Administration of vitamin D and high dose of omega 3 to sustain remission of type 1 diabetes.

OBJECTIVE: Two cases of Type 1 Diabetes (T1D) in pediatric subjects treated with supplementation with high dose vitamin D and omega 3 are reported. A similar pattern of remission of the disease was observed, resulting in restoration and subsequent persistence of optimal metabolic control, one and two years after T1D onset. Minimal basal insulin administration (0.1 IU/kg/die) in a single evening injection was required. The immunomodulatory and anti-inflammatory properties of the supplements were likely contributing to the observed effect. Similarities in genotyping and autoantibody patterns in these two cases could be of assistance to identify which subjects with T1D could benefit from this supplemental therapy. High dose vitamin D and omega 3 could be of assistance in childhood T1D therapy, to prolong preservation of endogenous insulin secretion in the absence of side effects. We do not know how long the state of remission can last, but these initial results are promising and represented a significant benefit for the two pediatric subjects treated. Larger controlled studies will determine the long-term effect of this proposed supplementation and its possible cost-benefits, including reduction of hypoglycemic episodes and complications.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

This corrects the article DOI: 10.1038/cdd.2016.22.

An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicentre studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients' cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity < 10% (compared to controls - 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts 'on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps.

BACKGROUND: Increasing evidence implicates both platelets and neutrophils in the formation, stabilization, and growth of peripheral and coronary thrombi. Neutrophil extracellular traps (NETs) play a key role. The early events in the deregulated cross-talk between platelets and neutrophils are poorly characterized. OBJECTIVES: To identify at the molecular level the mechanism through which platelets induce the generation of NETs in sterile conditions. PATIENTS/METHODS: The presence of NETs was determined in 26 thrombi from patients with acute myocardial infarction by immunohistochemistry and immunofluorescence and markers of NETs assessed in the plasma. In vitro NET generation was studied in static and in physiological flow conditions. RESULTS: Coronary thrombi mainly consist of activated platelets, neutrophils, and NETs in close proximity of platelets. Activated platelets commit neutrophils to NET generation. The event abates in the presence of competitive antagonists of the high mobility group box 1 (HMGB1) protein. Hmgb1(-/-) platelets fail to elicit NETs, whereas the HMGB1 alone commits neutrophils to NET generation. Integrity of the HMGB1 receptor, Receptor for Advanced Glycation End products (RAGE), is required for NET formation, as assessed using pharmacologic and genetic tools. Exposure to HMGB1 prevents depletion of mitochondrial potential, induces autophagosome formation, and prolongs neutrophil survival. These metabolic effects are caused by the activation of autophagy. Blockade of the autophagic flux reverts platelet HMGB1-elicited NET generation. CONCLUSIONS: Activated platelets present HMGB1 to neutrophils and commit them to autophagy and NET generation. This chain of events may be responsible for some types of thromboinflammatory lesions and indicates novel paths for molecular intervention.

Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator.

Mismanaged protein trafficking by the proteostasis network contributes to several conformational diseases, including cystic fibrosis, the most frequent lethal inherited disease in Caucasians. Proteostasis regulators, as cystamine, enable the beneficial action of cystic fibrosis transmembrane conductance regulator (CFTR) potentiators in ΔF508-CFTR airways beyond drug washout. Here we tested the hypothesis that functional CFTR protein can sustain its own plasma membrane (PM) stability. Depletion or inhibition of wild-type CFTR present in bronchial epithelial cells reduced the availability of the small GTPase Rab5 by causing Rab5 sequestration within the detergent-insoluble protein fraction together with its accumulation in aggresomes. CFTR depletion decreased the recruitment of the Rab5 effector early endosome antigen 1 to endosomes, thus reducing the local generation of phosphatidylinositol-3-phosphate. This diverts recycling of surface proteins, including transferrin receptor and CFTR itself. Inhibiting CFTR function also resulted in its ubiquitination and interaction with SQSTM1/p62 at the PM, favoring its disposal. Addition of cystamine prevented the recycling defect of CFTR by enhancing BECN1 expression and reducing SQSTM1 accumulation. Our results unravel an unexpected link between CFTR protein and function, the latter regulating the levels of CFTR surface expression in a positive feed-forward loop, and highlight CFTR as a pivot of proteostasis in bronchial epithelial cells.

Efficacy of budesonide therapy in the early phase of treatment of adult coeliac disease patients with malabsorption: an in vivo/in vitro pilot study.

1. Budesonide is a glucocorticosteroid with a local anti-inflammatory effect. Coeliac disease is an immune-mediated disease caused by gluten ingestion in intolerant patients. The aim of the present study was to investigate the efficacy of budesonide in malabsorptive coeliac patients and its effect in an in vitro gliadin challenge. 2. Twenty coeliac patients with malabsorption were enrolled in the present study and were randomly assigned to one of two 4 week treatments: (i) a gluten-free diet alone; or (ii) a gluten-free diet plus 6 mg budesonide daily. At the end of 4 weeks treatment, all patients underwent clinical evaluation, laboratory tests and self-evaluation of well-being using a visual analogue scale. Intestinal biopsies from five coeliac patients (selected randomly) and four non-coeliac disease controls who underwent upper endoscopy for intestinal bleeding were challenged with gliadin (0.5 mg/mL) and budesonide (10-30 microg/mL) for 3 and 24 h. Biopsies were tested by immunohistochemistry and immunofluorescence for known markers of inflammation. 3. Treatment of patients with 6 mg budesonide daily for 4 weeks resulted in increased bodyweight, a decreased number of evacuations and decreased stool weight compared with patients on a gluten-free diet alone for 4 weeks. Well-being scores were higher in patients treated with both a gluten-free diet and budesonide compared with those receiving a gluten-free diet alone. 4. In vitro studies showed that budesonide reduced epithelial tyrosine phosphorylation and expression of histocompatibility leucocyte antigen complex DR (HLA-DR) elicited by gliadin-derived peptides. In addition, the expression of cyclo-oxygenase (COX)-2 and intercellular adhesion molecule (ICAM)-1 in the lamina propria was reduced in patients treated with both gliadin and budesonide compared with patients treated with gliadin alone. Budesonide alone decreased HLA-DR in crypt enterocytes, as well as ICAM-1 and COX-2 expression in the lamina propria of biopsy specimen of coeliac patients. Budesonide had no effect in control samples. 5. In conclusion, the results of the present study indicate that budesonide shows efficacy in the treatment of symptoms in adult coeliac patients with overt malabsorption. The mechanism underlying the effects of budesonide in reducing symptoms was elucidated by in vitro studies involving a gliadin challenge.

Inhibition of p38 mitogen activated protein kinase controls airway inflammation in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) airways are characterised by chronic inflammation, increased interleukin (IL)-8 secretion, and neutrophil activation which are considered the principal factors of morbidity and mortality in CF patients. Optimising management of this chronic inflammatory response is therefore a key issue of basic and clinical CF research. Several reports have addressed ways to manage CF airways inflammation, and an attractive therapeutic strategy may be the inhibition of the p38-mitogen activated protein kinase (p38-MAP-k) pathway. METHODS: A new ex vivo model was used to study the mucosal inflammatory response to environmental airways stimuli. Nasal biopsy tissues from CF patients and controls were cultured ex vivo for 20 minutes, 4 hours, and 24 hours in the presence of lipopolysaccharide (LPS) from Pseudomonas aeruginosa (PA) with and without the p38-MAP-k inhibitor SB203580. Quantitative mRNA assessment, immunohistochemistry, and Western blots were used to detect the expression and modulation of inflammatory markers. RESULTS: PA-LPS challenge induced a time dependent mucosal inflammation indicated by rapid epithelial activation, IL-8 release, COX-2 upregulation, and neutrophil migration to the upper mucosal layers. Some of these LPS induced changes (IL-8 release and neutrophil migration) were specific to CF tissues. SB203580 significantly controlled all LPS induced mucosal changes in CF tissues. CONCLUSION: These findings provide a rationale and proof of principle for the potential use of p38-MAP-k inhibitors to control inflammation in patients with CF.

Celiac disease: a model autoimmune disease with gene therapy applications.

Gene therapy (GT) is still at the 'experimental' stage and some recent setbacks have cooled the potential use of this therapeutic tool even in life-threatening conditions. However, this therapeutic approach has a potential, which is not limited to disease for which we have not other option. There are increasing evidence that GT will be soon used in diseases that are not life threatening. One group of diseases that can benefit from GT is the autoimmune one. Several experimental animal models have indicated the efficacy (proof of principle) of GT. In the present review, we have addressed the possibility that even extremely benign autoimmune-like diseases such as Celiac Disease (CD) might one day profit from this type of therapy. We further point that in conditions such as CD, where the trigger is well known and the pathogenic cascade is relatively well defined, a situation not common in autoimmunity, we can even have a better situation where to explore and use GT to control disease initiation and progression. Once the risks that are still intrinsic to GT will have been reduced the therapeutic options we outline in the present review might not appear too far from reality.

Severe clinical onset of diabetes and increased prevalence of other autoimmune diseases in children with coeliac disease diagnosed before diabetes mellitus.

AIMS/HYPOTHESIS: To analyse whether the time of diagnosis of coeliac disease with respect to the clinical onset of diabetes could differentiate subgroups of varying severity in patients with both diseases. METHODS: We investigated 383 patients with Type I (insulin-dependent) diabetes mellitus for coeliac disease. Sex distribution, age at diagnosis of diabetes, prevalence of ketoacidosis at the onset of diabetes and prevalence of other autoimmune diseases were compared in patients. We divided these patients according to whether coeliac disease was diagnosed before (Group A, n=8) or after (Group B, n=24) diabetes onset and whether they had presented clinical symptoms of coeliac disease. Group C (n=351) included diabetic patients without coeliac disease. RESULTS: Out of 383 Type I diabetic patients we found 32 coeliac subjects (8.3%). There was a higher number of girls (p=0.003), but similar age and prevalence of ketoacidosis compared with Group C; 18.7% had a third autoimmune disorder. The higher number of girls was confirmed in Groups A and B in comparison to Group C (p=0.013), while higher prevalence of both ketoacidosis (p=0.009) and other autoimmune diseases (p=0.001) was found only in Group A. Compared with symptomatic patients, asymptomatic subjects in Group B had a lower number of girls, older age at diabetes onset, lower prevalence of ketoacidosis and no other associated autoimmune disease. CONCLUSIONS/INTERPRETATION: A wide clinical spectrum characterises the association of coeliac disease and diabetes mellitus, with a severe clinical presentation (higher prevalence of ketoacidosis at the onset and occurrence of other autoimmune diseases) when coeliac disease is diagnosed before diabetes. Distinct phenotypes might imply the contribution of a peculiar genetic background.

IL-15 drives the specific migration of CD94+ and TCR-gammadelta+ intraepithelial lymphocytes in organ cultures of treated celiac patients.

OBJECTIVES: Celiac disease (CD) is an under-diagnosed but extremely frequent disease, triggered by the ingestion of gliadin. The pathogenic mechanisms of CD are still poorly understood, but intraepithelial lymphocytes are considered to have a key role. We intended to define the subsets of T lymphocytes migrating upon gliadin challenge in organ cultures of treated celiac patients and establish the type of factor(s) driving such an infiltration. METHODS: Duodenum biopsies from 10 treated celiacs and 7 controls were cultured in vitro with/without gliadin digest (1 mg/ml) or interleukin (IL)-15 (10 ng/ml). In 7 treated celiacs IL-7, IL-4, and IL-2 were similarly tested. Intraepithelial CD3, CD8, TCR-gammadelta, and CD94 were detected by immunohistochemistry and numbered per mm epithelium. RESULTS: IL-15 but not IL-7, IL-4, or IL-2 induced intraepithelial increase of CD3+ and CD8+ cells in celiac and control intestine (p < 0.001 vs cultures with medium). IL-15 induced increases in the number of intraepithelial TCR- gammadelta+ and CD94+ cells only in celiacs (p < 0.001). IL-7 was also effective in increasing intraepithelial TCR-gammadelta+ (but not CD94+) cells in celiac biopsies (p < 0.001). Gliadin induced intraepithelial migration of CD3+, CD8+ (p < 0.001), and CD94+ (p < 0.05) cells in celiacs, but not in controls. CONCLUSIONS: The results we describe in this report indicate that IL-15 might have a key role in modulating and driving intraepithelial infiltration and ultimately in the pathogenesis of CD.

FAS engagement drives apoptosis of enterocytes of coeliac patients.

BACKGROUND: Villus atrophy is the most distinctive sign of untreated coeliac disease (CD) and epithelial apoptosis is considered to be involved in this stage of the coeliac lesion. The extent of villus atrophy is, however, not homogeneous and patients with patchy or mild lesions have been described. AIMS: To address: (a) the degree of "patchiness" in untreated CD patients; and (b) to clarify if apoptosis, and eventually which trigger drives it, causes epithelial damage. PATIENTS: Twenty of 40 untreated, 14 treated coeliac patients, and 15 controls received five or more multiple duodenal biopsies; the remaining 20 untreated CD patients had no more than three biopsies. METHODS: All biopsies were analysed to monitor the presence of a "flat" mucosa. Biopsies of 14 untreated, 10 treated coeliacs, and seven controls were cultured with or without gliadin. DNA fragmentation was studied by terminal deoxynucleotidyl transferase (TdT) mediated dUTP digoxigenin nick end labelling (TUNEL), and FAS and Ki67 expression by immunohistochemistry. Antiendomysium antibodies (EMA) were surveyed in biopsy culture supernatants. RESULTS: A pattern of patchy duodenal lesions was observed in all untreated CD patients biopsied up to five times. High enterocyte FAS expression, and a high number of TUNEL+ and Ki67+ enterocytes were detected in areas with villus atrophy but not in those with a normal morphology (p<0.001). Conversely, EMA in culture supernatants and signs of immunological activation were present in all untreated CD biopsies. In vitro gliadin challenge increased the number of TUNEL+ and Ki67+ enterocytes (p<0.001 v cultures with medium alone) only in "flat" biopsies. Neutralising anti-FAS monoclonal antibodies were found to control gliadin induced enterocyte apoptosis (p>0.01) while agonist anti-FAS monoclonal antibody increased it (p<0.001). CONCLUSIONS: Patchy lesions are observed in untreated CD mucosa and epithelial FAS engagement is a key trigger in driving villus atrophy in CD.

Interleukin 15 mediates epithelial changes in celiac disease.

BACKGROUND & AIMS: Villous atrophy and crypt proliferation are key epithelial features of untreated celiac disease. We tried to define whether cytokines such as interleukin (IL)-15, IL-2, IL-4, and IL-7, which share chains of their receptors, could influence the epithelial modifications. METHODS: Duodenal biopsy specimens (14 treated and 13 untreated celiac patients, 7 controls) were cultured in vitro for 24 hours with or without gliadin (1 mg/mL), IL-15, IL-7, IL-4, or IL-2 (10 ng/mL). Tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were also used in some specimens of untreated celiacs. Epithelial expression of Ki67, FAS, and transferrin receptor (TFR) was detected by immunohistochemistry, and apoptosis by TUNEL technique (percentage of positive enterocytes). IL-15-positive cells were detected by immunohistochemistry in celiac disease and control biopsy specimens; presence of IL-15 was also determined by semiquantitative polymerase chain reaction. RESULTS: Only IL-15 induced enterocyte expression of Ki67, TFR, and FAS in treated celiac (P<0.01 vs. medium) and enterocyte apoptosis in untreated celiac disease specimens. Anti-IL-15 monoclonal antibodies neutralized gliadin-induced enterocyte TFR and FAS expression in treated celiac and enterocyte apoptosis in untreated celiac disease specimens (P<0.05 vs. gliadin). IL-15-positive cells were increased in untreated celiacs (P<0.001 vs. treated celiacs and controls). CONCLUSIONS: IL-15 is involved in the modulation of epithelial changes in celiac disease, indicating that this cytokine has an unforeseen role in the pathologic manifestations of celiac disease.

Evidence of chronic inflammation in morphologically normal small intestine of cystic fibrosis patients.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator gene and characteristically leads to prominent lung and pancreatic malfunctions. Although an inflammatory reaction is normally observed in the CF airways, no studies have been performed to establish whether a chronic inflammatory response is also present in the CF intestine. We have investigated whether immunologic alterations and signs of inflammation are observed in CF small intestine. Fourteen CF, 20 negative, and four disease controls underwent duodenal endoscopy for diagnostic purposes. Two CF patients were rebiopsied, one after 3 mo of an elemental diet and the other after 2 wk of pancreatic enzyme withdrawal. In three CF and 10 controls, in vitro small intestine organ cultures were also performed. Expression of ICAM-1, IL-2 receptor, IL-2, IFN-gamma, CD80, and transferrin receptor was studied by immunohistochemistry before and after in vitro organ culture. In CF small intestine, an increased number of lamina propria mononuclear cells express ICAM-1 [mean 114 (SD 82.8), p < 0.001 versus controls], CD25 [20.2 (18.7), p < 0.01], IL-2 [23.6 (13.7), p < 0.05], and IFN-gamma [19 (15.9), p < 0.05], whereas villus enterocytes highly express transferrin receptor. Reduced expression of immunologic markers was observed after 24 h of in vitro culture in all three CF patients as well as in the patient kept on elemental diet for 3 mo. These results indicate that chronic inflammation is observed in CF duodenum and suggest that the perturbation of local mucosal immune response may contribute to the overall clinical picture in CF patients.

Imaging active lymphocytic infiltration in coeliac disease with iodine-123-interleukin-2 and the response to diet.

Coeliac disease is diagnosed by the presence of specific antibodies and a jejunal biopsy showing mucosal atrophy and mononuclear cell infiltration. Mucosal cell-mediated immune response is considered the central event in the pathogenesis of coeliac disease, and untreated coeliac patients show specific features of T-cell activation in the small intestine. Here we describe the use of iodine-123-interleukin-2 scintigraphy in coeliac patients as a non-invasive tool for detection of lymphocytic infiltration in the small bowel and its use for therapy follow-up, and we demonstrate the specificity of binding of labelled-IL2 to activated lymphocytes by ex-vivo autoradiography of jejunal biopsies. 123I-IL2 was administered i.v. [74 MBq (2 mCi)], and gamma camera images were acquired after 1 h. Ten patients were studied with 123I-IL2 scintigraphy at diagnosis and seven were also investigated after 12-19 months of gluten-free diet. Results were expressed as target-to-background radioactivity ratios in six different bowel regions before and after the diet. At the time of diagnosis all patients showed a significantly higher bowel uptake of 123I-IL2 than normal subjects (P < 0.003 in all regions). A significant correlation was found between jejunal radioactivity and the number of IL2R + ve lymphocytes per millimetre of jejunal mucosa as detected by immunostaining of jejunal biopsy (r2 = 0.66; P = 0.008). Autoradiography of jejunal biopsies confirmed that labelled-IL2 only binds to activated T-lymphocytes infiltrating the gut mucosa. After 1 year of the diet, bowel uptake of 123I-IL2 significantly decreased in five out of six regions (P < 0.03), although two patients still had a positive IL2 scintigraphy in one region. We conclude that 123I-IL2 scintigraphy is a sensitive non-invasive technique for assessing in vivo the presence of activated mononuclear cells in the bowel of patients affected by coeliac disease. Unlike jejunal biopsy, this method provides information from the whole intestine and gives a non-invasive measure of the effectiveness of the gluten-free diet.

Blockage of T-cell costimulation inhibits T-cell action in celiac disease.

BACKGROUND & AIMS: Celiac disease is an exemplary model of T cell-mediated pathology. Therefore, therapeutic approaches that target T cells may successfully control this disease. CTLA-4 immunoglobulin (CTLA-4Ig) can inhibit T-cell activation by blocking the engagement of CD28. We took advantage of this tool to define the pathogenic role of gliadin-specific T cells in the induction of celiac disease. METHODS: Duodenal biopsy specimens from 7 treated celiac patients were challenged in vitro with gliadin and CTLA-4Ig or CD40-Ig. After 24 hours, the biopsy specimens were analyzed for the presence of characteristic modifications induced by gliadin challenge. RESULTS: CTLA-4Ig down-regulated the expression of CD25, intercellular adhesion molecule 1, interleukin 2, and interferon gamma (stained lamina propria mononuclear cells/mm2; P < 0.05) induced by gliadin challenge, caused apoptosis of gliadin-specific T cells (apoptotic T cells/mm2; P < 0.05), and inhibited the production of antiendomysial antibody (P < 0.01). However, it did not control intraepithelial T-cell migration (P = NS) and Fas expression by enterocytes. Conversely, CD40-Ig only controlled production of antiendomysial antibody. CONCLUSIONS: In an organ culture model, CTLA-4Ig controls many but not all of the immunologic features of celiac disease.

DNA fragmentation is a feature of cystic fibrosis epithelial cells: a disease with inappropriate apoptosis?

Cystic fibrosis (CF) is a single-gene disease caused by mutations in the CFTR gene, which result in disrupted chloride secretions with inspissated mucous secretions by exocrine glands. Nick-end labelling was used to assess DNA fragmentation in 14 CF and 24 control duodenal samples, and in two CF and two control lung tissues. In CF small intestine median 46% (range: 30-82) villus enterocytes show DNA fragmentation (vs. 3% (range: 1-7) in controls P < 0.001) and median 37.5% (range: 23-79) crypt enterocytes show Ki67 antigen (P < 0.001). In CF airways 57% (range: 54-70) of epithelial cells show DNA fragmentation. Inappropriate high DNA fragmentation is a feature of various CF epithelia. This could have great impact in understanding the mechanisms leading to disease.

Lactase persistence versus decline in human adults: multifactorial events are involved in down-regulation after weaning.

BACKGROUND & AIMS: In nonhuman mammals, lactase activity declines during or after weaning. In contrast, about one half of the human species maintains high lactase activity even in adulthood. To clarify this difference, this study examined some parameters for which contrasting observations have been reported in connection with lactase decline. METHODS: Lactase activity, lactase messenger RNA (mRNA) levels, and in vitro lactase biosynthesis were determined in normal jejunal samples from a large group of white adults, all born in or near Naples. RESULTS: Of 44 individuals, 10 were lactase persistent and 34 were hypolactasic. Biosynthesis of prolactase correlated well with lactase mRNA levels, indicating transcriptional control; it did less so with steady-state lactase activity. Examination of lactase mRNA levels and lactase activity/lactase mRNA ratios revealed a heterogeneous pattern of lactase mRNA level, lactase synthesis, and activity in both lactase persistent and hypolactasic subjects. CONCLUSIONS: Both transcriptional and posttranscriptional factors cause the decline of intestinal lactase. This probably explains the multifarious observations that most studies on adult-type hypolactasia have reported. The single overriding factor distinguishing lactase-persistent subjects from hypolactasic subjects is the high rate of lactase biosynthesis.

Production of antiendomysial antibodies after in-vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease.

BACKGROUND: Diagnosis of many immune-mediated diseases is helped by detection of antibodies directed against the pathogenetic (self or foreign) antigen. In coeliac disease (CD), we have a situation in which antiendomysial antibodies (EMA), which are not specific for the pathogenetic antigen, reach a specificity and sensitivity of detection of CD approaching 100%, whereas detection of antibodies against gliadin (AGA), the pathogenetic antigen, is far less specific and sensitive in diagnosis. No direct evidence of a relation between gluten/gliadin challenge and EMA production exists. We tried to establish whether the small intestine of CD patients is the site of EMA production and whether gliadin challenge could induce their release. METHODS: Small intestine biopsy samples from treated (23) and untreated (16) CD patients and controls (18) were cultured in vitro for 24-48 h in the presence of gliadin, another alimentary antigen, or medium. EMA and AGA were detected in the supernatants of these organ culture biopsy samples by ELISA and immunofluorescence technique, respectively. FINDINGS: No EMA were found in the culture supernatants of biopsy samples of 18 controls, whereas they were detected in the culture supernatants of all 16 untreated CD patients irrespective of gliadin challenge. Conversely, EMA were not detected in supernatants of biopsy samples cultured in medium only from 23 treated CD patients, but were detected in 17 of the 23 biopsy samples challenged with gliadin. INTERPRETATION: Our results suggest that a more complex pathogenetic mechanism than normally accepted is involved in CD. Furthermore, our findings raise the possibility that EMA, or the antigen recognised by them, are involved directly in the pathogenesis of CD.

Gluten-sensitive disease with mild enteropathy.

BACKGROUND & AIMS: Celiac disease is a permanent gluten intolerance strongly associated with HLA class II antigens and possibly showing milder changes of mucosal architecture. Ten patients with symptoms suggesting celiac disease and serum antiendomysium antibodies with normal mucosal architecture were studied. METHODS: Immunohistochemical detection of mucosal immune activation and HLA typings were performed. RESULTS: Mucosal immune activation, with normal mucosal architecture and normal gamma/delta+ intraepithelial lymphocytes counts, was found on a gluten-containing diet. In 3 of 6 patients, multiple biopsy specimens showed one sample with severe villous atrophy. Clinical and immunomorphologic features were strictly gluten dependent. The mucosal immune activation was elicited in vitro by gliadin. Only 4 patients had the typical HLA typing of celiac disease. CONCLUSIONS: Gluten-sensitive celiac-like symptoms may occur in patients with serum antiendomysium antibodies, apparently normal intestinal mucosa, and HLA typing not commonly associated with celiac disease. These patients should undergo multiple biopsies, and signs of immunologic activation should be sought accurately; in the presence of mucosal immune activation, a trial with a gluten-free diet should be encouraged to detect gluten dependency. In vitro immunologic response of small intestinal mucosa to gliadin may support the diagnosis of gluten-sensitive enteropathy.