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 (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.

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.

Summary In this report, we describe 12 subpopulations of porcine γδ thymocytes based on their expression of CD1, CD2, CD4, CD8‐isoforms and CD45RC. Our data suggest that γδ thymocytes can be divided into two major families: (a) one large family of CD4–γδ thymocytes that could be further subdivided according to the CD2/CD8αα phenotype and (b) a small family of CD4+γδ thymocytes bearing CD8αβ and possessing certain unusual features in comparison with other γδ thymocytes. Maturation of γδ thymocytes within the CD4– family begins with proliferation of the CD2+ CD8– CD1+ CD45RC–γδ common precursor. This developmental stage is followed by diversification into the CD2+ CD8αα+, 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, γδ thymocytes within the CD4+ family bear exclusively CD8αβ, always express CD1, but may coexpress CD45RC. These cells have no counterpart in the periphery. Our observations suggest that all peripheral CD8+γδ T cells express CD8αα and that two subsets of these cells differing in major histocompatibility complex II expression, occur. We propose that one subset acquires CD8αα in the thymus while the second acquires CD8αα as a result of stimulation in the periphery.

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 (IC50 of ∼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 Ca2+ 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.

-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 γδ cells expressing a monomorphic Vδ rearrangement. Thereafter, αβ 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.

SummaryVS1 bearing T ceils comprise the major population of 3'/8 T cells in the human intestinal tract. To gain insight into mechanisms involved in the generation of these cells and the diversity of their repertoire, we have characterized the junctional sequences of V81 T cell receptor transcripts in the human small intestine and colon. Mucosal biopsies obtained from defined regions along the length of the small intestine or colon contained a high frequency of either one or a few identical in frame V81 sequences. Less abundant sequences were also detected repeatedly throughout the length of small intestine or colon. Moreover, the intestinal V61 repertoire in the small intestine and colon appeared compartmentalized and showed no overlap with the V81 repertoire in peripheral blood. Dominant V81 transcripts in each subject differed between the small intestine and colon, and the dominant transcripts within these sites differed among individuals. Analysis of small intestinal transcripts obtained at a 1-yr interval revealed that the V51 repertoire was stable over time. The fact that the majority of V81 transcripts, both dominant and rare, are distributed throughout a several meter length of the adult intestinal tract and are stable over time suggests they are not generated by an ongoing process of in situ VDJ gene rearrangement. Our results favor a model in which the repertoire of V81 T cells in the intestinal tract is shaped by positive selection in response to a limited array of ligands before the migration of V(51 cells throughout the small intestine or colon.

Developmental pathways of γδ 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 γδ thymocytes can be divided into 12 subsets of the following two major groups: 1) CD4− γδ thymocytes that can be further subdivided according to their CD2/CD8αα phenotype, and 2) CD4+ γδ thymocytes that are always CD1+CD2+CD8αβ+ and have no counterpart in the periphery. In this study, we have analyzed γδ thymocyte subsets with respect to behavior during cultivation, cell cycle status, and lymphocyte-specific transcripts. The group of CD4− γδ thymocytes gives rise to all γδ T cells found in the periphery. Proliferating CD2+CD8−CD1+CD45RC− γδ thymocytes are a common precursor of this group. These precursors differentiate into CD2+CD8αα+, CD2+CD8−, and CD2−CD8− γδ T cell subsets, which subsequently mature by loss of CD1 and by eventual gain of CD45RC expression. In contrast, the group of CD4+ γδ thymocytes represents transient and independent subsets that are never exported from thymus as TCRγδ+ T cells. In accordance with the following findings, we propose that CD4+CD8αβ+ γδ thymocytes extinguish their TCRγδ expression and differentiate along the αβ T cell lineage program: 1) CD4+ γδ thymocytes are actively dividing; 2) CD4+ γδ thymocytes do not die, although their numbers decreased with prolonged cultivation; 3) CD4+ γδ thymocytes express transcripts for RAG-1, TdT, and TCRβ; and 4) CD4+ γδ thymocytes are able to alter their phenotype to TCRαβ+ thymocytes under appropriate culture conditions.