins were analyzed in the absence and presence of alpha-MG using RNA sequencing and LC-MS/MS experiments. Bioinformatic tools were used to categorize the biological processes, molecular functions and KEGG pathways of differentially expressed genes/proteins. qRT-PCR was employed to validate the results obtained from these analyses. Results: Transcriptomic and proteomic profiling of alpha-MG treated cells indicated that genes/proteins affected by alpha-MG treatment were associated with diverse cellular functions. The greatest reduction in expression was observed in transcription of genes conferring cytoplasmic membrane integrity (yidC2, secA and mscL), cell division (ftsY and divlB), teichoic acid biosynthesis (tagG and dltA), fatty-acid biosynthesis (accB, accC, fabD, fabH, fabl, and fabZ), biofilm formation (icaA) and DNA replication and repair machinery (polA, polC, dnaE, and uvrA). Those with increased expression were involved in oxidative (katA and sodA) and cellular stress response (clpB, clpC, groEL, and asp23). The qRT-PCR analysis substantiated the results obtained from transcriptomic and proteomic profiling studies. Conclusion: Combining transcriptomic and proteomic methods provided comprehensive information about the antibacterial mode of action of alpha-MG. The obtained results suggest that alpha-MG targets S. epidermidis through multifarious mechanisms, and especially prompts that loss of cytoplasmic membrane integrity leads to rapid onset of bactericidal activity.

Objectives Immunotherapy and personalized medicine therapeutics are emerging as promising approaches in the management of head and neck squamous cell carcinoma (HNSCC). In spite of that, there is yet no assay that could predict individual response to immunotherapy. Methods We manufactured an in vitro 3D microfluidic chip to test the efficacy of immunotherapy. The assay was first tested using a tongue cancer cell line (HSC-3) embedded in a human tumour-derived matrix “Myogel/fibrin” and immune cells from three healthy donors. Next, the chips were used with freshly isolated cancer cells, patients' serum and immune cells. Chips were loaded with different immune checkpoint inhibitors, PD-L1 antibody and IDO 1 inhibitor. Migration of immune cells towards cancer cells and the cancer cell proliferation rate were evaluated. Results Immune cell migration towards HSC-3 cells was cancer cell density dependent. IDO 1 inhibitor induced immune cells to migrate towards cancer cells both in HSC-3 and in two HNSCC patient samples. Efficacy of PD-L1 antibody and IDO 1 inhibitor was patient dependent. Conclusion We introduced the first humanized in vitro microfluidic chip assay to test immunotherapeutic drugs against HNSCC patient samples. This assay could be used to predict the efficacy of immunotherapeutic drugs for individual patients.

Background: CCAAT enhancer-binding protein epsilon (C/EBP epsilon) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBP epsilon is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. Objective: The aim of this study was to molecularly characterize the effects of C/EBP epsilon transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. Methods: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. Results: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. Conclusion: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBP epsilon. Mutated C/EBPe acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBP epsilon. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.

Background and AimsIn nonresponsive celiac disease (NRCD), the symptoms and duodenal damage persist despite a gluten-free diet. Celiac disease patients with persistent symptoms are found to have a dysbiotic microbiota. We thus hypothesized that increased seroreactivity to the serum gluten-sensitive microbial antibodies Saccharomyces cerevisiae (ASCA), Pseudomonas fluorescens-associated sequence (I2), and Bacteroides caccae TonB-linked outer membrane protein (OmpW) is associated with NRCD.MethodsASCA, I2 and OmpW were measured in 20 seronegative CD patients with persistent villous damage despite strict dietary treatment (NRCD group). Fifty-eight responsive patients served as CD controls (55 on gluten-free treatment) and 80 blood donors as non-CD controls.ResultsAt least one microbial marker was positive in 80% of NRCD patients, in 97% of untreated CD and 87% of treated CD patients, and in 44% of controls. NRCD patients had the highest frequency of ASCA positivity (65% vs 52, 20, and 0%, respectively) and also significantly higher ASCA IgA (median 14.5 U/ml) and IgG (32.5 U/ml) titers than treated CD patients (7.0 U/ml, 13.0 U/ml) and non-CD controls (4.5 U/ml, 5.8 U/ml). The frequencies of I2 and OmpW were lower in NRCD than in untreated CD (65% and 45% vs 86% and 59%, respectively), and I2 titers were higher in NRCD (median absorbance 0.76) and untreated (1.0) and treated (0.83) CD than controls (0.32). OmpW was elevated in untreated (1.1) and treated (0.94) CD patients compared with controls (0.79).ConclusionsSeropositivity and high titers of ASCA are associated with NRCD and might serve as an additional follow-up tool in CD.

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. publishedVersion

Background & Aims Celiac disease could be treated, and potentially cured, by restoring T-cell tolerance to gliadin. We investigated the safety and efficacy of negatively charged 500-nm poly(lactide-co-glycolide) nanoparticles encapsulating gliadin protein (TIMP-GLIA) in 3 mouse models of celiac disease. Uptake of these nanoparticles by antigen-presenting cells was shown to induce immune tolerance in other animal models of autoimmune disease. Methods We performed studies with C57BL/6; RAG1–/– (C57BL/6); and HLA-DQ8, huCD4 transgenic Ab0 NOD mice. Mice were given 1 or 2 tail-vein injections of TIMP-GLIA or control nanoparticles. Some mice were given intradermal injections of gliadin in complete Freund's adjuvant (immunization) or of soluble gliadin or ovalbumin (ear challenge). RAG–/– mice were given intraperitoneal injections of CD4+CD62L–CD44hi T cells from gliadin-immunized C57BL/6 mice and were fed with an AIN-76A–based diet containing wheat gluten (oral challenge) or without gluten. Spleen or lymph node cells were analyzed in proliferation and cytokine secretion assays or by flow cytometry, RNA sequencing, or real-time quantitative polymerase chain reaction. Serum samples were analyzed by gliadin antibody enzyme-linked immunosorbent assay, and intestinal tissues were analyzed by histology. Human peripheral blood mononuclear cells, or immature dendritic cells derived from human peripheral blood mononuclear cells, were cultured in medium containing TIMP-GLIA, anti-CD3 antibody, or lipopolysaccharide (controls) and analyzed in proliferation and cytokine secretion assays or by flow cytometry. Whole blood or plasma from healthy volunteers was incubated with TIMP-GLIA, and hemolysis, platelet activation and aggregation, and complement activation or coagulation were analyzed. Results TIMP-GLIA did not increase markers of maturation on cultured human dendritic cells or induce activation of T cells from patients with active or treated celiac disease. In the delayed-type hypersensitivity (model 1), the HLA-DQ8 transgenic (model 2), and the gliadin memory T-cell enteropathy (model 3) models of celiac disease, intravenous injections of TIMP-GLIA significantly decreased gliadin-specific T-cell proliferation (in models 1 and 2), inflammatory cytokine secretion (in models 1, 2, and 3), circulating gliadin-specific IgG/IgG2c (in models 1 and 2), ear swelling (in model 1), gluten-dependent enteropathy (in model 3), and body weight loss (in model 3). In model 1, the effects were shown to be dose dependent. Splenocytes from HLA-DQ8 transgenic mice given TIMP-GLIA nanoparticles, but not control nanoparticles, had increased levels of FOXP3 and gene expression signatures associated with tolerance induction. Conclusions In mice with gliadin sensitivity, injection of TIMP-GLIA nanoparticles induced unresponsiveness to gliadin and reduced markers of inflammation and enteropathy. This strategy might be developed for the treatment of celiac disease.

The natural CD25 + FOXP3 + regulatory T cell (Treg) population is generated as a distinct lineage in the thymus, but the details of Treg development in humans remain unclear, and the timing of Treg commitment is also contested. Here we have analyzed the emergence of CD25 + cells at the CD4 + CD8 + double positive (DP) stage in the human thymus. We show that these cells share T cell receptor repertoire with CD25 + CD4 single-positive thymocytes, believed to be committed Tregs. They already have a fully demethylated FOXP3 enhancer region and thus display stable expression of FOXP3 and the associated Treg phenotype. Transcriptome analysis also grouped the DP CD25 + and CD4 CD25 + thymocytes apart from the CD25 - subsets. Together with earlier studies, our data are consistent with human Treg commitment already at the DP thymocyte stage. We suggest that the most important antigens and signals necessary for human Treg differentiation may be found in the thymic cortex.

The alternative pathway (AP) of complement is constantly active in plasma and can easily be activated on self surfaces and trigger local inflammation. Host cells are protected from AP attack by Factor H (FH), the main AP regulator in plasma. Although complement is known to play a role in atherosclerosis, the mechanisms of its contribution are not fully understood. Since FH via its domains 5-7 binds apoliporotein E (apoE) and macrophages produce apoE we examined how FH could be involved in the antiatherogenic effects of apoE. We used blood peripheral monocytes and THP-1 monocyte/macrophage cells which were also loaded with acetylated low-density lipoprotein (LDL) to form foam cells. Binding of FH and apoE on these cells was analyzed by flow cytometry. High-density lipoprotein (HDL)-mediated cholesterol efflux of activated THP-1 cells was measured and transcriptomes of THP-1 cells using mRNA sequencing were determined. We found that binding of FH to human blood monocytes and cholesterol-loaded THP-1 macrophages increased apoE binding to these cells. Preincubation of fluorescent cholesterol labeled THP-1 macrophages in the presence of FH increased cholesterol efflux and cholesterol-loaded macrophages displayed reduced transcription of proinflammatory/proatherogenic factors and increased transcription of anti-inflammatory/anti-atherogenic factors. Further incubation of THP-1 cells with serum reduced C3b/iC3b deposition. Overall, our data indicate that apoE and FH interact with monocytic cells in a concerted action and this interaction reduces complement activation and inflammation in the atherosclerotic lesions. By this way FH may participate in mediating the beneficial effects of apoE in suppressing atherosclerotic lesion progression.

Pharyngeal tonsillitis is one of the most common upper respiratory tract infections, and group A streptococcus is the most important bacterial pathogen causing it. While most patients experience tonsillitis only rarely, a subset of patients suffers from recurrent or chronic tonsillitis or pharyngitis. The predisposing factors for recurring or chronic forms of this disease are not yet fully understood, but genetic predisposition has been suggested. A genetic association study using Illumina's Immunochip single-nucleotide polymorphism (SNP) array was performed to search for new genetic biomarkers in pharyngeal tonsillitis. More than 100,000 SNPs relevant to immune-mediated diseases were analyzed in a cohort of 95 patients subjected to tonsillectomy due to recurrent/chronic tonsillitis and 504 controls. Genetic association between the cases and controls showed strongest association with two peaks in the HLA locus (odds ratio [OR], 3.7 to 4.7; P = 4.9 x 10(-6) to 5.7 x 10(-6)). Further analysis with imputed classical HLA alleles suggested the known psoriasis risk allele HLA-C*06:02 as a risk factor for tonsillitis (P = 4.8 x 10(-4); OR, 2.3). In addition, the imputed HLA haplotype HLA-C*06:02/HLA-B*57:01, a reported risk haplotype in psoriasis, had the strongest risk for tonsillitis (P = 3.2 x 10(-4); OR, 6.5). These findings further support the previously reported link between streptococcal throat infections and psoriasis.

As part of a broader collaborative network of exome sequencing studies, we developed a jointly called data set of 5,685 Ashkenazi Jewish exomes. We make publicly available a resource of site and allele frequencies, which should serve as a reference for medical genetics in the Ashkenazim (hosted in part at https://ibd.broadinstitute.org, also available in gnomAD at http://gnomad.broadinstitute.org). We estimate that 34% of protein-coding alleles present in the Ashkenazi Jewish population at frequencies greater than 0.2% are significantly more frequent (mean 15-fold) than their maximum frequency observed in other reference populations. Arising via a well-described founder effect approximately 30 generations ago, this catalog of enriched alleles can contribute to differences in genetic risk and overall prevalence of diseases between populations. As validation we document 148 AJ enriched protein-altering alleles that overlap with "pathogenic" ClinVar alleles (table available at https://github.com/macarthur-lab/clinvar/blob/master/output/clinvar.tsv), including those that account for 10–100 fold differences in prevalence between AJ and non-AJ populations of some rare diseases, especially recessive conditions, including Gaucher disease (GBA, p.Asn409Ser, 8-fold enrichment); Canavan disease (ASPA, p.Glu285Ala, 12-fold enrichment); and Tay-Sachs disease (HEXA, c.1421+1G>C, 27-fold enrichment; p.Tyr427IlefsTer5, 12-fold enrichment). We next sought to use this catalog, of well-established relevance to Mendelian disease, to explore Crohn's disease, a common disease with an estimated two to four-fold excess prevalence in AJ. We specifically attempt to evaluate whether strong acting rare alleles, particularly protein-truncating or otherwise large effect-size alleles, enriched by the same founder-effect, contribute excess genetic risk to Crohn's disease in AJ, and find that ten rare genetic risk factors in NOD2 and LRRK2 are enriched in AJ (p < 0.005), including several novel contributing alleles, show evidence of association to CD. Independently, we find that genomewide common variant risk defined by GWAS shows a strong difference between AJ and non-AJ European control population samples (0.97 s.d. higher, p, Author summary The Ashkenazim are a people with ancestry in northern-European Jewish groups. A founder effect caused a bottleneck in this population approximately one thousand years ago, resulting in a group of enriched alleles in their genetic makeup. A higher documented prevalence of Crohn’s Disease in the Ashkenazim indicates that some enriched alleles may confer risk of having this disease. By studying which genes are enriched, and which of these contribute to Crohn’s Disease risk, we are better able to understand the genetic architecture of the affected population, and of the disease itself. Further, we are able to develop a resource containing tables of significantly enriched alleles that are known or suspected to contribute to other disease.

Background The revised paediatric criteria for coeliac disease allow omission of duodenal biopsies in symptomatic children who have specific serology and coeliac disease-associated genetics. It remains unclear whether this approach is also applicable for adults with various clinical presentations. Aim To evaluate the accuracy of serology-based criteria in adults with variable pre-test probabilities for coeliac disease. Methods Three study cohorts comprised adults with high-risk clinical coeliac disease suspicion (n = 421), moderate-risk family members of coeliac disease patients (n = 2357), and low-risk subjects from the general population (n = 2722). Serological and clinical data were collected, and "triple criteria" for coeliac disease comprised transglutaminase 2 antibodies >10x the upper limit of normal, positive endomysium antibodies, and appropriate genetics without requirement of symptoms. The diagnosis was based on intestinal biopsy. Results The diagnosis of coeliac disease was established in 274 subjects. Of these, 59 high-risk subjects, 17 moderate-risk subjects, and 14 low-risk subjects fulfilled the "triple criteria". All had histologically proven coeliac disease, giving the criteria a positive predictive value of 100%. Altogether, 90 (33%) of all 274 newly diagnosed patients could have avoided biopsy, including 37% among high-risk, 20% among moderate-risk, and 48% among low-risk patients. No histological findings other than coeliac disease were found in the biopsies of "triple positive" subjects. Conclusions Coeliac disease can reliably and safely be diagnosed without biopsy in adults fulfilling the "triple criteria" regardless of the pre-test probability. Revised criteria would enable the number of endoscopies to be reduced by one-third.

Funding Information: Acknowledgments: Helsinki University Hospital special state subsidy for health science research grants, the Academy of Finland (project 1288701) and Jane & Aatos Erkko Foundation are acknowledged for funding (to MS). KL was supported by the Emil Aaltonen Foundation. Annamari Heikinheimo is thanked for providing the porcine S. aureus strain collection and Taeok Bae for sharing the TB4 and tagO strains. Arnab Bhattacharjee is acknowledged for his help with structural modelling and Joseph Michael Ochieng’ Oduor for critically reading the manuscript. Publisher Copyright: © 2017 by the authors. Staphylococcus aureus is a commensal and pathogenic bacterium that causes infections in humans and animals. It is a major cause of nosocomial infections worldwide. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the pathogen are necessary. In this respect, polyvalent staphylococcal myoviruses have been demonstrated to be excellent candidates for phage therapy. Here we present the characterization of the bacteriophage vB_SauM-fRuSau02 (fRuSau02) that was isolated from a commercial Staphylococcus bacteriophage cocktail produced by Microgen (Moscow, Russia). The genomic analysis revealed that fRuSau02 is very closely related to the phage MSA6, and possesses a large genome (148,464 bp), with typical modular organization and a low G+ C (30.22%) content. It can therefore be classified as a new virus among the genus Twortlikevirus. The genome contains 236 predicted genes, 4 of which were interrupted by insertion sequences. Altogether, 78 different structural and virion-associated proteins were identified from purified phage particles by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The host range of fRuSau02 was tested with 135 strains, including 51 and 54 Staphylococcus aureus isolates from humans and pigs, respectively, and 30 coagulase-negative Staphylococcus strains of human origin. All clinical S. aureus strains were at least moderately sensitive to the phage, while only 39% of the pig strains were infected. Also, some strains of Staphylococcus intermedius, Staphylococcus lugdunensis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus saprophyticus and Staphylococcus pseudointer were sensitive. We conclude that fRuSau02, a phage therapy agent in Russia, can serve as an alternative to antibiotic therapy against S. aureus.

Preeclampsia is a common vascular disease of pregnancy with genetic predisposition. Dysregulation of the complement system has been implicated, but molecular mechanisms are incompletely understood. In this study, we determined the potential linkage of severe preeclampsia to the most central complement gene, C3. Three cohorts of Finnish patients and controls were recruited for a genetic case-control study. Participants were genotyped using Sequenom genotyping and Sanger sequencing. Initially, we studied 259 Finnish patients with severe preeclampsia and 426 controls from the Southern Finland preeclampsia and the Finnish population based preeclampsia cohorts. We used a custom-made single nucleotide polymorphism (SNP) genotyping assay consisting of 98 SNPs in 18 genes that encode components of the complement system. Following the primary screening, C3 was selected as the candidate gene and consequently Sanger sequenced. Fourteen SNPs from C3 were also genotyped by a Sequenom panel in 960 patients with severe preeclampsia and 705 controls, including already sequenced individuals. Three of the 43 SNPs observed within C3 were associated with severe preeclampsia: rs2287845 (p=0.038, OR=1.158), rs366510 (p=0.039, OR=1.158), and rs2287848 (p=0.041, OR=1.155). We also discovered 16 SNP haplotypes with extreme linkage disequilibrium in the middle of the gene with a protective (p=0.044, OR=0.628) or a predisposing (p=0.011, OR=2.110) effect to severe preeclampsia depending on the allele combination. Genetic variants associated with preeclampsia are located in key domains of C3 and could thereby influence the function of C3. This is, as far as we are aware, the first candidate gene in the complement system with an association to a clinically relevant preeclampsia subphenotype, severe preeclampsia. The result highlights a potential role for the complement system in the pathogenesis of preeclampsia and may help in defining prognostic and therapeutic subgroups of preeclamptic women.

Inflammation is an important mediator of obesity-related complications such as the metabolic syndrome but its causes and mechanisms are unknown. As the complement system is a key mediator of inflammation, we studied whether it is activated in acquired obesity in subcutaneous adipose tissue and isolated adipocytes. We used a special study design of genetically matched controls of lean and heavy groups, rare monozygotic twin pairs discordant for BMI (n=26, within-pair difference (in body mass index, kg/m2with as much as 18 kg mean weight. Additionally, 14 BMI-concordant (kg/m2) served as a reference group. The detailed measurements included body composition (DEXA), fat distribution (MRI), glucose, insulin, adipokines, C3a and SC5b-9 levels and the expression of complement and insulin signaling pathway-related genes in adipose tissue and adipocytes. In both adipose tissue and isolated adipocytes, the classical and alternative pathway genes were upregulated, and the terminal pathway genes downregulated in the heavier co-twins of the BMI-discordant pairs. The up-regulated genes included C1q, C1s, C2, ficolin-1, factor H, receptors for C3a and C5a (C5aR1) and the iC3b receptor (CR3). While the terminal pathway components C5 and C6 were downregulated, its inhibitor clusterin was upregulated. Complement gene up-regulation in adipose tissue and adipocytes correlated positively with adiposity and hyperinsulinemia, and negatively with the expression of insulin signaling related genes. Plasma C3a, but not SC5b-9, levels were elevated in the heavier co-twins. There were no differences between the co-twins in BMI-concordant pairs. Obesity is associated with increased expression of the early, but not late, complement pathway components and of key receptors. The twins with acquired obesity have therefore an inflated inflammatory activity in the adipose tissue. The results suggest that complement is likely involved in orchestrating clearance of apoptotic debris and inflammation in the adipose tissue.

Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well- studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005-2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system.

Celiac disease (CD) patients mount an abnormal immune response to gluten. T-cell receptor (TCR) repertoires directed to some immunodominant gluten peptides have previously been described, but the global immune response to in vivo gluten exposure in CD has not been systematically investigated yet. Here, we characterized signatures associated with gluten directed immune activity and identified gluten-induced T-cell clonotypes from total blood and gut TCR repertoires in an unbiased manner using immunosequencing. CD patient total TCR repertoires showed increased overlap and substantially altered TRBV-gene usage in both blood and gut samples, and increased diversity in the gut during gluten exposure. Using differential abundance analysis, we identified gluten-induced clonotypes in each patient that were composed of a large private and an important public component. Hierarchical clustering of public clonotypes associated with dietary gluten exposure identified subsets of highly similar clonotypes, the most proliferative of which showing significant enrichment for the motif ASS[LF]R[SW][TD][DT][TE][QA][YF] in PBMC repertoires. These results show that CD-associated clonotypes can be identified and that common gluten associated immune response features can be characterized in vivo from total repertoires, with potential use in disease stratification and monitoring.

The emergence of multiresistant Gram-negative bacteria requires new therapies for combating bacterial infections. Targeting the biogenesis of virulence factors could be an alternative strategy instead of killing bacteria with antibiotics. The outer membrane (OM) of Gram-negative bacteria acts as a physical barrier. At the same time it facilitates the exchange of molecules and harbors a multitude of proteins associated with virulence. In order to insert proteins into the OM, an essential oligomeric membrane-associated protein complex, the ss-barrel assembly machinery (BAM) is required. Being essential for the biogenesis of outer membrane proteins (OMPs) the BAM and also periplasmic chaperones may serve as attractive targets to develop novel antiinfective agents. Herein, we aimed to elucidate which proteins belonging to the OMP biogenesis machinery have the most important function in granting bacterial fitness, OM barrier function, facilitating biogenesis of dedicated virulence factors and determination of overall virulence. To this end we used the enteropathogen Yersinia enterocolitica as a model system. We individually knocked out all non-essential components of the BAM (BamB, C and E) as well as the periplasmic chaperones DegP, SurA and Skp. In summary, we found that the most profound phenotypes were produced by the loss of BamB or SurA with both knockouts resulting in significant attenuation or even avirulence of Ye in a mouse infection model. Thus, we assume that both BamB and SurA are promising targets for the development of new antiinfective drugs in the future.

Human mesenchymal stem/stromal cells (hMSC) are increasingly used in advanced cellular therapies. The clinical use of hMSCs demands sequential cell expansions. As it is well established that membrane glycerophospholipids (GPL) provide precursors for signaling lipids that modulate cellular functions, we studied the effect of the donor's age and cell doublings on the GPL profile of human bone marrow MSC (hBMSC). The hBMSCs, which were harvested from five young and five old adults, showed clear compositional changes during expansion seen at the level of lipid classes, lipid species, and acyl chains. The ratio of phosphatidylinositol to phosphatidylserine increased toward the late-passage samples. Furthermore, 20:4n-6-containing species of phosphatidylcholine and phosphatidylethanolamine accumulated while the species containing monounsaturated fatty acids (FA) decreased during passaging. Additionally, in the total FA pool of the cells, 20:4n-6 increased, which happened at the expense of n-3 polyunsaturated FAs, especially 22:6n-3. The GPL and FA correlated with the decreased immunosuppressive capacity of hBMSCs during expansion. Our observations were further supported by alterations in the gene expression levels of several enzymes involved in lipid metabolism and immunomodulation. The results show that extensive expansion of hBMSCs harmfully modulates membrane GPLs, affecting lipid signaling and eventually impairing functionality.

Mesenchymal stromal cells (MSC) are currently used in many cell based therapies. Prior to use in therapy, extensive expansion is required. We used microarray profiling to investigate expansion induced miRNA and mRNA expression changes of bone marrow MSCs (BM-MSCs) derived from old and young donors. The expression levels of 36 miRNAs were altered in cells derived from the old and respectively 39 miRNAs were altered in cells derived from young donors. Of these, only 12 were differentially expressed in both young and old donor BM-MSCs, and their predicted target mRNAs, were mainly linked to cell proliferation and senescence. Further qPCR verification showed that the expression of miR-1915-3p, miR-1207, miR-3665, and miR-762 correlated with the expansion time at passage 8. Previously described BM-MSC-specific miRNA fingerprints were also detected but these remained unchanged during expansion. Interestingly, members of well-studied miR-17/92 cluster, involved in cell cycle regulation, aging and also development of immune system, were down regulated specifically in cells from old donors. The role of this cluster in MSC functionality is worth future studies since it links expansion, aging and immune system together.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare human autoimmune disorder caused by mutations in the AIRE (autoimmune regulator) gene. Loss of AIRE disrupts thymic negative selection and gives rise to impaired cytotoxic and regulatory T cell populations. To date, CD4(+) T helper (Th) cells remain little studied. This study aims to elucidate their role in APECED pathogenesis.Th cells were explored in ten APECED patients and ten healthy controls using cell culture assays, multiparameter flow cytometry, and transcriptome analysis.The proportions of effector/memory populations were increased while the fraction of naive cells was diminished. The naive population was abnormally activated, with an increased number of cells expressing characteristic Th1, Th2, and Th17 cytokines. No clear deviation to any Th subclass was observed, but transcriptome analysis suggested abnormalities in the Th1 cytokine interferon gamma (IFN-γ) pathway and flow cytometry showed that INF-γ had the highest expression. The augmented INF-γ signaling may promote the function of the putative pathogenic CD8(+) cytotoxic population in the patients. In addition, the frequency of CD4(+) recent thymic emigrants (RTEs) was decreased in the patients, and RTEs also contained cytokine-producing cells at an increased frequency.These data reveal abnormalities in the Th population and suggest that they may in part be traced to premature activation already in the thymus.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access. Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10(-7), odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain. National Institute of Diabetes and Digestive and Kidney Disease (NIDDK) DK064869 DK062432 National Human Genome Research Institute (NHGRI) DK064869 DK043351 HG005923 Crohns and Colitis Foundation 3765 Leona M. & Harry B. Helmsley Charitable Trust 2015PG-IBD001 Amgen 2013583217 CCFA 3765 Cedars-Sinai F. Widjaja Foundation, info:eu-repo/grantAgreement/EC/FP7/305479, European Union DK062413 AI067068 U54DE023789-01 Leona M. and Harry B. Helmsley Charitable Trust Crohn's and Colitis Foundation of America NIH DK062431 U01 DK062429 U01 DK062422 R01 DK092235 U01 DK062420 Medical Research Council, UK MR/J00314X/1 Wellcome Trust WT091310 098051 Inflammatory Bowel Disease Genetic Research Chair at the University of Pittsburgh PO1DK046763

Dermatitis herpetiformis (DH) is a blistering skin disease, which is regarded as an extra-intestinal manifestation of coeliac disease. Refractory cases of coeliac disease, that do not respond to a gluten-free diet and which carry an increased risk of lymphoma, are well-known in coeliac disease. To determine whether refractory cases of DH with active rash and persistent small bowel atrophy occur we analysed our series of 403 patients with DH. Seven (1.7%) patients, who had been on a gluten-free diet for a mean of 16 years, but who still required dapsone to treat the symptoms of DH, were identified. Of these, one patient died from mucinous adenocarcinoma before re-examination. At re-examination skin immunoglobulin A (IgA) deposits were found in 5/6 refractory and 3/16 control DH patients with good dietary response. Small bowel mucosa was studied at re-examination from 5 refractory and 8 control DH patients and was normal in all 5 refractory and 7/8 control DH patients. One refractory DH patient died from adenocarcinoma, but no lymphoma developed in any of the patients. This study documents for the first time refractory DH, in which the rash is non-responsive to a gluten-free diet, but the small bowel mucosa heals. This differs from refractory coeliac disease, in which the small bowel mucosa does not heal on a gluten-free diet.

Oxysterols are oxidized 27-carbon cholesterol derivatives or by-products of cholesterol biosynthesis, with a spectrum of biologic activities. Several oxysterols have cytotoxic and pro-apoptotic activities, the ability to interfere with the lateral domain organization, and packing of membrane lipids. These properties may account for their suggested roles in the pathology of diseases such as atherosclerosis, age-onset macular degeneration and Alzheimer’s disease. Oxysterols also have the capacity to induce inflammatory responses and play roles in cell differentiation processes. The functions of oxysterols as intermediates in the synthesis of bile acids and steroid hormones, and as readily transportable forms of sterol, are well established. Furthermore, their actions as endogenous regulators of gene expression in lipid metabolism via liver X receptors and the Insig (insulin-induced gene) proteins have been investigated in detail. The cytoplasmic oxysterol-binding protein (OSBP) homologues form a group of oxysterol/cholesterol sensors that has recently attracted a lot of attention. However, their mode of action is, as yet, poorly understood. Retinoic acid receptor-related orphan receptors (ROR) α and γ, and Epstein-Barr virus induced gene 2 (EBI2) have been identified as novel oxysterol receptors, revealing new physiologic oxysterol effector mechanisms in development, metabolism, and immunity, and evoking enhanced interest in these compounds in the field of biomedicine.

Gliadin triggers T-cell mediated immunity in celiac disease, and has cytotoxic effects on enterocytes mediated through obscure mechanisms. In addition, gliadin transport mechanisms, potential cell surface receptors and gliadin-activated downstream signaling pathways are not completely understood. In order to screen for novel downstream gliadin target genes we performed a systematic whole genome expression study on intestinal epithelial cells. Undifferentiated Caco-2 cells were exposed to pepsin- and trypsin- digested gliadin (PT-G), a blank pepsin-trypsin control (PT) and to a synthetic peptide corresponding to gliadin p31-43 peptide for six hours. RNA from four different experiments was used for hybridization on Agilent one color human whole genome DNA microarray chips. The microarray data were analyzed using the Bioconductor package LIMMA. Genes with nominal p

To investigate the association between serum antibody levels and a subsequent celiac disease diagnosis in a large series of children and adults.Besides subjects with classical gastrointestinal presentation of celiac disease, the study cohort included a substantial number of individuals with extraintestinal symptoms and those found by screening in at-risk groups. Altogether 405 patients underwent clinical, serological and histological evaluations. After collection of data, the antibody values were further graded as low [endomysial (EmA) 1:5-200, transglutaminase 2 antibodies (TG2-ab) 5.0-30.0 U/L] and high (EmA 1: ≥ 500, TG2-ab ≥ 30.0 U/L), and the serological results were compared with the small intestinal mucosal histology and clinical presentation.In total, 79% of the subjects with low and 94% of those with high serum EmA titers showed small-bowel mucosal villous atrophy. Furthermore, 96% of the 47 EmA positive subjects who had normal mucosal villi and remained on follow-up either subsequently developed mucosal atrophy while on a gluten-containing diet, or responded positively to a gluten-free diet.Irrespective of the initial serum titers or clinical presentation, EmA positivity as such is a very strong predictor of a subsequent celiac disease diagnosis.

BackgroundNarcolepsy results from immune-mediated destruction of hypocretin secreting neurons in hypothalamus, however the triggers and disease mechanisms are poorly understood. Vaccine-attributable risk of narcolepsy reported so far with the AS03 adjuvanted H1N1 vaccination Pandemrix has been manifold compared to the AS03 adjuvanted Arepanrix, which contained differently produced H1N1 viral antigen preparation. Hence, antigenic differences and antibody response to these vaccines were investigated.Methods and findingsIncreased circulating IgG-antibody levels to Pandemrix H1N1 antigen were found in 47 children with Pandemrix-associated narcolepsy when compared to 57 healthy children vaccinated with Pandemrix. H1N1 antigen of Arepanrix inhibited poorly these antibodies indicating antigenic difference between Arepanrix and Pandemrix. High-resolution gel electrophoresis quantitation and mass spectrometry identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix. Increased antibody levels to hemagglutinin (HA) and NP, particularly to detergent treated NP, was seen in narcolepsy. Higher levels of antibodies to NP were found in children with DQB1*06:02 risk allele and in DQB1*06:02 transgenic mice immunized with Pandemrix when compared to controls.ConclusionsThis work identified 1) higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix, 2) detergent-induced antigenic changes of viral NP, that are recognized by antibodies from children with narcolepsy, and 3) increased antibody response to NP in association of DQB1*06:02 risk allele of narcolepsy. These findings provide a link between Pandemrix and narcolepsy. Although detailed mechanisms of Pandemrix in narcolepsy remain elusive, our results move the focus from adjuvant(s) onto the H1N1 viral proteins.