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Helicobacter pylori disturbs the stomach lining during long-term colonization of its human host, with sequelae including ulcers and gastric cancer 1,2 . Numerous H. pylori virulence factors have been identified, showing extensive geographic variation 1 . Here we identify a 'Hardy' ecospecies of H. pylori that shares the ancestry of 'Ubiquitous' H. pylori from the same region in most of the genome but has nearly fixed single-nucleotide polymorphism differences in 100 genes, many of which encode outer membrane proteins and host interaction factors. Most Hardy strains have a second urease, which uses iron as a cofactor rather than nickel 3 , and two additional copies of the vacuolating cytotoxin VacA. Hardy strains currently have a limited distribution, including in Indigenous populations in Siberia and the Americas and in lineages that have jumped from humans to other mammals. Analysis of polymorphism data implies that Hardy and Ubiquitous coexisted in the stomachs of modern humans since before we left Africa and that both were dispersed around the world by our migrations. Our results also show that highly distinct adaptive strategies can arise and be maintained stably within bacterial populations, even in the presence of continuous genetic exchange between strains., (© 2024. The Author(s).)

Abstract: Mantle cell lymphoma (MCL) is dependent on a supportive tumor immune microenvironment (TIME) in which infiltration of CD163+ macrophages has a negative prognostic impact. This study explores how abundance and spatial localization of CD163+ cells are associated with the biology of MCL, using spatial multiomic investigations of tumor and infiltrating CD163+ and CD3+ cells. A total of 63 proteins were measured using GeoMx digital spatial profiling in tissue microarrays from 100 diagnostic MCL tissues. Regions of interest were selected in tumor-rich and tumor-sparse tissue regions. Molecular profiling of CD163+ macrophages, CD20+ MCL cells, and CD3+ T-cells was performed. To validate protein profiles, 1811 messenger RNAs were measured in CD20+ cells and 2 subsets of T cells. Image analysis was used to extract the phenotype and position of each targeted cell, thereby allowing the exploration of cell frequencies and cellular neighborhoods. Proteomic investigations revealed that CD163+ cells modulate their immune profile depending on their localization and that the immune inhibitory molecules, V-domain immunoglobulin suppressor of T-cell activation and B7 homolog 3, have higher expression in tumor-sparse than in tumor-rich tissue regions and that targeting should be explored. We showed that MCL tissues with more abundant infiltration of CD163+ cells have a higher proteomic and transcriptional expression of key components of the MAPK pathway. Thus, the MAPK pathway may be a feasible therapeutic target in patients with MCL with CD163+ cell infiltration. We further showed the independent and combined prognostic values of CD11c and CD163 beyond established risk factors., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Bariatric surgery is an effective treatment option for obesity and provides long-term weight loss and positive effects on metabolism, but the underlying mechanisms are poorly understood. Alterations in bile acid metabolism have been suggested as a potential contributing factor, but comprehensive studies in humans are lacking., Methods: In this study, we analysed the postprandial responses of bile acids, C4 and FGF19 in plasma, and excretion of bile acids in faeces, before and after bariatric surgery in patients (n = 38; 74% females) with obesity with or without type 2 diabetes from the BARIA cohort., Findings: We observed that total fasting plasma bile acid levels increased, and faecal excretion of bile acids decreased after surgery suggesting increased reabsorption of bile acids. Consistent with increased bile acid levels after surgery we observed increased postprandial levels of FGF19 and suppression of the bile acid synthesis marker C4, suggesting increased FXR activation in the gut. We also noted that a subset of bile acids had altered postprandial responses before and after surgery. Finally, fasting plasma levels of 6α-hydroxylated bile acids, which are TGR5 agonists and associated with improved glucose metabolism, were increased after surgery and one of them, HDCA, covaried with diabetes remission in an independent cohort., Interpretation: Our findings provide new insights regarding bile acid kinetics and suggest that bariatric surgery in humans alters bile acid profiles leading to activation of FXR and TGR5, which may contribute to weight loss, improvements in glucose metabolism, and diabetes remission., Funding: Novo Nordisk Fonden, Leducq Foundation, Swedish Heart-Lung Foundation, Knut and Alice Wallenberg Foundation, the ALF-agreement, ZonMw., Competing Interests: Declaration of interests F.B. receives research funding from Biogaia AB, is co-founder and shareholder of Roxbiosens Inc and Implexion AB, and is on the scientific advisory board of Bactolife A/S. L.O is co-founder and shareholder of Roxbiosens Inc. V.G. receives research funding from Spaarne Gastuis. M.N is member of the Scientific Advisory Board of and holds equity in Caelus Pharmaceuticals and receives research funding from and holds equity in Ami therapeutics, the Netherlands. However, none of these possible conflicts of interest bear direct relations to the content of the current paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

With the aim to advance the understanding of immune regulation in MCL and to identify targetable T-cell subsets, we set out to combine image analysis and spatial omic technology focused on both early and late differentiation stages of T cells. MCL patient tissue ( n = 102) was explored using image analysis and GeoMx spatial omics profiling of 69 proteins and 1812 mRNAs. Tumor cells, T helper (T H ) cells and cytotoxic (T C ) cells of early (CD57-) and late (CD57+) differentiation stage were analyzed. An image analysis workflow was developed based on fine-tuned Cellpose models for cell segmentation and classification. T C and CD57+ subsets of T cells were enriched in tumor-rich compared to tumor-sparse regions. Tumor-sparse regions had a higher expression of several key immune suppressive proteins, tentatively controlling T-cell expansion in regions close to the tumor. We revealed that T cells in late differentiation stages (CD57+) are enriched among MCL infiltrating T cells and are predictive of an increased expression of immune suppressive markers. CD47, IDO1 and CTLA-4 were identified as potential targets for patients with T-cell-rich MCL TIME, while GITR might be a feasible target for MCL patients with sparse T-cell infiltration. In subgroups of patients with a high degree of CD57+ T C -cell infiltration, several immune checkpoint inhibitors, including TIGIT, PD-L1 and LAG3 were increased, emphasizing the immune-suppressive features of this highly differentiated T-cell subset not previously described in MCL.

Despite milestones in preventive measures and treatment, cardiovascular disease (CVD) remains associated with a high burden of morbidity and mortality. The protracted nature of the development and progression of CVD motivates the identification of early and complementary targets that might explain and alleviate any residual risk in treated patients. The gut microbiota has emerged as a sentinel between our inner milieu and outer environment and relays a modified risk associated with these factors to the host. Accordingly, numerous mechanistic studies in animal models support a causal role of the gut microbiome in CVD via specific microbial or shared microbiota-host metabolites and have identified converging mammalian targets for these signals. Similarly, large-scale cohort studies have repeatedly reported perturbations of the gut microbial community in CVD, supporting the translational potential of targeting this ecological niche, but the move from bench to bedside has not been smooth. In this Review, we provide an overview of the current evidence on the interconnectedness of the gut microbiome and CVD against the noisy backdrop of highly prevalent confounders in advanced CVD, such as increased metabolic burden and polypharmacy. We further aim to conceptualize the molecular mechanisms at the centre of these associations and identify actionable gut microbiome-based targets, while contextualizing the current knowledge within the clinical scenario and emphasizing the limitations of the field that need to be overcome., (© 2022. Springer Nature Limited.)

Weight loss through bariatric surgery is efficient for treatment or prevention of obesity related diseases such as type 2 diabetes and cardiovascular disease. Long term weight loss response does, however, vary among patients undergoing surgery. Thus, it is difficult to identify predictive markers while most obese individuals have one or more comorbidities. To overcome such challenges, an in-depth multiple omics analyses including fasting peripheral plasma metabolome, fecal metagenome as well as liver, jejunum, and adipose tissue transcriptome were performed for 106 individuals undergoing bariatric surgery. Machine leaning was applied to explore the metabolic differences in individuals and evaluate if metabolism-based patients' stratification is related to their weight loss responses to bariatric surgery. Using Self-Organizing Maps (SOMs) to analyze the plasma metabolome, we identified five distinct metabotypes, which were differentially enriched for KEGG pathways related to immune functions, fatty acid metabolism, protein-signaling, and obesity pathogenesis. The gut metagenome of the most heavily medicated metabotypes, treated simultaneously for multiple cardiometabolic comorbidities, was significantly enriched in Prevotella and Lactobacillus species. This unbiased stratification into SOM-defined metabotypes identified signatures for each metabolic phenotype and we found that the different metabotypes respond differently to bariatric surgery in terms of weight loss after 12 months. An integrative framework that utilizes SOMs and omics integration was developed for stratifying a heterogeneous bariatric surgery cohort. The multiple omics datasets described in this study reveal that the metabotypes are characterized by a concrete metabolic status and different responses in weight loss and adipose tissue reduction over time. Our study thus opens a path to enable patient stratification and hereby allow for improved clinical treatments., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2023 Lappa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Temporal dynamics of the gut microbiota potentially limit the identification of microbial features associated with health status. Here, we used whole-genome metagenomic and 16S rRNA gene sequencing to characterize the intra- and inter-individual variations of gut microbiota composition and functional potential of a disease-free Swedish population (n = 75) over one year. We found that 23% of the total compositional variance was explained by intra-individual variation. The degree of intra-individual compositional variability was negatively associated with the abundance of Faecalibacterium prausnitzii (a butyrate producer) and two Bifidobacterium species. By contrast, the abundance of facultative anaerobes and aerotolerant bacteria such as Escherichia coli and Lactobacillus acidophilus varied extensively, independent of compositional stability. The contribution of intra-individual variance to the total variance was greater for functional pathways than for microbial species. Thus, reliable quantification of microbial features requires repeated samples to address the issue of intra-individual variations of the gut microbiota., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Lymph node metastasis is the single most important prognostic risk factor for recurrence in patients with colon cancer who have undergone curative surgery. The routine method for detecting disseminated tumor cells in lymph nodes is microscopic examination of one or a few hematoxylin and eosin-stained tissue sections by a trained pathologist. This method, however, is insensitive mainly because less than 1% of the lymph node volume is examined, leading to misclassification., Objective: This study aimed to investigate whether analysis of a selected group of biomarker mRNAs improves detection and characterization of lymph node metastases/micrometastases compared with the routine method., Design: This study is a side-by-side comparison of biomarker mRNA analysis and histopathology of 185 lymph nodes from patients with colon cancer representing stages I to IV, and an investigation of the importance of lymph node tissue volume for tumor cell detection., Settings: This is a collaborative study between a high-volume central hospital and a preclinical university institution., Patients: Fifty-seven patients who had undergone tumor resection for colon cancer were included., Main Outcome Measures: The primary outcomes measured were mRNA copies per 18S rRNA copy of CEACAM5, KLK6, SLC35D3, POSTN, and MUC2 by multiplex assay and metastases/micrometastases detected by histopathology., Results: The number of tumor cell-positive lymph nodes was 1.33-fold higher based on CEACAM5 mRNA levels compared with histopathological examination. Increasing the tissue volume analyzed for CEACAM5 levels from an 80-µm section to half a lymph node increased the number of positive nodes from 34 of 107 to 80 of 107 (p < 0.0001). Similarly, the number of positive nodes for the aggressiveness marker KLK6 increased from 9 of 107 to 24 of 107., Limitations: Only a limited number of individual lymph nodes per patient was available for analysis., Conclusions: mRNA analysis of CEACAM5, KLK6, and SLC35D3 improves the detection of tumor cells in lymph nodes from patients surgically treated for colon cancer, and, together with POSTN and MUC2, it further allows characterization of the tumor cells with respect to aggressiveness and the tumor cell environment. See Video Abstract at http://links.lww.com/DCR/B650., El Anlisis De Arnm De Ceacam, Klk, Slcd, Postn Y Muc Mejora La Deteccin Y Permite La Caracterizacin De Clulas Tumorales En Los Ganglios Linfticos De Pacientes Con Cncer De Colon: ANTECEDENTES:Las metástasis en los ganglios linfáticos son el factor de riesgo pronóstico más importante de recurrencia en pacientes con cáncer de colon que se han sometido a cirugía curativa. El método de rutina para detectar células tumorales diseminadas en los ganglios linfáticos es el examen microscópico de una o algunas secciones de tejido teñidas con hematoxilina-eosina por un patólogo capacitado. Sin embargo, este método es insensible principalmente porque se examina menos del 1% del volumen de los ganglios linfáticos, lo que conduce a una clasificación errónea.OBJETIVO:Investigar si el análisis de un grupo seleccionado de ARNm de biomarcadores mejora la detección y caracterización de metástasis / micrometástasis en los ganglios linfáticos en comparación con el método de rutina.DISEÑO:Una comparación en paralelo del análisis de ARNm de biomarcadores y la histopatología de 185 ganglios linfáticos de pacientes con cáncer de colon que representan las etapas I-IV, e investigación de la importancia del volumen de tejido de los ganglios linfáticos para la detección de células tumorales.ENTORNO CLINICO:Estudio colaborativo entre un hospital central de alto volumen y una institución universitaria preclínica.PACIENTES:Cincuenta y siete pacientes que han sido sometidos a resección tumoral por cáncer de colon.PRINCIPALES MEDIDAS DE VALORACION:copias de ARNm / copia de ARNr 18S de CEACAM5, KLK6, SLC35D3, POSTN y MUC2 mediante análisis múltiple y metástasis / micrometástasis detectadas por histopatología.RESULTADOS:El número de ganglios linfáticos con células tumorales positivas fue 1,33 veces mayor según los niveles de ARNm de CEACAM5 en comparación con el examen histopatológico. El aumento del volumen de tejido analizado para los niveles de CEACAM5 de una sección de 80 µm a la mitad de un ganglio linfático aumentó el número de ganglios positivos de 34/107 a 80/107 (p <0,0001). De manera similar, el número de nodos positivos para el marcador de agresividad KLK6 aumentó de 9/107 a 24/107.LIMITACIONES:Solo un número limitado de ganglios linfáticos individuales / paciente estuvo disponible para el análisis.CONCLUSIONES:El análisis de ARNm de CEACAM5, KLK6 y SLC35D3 mejora la detección de células tumorales en los ganglios linfáticos de pacientes con cáncer de colon tratados quirúrgicamente y, junto con POSTN y MUC2, permite además la caracterización de las células tumorales con respecto a la agresividad y el entorno celular tumoral. Consulte Video Resumen en http://links.lww.com/DCR/B650., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Society of Colon and Rectal Surgeons.)

The gut is inhabited by a densely populated ecosystem, the gut microbiota, that is established at birth. However, the succession by which different bacteria are incorporated into the gut microbiota is still relatively unknown. Here, we analyze the microbiota from 471 Swedish children followed from birth to 5 years of age, collecting samples after 4 and 12 months and at 3 and 5 years of age as well as from their mothers at birth using 16S rRNA gene profiling. We also compare their microbiota to an adult Swedish population. Genera follow 4 different colonization patterns during establishment where Methanobrevibacter and Christensenellaceae colonize late and do not reached adult levels at 5 years. These late colonizers correlate with increased alpha diversity in both children and adults. By following the children through age-specific community types, we observe that children have individual dynamics in the gut microbiota development trajectory., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

An important aspect of precision medicine is to probe the stability in molecular profiles among healthy individuals over time. Here, we sample a longitudinal wellness cohort with 100 healthy individuals and analyze blood molecular profiles including proteomics, transcriptomics, lipidomics, metabolomics, autoantibodies and immune cell profiling, complemented with gut microbiota composition and routine clinical chemistry. Overall, our results show high variation between individuals across different molecular readouts, while the intra-individual baseline variation is low. The analyses show that each individual has a unique and stable plasma protein profile throughout the study period and that many individuals also show distinct profiles with regards to the other omics datasets, with strong underlying connections between the blood proteome and the clinical chemistry parameters. In conclusion, the results support an individual-based definition of health and show that comprehensive omics profiling in a longitudinal manner is a path forward for precision medicine.

The link between the gut microbiota and type 2 diabetes (T2D) warrants further investigation because of known confounding effects from antidiabetic treatment. Here, we profiled the gut microbiota in a discovery (n = 1,011) and validation (n = 484) cohort comprising Swedish subjects naive for diabetes treatment and grouped by glycemic status. We observed that overall gut microbiota composition was altered in groups with impaired glucose tolerance, combined glucose intolerance and T2D, but not in those with impaired fasting glucose. In addition, the abundance of several butyrate producers and functional potential for butyrate production were decreased both in prediabetes and T2D groups. Multivariate analyses and machine learning microbiome models indicated that insulin resistance was strongly associated with microbial variations. Therefore, our study indicates that the gut microbiota represents an important modifiable factor to consider when developing precision medicine approaches for the prevention and/or delay of T2D., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: The gut microbiota has been implicated in the aetiology of obesity and associated comorbidities. Patients with Prader-Willi syndrome (PWS) are obese but partly protected against insulin resistance. We hypothesised that the gut microbiota of PWS patients differs from that of non-genetically obese controls and correlate to metabolic health. Therefore, here we used PWS as a model to study the role of gut microbiota in the prevention of metabolic complications linked to obesity., Design: We conducted a case-control study with 17 adult PWS patients and 17 obese subjects matched for body fat mass index, gender and age. The subjects were metabolically characterised and faecal microbiota was profiled by 16S ribosomal RNA gene sequencing. The patients' parents were used as a non-obese control group. Stool samples from two PWS patients and two obese controls were used for faecal microbiota transplantations in germ-free mice to examine the impact of the microbiota on glucose metabolism., Results: The composition of the faecal microbiota in patients with PWS differed from that of obese controls, and was characterised by higher phylogenetic diversity and increased abundance of several taxa such as Akkermansia , Desulfovibrio and Archaea, and decreased abundance of Dorea . Microbial taxa prevalent in the PWS microbiota were associated with markers of insulin sensitivity. Improved insulin resistance of PWS was partly transmitted by faecal microbiota transplantations into germ-free mice., Conclusion: The gut microbiota of PWS patients is similar to that of their non-obese parents and might play a role for the protection of PWS patients from metabolic complications., Competing Interests: Competing interests: FB is in the Scientific Advisory Board of MetaboGen, Sweden., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

​OBJECTIVES: A single nucleotide polymorphism in the NCF1 gene (NCF1-339, rs201802880), encoding NADPH oxidase type II subunit NCF1/p47 phox , reducing production of reactive oxygen species (ROS) is strongly associated with the development of systemic lupus erythematosus (SLE). This study aimed at characterising NCF1-339 effects on neutrophil extracellular trap (NET) formation, type I interferon activity and antibody profile in patients with SLE. ​METHODS: Neutrophil NET-release pathways (n=31), serum interferon (n=141) and finally antibody profiles (n=305) were investigated in SLE subjects from Lund, genotyped for NCF1-339. Then, 1087 SLE subjects from the rheumatology departments of four Swedish SLE centres, genotyped for NCF1-339, were clinically characterised to validate these findings. ​RESULTS: Compared with patients with normal-ROS NCF1-339 genotypes, neutrophils from patients with SLE with low-ROS NCF1-339 genotypes displayed impaired NET formation (p<0.01) and increased dependence on mitochondrial ROS (p<0.05). Low-ROS patients also had increased frequency of high serum interferon activity (80% vs 21.4%, p<0.05) and positivity for anti-β2 glycoprotein I (p<0.01) and anticardiolipin antibodies (p<0.05) but were not associated with other antibodies. We confirmed an over-representation of having any antiphospholipid antibody, OR 1.40 (95% CI 1.01 to 1.95), anti-β2 glycoprotein I, OR 1.82 (95% CI 1.02 to 3.24) and the antiphospholipid syndrome (APS), OR 1.74 (95% CI 1.19 to 2.55) in all four cohorts (n=1087). ​CONCLUSIONS: The NCF1-339 SNP mediated decreased NADPH oxidase function, is associated with high interferon activity and impaired formation of NETs in SLE, allowing dependence on mitochondrial ROS. Unexpectedly, we revealed a striking connection between the ROS deficient NCF1-339 genotypes and the presence of phospholipid antibodies and APS., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

A single nucleotide polymorphism in Ncf1 has been found with a major effect on chronic inflammatory autoimmune diseases in the rat with the surprising observation that a lower reactive oxygen response led to more severe diseases. This finding was subsequently reproduced in the mouse and the effect operates in many different murine diseases through different pathogenic pathways; like models for rheumatoid arthritis, encephalomyelitis, lupus, gout, psoriasis and psoriatic arthritis. The human gene is located in an unstable region with many variable sequence repetitions, which means it has not been included in any genome wide associated screens so far. However, identification of copy number variations and single nucleotide polymorphisms has now clearly shown that major autoimmune diseases are strongly associated with the Ncf1 locus. In systemic lupus erythematosus the associated Ncf1 polymorphism (leading to an amino acid substitution at position 90) is the strongest locus and is associated with a lower reactive oxidative burst response. In addition, more precise mapping analysis of polymorphism of other NOX2 genes reveals that these are also associated with autoimmunity. The identified genetic association shows the importance of redox control and that ROS regulate chronic inflammation instead of promoting it. The genetic identification of Ncf1 polymorphisms now opens for relevant studies of the regulatory mechanisms involved, effects that will have severe consequences in many different pathogenic pathways and understanding of the origin of autoimmune diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: Ncf1 polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases in animal models. The human NCF1 gene is very complex with both functional and non-functional gene copies and genotyping requires assays specific for functional NCF1 genes. We aimed at investigating association and function of the missense single nucleotide polymorphism (SNP), rs201802880 (here denoted NCF1-339) in NCF1 with systemic lupus erythematosus (SLE)., Methods: We genotyped the NCF1-339 SNP in 973 Swedish patients with SLE and 1301 controls, using nested PCR and pyrosequencing. ROS production and gene expression of type 1 interferon-regulated genes were measured in isolated cells from subjects with different NCF1-339 genotypes., Results: We found an increased frequency of the NCF1-339 T allele in patients with SLE, 11% compared with 4% in controls, OR 3.0, 95% CI 2.4 to 3.9, p=7.0×10 -20 . The NCF1-339 T allele reduced extracellular ROS production in neutrophils (p=0.004) and led to an increase expression of type 1 interferon-regulated genes. In addition, the NCF1-339 T allele was associated with a younger age at diagnosis of SLE; mean age 30.3 compared with 35.9, p=2.0×1 -6 ., Conclusions: These results clearly demonstrate that a genetically controlled reduced production of ROS increases the risk of developing SLE and confirm the hypothesis that ROS regulate chronic autoimmune inflammatory diseases., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Metformin is widely used in the treatment of type 2 diabetes (T2D), but its mechanism of action is poorly defined. Recent evidence implicates the gut microbiota as a site of metformin action. In a double-blind study, we randomized individuals with treatment-naive T2D to placebo or metformin for 4 months and showed that metformin had strong effects on the gut microbiome. These results were verified in a subset of the placebo group that switched to metformin 6 months after the start of the trial. Transfer of fecal samples (obtained before and 4 months after treatment) from metformin-treated donors to germ-free mice showed that glucose tolerance was improved in mice that received metformin-altered microbiota. By directly investigating metformin-microbiota interactions in a gut simulator, we showed that metformin affected pathways with common biological functions in species from two different phyla, and many of the metformin-regulated genes in these species encoded metalloproteins or metal transporters. Our findings provide support for the notion that altered gut microbiota mediates some of metformin's antidiabetic effects.

The current review on the function of neutrophil cytosolic factor 1 (NCF1) and induced reactive oxygen species (ROS) is based on a genetic search for the major genes controlling autoimmune inflammatory disorders. Surprisingly, the disease-promoting allele determined a lower ROS response and was therefore in complete contrast to the prevailing dogma. Once cloned, it opened the possibility to dissect this complex field from a new angle and with the possibilities to study the role of ROS in vivo. We found that NCF1 and NADPH oxidase 2 (NOX2) complex-derived ROS is an important regulator of several chronic inflammatory disorders by using models for rheumatoid arthritis, multiple sclerosis, psoriasis and psoriasis arthritis, gout, and lupus. ROS could therefore affect many different types of diseases and the common denominator seems to be that ROS regulate macrophages, which prevents inflammation from going chronic. The role of ROS is currently changing from being seen as toxic agents that will promote inflammation toward a more complex view with ROS as crucial regulators of immune and inflammatory pathways., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Significance: An unexpected finding, revealed by positional cloning of genetic polymorphisms controlling models for rheumatoid arthritis, exposed a new function of Ncf1 and NADPH oxidase (NOX) 2 controlled oxidative burst., Recent Advances: A decreased capacity to produce ROS due to a natural polymorphism was found to be the major factor leading to more severe arthritis and increased T cell-dependent autoimmunity., Critical Issues: In the vein of this finding, we here review a possible new role of ROS in regulating inflammatory cell and autoreactive T cell activity. It is postulated that peroxide is an immunologic transmitter secreted by antigen-presenting cells that downregulate the responses by autoreactive T cells., Future Directions: This may operate at different levels of T cell selection and activation: during negative selection in the thymus, priming of T cells in draining lymph nodes, and while interacting with macrophages in peripheral target tissues.

The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4(+) and CD8(+) T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development.

Copy number variation, namely regions of the genome that can be either deleted or duplicated in a variable way, has emerged as an important source of genetic variance in the human genome. Genes with immunological functions are particularly prone to copy number variation, in part because this is a mechanism to expand the recognition repertoire; however, immunological genes not directly involved in immune recognition are also copy number variable but, despite the link between immunological function and copy number variation, very few copy number variants (CNVs) have been found to be associated with autoimmune diseases, even in recent large genome-wide CNV-association studies. Nonetheless, CNVs in FCGR3B, DEFB4, CCL3L1, C4A/B and NCF1 have been suggested to be associated with autoimmune diseases, although there is conflicting evidence in all cases. The reasons for the lack of definitive data on CNV-autoimmunity associations, as well as the technical challenges for the field are the focus of this review., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: The aim of this study was to investigate genetic variants in the gene neutrophil cytosolic factor 1 (NCF1) for association with rheumatoid arthritis (RA). In rodent models, a single-nucleotide polymorphism (SNP) in Ncf1 has been shown to be a major locus regulating severity of arthritis. Ncf1 encodes one of five subunits of the NADPH oxidase complex. In humans the genomic structure of NCF1 is complex, excluding it from genome-wide association screens and complicating genetic analysis. In addition to copy number variation of NCF1, there are also two nonfunctional pseudogenes, nearly identical in sequence to NCF1. We have characterized copy number variation and SNPs in NCF1, and investigated these variants for association with RA., Results: We find that RA patients are less likely to have an increased copy number of NCF1, 7.6%, compared with 11.6% in controls; p=0.037. We also show that the T-allele of NCF1-339 (rs13447) is expressed in NCF1 and significantly reduces reactive oxygen species production., Innovation: This is the first finding of genetic association of NCF1 with RA. The detailed characterization of genetic variants in NCF1 also helps elucidate the complexity of the NCF1 gene., Conclusion: These data suggest that an increased copy number of NCF1 can be protective against developing RA and add support to previous findings of a role of NCF1 and the phagocyte NADPH oxidase complex in RA pathogenesis.

The Ncf1 gene, encoding the P47(PHOX) protein that regulates production of reactive oxygen species (ROS) by the phagocyte NADPH oxidase (NOX2) complex, is associated with autoimmunity and arthritis severity in rats. We have now identified that the single-nucleotide polymorphism (SNP) resulting in an M153T amino acid substitution mediates arthritis resistance and thus explains the molecular polymorphism underlying the earlier identified Ncf1 gene effect. We identified the SNP in position 153 to regulate ROS production using COS(PHOX) cells transfected with mutated Ncf1. To determine the role of this SNP for control of arthritis, we used the Wistar strain, identified to carry only the postulated arthritis resistant SNP in position 153. When this Ncf1 allele was backcrossed to the arthritis susceptible DA strain, both granulocyte ROS production and arthritis resistance were restored. Position 153 is located in the hinge region between the PX and SH3 domains of P47(PHOX). Mutational analysis of this position revealed a need for an -OH group in the side chain but we found no evidence for phosphorylation. The polymorphism did not affect assembly of the P47(PHOX)/P67(PHOX) complex in the cytosol or membrane localization, but is likely to operate downstream of assembly, affecting activity of the membrane NOX2 complex.

Objective: The severity of joint destruction in rheumatoid arthritis (RA) is highly variable from patient to patient and is influenced by genetic factors. Genome-wide association studies have enormously boosted the field of the genetics of RA susceptibility, but risk loci for RA severity remain poorly defined. A recent meta-analysis of genome-wide association studies identified 6 genetic regions for susceptibility to autoantibody-positive RA: CD40, KIF5A/PIP4K2C, CDK6, CCL21, PRKCQ, and MMEL1/TNFRSF14. The purpose of this study was to investigate whether these newly described genetic regions are associated with the rate of joint destruction., Methods: RA patients enrolled in the Leiden Early Arthritis Clinic were studied (n=563). Yearly radiographs were scored using the Sharp/van der Heijde method (median followup 5 years; maximum followup 9 years). The rate of joint destruction between genotype groups was compared using a linear mixed model, correcting for age, sex, and treatment strategies. A total of 393 anti-citrullinated protein antibody (ACPA)-positive RA patients from the North American Rheumatoid Arthritis Consortium (NARAC) who had radiographic data available were used for the replication study., Results: The TT and CC/CG genotypes of 2 single-nucleotide polymorphisms, rs4810485 (CD40) and rs42041 (CDK6), respectively, were associated with a higher rate of joint destruction in ACPA-positive RA patients (P=0.003 and P=0.012, respectively), with rs4810485 being significant after Bonferroni correction for multiple testing. The association of the CD40 minor allele with the rate of radiographic progression was replicated in the NARAC cohort (P=0.021)., Conclusion: A polymorphism in the CD40 locus is associated with the rate of joint destruction in patients with ACPA-positive RA. Our findings provide one of the first non-HLA-related genetic severity factors that has been replicated.

For a long time, reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) complex have been considered harmful mediators of inflammation owing to their highly reactive nature. However, there are an increasing number of findings suggesting that ROS produced by the NOX2 complex are anti-inflammatory and prevent autoimmune responses, thus challenging existing dogma. ROS might not only be produced as a mechanism to eradicate invading pathogens, but rather as a means by which to fine-tune the inflammatory response, depending on when, where and at what amounts they are produced. In this review, we aim to describe the current findings highlighting ROS as regulators of autoimmune inflammation, focusing on autoimmune arthritis.

Extraordinary technical advances in the field of human genetics over the past few years have catalyzed an explosion of new information about the genetics of human autoimmunity. In particular, the ability to scan the entire genome for common polymorphisms that associate with disease has led to the identification of numerous new risk genes involved in autoimmune phenotypes. Several themes are emerging. Autoimmune disorders have a complex genetic basis; multiple genes contribute to disease risk, each with generally modest effects independently. In addition, it is now clear that common genes underlie multiple autoimmune disorders. There is also heterogeneity among subphenotypes within a disease and across major racial groups. The current crop of genetic associations are only the start of a complete catalog of genetic factors for autoimmunity, and it remains unclear to what extent common variation versus multiple rare variants contribute to disease susceptibility. The current review focuses on recent discoveries within functionally related groups of genes that provide clues to novel pathways of pathogenesis for human autoimmunity.

Autoimmune diseases such as rheumatoid arthritis (RA) are chronic diseases that cannot be prevented or cured If the pathologic basis of such disease would be known, it might be easier to develop new drugs interfering with critical pathway. Genetic analysis of animal models for autoimmune diseases can result in discovery of proteins and pathways that play key function in pathogenesis, which may provide rationales for new therapeutic strategies. Currently, only the MHC class II is clearly associated with human RA and animal models for RA. However, recent data from rats and mice with a polymorphism in Ncf1, a member of the NADPH oxidase complex, indicate a role for oxidative burst in protection from arthritis. Oxidative burst-activating substances can treat and prevent arthritis in rats, as efficiently as clinically applied drugs, suggesting a novel pathway to a therapeutic target in human RA. Here, the authors discuss the role of oxygen radicals in regulating the immune system and autoimmune disease. It is proposed that reactive oxygen species set the threshold for T cell activation and thereby regulate chronic autoimmune inflammatory diseases like RA. In the light of this new hypothesis, new possibilities for preventive and therapeutic treatment of chronic inflammatory diseases are discussed.

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a heritability of 60%. Genetic contributions to RA are made by multiple genes, but only a few gene associations have yet been confirmed. By studying animal models, reduced capacity of the NADPH-oxidase (NOX) complex, caused by a single nucleotide polymorphism (SNP) in one of its components (the NCF1 gene), has been found to increase severity of arthritis. To our knowledge, however, no studies investigating the potential role played by reduced reactive oxygen species production in human RA have yet been reported. In order to examine the role played by the NOX complex in RA, we investigated the association of 51 SNPs in five genes of the NOX complex (CYBB, CYBA, NCF4, NCF2, and RAC2) in a Swedish case-control cohort consisting of 1,842 RA cases and 1,038 control individuals. Several SNPs were found to be mildly associated in men in NCF4 (rs729749, P = 0.001), NCF2 (rs789181, P = 0.02) and RAC2 (rs1476002, P = 0.05). No associations were detected in CYBA or CYBB. By stratifying for autoantibody status, we identified a strong association for rs729749 (in NCF4) in autoantibody negative disease, with the strongest association detected in rheumatoid factor negative men (CT genotype versus CC genotype: odds ratio 0.34, 95% confidence interval 0.2 to 0.6; P = 0.0001). To our knowledge, this is the first genetic association identified between RA and the NOX complex, and it supports previous findings from animal models of the importance of reactive oxygen species production capacity to the development of arthritis.

One of the major quantitative trait loci (QTLs) associated with arthritis in crosses between B10.RIII and RIIIS/J mice is the Cia5 on chromosome 3. Early in the congenic mapping process it was clear that the locus was complex, consisting of several subloci with small effects. Therefore, we developed two novel strategies to dissect a QTL: the partial advanced inter-cross (PAI) strategy, with which we recently found the Cia5 region to consist of three loci, Cia5, Cia21 and Cia22, and now we introduce the QTL-chip strategy, where we have combined congenic mapping with a QTL-restricted expression profiling using a novel microarray design. The expression of QTL genes was compared between parental and congenic mice in lymph node, spleen and paw samples in five biological replicates and in dye-swapped experiments at three time points during the induction phase of arthritis. The QTL chip approach revealed 4 genes located in Cia21, differently expressed in lymph nodes, and 14 genes in Cia22, located within two clusters. One cluster contains six genes, differently expressed in spleen, and the second cluster contains eight genes, differently expressed in paws. We conclude the QTL-chip strategy to be valuable in the selection of candidate genes to be prioritized for further investigation., (Genes and Immunity (2005) 6, 575-583. doi:10.1038/sj.gene.6364242; published online 14 July 2005.)