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High-intensity interval training (HIIT) has been shown to improve cardiorespiratory fitness (CRF) and health-related outcomes in various chronic diseases, including cancer. However, data on feasibility and efficacy of HIIT in advanced cancer patients are still sparse, presumably because of safety concerns, like suspected immunosuppression after vigorous exercise. This randomized, sham-intervention controlled study aimed to investigate feasibility, safety, and preliminary efficacy of very low-volume HIIT (LOW-HIIT) in advanced cancer patients.Twenty-seven patients (55.4 ± 13.2 yr) with different advanced cancers (Union for International Cancer Control [UICC] III/IV) were randomly allocated to LOW-HIIT ( n = 13), consisting of 5 × 1 min cycle ergometer intervals (14 min per session total duration) at 80% to 95% HR peak (two sessions per week for 12 wk), or a sham intervention ( n = 14) performing light physical mobilization exercises (SHAM). Primary outcomes were attrition and attendance rates, with values of ≤25% and ≥80%, respectively, considered acceptable. Secondary outcomes were safety, protocol fidelity, physiological (including CRF measures) and patient-reported outcomes (including fatigue and quality of life).One of 13 patients (8%) receiving LOW-HIIT dropped out. Mean attendance rate was ~93%. The prescribed minimum exercise intensity was consistently reached by all patients. Low-volume HIIT was well tolerated and not associated with any serious adverse event nor with increased infection susceptibility. There were no biochemical signs of acute immunosuppression after LOW-HIIT. Contrarily, differentiation and degranulation of natural killer cells was acutely increased postexercise. Low-volume HIIT improved CRF measures including peak oxygen uptake, self-reported fatigue, physical, and social functioning. No significant changes occurred in the SHAM group.Low-volume HIIT can be regarded as feasible and safe in advanced cancer patients. Our preliminary data indicate favorable acute effects on NK-cells and beneficial chronic adaptations in CRF, fatigue, and aspects of quality of life.

Key points Physical activity is known to protect against cancer. The resistance exercise method whole-body electromyostimulation (WB-EMS) has a significant anti-cancer effect. WB-EMS-conditioned serum from advanced prostate cancer patients decreased human prostate carcinoma cell growth and viability in vitro. Multiplex analysis revealed that genes associated with human prostate cancer cell proliferation and apoptosis are sensitive for exercise. Feasible exercise should be part of multimodal anti-cancer therapies, also for physically weakened patients. Abstract Regular physical activity is known to protect against cancer development. In cancer survivors, exercise reduces the risk of cancer recurrence and mortality. However, the link between exercise and decreased cancer risk and improved survival is still not well understood. Serum from exercising healthy individuals inhibits proliferation and activates apoptosis in various cancer cells, suggesting that mechanisms regulating cancer cell growth are affected by exercise. For the first time, we analysed serum from advanced-stage cancer patients with prostate (exercise group n = 8; control group n = 10) or colorectal (exercise n = 6; control n = 6) cancer, after a 12-week whole-body electromyostimulation training (20 min/session, 2×/week; frequency 85 Hz; pulse width 350 µs; 6 s stimulation, 4 s rest), a tolerable, yet effective, resistance exercise for physically weakened patients. We report that serum from these advanced cancer patients inhibits proliferation and enhances apoptosis of human prostate and colon cancer cells in vitro using cell growth and death assays (5-bromo-2'-deoxyuridine incorporation, cell counting, DNA fragmentation). Exercise-mimicking electric pulse stimulation of human primary myotubes showed that electric pulse stimulation-conditioned myotube medium also impairs human cancer cell viability. Gene expression analysis using a multiplex array of cancer-associated genes and subsequent quantitative RT-PCR revealed the presence of exercise-sensitive genes in human prostate cancer cells that potentially participate in the exercise-mediated regulation of malignant cell growth and apoptosis. Our data document the strong efficiency of the anti-oncogenic effects of physical activity and will further support the application of regular therapeutic exercise during cancer disease.

Simple Summary Pancreatic cancer (PC) is a highly fatal malignancy. A major reason for the poor prognosis of patients with PC is the insensitivity to most oncological treatment approaches. It is known that regular exercise reduces the cancer risk. We have already shown that serum from advanced prostate and colon cancer patients after exercise reduces growth and viability of cancer cells. The aim of this study was to identify exercise-induced cytokines in serum from patients with advanced-stage PC that regulate cancer cell proliferation and apoptosis. Our data suggest that a mild resistance exercise training in advanced PC patients induces the release of CXCL1, IL10 and CCL4 from contracting skeletal muscle. We demonstrate that these myokines inhibit growth and migration of PC cells, and induce PC cell death. With this report we provide new knowledge on the cancer-protective function of exercise in PC. Our data strongly support sport therapies for cancer patients. Abstract The evidence that regular physical exercise reduces the risk of developing cancer is well described. However, the interaction between physical exercise and cancer is not fully clarified yet. Several myokines released by skeletal muscle appear to have a direct anti-tumour function. There are few data on myokine secretion after exercise in patients with advanced tumours. Pancreatic cancer (PC) is a very aggressive and usually fatal cancer. To investigate the effects of exercise in PC, the blood of advanced-stage PC patients was analysed after 12 weeks of resistance training using whole-body electromyostimulation. After the 12-week training period, the patient serum inhibited the proliferation and the motility of PC cells and enhanced PC cell apoptosis. The impact of exercise training was also investigated in an exercise-mimicking in vitro model using electric pulse stimulation of human myotubes and revealed similar anti-tumour effects on PC cells, clearly indicating direct cancer-protective properties of activated skeletal muscle. Protein and gene expression analyses in plasma from exercise-trained patients and in myotube cultures after in vitro exercise showed that interleukin 10 (IL10), C-X-C motif ligand 1 (CXCL1) and C-C motif chemokine ligand 4 (CCL4) are myokines released from activated skeletal muscle. In accordance with the effects of serum from exercise-trained patients, the supplementation with recombinant IL10, CXCL1 and CCL4 impaired growth and migration of PC cells. Treatment of PC cells with these myokines upregulated caspase 3/7 expression and the cleavage of poly(ADP-ribose) polymerase, leading to enhanced PC cell death. The identification of myokines with anti-tumour properties in advanced-stage PC patients after exercise opens a new perspective in supportive therapy with sports and exercise for cancer patients.

Zusammenfassung Hintergrund Die Existenz der Histaminintoleranz (HIT) ist umstritten. Als Ursache wird ein gestörter Histaminabbau infolge einer Diaminooxidase(DAO)-Defizienz vermutet. Doch auch andere Faktoren, z. B. eine erhöhte Histaminproduktion durch Darmbakterien, können zu erhöhten Histaminspiegel beitragen. Ziel dieser Untersuchung war es, eine mögliche Veränderung der Darmbakterien in histaminintoleranten Patienten im Vergleich zu gesunden Personen zu detektieren. Material und Methodik Bei allen Studienprobanden erfolgte die Bestimmung von Gesamt-IgE, nahrungsmittelspezifischen IgEs, TNF-α, Plasmahistamin sowie der Serum-DAO-Aktivität. Anhand von Stuhlproben wurde die Histamin- und Zonulinkonzentration sowie die bakterielle Zusammensetzung mittels 16S-rRNA-Sequenzierung unter Verwendung der Illumina Plattform analysiert. Ergebnisse Insgesamt 18 Probanden (30,2 ± 12,5 Jahre, 94,4 % weiblich) wurden in die Studie eingeschlossen. Diese umfassten Patienten mit HIT (n = 8; 28,9 ± 11,2 Jahre) und gesunde Kontrollen (n = 10; 31,3 ± 13,9 Jahre). Innerhalb der Studiengruppen wurden keine signifikanten Unterschiede in der Histaminkonzentration im Stuhl festgestellt, wohingegen die HIT-Patienten tendenziell erhöhte Zonulinkonzentration im Stuhl aufwiesen. Die Mikrobiomanalyse zeigte eine erniedrigte bakterielle α-Diversität (p = 0,05) und ein erhöhtes Vorkommen von Proteobacteria in der HIT-Gruppe. Weiterhin wiesen die HIT-Patienten ein signifikant geringeres Vorkommen der Butyrat-produzierenden Bakteriengattungen Faecalibacterium (p = 0,045) und Butyricimonas (p = 0,003) auf. Zudem waren die Anteile an Sutterella (p = 0,043) und Hespellia (p = 0,043) erniedrigt. In keiner Studiengruppe wurden vermehrt histaminproduzierende Bakterien detektiert. Schlussfolgerung Unsere Ergebnisse deuten auf eine veränderte Darm-Mikrobiota in Patienten mit HIT hin. Der erhöhte Anteil von Proteobacteria und der erniedrigte Anteil an Butyrat-produzierenden Bakterien sowie höhere Zonulinwerte im Stuhl weisen dabei auf eine mögliche Dysbiose oder eine gestörte Darmbarriere hin. Eine erhöhte intestinale Histaminexposition durch Darmbakterien scheint in der Pathogenese der Histaminintoleranz hingegen unwahrscheinlich.

Summary Background & aims Non-celiac gluten sensitivity (NCGS) is characterized by intestinal and extra-intestinal symptoms triggered by ingestion of gluten. However, non-gluten triggers have recently been implicated, and a FODMAP (fermentable oligo-, di-, monosaccharides and polyols)-reduced diet can partially improve symptoms in NCGS. Our aim was to analyze the effect of a low FODMAP versus a gluten-free diet (GFD) on clinical symptoms, psychological well-being, intestinal inflammation and integrity, and stool microbiota. Methods Nineteen patients with NCGS and ten healthy controls consumed a gluten-containing standard diet before starting a two-week low FODMAP diet; after a five day transition period, participants ingested a GFD for another two weeks. The primary outcome measure was the improvement of clinical symptoms in NCGS patients under the different diets. Secondary outcomes were the determination of dietary effects on intestinal inflammation, psychological well-being, and differences in stool microbiota between NCGS patients and controls. Results The low FODMAP diet and especially the GFD led to a significant improvement of clinical and psychological symptoms in NCGS. A clear reduction in duodenal intraepithelial lymphocytes and mucin-producing Goblet cells was found after the GFD in these patients. Significant microbial differences between NCGS patients and controls were noticed in stool samples at every time point. Both diets caused microbial shifts in all participants, with a greater variability on genus level and metabolisms groups in NCGS patients. Conclusions Our findings suggest a multifactorial etiology of NCGS, due to a functional effect caused by FODMAPs, combined with a mild gluten-triggered immune reaction, and a microbiota dysbalance. ClinicalTrial.gov ID : NCT03268720 .

Aim: The aim of this clinical audit was to assess patient-reported outcomes on the effect of dietary intervention, to enhance our understanding of possible treatment options in irritable bowel syndrome (IBS). Background: A large number of food-related gastro-intestinal disorders have been attributed to IBS for decades. Methods: Patient-reported outcomes from the records of 149 IBS patients treated at secondary and tertiary Gastroenterology outpatients in two UK hospitals between January 2014 and July 2016 were audited. Patients all presented with symptoms fulfilling Rome III-IV criteria for IBS had negative coeliac serology and did not have other gastrointestinal (GI) conditions. A modified version of a low FODMAP diet had been recommended (gluten and lactose free diet (G/LFD)) and was implemented for 6 weeks. Outcomes and dietary adherence were recorded during outpatient’s consultations. Results: A total of 134 patients complied with the diet optimally. The majority had an improvement rate >70% and continued with the diet. Fifty-three percent became completely or almost asymptomatic, while 27.6% had a poor response to the diet (scoring < 30%) to G/LFD. The improvement was excellent in patients with normal BMI and good in overweight and obese and where BMI

Introduction The prevalence of patients with food intolerance (FI) has increased significantly. Immunoglobulin (Ig)E-mediated food allergies (FAs) are detected by determining IgE antibodies and skin prick test. Carbohydrate malabsorptions are clarified with breath tests. However, these diagnostic measures cannot capture all intolerances and have limitations in case of gut-mediated FI. The aims of this pilot study were to evaluate different methods to determine intestinal mucosal IgE in patients with FA and to characterize the intestinal mucosa in patients with FI of unknown origin (FH). Methods Patients with FA and FH were compared with healthy controls. To determine the IgE antibodies and the cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ of the intestinal mucosal, a lavage was performed as part of an ileocolonoscopy and samples were taken using the cytobrush and biopsy forceps. In a subgroup, mucosal samples were also taken from the duodenum. Results Data in homogenates of intestinal mucosal samples yielded the highest sensitivity for IgE antibody titers compared with lavage and cytobrush. Patients with FA presented increased intestinal TNF-α and low IFN-γ values. This was in contrast to FH patients, who showed low intestinal IgE antibodies and TNF-α levels, but increased IFN-γ values. Discussion The determination of IgE antibodies to diagnose intestinal IgE-mediated FA is most reliable in intestinal mucosal samples. Increased TNF-α and low IFN-γ levels in patients with FA characterize an allergic reaction. Decreased TNF-α and increased IFN-γ levels in patients with FH indicate an inflammation-related intolerance reaction (see Visual Abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A520).

Anti-tumor necrosis factor (TNF) antibodies are successfully used in the therapy of inflammatory bowel diseases (IBD). However, the molecular mechanism of action of these agents is still a matter of debate. Apart from neutralization of TNF, influence on the intestinal barrier function, induction of apoptosis in mucosal immune cells, formation of regulatory macrophages as well as other immune modulating properties have been discussed as central features. Nevertheless, clinically effective anti-TNF antibodies were shown to differ in their mode-of-action in vivo and in vitro. Furthermore, the anti-TNF agent etanercept is effective in the treatment of rheumatoid arthritis but failed to induce clinical response in Crohn's disease patients, suggesting different contributions of TNF in the pathogenesis of these inflammatory diseases. In the following, we will review different aspects regarding the mechanism of action of anti-TNF agents in general and analyze comparatively different effects of each anti-TNF agent such as TNF neutralization, modulation of the immune system, reverse signaling and induction of apoptosis. We discuss the relevance of the membrane-bound form of TNF compared to the soluble form for the immunopathogenesis of IBD. Furthermore, we review reports that could lead to personalized medicine approaches regarding treatment with anti-TNF antibodies in chronic intestinal inflammation, by predicting response to therapy.

Objective Patients with chronic inflammatory diseases and malignant tumors have an increased risk of cachexia. No consistent definition exists to rapidly identify cachexia in older patients with and without cancer. Methods One-hundred patients (53% male) aged 70 + years were included in the study by a university hospital. In addition to the detection of malnutrition and determination of body composition by bioelectrical impedance analysis, cachexia was assessed according to the well-established definitions of Evans (weight loss ≥ 5% within the last 12 months plus additional clinical parameters), Fearon (weight loss > 5% in 6 months) and Bozzetti (weight loss ≥ 10% of habitual weight). After a follow-up of 3.5 years, the mortality rate was recorded. Results Thirty-three patients had a malignant tumor disease. The patients with a non-malignant underlying disease did not differ in their mental state, physical condition and state of health compared to patients with cancer. A higher percentage of patients with underlying malignancy had cachexia. There were significant differences in the body composition between the patients with or without cachexia. Cachectic patients exhibited a significantly lower skeletal muscle mass and fat mass. The risk of death was increased in cachectic patients of all three cachexia definitions. Conclusion For clinical daily routine, the assessments by a weight loss according to Fearon and Bozzetti are suggested to be practicable methods to detect cachexia in older patients with and without cancer.

A well-balanced diet is the basis for a healthy life. Both the western diet and special diets can have a relevant impact on the microbiome and promote the development of various diseases. There has been an increase in food-related disorders in recent years, largely associated with dramatic changes in food consumption trends and main nutrients. A major response to food intolerances has been the adoption of new dietary trends involving the reduction or exclusion of specific food ingredients. Especially gluten-containing, but also gluten-free cereals are in the cross-fire. Supporters of the gluten-free diet argue that gluten triggers inflammation and related diseases, while followers of the Paleo diet drastically impeach all cereals as dangerous for human health. To date, no controlled studies support or reject a positive health effect of a gluten-free or cereal-free diet. Future large-scale studies need to evaluate the effect of gluten-containing and gluten-free cereals and the various diets on human health, inflammatory parameters, clinical symptoms, and the gut microbiota (including the bacteria, fungi, and viruses). Dietary-associated changes in compositional and functional microbiota traits should be correlated with the health status for the future development of dietary recommendations and potential clinical interventions.

Die Nicht-Zoliakie-Nicht-Weizenallergie-Weizensensitivitat stellt eine Glutenunvertraglichkeit dar und wird nach Abklarung einer Zoliakie und Weizenallergie als Ausschlussdiagnose gestellt. Aufgrund fehlender diagnostischer Biomarker existieren keine genauen Zahlen zur Haufigkeit und Pravalenzen von 0,5–6 % werden beschrieben. Die Patienten klagen nach dem Verzehr von Weizenprodukten uber sehr rasch auftretende intestinale Beschwerden, wie Blahungen, Bauchschmerzen, Durchfalle oder Ubelkeit, die sich nach Glutenelimination schnell verbessern. Extraintestinal werden vor allem Mudigkeit, Muskelkrampfe, Gelenkschmerzen, Kopfschmerzen und Depressionen beschrieben. Serologische Marker sind nicht bekannt und die Patienten weisen keine Mukosaschadigung auf. Die Ursache der Nicht-Zoliakie-Nicht-Weizenallergie-Weizensensitivitat ist vollig unklar und neben dem Klebereiweis Gluten werden weitere Weizenproteine, Amylase-Trypsin-Inhibitoren oder kurzkettige Kohlenhydrate sowie das intestinale Mikrobiom als auslosende Faktoren diskutiert.

Gluten-related disorders have recently been reclassified with an emerging scientific literature supporting the concept of non-celiac gluten sensitivity (NCGS). New research has specifically addressed prevalence, immune mechanisms, the recognition of non-immunoglobulin E (non-IgE) wheat allergy and overlap of NCGS with irritable bowel syndrome (IBS)-type symptoms. This review article will provide clinicians with an update that directly impacts on the management of a subgroup of their IBS patients whose symptoms are triggered by wheat ingestion.

ObjectiveAnti-tumour necrosis factor (TNF) antibodies are successfully used for treatment of Crohn’s disease. Nevertheless, approximately 40% of patients display failure to anti-TNF therapy. Here, we characterised molecular mechanisms that are associated with endoscopic resistance to anti-TNF therapy.DesignMucosal and blood cells were isolated from patients with Crohn’s disease prior and during anti-TNF therapy. Cytokine profiles, cell surface markers, signalling proteins and cell apoptosis were assessed by microarray, immunohistochemistry, qPCR, ELISA, whole organ cultures and FACS.ResultsResponders to anti-TNF therapy displayed a significantly higher expression of TNF receptor 2 (TNFR2) but not IL23R on T cells than non-responders prior to anti-TNF therapy. During anti-TNF therapy, there was a significant upregulation of mucosal IL-23p19, IL23R and IL-17A in anti-TNF non-responders but not in responders. Apoptosis-resistant TNFR2+IL23R+ T cells were significantly expanded in anti-TNF non-responders compared with responders, expressed the gut tropic integrins α4β7, and exhibited increased expression of IFN-γ, T-bet, IL-17A and RORγt compared with TNFR2+IL23R− cells, indicating a mixed Th1/Th17-like phenotype. Intestinal TNFR2+IL23R+ T cells were activated by IL-23 derived from CD14+ macrophages, which were significantly more present in non-responders prior to anti-TNF treatment. Administration of IL-23 to anti-TNF-treated mucosal organ cultures led to the expansion of CD4+IL23R+TNFR2+ lymphocytes. Functional studies demonstrated that anti-TNF-induced apoptosis in mucosal T cells is abrogated by IL-23.ConclusionsExpansion of apoptosis-resistant intestinal TNFR2+IL23R+ T cells is associated with resistance to anti-TNF therapy in Crohn’s disease. These findings identify IL-23 as a suitable molecular target in patients with Crohn’s disease refractory to anti-TNF therapy.

Patients with inflammatory bowel diseases (IBD) are often affected by nutrient deficiencies. Besides a limited nutrient intake by disease-related side effects and malabsorption, the influence of diets and dietary changes is much discussed. However, data about food patterns are mostly based on subjective declarations. Therefore, this study aimed to achieve a detailed and objective analysis of nutrient intake and individual food habits in IBD, a prerequisite for an appropriate nutritional intervention in risk patients. One hundred IBD patients (n=70 Crohn’s disease (CD), n=30 ulcerative colitis (UC)) and 30 healthy controls (HC) were included in the study. All participants filled in a disease-related and food frequency questionnaire. Fifteen healthy controls and 71 IBD patients additionally completed a questionnaire for self-imposed food restrictions. Most IBD patients and healthy controls showed a normal body mass index. However, within the IBD group, UC patients were more often overweight than CD patients (p=0.007). Food restrictions were common in all participants, but significantly more IBD patients restricted their consumption of fast food (p=0.037), fruits and vegetables (p=0.019). Especially CD patients with acute relapse consumed less fruits and vegetables (p=0.035). Contrary to subjective assessment, an objective survey of dietary intake revealed a significantly higher sugar intake of IBD patients compared to healthy controls (p

Patients with celiac disease display autoantibodies against tissue transglutaminase (TG2), and the high sensitivity and specificity of these autoantibodies render them a reliable tool for diagnosis. However, we found that denatured sera from healthy persons also showed reactivity against TG2.To further examine the specificity of this phenomenon, sera of healthy individuals and celiac patients were denatured by heat or pH shift.Denatured sera of all individuals showed autoantibodies against TG2 in ELISA that could be specifically inhibited by TG2, but the biological role of these autoantibodies remains unknown. The alpha fibrinogen precursor could be isolated as serum protein that reacts with TG2 antibodies and treated sera reacted with fibrinogen in Western blotting. Cross-reactivity of TG2 antibodies with fibrinogen and vice versa was observed.We hypothesise that denaturation of sera reveals hidden autoantibodies against TG2, which might be normally masked by fibrinogen.

This study was done to further reveal the role of the innate immune system in celiac disease.Dendritic cells were matured from venous blood of patients with active or treated celiac disease and DQ2-DQ8-positive or negative controls. Dendritic cells were treated with a peptic-tryptic digest of gliadin (500 microg/ml) and their activation was analyzed by fluorescent-activated cell sorting analysis, cytokine secretion, and their ability to elicit T cell proliferation.Gliadin upregulated interleukin (IL)-6, IL-8, and IL-12 (p40) secretion in dendritic cells and induced strong expression of the maturation markers human leukocyte antigen (HLA)-DR, CD25, CD83, and CD86 of all subjects irrespective of their genotype or the presence of disease, whereas the digest of bovine serum albumin showed no effect. However, gliadin-stimulated dendritic cells from active celiac showed enhanced stimulation of autologous T cells compared to the other groups.Further research should be aimed at identifying the mechanisms that control inflammation in healthy individuals.

Non-celiac non-wheat allergy wheat sensitivity is regarded as discrete glutensensitivity diagnosed after the exclusion of celiac disease and wheat allergy. Due to the absence of reliable biomarkers no exact prevalence rates are known and estimations range between 0,5-6 %. Soon after ingestion of wheat, patients complain of intestinal symptoms mainly bloating, abdominal pain, diarrhea or nausea which improve fast under glutenfree diet. Often extraintestinal manifestation as tiredness, muscle or joint pain, headache and depression are reported. Actually, there are no serological markers and no intestinal mucosal damage was found in patients. The underlying mechanism of the disease is completely unknown and beside of gluten other wheat proteins as well as amylase-trypsin-inhibitor or short chain sugars are discussed as triggers. In addition, the involvement of the intestinal microbiome in pathology of glutensensitivity must be considered.

Non-Celiac Gluten Sensitivity (NCGS) is a syndrome characterized by intestinal and extra-intestinal symptoms related to the ingestion of gluten-containing food, in subjects that are not affected by either celiac disease or wheat allergy. Given the lack of a NCGS biomarker, there is the need for standardizing the procedure leading to the diagnosis confirmation. In this paper we report experts' recommendations on how the diagnostic protocol should be performed for the confirmation of NCGS. A full diagnostic procedure should assess the clinical response to the gluten-free diet (GFD) and measure the effect of a gluten challenge after a period of treatment with the GFD. The clinical evaluation is performed using a self-administered instrument incorporating a modified version of the Gastrointestinal Symptom Rating Scale. The patient identifies one to three main symptoms that are quantitatively assessed using a Numerical Rating Scale with a score ranging from 1 to 10. The double-blind placebo-controlled gluten challenge (8 g/day) includes a one-week challenge followed by a one-week washout of strict GFD and by the crossover to the second one-week challenge. The vehicle should contain cooked, homogeneously distributed gluten. At least a variation of 30% of one to three main symptoms between the gluten and the placebo challenge should be detected to discriminate a positive from a negative result. The guidelines provided in this paper will help the clinician to reach a firm and positive diagnosis of NCGS and facilitate the comparisons of different studies, if adopted internationally.

Mammalian transglutaminases (TGs) catalyze the irreversible post-translational modifications of proteins, the most prominent of which is the calcium-dependent formation of covalent acyl transfers between the γ-carboxamide group of glutamine and the e-amino-group of lysine (GGEL-linkage). In the central nervous system, at least four TG isoforms are present and some of them are differentially expressed under pathological conditions in human patients. However, the precise TG-isoform-dependent enzymatic activities in the brain as well as their anatomical distribution are unknown. Specificity of the used biotinylated peptides was analyzed using an in vitro assay. Isoform-specific TG activity was evaluated in in vitro and in situ studies, using brain extracts and native brain tissue obtained from rodents. Our method allowed us to reveal in vitro and in situ TG-isoform-dependent enzymatic activity in brain extracts and tissue of rats and mice, with a specific focus on TG6. In situ activity of this isoform varied between BACHD mice in comparison to their wt controls. TG isozyme-specific activity can be detected by isoform-specific biotinylated peptides in brain tissue sections of rodents to reveal differences in the anatomical and/or subcellular distribution of TG activity. Our findings yield the basis for a broader application of this method for the screening of pathological expression and activity of TGs in a variety of animal models of human diseases, as in the case of neurodegenerative conditions such as Huntington׳s, Parkinson׳s and Alzheimer׳s, where protein modification is involved as a key mechanism of disease progression.

Background: Dermatitis herpetiformis (DH) is highly associated with celiac disease. Recently, tissue transglutaminase (tTG) was identified as the autoantigen of IgA antibodies in celiac disease. The prevalence of antibodies to tTG in DH patients is not known. Objective: The purpose of this study was to determine whether DH patients show circulating IgA autoantibodies to tTG, to compare their serum levels with those from patients with other subepidermal autoimmune bullous diseases, and to correlate levels of antibodies to tTG with the extent of small bowel disease. Methods: Sera were obtained from consecutive patients with DH (n = 11), linear IgA disease (n = 15), bullous pemphigoid (n = 10), and epidermolysis bullosa acquisita (n = 6) and were studied by indirect immunofluorescence on monkey esophagus and NaCl-split human skin. IgA reactivity to tTG was measured by enzyme-linked immunosorbent assay. The extent of mucosal involvement in jejunal biopsy specimens was assessed according to the grading of Marsh. Results: All untreated DH patients showed circulating IgA autoantibodies to tTG. One DH patient already undergoing therapy, the 15 patients with linear IgA disease, and all other controls revealed no autoantibodies to tTG. In addition, serum levels of IgA anti-tTG antibodies reflected the degree of histopathologic changes in jejunal mucosa from biopsy specimens in DH patients. Conclusion: Our results show that circulating IgA autoantibodies to tTG are detectable in DH but not in linear IgA disease or other subepidermal autoimmune bullous diseases. The levels of IgA anti-tTG antibodies reflect the extent of histopathologic changes of the jejunal mucosa in DH. (J Am Acad Dermatol 1999;41:957-61.)

Background & Aims: Immunoglobulin A (IgA) autoantibodies to endomysium (EMA) are highly specific and sensitive markers for celiac disease. Recently, we identified tissue transglutaminase (tTG) as the major if not sole endomysial autoantigen. Methods: An enzyme-linked immunosorbent assay (ELISA) was established to measure IgA anti-tTG titers in serum samples from 106 celiac patients with partial or subtotal villous atrophy, 43 celiac patients on a gluten-free diet, and 114 diseased and healthy controls. Results were correlated with clinical and histological data and with EMA titers. Results: In patients with biopsy-proven celiac disease consuming a normal, gluten-containing diet, 98.1% of the serum samples had elevated IgA titers against tTG, whereas 94.7% of the control sera were negative. IgA anti-tTG correlated positively with semiquantitative IgA EMA titers ( r = 0.862; P Conclusions: An ELISA based on tTG allows diagnosis of celiac disease with a high sensitivity and specificity. IgA anti-tTG and IgA EMA show an excellent correlation, further confirming the enzyme as the celiac disease autoantigen. Because the assay is quantitative, not subjected to interobserver variation, and easy to perform, it will be a useful tool for population screening of a hitherto underdiagnosed disease. GASTROENTEROLOGY 1998;115:1317-1321

Tissue transglutaminase is demonstrated to be the unknown endomysial autoantigen by means of immunoprecipitations from a fibrosarcoma cell culture. A novel hypothesis for the pathogenesis of celiac disease is formulated: The mainly intracellular tissue transglutaminase is released from cells during wound healing where it aids in stabilizing the wound area by cross-linking a small set of extracellular matrix components.

Cell adhesion to collagen XIV is implied to be mediated by proteoglycans as cellular receptors (T. Ehniset al.,1996,Exp. Cell Res.229, 388–397). In order to define the cell binding region(s), fusion proteins expressed inEscherichia coliand covering the large noncollagenous domain NC3 of collagen XIV were used as substrates for the adhesion of skin fibroblasts. A prominent cell binding site could be localized in the N-terminal fibronectin type III repeat of collagen XIV and its immediate C-terminal extension. Since this region also mediates the binding of the small chondroitin/dermatan sulfate proteoglycan decorin (T. Ehniset al.,1997,J. Biol. Chem.272, 20414–20419), our finding could provide the molecular basis for the observation that decorin serves as inhibitor and potential modulator of cellular interactions with collagen XIV.

The substitution of a glutamine residue with 6-diazo-5-oxo-norleucine (DON) transforms an immunodominant gluten peptide into a potent inhibitor of tissue transglutaminase. DON-modified peptides could be useful for the study and therapy of celiac sprue.

Background & Aims The ubiquitous cross-linking enzyme tissue transglutaminase (TG2) has been implicated in irreversible collagen stabilization in liver fibrosis, although functional evidence is lacking. We studied the contribution of TG2 to hepatic fibrotic matrix stability, as well as liver fibrosis progression and regression in TG2-deficient mice. Methods Advanced liver fibrosis was induced by carbon tetrachloride or thioacetamide in TG2 −/− mice and their wild-type littermates to study fibrosis progression and its spontaneous regression for up to 36 weeks. Pattern and extent of fibrosis were analyzed by histology and hepatic hydroxyproline quantification. Dynamic changes in hepatic matrix cross-linking were assessed by stepwise collagen extraction. Expression of 7 TGs and fibrosis-related genes was determined by quantitative reverse-transcription polymerase chain reaction. Results Transglutaminase activity was increased in fibrosis, and the level of TG2 messenger RNA correlated with the expression of fibrosis-related genes. Biochemical analysis revealed progressive collagen stabilization, with an up to 6-fold increase in the highly cross-linked, pepsin-insoluble fraction (26%). In TG2 −/− mice, hepatic TG activity was significantly decreased, but chronic administration of carbon tetrachloride or thioacetamide led to a comparable extent and pattern of liver fibrosis, as in wild-type mice. In TG2 −/− mice, the composition of hepatic collagen fractions and levels of fibrosis-related transcripts were unchanged, and fibrosis reversal was not facilitated. Conclusions TG2 and TG activity are up-regulated during hepatic fibrosis progression, but do not contribute to fibrogenesis or stabilization of the collagen matrix. TG2 deletion does not promote regression of liver fibrosis. TG2-independent collagen cross-linking is a remarkable feature of progressing hepatic fibrosis and represents an important therapeutic target for liver fibrosis.

Tissue transglutaminase (TG2) was identified as the humoral autoantigen in coeliac disease, but whether it can also serve as T cell autoantigen is still unknown. We aimed, therefore, to firstly explore the presence of TG2-specific T cells in peripheral blood of ten adult patients (four active, i.e. carrying both serological and histological features of the disease; four treated, i.e. with proven mucosal recovery and disappearance of specific antibodies after an adequate period of gluten free diet; and two potential coeliacs, i.e. carrying the serological stigmata of the disease, but not the intestinal lesions), and four healthy controls (two carrying the HLA-DQ2 haplotype of susceptibility to the disease), and secondly to carry out a detailed in vitro characterization of the isolated antigen-specific T cells. T cell lines were first established by means of weekly stimulation with human recombinant TG2 followed by generation of T cell clones through distribution of T cells on plates at one cell/well limiting dilution and further rounds of stimulation. Antigen specificity and HLA-DQ2 restriction were both assessed by evaluating the proliferative response to TG2 in the absence and presence of human sera blocking HLA-DQ2 molecules, after exclusion of impurities in the antigen preparation. Immune phenotyping of T cell clones was performed by flow cytometry, and the expression of IL-1β, IL-4, IL-6, IL-10, IL-12, TGF-β, IFN-γ and TNF-α was determined by ELISA assay on the supernatants of these clones. A total of 91 T cell clones were isolated from the three HLA-DQ2-positive, active patients, but none from the other patients and controls. The immune phenotyping showed that the majority of them (85.7%) were CD3/CD4+ and only a small percentage (14.3%) were CD3/CD8+, all carried the TCR αβ, and had a memory phenotype. The cytokine profile showed high levels of IFN-γ and IL-6 that, together with the absence of IL-4, placed these T cell clones in the T helper type 1-like category. Further in vitro analysis was carried out on 32/91 CD4+ clones and showed a specific and dose-dependent proliferative response towards TG2 and an HLA-DQ2 restriction. Finally, when incubating duodenal mucosal specimens of treated patients with the supernatant of TG2-specific T cell clones, characteristic disease lesions were found, indicating a role for TG2-specific cellular immune response in the pathogenesis of coeliac disease.

Background and aims: Intestinal inflammation in coeliac disease is driven by the gluten fraction of wheat proteins. Deamidation or cross linking of gluten peptides by tissue transglutaminase (tTG), the coeliac disease autoantigen, creates potent T cell stimulatory peptides. Therefore, our aim was to identify the reaction patterns of gluten peptides, intestinal extracellular matrix proteins, and tTG. Methods: tTG activity was analysed by incorporation of monodansyl cadaverine into gliadins. Fluorescence labelled tTG reactive short gliadin peptides were used to demonstrate their deamidation and explore their cross linking patterns with tTG itself or extracellular matrix proteins. Patient sera and controls were checked for autoantibodies to matrix proteins. Results: Gliadins α1–α11, γ1–γ6, ω1–ω3, and ω5 were substrates for tTG. tTG catalysed the cross linking of gliadin peptides with interstitial collagen types I, III, and VI. Coeliac patients showed increased antibody titres against the collagens I, III, V, and VI. Conclusions: tTG formed high molecular weight complexes with all tested gliadins. As all tested gliadins were substrates for tTG, the tTG catalysed modifications were not restricted to single gliadin types and epitopes. Furthermore, haptenisation and long term immobilisation of gliadin peptides by tTG catalysed binding to abundant extracellular matrix proteins could be instrumental in the perpetuation of intestinal inflammation and some associated autoimmune diseases in coeliac disease.

Collagen XIV (CXIV) is a fibril-associated collagen that is mainly expressed in well differentiated tissues and in late embryonic development. Because CXIV is almost absent in proliferating and/or dedifferentiated tissues, a functional role in maintaining cell differentiation is suspected. We demonstrate antiproliferative, quiescence- and differentiation-inducing effects of human CXIV and its recombinant fragments on mesenchymal cells. In primary human fibroblasts, in mouse 3T3 fibroblasts and in 3T3-L1 preadipocytes, CXIV reduced de novo DNA synthesis by 75%, whereas cell numbers and viability remained unaltered. Cells showed no signs of apoptosis, and maximal proliferation was restored when serum was supplemented, thus indicating that CXIV induced reversible cellular quiescence. Exposure of fibroblasts to CXIV in vitro led to cellular bundles and clusters. CXIV also triggered trans-differentiation of 3T3-L1 preadipocytes into adipocytes, as could be shown by lipid accumulation and by expression of the glucose transporter Glut4. These effects were also observed with the amino-terminal recombinant fragment Gln(29)-Pro(154) that harbors the first fibronectin type III domain and a 39-amino-acid extension, whereas no activity was found for all other recombinant CXIV fragments. Based on these finding the development of small molecular analogs that modulate fibroblast cell growth and differentiation, e.g. in wound healing and fibrosis, seems feasible.

Celiac disease is characterized by mucosal changes in the small intestine, ranging from increased numbers of intraepithelial lymphocytes to complete villus effacement with various signs of malabsorption (1,2). Celiac disease is diagnosed by the demonstration of an altered intestinal mucosa in a jejunal biopsy specimen (3). Celiac disease patients invariably have antibodies directed against gliadin and endomysium, a structural component of the extracellular matrix: both antibodies disappear under a gluten-free diet. Therefore, these antibodies are useful tools for diagnosis, and in the dietary control of coeliac disease (3-7). Serum IgA antibodies against endomysial antibodies (EMAs) are especially considered to be sensitive and highly specific markers for celiac disease (8)(9). Previously, EMAs were detected by indirect immunofluorescence on tissue slides of monkey esophagus or human umbilical cord (10-12).

Celiac disease is a complex autoimmune disease which is characterized by a strong genetic association (HLA-DQ2 or -DQ8), gluten as nutritional etiological factor, and the enzyme tissue transglutaminase as endomysial autoantigen. Patients develop highly predictive IgA autoantibodies to tTG. Certain gluten peptides are presented by the disease-associated HLA-DQ2/DQ8 molecules leading to stimulation of gluten-specific T cells. This immune response which is driven in the lamina propria causes the mucosal transformation characteristic for celiac disease. Increased intestinal expression of tTG in patients with CD appears to play an important role in the pathogenesis of CD. Thus, modification of gluten peptides by tTG, especially deamidation of certain glutamine residues, can enhance their binding to HLA-DQ2 or -DQ8 and potentiate T cell stimulation. Furthermore, tTG-catalyzed cross-linking and consequent haptenization of gluten with extracellular matrix proteins allows for storage and extended availability of gluten in the mucosa. New therapeutic approaches aim at proteolytic destruction of immunodominant gliadin peptides that are resistant to intestinal enzymes by bacterial prolyl endopeptidases, the inhibition of tTG activity with highly specific enzyme inhibitors or at HLA-DQ2/DQ8 blocking peptide analogues.