Transcriptomic analysis of intestine following administration of a transglutaminase 2 inhibitor to prevent gluten-induced intestinal damage in celiac disease.

Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2/HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.

How Future Pharmacologic Therapies for Celiac Disease Will Complement the Gluten-Free Diet.

The only proven treatment for celiac disease is adherence to a strict, lifelong, gluten-free diet. However, complete dietary gluten avoidance is challenging and a substantial number of patients do not respond fully, clinically, or histologically, despite their best efforts. As celiac disease is common and its central pathophysiology is well elucidated, it has become attractive for drug development to address the limitations of dietary treatment. Most efforts address nonresponsive celiac disease, defined as continued symptoms and/or signs of disease activity despite a gluten-free diet, and the more severe forms of refractory celiac disease, types I and II. An increasing spectrum of therapeutic approaches target defined mechanisms in celiac disease pathogenesis and some have advanced to current phase 2 and 3 clinical studies. We discuss these approaches in terms of potential efficiency, practicability, safety, and need, as defined by patients, regulatory authorities, health care providers, and payors.

pH-degradable, bisphosphonate-loaded nanogels attenuate liver fibrosis by repolarization of M2-type macrophages.

Immune-suppressive (M2-type) macrophages can contribute to the progression of cancer and fibrosis. In chronic liver diseases, M2-type macrophages promote the replacement of functional parenchyma by collagen-rich scar tissue. Here, we aim to prevent liver fibrosis progression by repolarizing liver M2-type macrophages toward a nonfibrotic phenotype by applying a pH-degradable, squaric ester­based nanogel carrier system. This nanotechnology platform enables a selective conjugation of the highly water-soluble bisphosphonate alendronate, a macrophage-repolarizing agent that intrinsically targets bone tissue. The covalent delivery system, however, promotes the drug's safe and efficient delivery to nonparenchymal cells of fibrotic livers after intravenous administration. The bisphosphonate payload does not eliminate but instead reprograms profibrotic M2- toward antifibrotic M1-type macrophages in vitro and potently prevents liver fibrosis progression in vivo, mainly via induction of a fibrolytic phenotype, as demonstrated by transcriptomic and proteomic analyses. Therefore, the alendronate-loaded squaric ester­based nanogels represent an attractive approach for nanotherapeutic interventions in fibrosis and other diseases driven by M2-type macrophages, including cancer.

Artificial intelligence-assisted digital pathology for non-alcoholic steatohepatitis: current status and future directions.

The worldwide prevalence of non-alcoholic steatohepatitis (NASH) is increasing, causing a significant medical burden, but no approved therapeutics are currently available. NASH drug development requires histological analysis of liver biopsies by expert pathologists for trial enrolment and efficacy assessment, which can be hindered by multiple issues including sample heterogeneity, inter-reader and intra-reader variability, and ordinal scoring systems. Consequently, there is a high unmet need for accurate, reproducible, quantitative, and automated methods to assist pathologists with histological analysis to improve the precision around treatment and efficacy assessment. Digital pathology (DP) workflows in combination with artificial intelligence (AI) have been established in other areas of medicine and are being actively investigated in NASH to assist pathologists in the evaluation and scoring of NASH histology. DP/AI models can be used to automatically detect, localise, quantify, and score histological parameters and have the potential to reduce the impact of scoring variability in NASH clinical trials. This narrative review provides an overview of DP/AI tools in development for NASH, highlights key regulatory considerations, and discusses how these advances may impact the future of NASH clinical management and drug development. This should be a high priority in the NASH field, particularly to improve the development of safe and effective therapeutics.

A Randomized Trial of a Transglutaminase 2 Inhibitor for Celiac Disease.

BACKGROUND: In celiac disease, small intestinal transglutaminase 2 causes deamidation of glutamine residues in gluten peptides, which enhances stimulation of T cells and leads to mucosal injury. Inhibition of transglutaminase 2 is a potential treatment for celiac disease. METHODS: In a proof-of-concept trial, we assessed the efficacy and safety of a 6-week treatment with ZED1227, a selective oral transglutaminase 2 inhibitor, at three dose levels as compared with placebo, in adults with well-controlled celiac disease who underwent a daily gluten challenge. The primary end point was the attenuation of gluten-induced mucosal damage, as measured by the ratio of villus height to crypt depth. Secondary end points included intraepithelial lymphocyte density, the Celiac Symptom Index score, and the Celiac Disease Questionnaire score (for assessment of health-related quality of life). RESULTS: Of the 41 patients assigned to the 10-mg ZED1227 group, the 41 assigned to the 50-mg group, the 41 assigned to the 100-mg group, and the 40 assigned to the placebo group, 35, 39, 38, and 30 patients, respectively, had adequate duodenal-biopsy samples for the assessment of the primary end point. Treatment with ZED1227 at all three dose levels attenuated gluten-induced duodenal mucosal injury. The estimated difference from placebo in the change in the mean ratio of villus height to crypt depth from baseline to week 6 was 0.44 (95% confidence interval [CI], 0.15 to 0.73) in the 10-mg group (P = 0.001), 0.49 (95% CI, 0.20 to 0.77) in the 50-mg group (P<0.001), and 0.48 (95% CI, 0.20 to 0.77) in the 100-mg group (P<0.001). The estimated differences from placebo in the change in intraepithelial lymphocyte density were -2.7 cells per 100 epithelial cells (95% CI, -7.6 to 2.2) in the 10-mg group, -4.2 cells per 100 epithelial cells (95% CI, -8.9 to 0.6) in the 50-mg group, and -9.6 cells per 100 epithelial cells (95% CI, -14.4 to -4.8) in the 100-mg group. Use of the 100-mg dose may have improved symptom and quality-of-life scores. The most common adverse events, the incidences of which were similar across all groups, were headache, nausea, diarrhea, vomiting, and abdominal pain. Rash developed in 3 of 40 patients (8%) in the 100-mg group. CONCLUSIONS: In this preliminary trial, treatment with ZED1227 attenuated gluten-induced duodenal mucosal damage in patients with celiac disease. (Funded by Dr. Falk Pharma; CEC-3 EudraCT number, 2017-002241-30.).

Machine learning algorithm improves the detection of NASH (NAS-based) and at-risk NASH: A development and validation study.

BACKGROUND AND AIMS: Detecting NASH remains challenging, while at-risk NASH (steatohepatitis and F≥ 2) tends to progress and is of interest for drug development and clinical application. We developed prediction models by supervised machine learning techniques, with clinical data and biomarkers to stage and grade patients with NAFLD. APPROACH AND RESULTS: Learning data were collected in the Liver Investigation: Testing Marker Utility in Steatohepatitis metacohort (966 biopsy-proven NAFLD adults), staged and graded according to NASH CRN. Conditions of interest were the clinical trial definition of NASH (NAS ≥ 4;53%), at-risk NASH (NASH with F ≥ 2;35%), significant (F ≥ 2;47%), and advanced fibrosis (F ≥ 3;28%). Thirty-five predictors were included. Missing data were handled by multiple imputations. Data were randomly split into training/validation (75/25) sets. A gradient boosting machine was applied to develop 2 models for each condition: clinical versus extended (clinical and biomarkers). Two variants of the NASH and at-risk NASH models were constructed: direct and composite models.Clinical gradient boosting machine models for steatosis/inflammation/ballooning had AUCs of 0.94/0.79/0.72. There were no improvements when biomarkers were included. The direct NASH model produced AUCs (clinical/extended) of 0.61/0.65. The composite NASH model performed significantly better (0.71) for both variants. The composite at-risk NASH model had an AUC of 0.83 (clinical and extended), an improvement over the direct model. Significant fibrosis models had AUCs (clinical/extended) of 0.76/0.78. The extended advanced fibrosis model (0.86) performed significantly better than the clinical version (0.82). CONCLUSIONS: Detection of NASH and at-risk NASH can be improved by constructing independent machine learning models for each component, using only clinical predictors. Adding biomarkers only improved the accuracy of fibrosis.

Dietary wheat amylase trypsin inhibitors exacerbate CNS inflammation in experimental multiple sclerosis.

OBJECTIVE: Wheat has become a main staple globally. We studied the effect of defined pro-inflammatory dietary proteins, wheat amylase trypsin inhibitors (ATI), activating intestinal myeloid cells via toll-like receptor 4, in experimental autoimmune encephalitis (EAE), a model of multiple sclerosis (MS). DESIGN: EAE was induced in C57BL/6J mice on standardised dietary regimes with defined content of gluten/ATI. Mice received a gluten and ATI-free diet with defined carbohydrate and protein (casein/zein) content, supplemented with: (a) 25% of gluten and 0.75% ATI; (b) 25% gluten and 0.19% ATI or (c) 1.5% purified ATI. The effect of dietary ATI on clinical EAE severity, on intestinal, mesenteric lymph node, splenic and central nervous system (CNS) subsets of myeloid cells and lymphocytes was analysed. Activation of peripheral blood mononuclear cells from patients with MS and healthy controls was compared. RESULTS: Dietary ATI dose-dependently caused significantly higher EAE clinical scores compared with mice on other dietary regimes, including on gluten alone. This was mediated by increased numbers and activation of pro-inflammatory intestinal, lymph node, splenic and CNS myeloid cells and of CNS-infiltrating encephalitogenic T-lymphocytes. Expectedly, ATI activated peripheral blood monocytes from both patients with MS and healthy controls. CONCLUSIONS: Dietary wheat ATI activate murine and human myeloid cells. The amount of ATI present in an average human wheat-based diet caused mild intestinal inflammation, which was propagated to extraintestinal sites, leading to exacerbation of CNS inflammation and worsening of clinical symptoms in EAE. These results support the importance of the gut-brain axis in inflammatory CNS disease.

NAFLD and NASH biomarker qualification in the LITMUS consortium - Lessons learned.

Biomarkers have the potential to accelerate drug development, as early indicators of improved clinical response, to improve patient safety, and for personalised medicine. However, few have been approved through the biomarker qualification pathways of the regulatory agencies. This paper outlines how biomarkers can accelerate drug development, and reviews the lessons learned by the EU IMI2-funded LITMUS consortium, which has had several interactions with regulatory agencies in both the US and EU regarding biomarker qualification in patients with non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Sharing knowledge of such interactions with the scientific community is of paramount importance to increase the chances of qualification of relevant biomarkers that may accelerate drug development, and thereby help patients, across disease indications. A qualified biomarker enables a decision to be made that all understand and support in a common framework.

Dual roles of B lymphocytes in mouse models of diet-induced nonalcoholic fatty liver disease.

BACKGROUND AND AIMS: Growing evidence suggests an important role of B cells in the development of NAFLD. However, a detailed functional analysis of B cell subsets in NAFLD pathogenesis is lacking. APPROACH AND RESULTS: In wild-type mice, 21 weeks of high fat diet (HFD) feeding resulted in NAFLD with massive macrovesicular steatosis, modest hepatic and adipose tissue inflammation, insulin resistance, and incipient fibrosis. Remarkably, Bnull (JHT) mice were partially protected whereas B cell harboring but antibody-deficient IgMi mice were completely protected from the development of hepatic steatosis, inflammation, and fibrosis. The common feature of JHT and IgMi mice is that they do not secrete antibodies, whereas HFD feeding in wild-type mice led to increased levels of serum IgG2c. Whereas JHT mice have no B cells at all, regulatory B cells were found in the liver of both wild-type and IgMi mice. HFD reduced the number of regulatory B cells and IL-10 production in the liver of wild-type mice, whereas these increased in IgMi mice. Livers of patients with advanced liver fibrosis showed abundant deposition of IgG and stromal B cells and low numbers of IL-10 expressing cells, compatible with our experimental data. CONCLUSIONS: B lymphocytes have both detrimental and protective effects in HFD-induced NAFLD. The lack of secreted pathogenic antibodies protects partially from NAFLD, whereas the presence of certain B cell subsets provides additional protection. IL-10-producing regulatory B cells may represent such a protective B cell subset.

Multiomics Based Association Mapping in Wheat Reveals Genetic Architecture of Quality and Allergenic Related Proteins.

Wheat is an important staple crop since its proteins contribute to human and animal nutrition and are important for its end-use quality. However, wheat proteins can also cause adverse human reactions for a large number of people. We performed a genome wide association study (GWAS) on 114 proteins quantified by LC-MS-based proteomics and expressed in an environmentally stable manner in 148 wheat cultivars with a heritability > 0.6. For 54 proteins, we detected quantitative trait loci (QTL) that exceeded the Bonferroni-corrected significance threshold and explained 17.3−84.5% of the genotypic variance. Proteins in the same family often clustered at a very close chromosomal position or the potential homeolog. Major QTLs were found for four well-known glutenin and gliadin subunits, and the QTL segregation pattern in the protein encoding the high molecular weight glutenin subunit Dx5 could be confirmed by SDS gel-electrophoresis. For nine potential allergenic proteins, large QTLs could be identified, and their measured allele frequencies open the possibility to select for low protein abundance by markers as long as their relevance for human health has been conclusively demonstrated. A potential allergen was introduced in the beginning of 1980s that may be linked to the cluster of resistance genes introgressed on chromosome 2AS from Triticum ventricosum. The reported sequence information for the 54 major QTLs can be used to design efficient markers for future wheat breeding.

The Oral Transglutaminase 2 Inhibitor ZED1227 Accumulates in the Villous Enterocytes in Celiac Disease Patients during Gluten Challenge and Drug Treatment.

The enzyme transglutaminase 2 (TG2) plays a key role in celiac disease (CeD) pathogenesis. Active TG2 is located mainly extracellularly in the lamina propria but also in the villous enterocytes of the duodenum. The TG2 inhibitor ZED1227 is a promising drug candidate for treating CeD and is designed to block the TG2-catalyzed deamidation and crosslinking of gliadin peptides. Our aim was to study the accumulation of ZED1227 after oral administration of the drug. We studied duodenal biopsies derived from a phase 2a clinical drug trial using an antibody that detects ZED1227 when bound to the catalytic center of TG2. Human epithelial organoids were studied in vitro for the effect of ZED1227 on the activity of TG2 using the 5-biotin-pentylamine assay. The ZED1227-TG2 complex was found mainly in the villous enterocytes in post-treatment biopsies. The signal of ZED1227-TG2 was strongest in the luminal epithelial brush border, while the intensity of the signal in the lamina propria was only ~20% of that in the villous enterocytes. No signal specific to ZED1227 could be detected in pretreatment biopsies or in biopsies from patients randomized to the placebo treatment arm. ZED1227-TG2 staining co-localized with total TG2 and native and deamidated gliadin peptides on the enterocyte luminal surface. Inhibition of TG2 activity by ZED1227 was demonstrated in epithelial organoids. Our findings suggest that active TG2 is present at the luminal side of the villous epithelium and that inhibition of TG2 activity by ZED1227 occurs already there before gliadin peptides enter the lamina propria.

A structurally engineered fatty acid, icosabutate, suppresses liver inflammation and fibrosis in NASH.

BACKGROUND & AIMS: Although long-chain omega-3 fatty acids (LCn-3FAs) regulate inflammatory pathways of relevance to non-alcoholic steatohepatitis (NASH), their susceptibility to peroxidation may limit their therapeutic potential. We compared the metabolism of eicosapentaenoic acid (EPA) with an engineered EPA derivative (icosabutate) in human hepatocytes in vitro and their effects on hepatic glutathione metabolism, oxidised lipids, inflammation, and fibrosis in a dietary mouse model of NASH, and in patients prone to fatty liver disease. METHODS: Oxidation rates and cellular partitioning of EPA and icosabutate were compared in primary human hepatocytes. Comparative effects of delayed treatment with either low- (56 mg/kg) or high-dose (112 mg/kg) icosabutate were compared with EPA (91 mg/kg) or a glucagon-like peptide 1 receptor agonist in a choline-deficient (CD), L-amino acid-defined NASH mouse model. To assess the translational potential of these findings, effects on elevated liver enzymes and fibrosis-4 (FIB-4) score were assessed in overweight, hyperlipidaemic patients at an increased risk of NASH. RESULTS: In contrast to EPA, icosabutate resisted oxidation and incorporation into hepatocytes. Icosabutate also reduced inflammation and fibrosis in conjunction with a reversal of CD diet-induced changes in the hepatic lipidome. EPA had minimal effect on any parameter and even worsened fibrosis in association with depletion of hepatic glutathione. In dyslipidaemic patients at risk of NASH, icosabutate rapidly normalised elevated plasma ALT, GGT and AST and reduced FIB-4 in patients with elevated ALT and/or AST. CONCLUSION: Icosabutate does not accumulate in hepatocytes and confers beneficial effects on hepatic oxidative stress, inflammation and fibrosis in mice. In conjunction with reductions in markers of liver injury in hyperlipidaemic patients, these findings suggest that structural engineering of LCn-3FAs offers a novel approach for the treatment of NASH. LAY SUMMARY: Long-chain omega-3 fatty acids are involved in multiple pathways regulating hepatic inflammation and fibrosis, but their susceptibility to peroxidation and use as an energy source may limit their clinical efficacy. Herein, we show that a structurally modified omega-3 fatty acid, icosabutate, overcame these challenges and had markedly improved antifibrotic efficacy in a mouse model of non-alcoholic steatohepatitis. A hepatoprotective effect of icosabutate was also observed in patients with elevated circulating lipids, in whom it led to rapid reductions in markers of liver injury.

Evaluation of Laboratory and Sonographic Parameters for Detection of Portal Hypertension in Patients with Common Variable Immunodeficiency.

Timely detection of portal hypertension as a manifestation in a subgroup of patients with common variable immunodeficiency (CVID) represents a challenge since it is usually not associated with liver cirrhosis. To identify relevant markers for portal hypertension, we evaluated clinical history, laboratory parameters, and abdominal ultrasound including liver elastography and biomarkers of extracellular matrix formation. Twenty seven (6%) of 479 CVID patients presented with clinically significant portal hypertension as defined by either the presence of esophageal varices or ascites. This manifestation occurred late during the course of the disease (11.8 years after first diagnosis of CVID) and was typically part of a multiorgan disease and associated with a high mortality (11/27 patients died during follow up). The strongest association with portal hypertension was found for splenomegaly with a longitudinal diameter of > 16 cm. Similarly, most patients presented with a liver stiffness measurement (LSM) of above 6.5 kPa, and a LSM above 20 kPa was always indicative of manifest portal hypertension. Additionally, many laboratory parameters including Pro-C4 were significantly altered in patients with portal hypertension without clearly increasing the discriminatory power to detect non-cirrhotic portal hypertension in CVID. Our data suggest that a spleen size above 16 cm and an elevated liver stiffness above 6.5 kPa should prompt further evaluation of portal hypertension and its sequelae, but earlier and better liquid biomarkers of this serious secondary complication in CVID are needed.

Co-factors, Microbes, and Immunogenetics in Celiac Disease to Guide Novel Approaches for Diagnosis and Treatment.

Celiac disease (CeD) is a frequent immune-mediated disease that affects not only the small intestine but also many extraintestinal sites. The role of gluten proteins as dietary triggers, HLA-DQ2 or -DQ8 as major necessary genetic predisposition, and tissue transglutaminase (TG2) as mechanistically involved autoantigen, are unique features of CeD. Recent research implicates many cofactors working in synergism with these key triggers, including the intestinal microbiota and their metabolites, nongluten dietary triggers, intestinal barrier defects, novel immune cell phenotypes, and mediators and cytokines. In addition, apart from HLA-DQ2 and -DQ8, multiple and complex predisposing genetic factors and interactions have been defined, most of which overlap with predispositions in other, usually autoimmune, diseases that are linked to CeD. The resultant better understanding of CeD pathogenesis, and its manifold manifestations has already paved the way for novel therapeutic approaches beyond the lifelong strict gluten-free diet, which poses a burden to patients and often does not lead to complete mucosal healing. Thus, supported by improved mouse models for CeD and in vitro organoid cultures, several targeted therapies are in phase 2-3 clinical studies, such as highly effective gluten-degrading oral enzymes, inhibition of TG2, cytokine therapies, induction of tolerance to gluten ingestion, along with adjunctive and preventive approaches using beneficial probiotics and micronutrients. These developments are supported by novel noninvasive markers of CeD severity and activity that may be used as companion diagnostics, allow easy-to perform and reliable monitoring of patients, and finally support personalized therapy for CeD.

Osteoprotegerin Expression in Liver is Induced by IL13 through TGFß.

BACKGROUND/AIMS: Osteoprotegerin (OPG) is a profibrotic mediator produced by myofibro-blasts under influence of transforming growth factor ß (TGFß). Its expression in experimental models of liver fibrosis correlates well with disease severity and treatment responses. The regulation of OPG in liver tissue is largely unknown and we therefore set out to elucidate which growth factors/interleukins associated with fibrosis induce OPG and through which pathways. METHODS: Precision-cut liver slices of wild type and STAT6-deficient mice and 3T3 fibroblasts were used to investigate the effects of TGFß, interleukin (IL) 13 (IL13), IL1ß, and platelet-derived growth factor BB (PDGF-BB) on expression of OPG. OPG protein was measure by ELISA, whereas OPG mRNA and expression of other relevant genes was measured by qPCR. RESULTS: In addition to TGFß, only IL13 and not PDGF-BB or IL1ß could induce OPG expression in 3T3 fibroblasts and liver slices. This IL13-dependent induction was not shown in liver slices of STAT6-deficient mice and when wild type slices were cotreated with TGFß receptor 1 kinase inhibitor galunisertib, STAT6 inhibitor AS1517499, or AP1 inhibitor T5224. This suggests that the OPG-inducing effect of IL13 is mediated through IL13 receptor α1-activation and subsequent STAT6-dependent upregulation of IL13 receptor α2, which in turn activates AP1 and induces production of TGFß and subsequent production of OPG. CONCLUSION: We have shown that IL13 induces OPG release by liver tissue through a TGFß-dependent pathway involving both the α1 and the α2 receptor of IL13 and transcription factors STAT6 and AP1. OPG may therefore be a novel target for the treatment liver fibrosis as it is mechanistically linked to two important regulators of fibrosis in liver, namely IL13 and TGFß1.

Fluorescence Correlation Spectroscopy Monitors the Fate of Degradable Nanocarriers in the Blood Stream.

The use of nanoparticles as carriers to deliver pharmacologically active compounds to specific parts of the body via the bloodstream is a promising therapeutic approach for the effective treatment of various diseases. To reach their target sites, nanocarriers (NCs) need to circulate in the bloodstream for prolonged periods without aggregation, degradation, or cargo loss. However, it is very difficult to identify and monitor small-sized NCs and their cargo in the dense and highly complex blood environment. Here, we present a new fluorescence correlation spectroscopy-based method that allows the precise characterization of fluorescently labeled NCs in samples of less than 50 µL of whole blood. The NC size, concentration, and loading efficiency can be measured to evaluate circulation times, stability, or premature drug release. We apply the new method to follow the fate of pH-degradable fluorescent cargo-loaded nanogels in the blood of live mice for periods of up to 72 h.

Dietary conjugated linoleic acid links reduced intestinal inflammation to amelioration of CNS autoimmunity.

A close interaction between gut immune responses and distant organ-specific autoimmunity including the CNS in multiple sclerosis has been established in recent years. This so-called gut-CNS axis can be shaped by dietary factors, either directly or via indirect modulation of the gut microbiome and its metabolites. Here, we report that dietary supplementation with conjugated linoleic acid, a mixture of linoleic acid isomers, ameliorates CNS autoimmunity in a spontaneous mouse model of multiple sclerosis, accompanied by an attenuation of intestinal barrier dysfunction and inflammation as well as an increase in intestinal myeloid-derived suppressor-like cells. Protective effects of dietary supplementation with conjugated linoleic acid were not abrogated upon microbiota eradication, indicating that the microbiome is dispensable for these conjugated linoleic acid-mediated effects. Instead, we observed a range of direct anti-inflammatory effects of conjugated linoleic acid on murine myeloid cells including an enhanced IL10 production and the capacity to suppress T-cell proliferation. Finally, in a human pilot study in patients with multiple sclerosis (n = 15, under first-line disease-modifying treatment), dietary conjugated linoleic acid-supplementation for 6 months significantly enhanced the anti-inflammatory profiles as well as functional signatures of circulating myeloid cells. Together, our results identify conjugated linoleic acid as a potent modulator of the gut-CNS axis by targeting myeloid cells in the intestine, which in turn control encephalitogenic T-cell responses.

End Group Dye-Labeled Polycarbonate Block Copolymers for Micellar (Immuno-)Drug Delivery.

Defined conjugation of functional molecules to block copolymer end groups is a powerful strategy to enhance the scope of micellar carriers for drug delivery. In this study, an approach to access well-defined polycarbonate-based block copolymers by labeling their end groups with single fluorescent dye molecules is established. Following controlled polymerization conditions, the block copolymers' primary hydroxy end group can be converted into activated pentafluorophenyl ester carbonates and subsequently aminolyzed with fluorescent dyes that are equipped with primary amines. During a solvent-evaporation process, the resulting end group dye-labeled block copolymers self-assemble into narrowly dispersed ∼25 nm-sized micelles and simultaneously encapsulate hydrophobic (immuno-)drugs. The covalently attached fluorescent tracer can be used to monitor both uptake into cells and stability under biologically relevant conditions, including incubation with blood plasma or during blood circulation in zebrafish embryos. By encapsulation of the toll-like receptor 7/8 (TLR7/8) agonist CL075, immune stimulatory polymeric micelles are generated that get internalized by various antigen-presenting dendritic cells and promote their maturation. Generally, such end group dye-labeled polycarbonate block copolymers display ideal features to permit targeted delivery of hydrophobic drugs to key immune cells for vaccination and cancer immunotherapy.

Depletion of CD56+CD3+ invariant natural killer T cells prevents allergen-induced inflammation in humanized mice.

BACKGROUND: CD56-expressing natural killer (NK) cells as well as invariant NK T (iNKT) cells have been shown to either promote or inhibit allergic immune responses. OBJECTIVE: The aim of the present study was to investigate the impact of these cells in a recently developed humanized mouse model of allergen-induced IgE-dependent gut and lung inflammation. METHODS: Nonobese diabetic-severe combined immunodeficiency γ-chain knockout mice were injected intraperitoneally with human PBMCs or CD56-depleted (CD56neg) PBMCs from highly sensitized donors with birch or grass pollen allergy together with the respective allergen or with NaCl as a control. Three weeks later, the mice were challenged with the allergen rectally and gut inflammation was monitored by video miniendoscopy and by histology. Furthermore, airway inflammation was measured after an additional intranasal allergen challenge. RESULTS: Allergen-specific human IgE in mouse sera, detectable only after coinjection of the respective allergen, was reduced in mice being injected with CD56neg PBMCs compared with in mice receiving nondepleted PBMCs. Consequently, allergen-induced IgE-dependent colitis, airway hyperreactivity, and mucus-producing goblet cells were significantly inhibited in these mice. Interestingly, reconstitution of CD56neg PBMCs with nondepleted CD56+ cells and with CD56+CD3+ iNKT cells restored gut as well as lung inflammation, whereas addition of CD3-depleted CD56+ cells did not. CONCLUSION: These results demonstrate that allergen-specific gut and lung inflammation in PBMC-engrafted humanized mice is promoted by CD56+CD3+ iNKT cells, which opens new possibilities of therapeutic intervention in allergic diseases.

Hybrid QconCAT-Based Targeted Absolute and Data-Independent Acquisition-Based Label-Free Quantification Enables In-Depth Proteomic Characterization of Wheat Amylase/Trypsin Inhibitor Extracts.

Wheat amylase/trypsin inhibitors (ATIs) have gained significant relevance as inducers of intestinal and extra-intestinal inflammation. In this study, we present a novel hybrid data-independent acquisition (DIA) liquid chromatography-mass spectrometry (LC-MS) approach, combining QconCAT technology with short microflow LC gradients and DIA and apply the method toward the quantitative proteome analysis of ATI extracts. The presented method is fast, robust, and reproducible and provides precise QconCAT-based absolute quantification of major ATI proteins while simultaneously quantifying the proteome by label-free quantification (LFQ). We analyzed extracts of 60 varieties of common wheat grown in replication and evaluated the reproducibility and precision of the workflow for the quantification of ATIs. Applying the method to analyze different wheat species (i.e., common wheat, spelt, durum wheat, emmer, and einkorn) and comparing the results to published data, we validated inter-laboratory and cross-methodology reproducibility of ATI quantification, which is essential in the context of large-scale breeding projects. Additionally, we applied our workflow to assess environmental effects on ATI expression, analyzing ATI content and proteome of same varieties grown at different locations. Finally, we explored the potential of combining QconCAT-based absolute quantification with DIA-based LFQ proteome analysis for the generation of new hypotheses or assay development.