Wheat-Triggered Food Protein-Induced Enterocolitis Syndrome in Celiac Children on Gluten-Free Diet: A New Clinical Association.

INTRODUCTION: The association between food protein-induced enterocolitis syndrome (FPIES) and wheat ingestion in children with celiac disease is unknown at this time. METHODS: We present seven cases of children with celiac disease who presented with symptoms of wheat-triggered acute FPIES (a-FPIES). An oral food challenge (OFC) with wheat allergen followed by 4 h of observation was performed. Activation of innate system cells was measured at baseline (T0), during symptoms (Ts), and 4 h after symptom onset (Ts + 4). A panel of human inflammatory cytokines was also performed. RESULTS: All patients reacted to the first allergen dose. Three patients experienced a decrease of 30 mm Hg in systolic blood pressure and tachycardia and required hemodynamic resuscitation. Neutrophilia and a decrease in eosinophil count were evident at 4 h after symptom onset. At 4 h after symptom onset, cytokines (IL-6 and IL-8, and to a lesser degree, IL-10) were elevated. CONCLUSION: In a small sample of celiac patients with wheat exposure in an OFC, symptoms and acute immunological changes in serum inflammatory cytokine profile were consistent with a-FPIES.

Intraepithelial Lymphogram in the Diagnosis of Celiac Disease in Adult Patients: A Validation Cohort.

BACKGROUND: Celiac disease is a gluten-related pathology, highly prevalent and heterogeneous in its clinical presentation, which leads to delays in diagnosis and misdiagnosis. The analysis of duodenal intraepithelial lymphocytes (IELs) by flow cytometry (lymphogram) is emerging as a discriminative tool in the diagnosis of various forms of celiac disease (CD). AIMS: The aim of this study was to validate IEL lymphogram performance in the largest adult series to our knowledge, in support of its use as a diagnostic tool and as a biomarker of the dynamic celiac process. METHODS: This was a retrospective study including 768 adult patients (217 with active CD, 195 on a gluten-free diet, 15 potential CD patients, and 411 non-celiac controls). The IEL subset cut-off values were established to calculate the diagnostic accuracy of the lymphogram. RESULTS: A complete celiac lymphogram profile (≥14% increase in T cell receptor [TCR]γδ IELs and simultaneous ≤4% decrease in surface-negative CD3 [sCD3-] IELs) was strongly associated with active and potential forms in over 80% of the confirmed patients with CD, whereas the remaining patients with CD had partial lymphogram profiles (≥14% increase in TCRγδ or ≤4% decrease in sCD3- IELs), with lower diagnostic certainty. None of these patients had a non-celiac lymphogram. Quantifying the TCRγδ versus sCD3- imbalance as a ratio (≥5) is a discriminative index to discard or suspect CD at diagnosis. CONCLUSIONS: We have validated the IEL lymphogram's diagnostic efficiency (79% sensitivity, 98% specificity), with an LR+ accuracy of 36.2. As expected, the increase in TCRγδ IELs is a reliable marker for celiac enteropathy, while changes in sCD3- IEL levels throughout the dynamic CD process are useful biomarkers of mucosal lesions.

Improving the Diagnosis of Dermatitis Herpetiformis Using the Intraepithelial Lymphogram.

Dermatitis herpetiformis is a cutaneous manifestation of celiac disease. Phenotyping of intraepithelial lymphocytes in the small bowel mucosa can strengthen the diagnosis of celiac disease when it is not clear-cut. We aim to evaluate the usefulness of the intraepithelial lymphogram to confirm dermatitis herpetiformis in equivocal cases. We performed a retrospective multicenter study on patients diagnosed with dermatitis herpetiformis and collected data from the intraepithelial lymphogram assessed by flow cytometry. A total of 36 patients were analyzed in relation to the severity of intestinal damage (18 had non-atrophic mucosa) at baseline (N = 28) and/or after the adoption of a gluten-free diet (median follow-up of three years, N = 16). We observed that patients with atrophy more often had positive celiac serology (p = 0.019), celiac clinical symptoms (p = 0.018), and iron-deficiency anemia (p = 0.018), but the severity of skin damage was similar in both groups (p = 0.79). At baseline, increased TCRγδ+ cells were present in 94% of patients with atrophy and 67% with non-atrophic lesions (p = 0.13). After a gluten-free diet, increased TCRγδ+ cells persisted in 100% and 63% of cases, respectively (p = 0.21). We concluded that increased TCRγδ+ cells may be helpful in confirming the diagnosis of dermatitis herpetiformis in equivocal cases, even in patients who were started on a gluten-free diet.

Recommendations for the use of anti-dsDNA autoantibodies in the diagnosis and follow-up of systemic lupus erythematosus - A proposal from an expert panel.

Anti-dsDNA autoantibodies are listed as one of the classification criteria for systemic lupus erythematosus (SLE) and are relatively effective indicators for monitoring disease activity and treatment response. Therefore, clinicians rely on them to diagnose and adjust medication and treatment strategies for SLE patients. However, the use of anti-dsDNA antibodies is not free from controversy. Part of this controversy stems from the fact that anti-dsDNA antibodies are found in several disorders, besides SLE. In addition to this, anti-dsDNA antibodies are a heterogeneous group of antibodies, and their determination still lacks proper standardization. Moreover, anti-dsDNA testing specificity and diagnostic performance change depending on the population under study. These and other issues result in inconsistency and encumber the clinical use of anti-dsDNA antibodies. A panel of medical laboratory and clinical experts on SLE discussed such issues based on their clinical experience in a first meeting, establishing a series of recommendations. The proceedings of this first meeting, plus an exhaustive review of the literature, were used to compose a paper draft. The panel subsequently discussed and refined this draft in a second meeting, the result of which is this paper. This document is relevant to clinical laboratories as it guides to improving diagnosis and monitoring of SLE. Simultaneously, it will help laboratories compile more informative reports, not limited to a mere number. It is also relevant to clinical doctors who wish to better understand laboratory methods so that they can do a more efficient, better-aimed laboratory test ordering.

Laboratory and clinical practices in antinuclear antibody detection and related antigens: recommendations from a Spanish multicentre survey.

Antinuclear antibodies (ANA) are the most widely used immunological test for the diagnosis of autoimmune diseases. Despite the recommendations of experts, there is some variability in performing and interpreting this test in routine practice. In this context, the Spanish Group on Autoimmune Diseases (GEAI) of the Spanish Society of Immunology (SEI) conducted a national survey of 50 autoimmunity laboratories. Here we report the survey results on ANA testing, detection of related antigens, and our recommendations. The survey showed that most of the participating laboratories use a similar approach for most key practices: 84% perform ANA by indirect immunofluorescence (IIF) on HEp-2 cells as the screening methodology while the other laboratories use IIF to confirm positive screens; 90% report ANA test results as either negative or positive with titer and pattern; 86% indicated that the ANA pattern conditioned follow-up testing for specific antigen-related antibodies; and 70% confirm positive anti-dsDNA. However, testing practices were highly heterogeneous for certain items, such as sera dilutions and the minimum time period for repeating ANA and related antigen determinations. Overall, this survey shows that most autoimmune laboratories in Spain use a similar approach but that further standardization of testing and reporting protocols is needed.

Folliculitis Decalvans Has a Heterogeneous Microbiological Signature and Impaired Immunological Response.

BACKGROUND: Folliculitis decalvans (FD) is a rare primary neutrophilic scarring alopecia whose etiology has not been completely elucidated yet. OBJECTIVE: The aim of the study was to determine if the follicular microbiota residing in FD-affected hair follicles had a distinct microbiological signature and if an aberrant immune response was present in the pathogenesis of FD. METHODS: We conducted a cross-sectional study of 10 patients affected by FD. Trichoscopy-guided follicular biopsies were taken from affected and healthy scalp to identify the follicular microbiome using next-generation sequencing. We searched for microbiological biomarkers of FD-affected follicles using the linear discriminant analysis (LDA) effect size (LEfSe) tool. Additionally, peripheral blood mononuclear cells were obtained, and their cytokine production was quantified after incubation with pathogen-associated molecular patterns isolated from patients' biopsies and compared with healthy controls. RESULTS: ß-diversity analysis showed statistically significant differences regarding bacteria comparing follicular microbiota of healthy and FD-affected hairs. Ruminococcaceae, Agathobacter sp., Tyzzerella sp., and Bacteriodales vadin HA21 family were good predictors of disease status. IL-10, TNF-α, and IL-6 levels were significantly decreased in patients after incubation with various strains of bacteria compared with controls. CONCLUSION: FD hair follicles have a specific heterogenous follicular bacterial microbiota signature. Additionally, these patients seem to have an impaired immunological response.

Respiratory mechanics in late COVID-19 ARDS - a restrictive pattern is strongly associated with death. A cohort study.

INTRODUCTION: COVID-19 is associated with severe respiratory distress and high mortality. We investigated the evolution of the respiratory mechanics in COVID-19 acute respiratory distress syndrome (ARDS) and the occurrence of a restrictive respiratory pattern. MATERIAL AND METHODS: A retrospective, single-centre study including patients admitted to the ICU during the first wave of the pandemic (March-April 2020). RESULTS: A total of 141 consecutive patients were included. Many patients developed a restrictive pattern of respiratory mechanics during the course of the disease. Fifty-two patients died in the hospital (36.8%). In 29 cases (58% of the deceased) death was associated with a pattern of pulmonary mechanics, indicating a restrictive evolution of ARDS. Other diagnoses related to death were pulmonary embolism (n = 7, 14%), septic shock (n = 17, 33%), and other causes (n = 10, 20%), with some patients combining at least 2 of these diagnoses. In a multivariate analysis, age (OR = 1.06; 95% CI: 1.01-1.12; P = 0.029) and the administration of steroid pulses (OR = 2.7; 95% CI: 1.1-6.8; P = 0.03) were associated with the development of a restrictive pulmonary pattern and a higher level of plasmatic interleukin-6. CONCLUSIONS: COVID-19 ARDS is associated with high mortality associated with a specific pattern of respiratory mechanics and sustained activation of innate immunological response. Age and administration of high-dose steroid pulses are associated with this clinical picture.

Intestinal and blood lymphograms as new diagnostic tests for celiac disease.

Accurate celiac disease (CD) diagnosis is still challenging for some specific patients or circumstances. Thus, much effort has been expended last decades focused on seronegative or low grade enteropathy CD and, especially, on enable early diagnosis of individuals on a gluten-free diet (GFD). We discuss here two diagnostic approaches based on immunophenotyping by flow cytometry that we expect to reduce the persistent low diagnostic rates and the common diagnostic delay. The intraepithelial lymphogram is based on determining the percentage of TCRγδ+ and surface CD3- lymphocytes in the intestinal epithelium. The concomitant increase in TCRγδ+ and decrease in surface CD3- intraepithelial lymphocytes has been termed the celiac lymphogram and has been proved to be discriminative in seronegative, low grade enteropathy and potential CD, as well as in most CD patients on a GFD. A blood lymphogram based on the analysis of activated gut-homing CD8+ T cells combined with a 3-day gluten challenge is also considered, which has shown high sensitivity and specificity to diagnose seropositive Marsh 1 and Marsh 3 CD in individuals following a GFD. In addition, flow cytometry can be extremely useful in cases of refractory CD type II to identify aberrant cells. Those approaches represent highly accurate methods for CD diagnosis, being simple, fast, highly reproducible and of easy implementation in clinical practice.

Assessment of Duodenal Intraepithelial Lymphocyte Composition (Lymphogram) for Accurate and Prompt Diagnosis of Celiac Disease in Pediatric Patients.

INTRODUCTION: Quantitative and phenotypic analyses of duodenal intraepithelial lymphocytes (IELs) by flow cytometry (IEL lymphogram) confer specificity and enable the diagnosis even in unconventional presentations of celiac disease (CD). To evaluate the validity of the IEL lymphograms in the pediatric population for new insights into their use as biomarkers in the natural history of CD. METHODS: We retrospectively included 1,211 children (602 with active CD, 92 on a gluten-free diet, 47 with potential CD, and 470 nonceliac controls) who required duodenal biopsies in this study. The cutoff values for IEL subsets were established to calculate the probability of disease according to the lymphogram. RESULTS: A celiac lymphogram (a ≥15% increase in gamma-delta T-cell receptor IELs and a simultaneous ≤6% decrease in CD3 surface-negative [sCD3-]) IELs was strongly associated with the diagnosis of active CD, which was present in 89.7% of the confirmed patients. The remaining 10% of the celiac patients had a partial celiac lymphogram (≥15% increase gamma-delta T-cell receptor IELs or ≤6% decrease in sCD3- IELs), with lower diagnostic certainty. On a gluten-free diet, nearly 20% of the patients were indistinguishable from nonceliac subjects based on the lymphogram. In potential CD, a decrease in sCD3- IELs was a risk marker of progression to villous atrophy and a diagnosis of active CD. DISCUSSION: If a biopsy is clinically indicated, the IEL lymphogram adds specificity to the histological findings, reducing diagnostic delays and misdiagnoses. The lymphogram is useful for monitoring the natural progression of the disease and predicting the transition from potential celiac to overt CD.

Remission Induced by TNF Inhibitors Plus Methotrexate is Associated With Changes in Peripheral Naïve B Cells in Patients With Rheumatoid Arthritis.

Biological therapies, such as TNF inhibitors (TNFi), are increasing remission (REM) rates in rheumatoid arthritis (RA) patients, although these are still limited. The aim of our study was to analyze changes in the profile of peripheral blood mononuclear cells (PBMC) in patients with RA treated with TNFi in relation to the clinical response. This is a prospective and observational study including 78 RA patients starting the first TNFi. PBMC were analyzed by flow cytometry both at baseline and at 6 months. Disease activity at the same time points was assessed by DAS28, establishing DAS28 ≤ 2.6 as the criteria for REM. Logistic regression models were employed to analyze the association between the changes in PBMC and REM. After 6 months of TNFi treatment, 37% patients achieved REM by DAS28. Patients who achieved REM showed a reduction in the percentage of naive B cells, but only when patients had received concomitant methotrexate (MTX) (OR: 0.59; 95% CI: 0.39-0.91). However, no association was found for patients who did not receive concomitant MTX (OR: 0.85; 95% CI: 0.63-1.16). In conclusion, PBMC, mainly the B-cell subsets, are modified in RA patients with TNFi who achieve clinical REM. A significant decrease in naive B-cell percentage is associated with achieving REM after 6 months of TNFi treatment in patients who received concomitant therapy with MTX.

An Immunologic Compatibility Testing Was Not Useful for Donor Selection in Fecal Microbiota Transplantation for Ulcerative Colitis.

Fecal microbiota transplantation (FMT) is an effective procedure against Clostridioides difficile infection (CDI), with promising but still suboptimal performance in other diseases, such as ulcerative colitis (UC). The recipient's mucosal immune response against the donor's microbiota could be relevant factor in the effectiveness of FMT. Our aim was to design and validate an individualized immune-based test to optimize the fecal donor selection for FMT. First, we performed an in vitro validation of the test by co-culturing lymphocytes obtained from the small intestine mucosa of organ donor cadavers (n=7) and microbe-associated molecular patterns (MAMPs) obtained from the feces of 19 healthy donors. The inflammatory response was determined by interleukin supernatant quantification using the Cytometric Bead Array kit (B&D). We then conducted a clinical pilot study with 4 patients with UC using immunocompetent cells extracted from rectal biopsies and MAMPs from 3 donor candidates. We employed the test results to guide donor selection for FMT, which was performed by colonoscopy followed by 4 booster instillations by enema in the following month. The microbiome engraftment was assessed by 16S rDNA massive sequencing in feces, and the patients were clinically followed-up for 16 weeks. The results demonstrated that IL-6, IL-8, and IL-1ß were the most variable markers, although we observed a general tolerance to the microbial insults. Clinical and colonoscopy remission of the patients with UC was not achieved after 16 weeks, although FMT provoked enrichment of the Bacteroidota phylum and Prevotella genus, with a decrease in the Actinobacteriota phylum and Agathobacter genus. The most relevant result was the lack of Akkermansia engraftment in UC. In summary, the clinical success of FMT in patients with UC appears not to be influenced by donor selection based on the explored recipient's local immunological response to FMT, suggesting that this approach would not be valid for FMT fecal donor optimization in such patients.

Long-term docosahexaenoic acid (DHA) supplementation in cystic fibrosis patients: a randomized, multi-center, double-blind, placebo-controlled trial.

BACKGROUND: Cystic fibrosis (CF) patients have an alteration in fatty acid (FA) metabolism, associated with increased omega-6 and low omega-3 FA. Previous studies on supplementation with omega-3 FA in CF had contradictory results, and to date there is no evidence to recommend routine use of omega-3 supplements in CF patients. We hypothesized that long-term supplementation with docosahexaenoic acid (DHA) will have beneficial effects in these patients, by reducing pulmonary, systemic and intestinal inflammation. METHODS: This was a randomized, double-blind, parallel, placebo-controlled trial. CF patients (age >2 months) were randomized to receive a seaweed DHA oil solution (50 mg/Kg/day) or matching placebo for 48 weeks. Primary outcomes were pulmonary (interleukin [IL]-8), systemic (IL-8) and intestinal (calprotectin) inflammatory biomarkers. Secondary outcomes included other pulmonary (IL-1ß, IL-6, neutrophil elastase, lactate and calprotectin) and systemic (serum-IL-1ß, IL-6) inflammatory biomarkers, as well as clinical outcomes (FEV1, pulmonary exacerbations, antibiotic use, nutritional status and quality of life). RESULTS: Ninety six CF patients, 44 female, age 14.6±11.9 years (48 DHA and 48 placebo) were included. At trial completion, there were no differences in all primary outcomes [serum-IL-8 (p=0.909), respiratory-IL-8 (p=0.384) or fecal calprotectin (p=0.948)], all secondary inflammatory biomarkers, or in any of the clinical outcomes evaluated. There were few adverse events, with similar incidence in both study groups. CONCLUSION: In this study, long-term DHA supplementation in CF patients was safe, but did not offer any benefit on inflammatory biomarkers, or in clinical outcomes compared with placebo. (NCT01783613).

Blood Lymphocyte Subsets for Early Identification of Non-Remission to TNF Inhibitors in Rheumatoid Arthritis.

Background: TNF inhibitors (TNFis) are widely used for the treatment of rheumatoid arthritis (RA), although the response rates to this therapy in patients with RA remains heterogeneous and < 50% achieve remission (REM). Objective: To analyze baseline peripheral blood leukocytes profiles in order to search for biomarkers identifying patients who will most likely not achieve REM under TNFi treatment. Methods: A prospective bi-center pilot study including 98 RA patients treated with TNFis and followed-up during 6 months. Patients were classified according to DAS28 as follows: those who achieved REM (DAS28 ≤ 2.6) and those who did not (DAS28 > 2.6) at 6 months after starting TNFis. These rates were also assessed by simplified disease activity index (SDAI ≤ 3.3 and SDAI > 3.3, respectively). Peripheral blood immune cells were studied by flow cytometry before treatment initiation. Results: At 6 months, 61 or 80% of patients did not achieve REM by DAS28 or SDAI, respectively. Basal leukocyte profiles differed between REM vs. non-REM patients. Non-REM patients showed lower percentages of total and naïve B cells at baseline than REM subjects. A B lymphocyte/CD4+ lymphocyte ratio (BL/CD4 ratio) <0.2 clearly associated with a higher probability of non-REM status based on DAS28 at 6 months (OR = 9.2, p = 0.006). These data were confirmed when patient response was evaluated by SDAI index. Conclusion: Our results strongly suggest that BL/CD4 ratio could be considered as a useful biomarker for the early identification of non-remitters to TNFi in clinical practice.

Gamma delta+ intraepithelial lymphocytes and coeliac lymphogram in a diagnostic approach to coeliac disease in patients with seronegative villous atrophy.

BACKGROUND: The causes of seronegative villous atrophy can be grouped as coeliac or noncoeliac related. There is no consensus on how to approach subjects with seronegative coeliac disease. AIM: To evaluate the accuracy of both an increase in CD3+ T-cell receptor gamma delta+ (TCRγδ+ ) intraepithelial lymphocytes and coeliac lymphogram for the diagnosis of coeliac disease in patients with seronegative villous atrophy. METHODS: Sixty-seven consecutive patients with seronegative villous atrophy were included. Duodenal biopsies to assess TCRγδ+ and CD3- by flow cytometry were performed at the index endoscopy. Coeliac lymphogram was defined as an increase in TCRγδ+ plus a decrease in CD3- intraepithelial lymphocytes. Sensitivity, specificity and Fagan's nomogram were calculated. RESULTS: Coeliac disease was diagnosed in 37 patients and noncoeliac villous atrophy in 30. Coeliac patients were younger (39 ± 3 vs 55 ± 3 years; P = 0.001), more often showed HLA-DQ2/8 (97.6% vs 61%; P = 0.002) and had a more severe histology (61% vs 32% Marsh 3b-c; P = 0.055), as compared to noncoeliac ones. Coeliac lymphogram was associated with a sensitivity of 87% (CI, 73.7-95) and specificity of 96.7% (82.7-99.9), whereas evaluating only TCRγδ+ yielded a sensitivity of 91.3% (79.2-97.6) and specificity of 83.3% (65.3-94.3). Among patients with a pre-test coeliac disease probability of 30%, post-test probabilities were 92% and 5% for positive and negative coeliac lymphogram, and 70% and 4% for positive and negative TCRγδ+ . CONCLUSIONS: Coeliac lymphogram was associated with a high level of diagnostic evidence either against or in favour of coeliac disease in patients with seronegative villous atrophy.

Distal duodenum versus duodenal bulb: intraepithelial lymphocytes have something to say in celiac disease diagnosis.

BACKGROUND AND AIM: After clinical screening and the serological test, many patients still require a duodenal biopsy for celiac disease diagnosis. Mild histological lesions, unspecific findings and patchiness are frequent outcomes of this mandatory diagnostic tool, thus complicating clinical decisions. METHODS: We analyzed the lymphoid components [number of total intraepithelial lymphocytes (IELs), TcR-γδ and CD3(-)IELs] of the duodenal epithelium by flow cytometry in samples obtained from bulb and distal duodenum during upper gastrointestinal endoscopies performed for diagnostic purposes. RESULTS: IEL counts and IEL subset distribution (IEL lymphogram) remain invariant along duodenal mucosa revealing a specific profile (immunophenotype) that characterizes either a healthy mucosa or a celiac mucosa. The celiac immunophenotype persists regardless of the biopsy's anatomical location or the corresponding histological findings. CONCLUSIONS: We propose the IEL lymphogram by flow cytometry as an immunological parameter to discern celiac condition from healthy mucosa. This obviates not only misinterpretation of minor histological changes, but also patchiness and the concerns about the location and number of biopsies.

Improvement of digestive health and reduction in proteobacterial populations in the gut microbiota of cystic fibrosis patients using a Lactobacillus reuteri probiotic preparation: a double blind prospective study.

BACKGROUND: Although scientific knowledge about the benefits of probiotic use in cystis fibrosis (CF) is scarce, their expectative is promising. The aim of this work was to analyze the effect of a Lactobacillus reuteri probiotic preparation versus placebo in CF patients. METHODS: A prospective, double blind, crossover and with placebo study was carried out in 30 CF patients from two Spanish hospitals. Patients were randomized in Group A (6 months of probiotic followed by 6 months of placebo) and Group B (6 months of placebo followed by 6 months of probiotic). GIQLI (gastrointestinal) and SF-12 (general) health tests were performed after probiotic and placebo intakes. Fat absorption coefficient, calprotectin, and inflammatory interleukin quantification were determined in fecal samples. Total fecal DNA was obtained and metagenomic 454-pyrosequencing was applied to analyze the microbiome composition. STATA v12 MP software was used for statistical analyses. RESULTS: Statistically significant improvement in the gastrointestinal health and decrease of the calprotectin levels were demonstrated in patients after probiotic exposure, in comparison with placebo. All CF subjects reported good tolerance to L. reuteri without secondary effects. Metagenomic analysis showed an important dysbiosis in CF gut microbiota associated with a high concentration of Proteobacteria. Probiotic intake was followed by a reduction in the total bacterial density, mostly due to a considerable reduction in the γ-Proteobacteria phylum; and an important increase of the microbial diversity with a higher representation of Firmicutes. CONCLUSIONS: Probiotics might ameliorate the dysbiosis of CF gut microbiota, characterized by a high density of Proteobacterial organisms. L. reuteri significantly decrease intestinal inflammation and increase digestive comfort.

Flow cytometric analysis of intestinal intraepithelial lymphocytes in the diagnosis of refractory celiac sprue.

The etiology of refractory celiac sprue (RCS) is unclear. In a high proportion of cases, the clonal nature of intestinal intraepithelial lymphocytes (IEL) can be demonstrated and a pathogenetic implication of intestinal IEL has been postulated. The prognosis of this subgroup of RCS is poor, with a high risk to develop an overt lymphoma and uncontrolled malabsorption despite steroid/immunosuppressive therapy. Cases with a relatively indolent clinical course, however, exist and their early diagnosis may be difficult. To gain insight into the pathogenic implication of intestinal IEL in refractory celiac sprue, we have performed an extensive phenotypic and functional characterization of clonal intestinal IEL in a patient with an indolent form of refractory celiac sprue, using multiparametric flow cytometry. The abnormal lymphocyte infiltrate lacked surface membrane expression of CD3/T-cell receptor (TCR) complexes (TCR(-), CD4(-), CD8(-), sCD3(-)), but contained intracellular CD3(epsilon) (CyCD3(+)) and surface CD103(+) and CD7(+). In particular, these cells showed a unique spontaneous ex-vivo cytokine secretion profile with an increased percentage of CD3(-) IEL containing TNF-alpha and IL-10, in the absence of IL-2, IL-4 and IFN-gamma. Altogether our results suggest that flow cytometry immunophenotyping of intestinal IEL, in cases suspected of celiac disease and their complicated forms, could be of great help in the correct diagnosis of RCS and the understanding of the immunopathogenic mechanisms of the disease and their clinical and/or therapeutical implications.

Age-related variation of intraepithelial lymphocytes subsets in normal human duodenal mucosa.

The enumeration of intestinal intraepithelial lymphocytes (IELs), and the phenotyping of CD3+CD 103+ (TcRalphabeta, TcRgammadelta) and CD3-CD103+ IEL subsets constitute useful diagnostic tools for the correct interpretation of the mucosal histology of duodenal/jejunal biopsies in many pathological conditions of the small intestine, particularly celiac disease (CD). This work evaluates the ranges of duodenal IEL counts by flow cytometry in healthy mucosa from pediatric and adult controls, establishing normal reference values for CD3+ TcRgammadelta and CD3- subsets and their variation with age. Seventy-four pediatric controls and 36 adult controls were identified on the basis of their normal histology from more than 1,000 duodenal diagnostic biopsies performed in Caucasian subjects. Total IEL counts and IEL subsets ("IEL lymphogram") were analyzed by four-color flow cytometry (FCM). IEL represent 7.7% +/- 0.4 (mean +/- SE) and 8.5% +/- 0.5 of the cells isolated from the epithelium in the pediatric and adult series, respectively. The upper normal range, considered as the 97 percentile, is 14% in pediatrics and 15% in adults. No significant difference was observed between TcRgammadeltaIEL percentages in children (6.9% +/- 0.5 of the total IELs) and adults (6.6% +/- 0.8). However, the density of CD3- IELs is significantly higher (p < 0.001) in the mucosa from controls under 3 years (50.2% +/- 2.6) than in adults (25.5% +/- 2.1). IEL lymphogram by flow cytometry is an easy, quick and reliable analysis performed in one of the biopsy specimens obtained during a diagnostic endoscopy, and confers specificity to the histopathological findings. IEL counts below 14% in children and 15% in adults should be considered within a normal range in the evaluation of duodenal mucosa by FCM. No differences with age were observed with respect to TcRgammadeltaIEL, while the CD3- IEL fraction was significantly higher on children under 3 years, with a trend to increase again in the elderly.