Diagnosis of Non-Celiac Gluten Sensitivity (NCGS): The Salerno Experts' Criteria.

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.

Non-Celiac Gluten sensitivity: the new frontier of gluten related disorders.

Non Celiac Gluten sensitivity (NCGS) was originally described in the 1980s and recently a "re-discovered" disorder characterized by intestinal and extra-intestinal symptoms related to the ingestion of gluten-containing food, in subjects that are not affected with either celiac disease (CD) or wheat allergy (WA). Although NCGS frequency is still unclear, epidemiological data have been generated that can help establishing the magnitude of the problem. Clinical studies further defined the identity of NCGS and its implications in human disease. An overlap between the irritable bowel syndrome (IBS) and NCGS has been detected, requiring even more stringent diagnostic criteria. Several studies suggested a relationship between NCGS and neuropsychiatric disorders, particularly autism and schizophrenia. The first case reports of NCGS in children have been described. Lack of biomarkers is still a major limitation of clinical studies, making it difficult to differentiate NCGS from other gluten related disorders. Recent studies raised the possibility that, beside gluten, wheat amylase-trypsin inhibitors and low-fermentable, poorly-absorbed, short-chain carbohydrates can contribute to symptoms (at least those related to IBS) experienced by NCGS patients. In this paper we report the major advances and current trends on NCGS.

Randomized, placebo-controlled, double-blind trial of Boswellia serrata in maintaining remission of Crohn's disease: good safety profile but lack of efficacy.

BACKGROUND: Complementary therapies are frequently used by patients with inflammatory bowel disease (IBD). The aim of this study was to evaluate the efficacy and safety of long-term therapy with a new Boswellia serrata extract (Boswelan, PS0201Bo) in maintaining remission in patients with Crohn's disease (CD). METHODS: In 22 German centers a double-blind, placebo-controlled, randomized, parallel study was performed. In all, 108 outpatients with CD in clinical remission were included. Patients were randomized to Boswelan (3×2 capsules/day; 400 mg each) or placebo for 52 weeks. The primary endpoint was the proportion of patients in whom remission was maintained throughout the 52 weeks. Secondary endpoints were time to relapse, changes of Crohn's Disease Activity Index (CDAI), and IBD Questionnaire (IBDQ) scores. RESULTS: The trial was prematurely terminated due to insufficient discrimination of drug and placebo with regard to the primary efficacy endpoint. A total of 82 patients were randomized to Boswelan (n=42) or placebo (n=40). Sixty-six patients could be analyzed for efficacy. 59.9% of the actively treated patients and 55.3% of the placebo group stayed in remission (P=0.85). The mean time to diagnosis of relapse was 171 days for the active group and 185 days for the placebo group (P=0.69). With respect to CDAI, IBDQ, and laboratory measurements of inflammation, no advantages in favor of active treatment were detected. Regarding safety concerns, no disadvantages of taking the drug compared to placebo were observed. CONCLUSIONS: The trial confirmed good tolerability of a new Boswellia serrata extract, Boswelan, in long-term treatment of CD. However, superiority versus placebo in maintenance therapy of remission could not be demonstrated.

Distribution and development of the postnatal murine Vδ1 T-cell receptor repertoire.

Murine γ/δ T cells express canonical Vγ5Vδ1 chains in the epidermis and Vγ6Vδ1 chains at reproductive sites. Both subsets carry an identical Vδ1-Dδ2-Jδ2 chain which completely lacks junctional diversity. These cells are thought to monitor tissue integrity via recognition of stress-induced self antigens. In this study, we showed by reverse transcription-polymerase chain reaction (RT-PCR), complementarity determining region 3 (CDR3) spectratyping and sequencing of the junctional regions of Vδ1 chains from C57BL/6 mice (aged 1 day to 14 months) that the canonical Vδ1-Dδ2-Jδ2 chain is also consistently present at other sites such as the thymus, gut, lung, liver, spleen and peripheral blood. In addition, we found multiple Vδ1 chains with fetal type rearrangements which were also shared among organs and among animals. These Vδ1 chains were typically characterized by a conserved amino acid motif, 'GGIRA'. Furthermore, by analysing the early postnatal period at days 10 and 16, we demonstrated that the diversification of the thymic Vδ1 repertoire is not paralleled by a diversification of extrathymic Vδ1+γ/δ T cells. This indicates that only fetal type rearrangements survive at extrathymic sites. In conclusion, γ/δ T cells expressing the canonical Vδ1-Dδ2-Jδ2 chain are not unique to the skin and reproductive sites. Furthermore, we found other γ/δ T cells expressing fetal type Vδ1 chains which were shared among different organs and animals. Thus, γ/δ T cells expressing conserved Vδ1 chains are likely to have important functions. We suggest a model in which this subset continuously recirculates throughout the organism and rapidly responds to stress-induced self antigens.

Identification of human cathepsin G as a functional target of boswellic acids from the anti-inflammatory remedy frankincense.

Frankincense preparations, used in folk medicine to cure inflammatory diseases, showed anti-inflammatory effectiveness in animal models and clinical trials. Boswellic acids (BAs) constitute major pharmacological principles of frankincense, but their targets and the underlying molecular modes of action are still unclear. Using a BA-affinity Sepharose matrix, a 26-kDa protein was selectively precipitated from human neutrophils and identified as the lysosomal protease cathepsin G (catG) by mass spectrometry (MALDI-TOF) and by immunological analysis. In rigid automated molecular docking experiments BAs tightly bound to the active center of catG, occupying the same part of the binding site as the synthetic catG inhibitor JNJ-10311795 (2-[3-[methyl[1-(2-naphthoyl)piperidin-4-yl]amino]carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid). BAs potently suppressed the proteolytic activity of catG (IC(50) of approximately 600 nM) in a competitive and reversible manner. Related serine proteases were significantly less sensitive against BAs (leukocyte elastase, chymotrypsin, proteinase-3) or not affected (tryptase, chymase). BAs inhibited chemoinvasion but not chemotaxis of challenged neutrophils, and they suppressed Ca(2+) mobilization in human platelets induced by isolated catG or by catG released from activated neutrophils. Finally, oral administration of defined frankincense extracts significantly reduced catG activities in human blood ex vivo vs placebo. In conclusion, we show that catG is a functional and pharmacologically relevant target of BAs, and interference with catG could explain some of the anti-inflammatory properties of frankincense.

Microtubule-targeted drugs inhibit VEGF receptor-2 expression by both transcriptional and post-transcriptional mechanisms.

Cytoskeletal polymers control a wide range of cellular functions, including proliferation, migration, and gene expression. As changes in endothelial cell shape and motility are required to form vascular networks, we hypothesized that disassembly of actin filaments or microtubules may impact endothelial vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) expression as a critical determinant of angiogenesis. We therefore investigated the effect of actin filament- and microtubule-disrupting agents on VEGFR1 and VEGFR2 expression by endothelial cells. Microtubule (MT) disassembly greatly inhibited endothelial VEGFR2 expression, whereas VEGFR1 expression levels remained largely unchanged. These suppressive effects were neither conveyed by increased VEGFR2 shedding nor by shortened protein half-life, suggesting that transcriptional mechanisms account for the observed effects. In line with this conclusion, MT disruption significantly suppressed endothelial VEGFR2 mRNA accumulation. The treatment considerably decreased transcriptional activity of 5'-deletional VEGFR2 promoter gene constructs. MT disruption-mediated repression was conveyed by a GC-rich region harboring two consensus Sp1-binding sites. Electrophoretic mobility-shift assay analysis demonstrated that constitutive Sp1-dependent DNA binding is decreased by MT disassembly. In addition, we provide evidence for additional post-transcriptional regulatory mechanisms, as the VEGFR2 mRNA half-life is significantly reduced by MT-disrupting agents. Hence, both inhibition of the rate of gene transcription and increased mRNA turnover represent critical molecular mechanisms by which MT disruption inhibits VEGFR2 expression.

[Crohn's disease and cancer risk. Incidence/prevalence, clinical characteristics, and potential prevention strategies]. / Morbus Crohn und Malignomrisiko: Häufigkeit, klinische Charakteristika und mögliche Präventionsstrategien.

Epidemiologic data concerning the risk for colorectal cancer in ulcerative colitis are based on a variety of large and well-conducted cohort studies. Guidelines for tumor prevention have been published by the German Gastroenterology Society (DGVS) in 2004. By contrast, the risk constellations in Crohn's disease are less clear and guidelines are lacking. Based on a recent literature search (PubMed March 2007), the published data predominantly expressed as relative risk and/or standardized incidence/mortality ratio are critically reviewed with recommendations for tumor prevention strategies based on individual risk constellations.

On-demand treatment of acute heartburn with the antacid hydrotalcite compared with famotidine and placebo: randomized double-blind cross-over study.

BACKGROUND: Antacids are widely used in the self-treatment of gastroesophageal reflux-induced complaints, but respective studies are lacking. GOALS: To compare the efficacy and safety of hydrotalcite with the H2 receptor antagonist famotidine and placebo in the on-demand treatment of acute heartburn under daily practice conditions. STUDY: Five hundred sixty-two individuals in 4 centers were randomized in a double-blind, double-dummy, 3-fold cross-over study to single-dose treatments of 1000 mg hydrotalcite, 10 mg famotidine, or placebo. Heartburn severity and relief was measured with numerical and visual rating scales. RESULTS: A significantly better heartburn relief score was achieved 60 minutes after administration of hydrotalcite compared with placebo (primary end point, P<0.0001). Better efficacy was also observed 30 minutes and 3 hours after the intake of hydrotalcite in comparison with famotidine or placebo. A significant decrease in heartburn severity compared with placebo occurred within 10 minutes for hydrotalcite and was faster compared with both controls. CONCLUSIONS: For subjects self-administering antacids for episodic heartburn, antacid hydrotalcite provides symptom relief significantly faster and, within the first 3 hours postdosing, more effective than famotidine or placebo. These results suggest that on-demand treatment of hydrotalcite is an effective and well-tolerated therapy for heartburn.

Two groups of porcine TCRgammadelta+ thymocytes behave and diverge differently.

Developmental pathways of gammadelta T cells are still unknown, largely because of the absence of recognized lineage-specific surface markers other than the TCR. We have shown that porcine gammadelta thymocytes can be divided into 12 subsets of the following two major groups: 1) CD4(-) gammadelta thymocytes that can be further subdivided according to their CD2/CD8alphaalpha phenotype, and 2) CD4(+) gammadelta thymocytes that are always CD1(+)CD2(+)CD8alphabeta(+) and have no counterpart in the periphery. In this study, we have analyzed gammadelta thymocyte subsets with respect to behavior during cultivation, cell cycle status, and lymphocyte-specific transcripts. The group of CD4(-) gammadelta thymocytes gives rise to all gammadelta T cells found in the periphery. Proliferating CD2(+)CD8(-)CD1(+)CD45RC(-) gammadelta thymocytes are a common precursor of this group. These precursors differentiate into CD2(+)CD8alphaalpha(+), CD2(+)CD8(-), and CD2(-)CD8(-) gammadelta T cell subsets, which subsequently mature by loss of CD1 and by eventual gain of CD45RC expression. In contrast, the group of CD4(+) gammadelta thymocytes represents transient and independent subsets that are never exported from thymus as TCRgammadelta(+) T cells. In accordance with the following findings, we propose that CD4(+)CD8alphabeta(+) gammadelta thymocytes extinguish their TCRgammadelta expression and differentiate along the alphabeta T cell lineage program: 1) CD4(+) gammadelta thymocytes are actively dividing; 2) CD4(+) gammadelta thymocytes do not die, although their numbers decreased with prolonged cultivation; 3) CD4(+) gammadelta thymocytes express transcripts for RAG-1, TdT, and TCRbeta; and 4) CD4(+) gammadelta thymocytes are able to alter their phenotype to TCRalphabeta(+) thymocytes under appropriate culture conditions.

Celiac disease.

Celiac disease is a chronic intestinal disease caused by intolerance to gluten. It is characterized by immune-mediated enteropathy, associated with maldigestion and malabsorption of most nutrients and vitamins. In predisposed individuals, the ingestion of gluten-containing food such as wheat and rye induces a flat jejunal mucosa with infiltration of lymphocytes. The main symptoms are: stomach pain, gas, and bloating, diarrhea, weight loss, anemia, edema, bone or joint pain. Prevalence for clinically overt celiac disease varies from 1:270 in Finland to 1:5000 in North America. Since celiac disease can be asymptomatic, most subjects are not diagnosed or they can present with atypical symptoms. Furthermore, severe inflammation of the small bowel can be present without any gastrointestinal symptoms. The diagnosis should be made early since celiac disease causes growth retardation in untreated children and atypical symptoms like infertility or neurological symptoms. Diagnosis requires endoscopy with jejunal biopsy. In addition, tissue-transglutaminase antibodies are important to confirm the diagnosis since there are other diseases which can mimic celiac disease. The exact cause of celiac disease is unknown but is thought to be primarily immune mediated (tissue-transglutaminase autoantigen); often the disease is inherited. Management consists in life long withdrawal of dietary gluten, which leads to significant clinical and histological improvement. However, complete normalization of histology can take years.

Development of the neonatal B and T cell repertoire in swine: implications for comparative and veterinary immunology.

Birth in all higher vertebrates is at the center of the critical window of development in which newborns transition from dependence on innate immunity to dependence on their own adaptive immunity, with passive maternal immunity bridging this transition. Therefore we have studied immunological development through fetal and early neonatal life. In swine, B cells appear earlier in fetal development than T cells. B cell development begins in the yolk sac at the 20th day of gestation (DG20), progresses to fetal liver at DG30 and after DG45 continues in bone marrow. The first wave of developing T cells is gammadelta cells expressing a monomorphic Vdelta rearrangement. Thereafter, alphabeta T cells predominate and at birth, at least 19 TRBV subgroups are expressed, 17 of which appear highly homologous with those in humans. In contrast to the T cell repertoire and unlike humans and mice, the porcine pre-immune VH (IGHV-D-J) repertoire is highly restricted, depending primarily on CDR3 for diversity. The V-KAPPA (IGKV-J) repertoire and apparently also the V-LAMBDA (IGLV-J) repertoire, are also restricted. Diversification of the pre-immune B cell repertoire of swine and the ability to respond to both T-dependent and T-independent antigen depends on colonization of the gut after birth in which colonizing bacteria stimulate with Toll-like receptor ligands, especially bacterial DNA. This may explain the link between repertoire diversification and the anatomical location of primary lymphoid tissue like the ileal Peyers patches. Improper development of adaptive immunity can be caused by infectious agents like the porcine reproductive and respiratory syndrome virus that causes immune dysregulation resulting in immunological injury and autoimmunity.

Lymphocyte development in fetal piglets: facts and surprises.

The developing porcine fetus offers an excellent opportunity for the study of lymphocyte development. Studies on B cell, alphabeta T cells and gammadelta T cells in the last decade have expanded our knowledge of lymphocyte development in pigs. These studies have revealed several interesting differences between swine, mice and humans. For example, porcine peripheral lymphocytes include CD4+CD8+ alphabeta T cells and an abundance of gammadelta T cells that may even prevail over the alphabeta population. There are numerous CD2- gammadelta T cells in the blood and a large number of CD8alphaalpha-bearing cells that include NK cells, conventional gammadelta and alphabeta T cells. All porcine B lymphocytes are CD25(lo) and sIgM+ B cells may differ in the expression of CD2 antigen. Unlike mice, porcine B cells appear approximately 2 weeks before T cells and progenitors undergo VDJH rearrangement at 20th day of gestation (DG20) in the yolk sac and DG30 in the fetal liver before consummating high level lymphogenesis in the bone marrow after DG45. Early B cells show an unexpectedly high proportion of in-frame rearrangements, undergo switch recombination in thymus on DG60 and use N-region insertion from the time of the earliest VDJ rearrangement. The genomic repertoire of VH, DH and JH genes is small compared to mice and humans and swine appear to depend on junctional diversity for the majority of their repertoire. The limited VH repertoire of swine contrasts sharply with the porcine TCRbeta repertoire, which is extensive, extraordinarily conserved and nearly identical to that in humans. Therefore, swine present an example of two highly related receptor systems that have diverged in the same species.

Development of gammadelta thymocyte subsets during prenatal and postnatal ontogeny.

In this report, we describe 12 subpopulations of porcine gammadelta thymocytes based on their expression of CD1, CD2, CD4, CD8- isoforms and CD45RC. Our data suggest that gammadelta thymocytes can be divided into two major families: (a) one large family of CD4-gammadelta thymocytes that could be further subdivided according to the CD2/CD8alphaalpha phenotype and (b) a small family of CD4+ gammadelta thymocytes bearing CD8alphabeta and possessing certain unusual features in comparison with other gammadelta thymocytes. Maturation of gammadelta thymocytes within the CD4- family begins with proliferation of the CD2+ CD8- CD1+ CD45RC- gammadelta common precursor. This developmental stage is followed by diversification into the CD2+ CD8alphaalpha+, CD2+ CD8- and CD2- CD8- subsets. Their further maturation is accompanied by a loss of expression of CD1 and by increased expression of CD45RC. Therefore, individual subsets develop from CD1+ CD45RC- through CD1- CD45RC- into CD1- CD45RC+ cells. On the other hand, gammadelta thymocytes within the CD4+ family bear exclusively CD8alphabeta, always express CD1, but may coexpress CD45RC. These cells have no counterpart in the periphery. Our observations suggest that all peripheral CD8+ gammadelta T cells express CD8alphaalpha and that two subsets of these cells differing in major histocompatibility complex II expression, occur. We propose that one subset acquires CD8alphaalpha in the thymus while the second acquires CD8alphaalpha as a result of stimulation in the periphery.

gammadelta T cells link innate and adaptive immune responses.

While most T cells use a CD3-associated alpha/beta T cell receptor as antigen recognition structure, a second population of T cells expresses the alternative gamma/delta T cell receptor. gamma/delta T cells are a minor population in the peripheral blood but constitute a major population among intestinal intraepithelial lymphocytes. Most gamma/delta T cells recognize ligands which are fundamentally different from the short peptides that are seen by alpha/beta T cells in the context of MHC class I or class II molecules. Thus, human Vdelta2 T cells recognize small bacterial phosphoantigens, alkylamines and synthetic aminobisphosphonates, whereas Vdelta1 T cells recognize stress-inducible MHC-related molecules MICA/B as well as several other ligands. At the functional level, gamma/delta T cells rapidly produce a variety of cytokines and usually exert potent cytotoxic activity, also towards many tumor cells. In this article, we discuss the role of gamma/delta T cells as a bridge between the innate and the adaptive immune system, based on the interpretation that gamma/delta T cells use their T cell receptor as a pattern recognition receptor. Our increasing understanding of the ligand recognition and activation mechanisms of gamma/delta T cells also opens new perspectives for the development of gamma/delta T cell-based immunotherapies.

Epithelial defence by gamma delta T cells.

Gamma delta T cells constitute a separate lineage of T lymphocytes which differ from conventional alpha beta T cells with regard to T cell receptor (TCR) repertoire and tissue localization. In murine skin, gamma delta T cells expressing a canonical V gamma5 TCR are abundant and contribute as so-called dendritic epidermal T cells to local immune surveillance. In humans, major subsets of gammadelta T cells are recognized on the basis of their TCR V delta usage. While V delta2 cells dominate in the peripheral blood, V delta1 cells are preferentially localized in mucosal tissue including the intestinal epithelia. In this article we summarize basic features of intraepithelial gamma delta T cells and discuss their possible role in epithelial defence.

Prenatal development of the porcine TCR delta repertoire: dominant expression of an invariant T cell receptor Vdelta3-Jdelta3 chain.

The prenatal development of the porcine gamma/delta TCR repertoire was studied by complementarity-determining region 3 (CDR3) spectratyping and sequencing of TRDV1-DV5 transcripts. Specimens from the small and large intestine, spleen, thymus, liver, bone marrow and PBMC from fetal piglets between 38 and 114 days of gestation (DG) were examined. The TCR delta repertoire was highly restricted early in gestation (DG38-DG57) and an invariant TRDV3 transcript, lacking the N/D region, was found in different fetuses throughout gestation and dominated the TRDV3 repertoires of all organs at mid gestation ( approximately DG55). Near the end of gestation, this invariant TRDV3 transcript was absent from the thymus but was still present, in a less dominant manner, in the intestine and spleen. The average CDR3 length of all Vdelta subgroups increased with ontogeny, suggesting an increase in activity of TdT. Thus, the persistence of fetal gamma/delta T cells expressing an invariant TRDV3 chain throughout development is especially surprising since TdT is active early in gestation in swine. We speculate that these gamma/delta T cells might have been selectively expanded by (self)-ligands and may have an important function throughout fetal development.

Proliferating intestinal gamma/delta T cells recirculate rapidly and are a major source of the gamma/delta T cell pool in the peripheral blood.

The proliferation, recirculation and repertoire of gut-derived gamma/delta T cells were studied in pigs in vivo. Proliferating gamma/delta T cells (detected by BrdU labeling) are present in all intestinal compartments. In the gut lymph approximately 0.5% of all gamma/delta T cells were proliferating. These gut-derived BrdU(+) gamma/delta T cells re-enter the intestinal tissues, and re-appear in the intestinal lymph far more often than other cells: about 22% of i.v.-injected BrdU(+) gamma/delta T cells were recovered again from the intestinal lymph within 72 h (compare with BrdU(+) B cells 2%, and other BrdU(+) T cells 10%). The contribution of the gut to the migrating gamma/delta T cell pool in the blood became obvious: the proportion of BrdU(+) gamma/delta T cells was three-times larger in control versus cannulated pigs. In 9-month-old pigs, clonally expanded T cells were identified in the intestine by complementarity-determining region 3 spectratyping of TCR-delta transcripts. Such expansions were not visible in the blood or intestinal lymph. The distribution of gamma/delta T cells within the intestinal tract is likely to depend to a large degree on the proliferation and the migratory properties of these cells which are different to those of alpha/beta T cells and B lymphocytes.

Herpes simplex virus type 2-associated eosinophilic cellulitis (Wells' syndrome).

Eosinophilic cellulitis (Wells' syndrome) is a recurrent inflammatory dermatosis characterized by massive infiltration of eosinophils into the skin. Drugs and pathogens have been recognized causes of eosinophilic cellulitis. We report the repeated association of eosinophilic cellulitis with herpes simplex virus type 2 infections. Antiviral therapy led to a complete remission of eosinophilic cellulitis, indicating that causative treatment of underlying diseases can be effective in controlling eosinophilic cellulitis.