A. Gallia

Ex vivo immunosuppressive effects of mesenchymal stem cells on Crohn’s disease mucosal T cells are largely dependent on indoleamine 2,3-dioxygenase activity and cell-cell contact

IntroductionCrohn’s disease (CD) is a disabling chronic enteropathy sustained by a harmful T-cell response toward antigens of the gut microbiota in genetically susceptible subjects. Growing evidence highlights the safety and possible efficacy of mesenchymal stem cells (MSCs) as a new therapeutic tool for this condition. Therefore, we aimed to investigate the effects of bone marrow-derived MSCs on pathogenic T cells with a view to clinical application.MethodsT-cell lines from both inflamed and non-inflamed colonic mucosal specimens of CD patients and from healthy mucosa of control subjects were grown with the antigen muramyl-dipeptide in the absence or presence of donors’ MSCs. The MSC effects were evaluated in terms of T-cell viability, apoptotic rate, proliferative response, immunophenotype, and cytokine profile. The role of the indoleamine 2,3-dioxygenase (IDO) was established by adding a specific inhibitor, the 1-methyl-DL-tryptophan, and by using MSCs transfected with the small interfering RNA (siRNA) targeting IDO. The relevance of cell-cell contact was evaluated by applying transwell membranes.ResultsA significant reduction in both cell viability and proliferative response to muramyl-dipeptide, with simultaneous increase in the apoptotic rate, was found in T cells from both inflamed and non-inflamed CD mucosa when co-cultured with MSCs and was reverted by inhibiting IDO activity and expression. A reduction of the activated CD4+CD25+ subset and increase of the CD3+CD69+ population were also observed when T-cell lines from CD mucosa were co-cultured with MSCs. In parallel, an inhibitory effect was evident on the expression of the pro-inflammatory cytokines tumor necrosis factor-α, interferon-γ, interleukin-17A and -21, whereas that of the transforming growth factor-β and interleukin-6 were increased, and production of the tolerogenic molecule soluble HLA-G was high. These latter effects were almost completely eliminated by blocking the IDO, whose activity was upregulated in MSCs co-cultured with CD T cells. The use of a semipermeable membrane partially inhibited the MSC immunosuppressive effects. Finally, hardly any effects of MSCs were observed when T cells obtained from control subjects were used.ConclusionMSCs exert potent immunomodulant effects on antigen-specific T cells in CD through a complex paracrine and cell-cell contact-mediated action, which may be exploited for widespread therapeutic use.

A laboratory score in the diagnosis of autoimmune atrophic gastritis: a prospective study.

Background: Several biomarkers have been proposed for the diagnosis of autoimmune atrophic gastritis (AAG), but at the present there is no appropriate testing strategy for the disease. Goals: The aim of this study was to develop and validate a laboratory score able to address the diagnosis of AAG in a general practice setting. Study: We prospectively evaluated a number of serum biomarkers (vitamin B12, mean corpuscular volume, hemoglobin, gastrin, and chromogranin A levels) in a case-control population and built 2 biochemical scores, the first with all the parameters [Global Score (GS)], and the second as the best statistical combination of them [Simple Score (SS)]. In the second phase we validated the score that proved to be more efficient on a random population referred to our center (Gastroenterology Outpatient Clinic). Results: Both models turned out to be reliable in detecting patients with suspected AAG, showing excellent accuracy [area under the receiver operating curve (AUC-ROC) 0.94; 95% confidence interval (CI), 0.91-0.97 for GS and AUC-ROC 0.93; 95% CI, 0.89-0.86 for SS]. The SS proved to be more convenient because of its accessibility and availability in a general setting and its low cost. The validation of the SS showed a sensitivity of 85.7% (95% CI, 57.2-98.2) and a specificity of 83.7% (95% CI, 74.2-90.89). Conclusions: Herein, we describe 2 nonexpensive and reliable score models, particularly the SS, that can be applied in daily medical practice for identifying patients potentially affected by AAG.

Autologous Human Cytomegalovirus-Specific Cytotoxic T Cells as Rescue Therapy for Ulcerative Enteritis in Primary Immunodeficiency

PurposePatients affected by primary immunodeficiency usually undergo a wide range of infections, including reactivation of latent ones. Here we report two cases suffering from late-onset combined immunodeficiency in which ulcerative enteritis due to human Cytomegalovirus caused a life-threatening malabsorption syndrome.MethodsThe assessment of the viral load was carried out on both blood and mucosal samples by quantitative real-time polymerase chain reaction assay. The generation of autologous virus-specific cytotoxic T cell lines was performed according to Good Manufacturing Practice protocol after peripheral blood mononuclear cells were collected through a single leukapheresis.ResultsIn both patients, the viral load resulted negligible in peripheral blood, but very high in mucosal specimens (range 1.064 - 1.031.692 copies/105 cells). After two rounds of antiviral therapy proved unsuccessful, the generation of virus-specific cytotoxic T cell lines was carried out despite severe lymphopenia, and their infusion resulted safe and durably effective in healing intestinal ulcerations and resetting the viral load.ConclusionsVirus-specific cellular therapy was useful in reconstituting specific immunity and treating severe human Cytomegalovirus-related enteritis in patients with primary immunodeficiency.

An unconventional case of scurvy.

In April 2012, a 29-year-old Caucasian man was admitted to our Department for worsening of chronic diarrhoea and the appearance of fatigue and lower limb oedema. During the previous 5 years, he had lost 50 kilos body weight (from 110 to 60, achieving a body mass index of 21.4) probably due to the malabsorption syndrome for which he had undergone extensive diagnostic workup, including serological screening for endocrinopathies and immune-mediated enteropathies, stool tests, upper and lower endoscopy with biopsy sampling, despite which the underlying disease had not been identified. Moreover, previous tentative treatments with mesalamine, antibiotics and systemic steroids resulted only in transient benefit. No concomitant psychiatric or chronic illnesses, drug abuse, smoking or alcohol intake were reported. At admission, skin examination revealed multiple purpuric, non-scaling, follicular papules spread over the body and some ecchymotic patches with muscle wasting of the lower extremities and oedema of the calves (Figure 1). No organomegaly or lymphadenopathy were found. Blood tests showed moderate anaemia (haemoglobin 9.5 g/dl, red cell count 2.8 10, mean corpuscular volume 99.5 fl), lymphopenia (850 cells/ml, whose immunophenotype assessment pointed to a depletion of CD3þ T cells), hypo-albuminaemia (2.7 g/dl), hypo-g-globulinemia (0.9 g/dl), hypo-ferritinaemia (12.0 ng/ml), increase of the erythrocyte sedimentation rate (34 mm/h), normal levels of folic acid, vitamin B12 and negativity of the D-xylose test (36 mg/dl, normal value X30 mg). Other investigations concerning systemic and organrelated autoantibodies, viral and bacterial tests, cryoglobulins, complement levels and coagulation (including prothrombin time, international normalized ratio and factors II, V, VII, IX and X) were all within normal ranges. Faecal occult blood test, calprotectin and stool tests for pathogens were repeatedly negative, while human immunodeficiency virus infection was ruled out by both specific antibody search and RNA analysis. A colour-Doppler ultrasound excluded deep vein thrombosis. The dermatologist consultation highlighted the presence of perifollicular haemorrhages and corkscrew hairs on careful inspection, while oral mucosa had normal appearance, thus raising the suspicion of scurvy, which was confirmed through a skin biopsy whose histological examination showed a scant, perifollicular and perivascular lymphocytic infiltrate in the superficial dermis with extravasated erythrocytes, and an overlying follicular hyperkeratosis (Figure 2). Meanwhile, following a further unremarkable upper and lower endoscopy, a wireless capsule enteroscopy was carried out which showed several areas with blunted and whitish villi scattered throughout the jejunum supporting the diagnosis of protein-losing enteropathy due to intestinal lymphangiectasia. Subsequent direct questioning revealed a two-year self-imposed diet completely devoid of vegetables and fruit in an attempt to reduce the number of bowel movements. Measurement of plasma level of vitamin C was not performed due to the unavailability in the laboratory; however, it should be considered that it is affected by recent intakes of vitamin C and may be normal despite a depleted body store. Moreover, laboratory investigations are usually not necessary in the diagnosis of scurvy, especially if histologic findings show the characteristic lesions and vitamin C administration promptly recovers the lesions. High-dosage supplementation was then administered (500 mg intravenously twice daily) for two weeks in order to replace the body store, followed by oral replacement at the same dose for a further two weeks, and was maintained with a daily intake of citrus fruits and green leafy vegetables. The follicular purpura improved rapidly after 2–3 days, and the ecchymotic patches faded almost completely within 7 days, while the lower limb oedema had already disappeared following a mild diuretic therapy and anaemia ameliorated after a month, when the patient was referred to a tertiary centre of intestinal microsurgery for the appropriate treatment of lymphangiectasia. Unlike most animals that do not require exogenous source of ascorbic acid since they derive it by the glucose metabolism, humans lack this skill, so they need a regular dietary intake with the citrus fruits, green vegetables, tomatoes, and peppers being very rich in vitamin C. This latter plays a crucial role in collagen biosynthesis where it is needed for proline hydroxylation. Mature collagen, indeed, is composed of three polypeptide molecules arranged in a triple helix, which are initially synthesized as