BACKGROUND: Coeliac disease (CD) is a chronic condition requiring a gluten-free diet, which is a very demanding diet to maintain on a life-long basis. For this reason it is a condition that can have serious repercussions on the quality of life (QOL). Therefore the need to elaborate a questionnaire on QOL specifically for patients with CD (CDQ): its original language is German, and the translation/validation process represents a considerable challenge involving not only a translation into Italian but also an adaptation to the country's specific cultural differences. METHODS: The questionnaire has been translated according to a "German→Italian→Italian→German" algorithm with reconciliation of the differences. Scores for CDQ are computed overall and over four areas of four items each: emotion, gastrointestinal symptoms, gastrointestinal worries, social problems. RESULTS: CDQ was administered to 171 coeliacs (F 132, mean age 38yrs±14). Completeness was optimal. Item internal consistency was satisfied for 100% and 97% of patients for the specific and generic part, respectively. Cronbach's α coefficient was 0.7 for all scales. The general CDQ was higher in patients reporting subjective well-being (discriminant validity). CONCLUSIONS: The Italian translation of CDQ sounds natural, is easy to understand and reduces possible cultural biases to a minimum. A field test gave results comparable to the original validation, supporting the use of CDQ in cross-national surveys.

Gastrointestinal manifestations and villous atrophy can be seen in patients with common variable immunodeficiency (CVID). In some patients, infectious agents may be responsible, whereas in others, celiac disease (CD) may be the cause. In this study, we investigate the causes and the histopathologic features seen in patients with CVID. Eleven patients with CVID and villous atrophy underwent duodenal biopsies, human leukocyte antigen (HLA) typing, and testing for all celiac antibodies. Fifteen patients with CVID and normal villi and 6 patients with CD but without CVID served as controls. Histologic response to a gluten-free diet (GFD) allowed a diagnosis of CD in 3 of 11 patients. In the remaining 8, the lack of a histologic response to a GFD or HLA typing excluded CD. Celiac antibodies gave conflicting results and were of no help. Polymorphonuclear infiltrates and lesions like graft-versus-host disease are seen more often in flat mucosa unresponsive to a GFD. However, the specificity of these findings remains to be determined and response to a GFD remains the only diagnostic criteria for CD in these patients. Villous atrophy was gluten-sensitive in 3 of 11 patients with CVID. It was not related to gluten-responsive CD in most patients.

Background: Wheat gluten comprises gliadins and glutenins. The high-molecular-weight (HMW) glutenin subunits (GS)-1Dy10 are toxic for patients with celiac disease (CD). This study aimed to assess whether CD patients mount a serological response to HMW-GS-1Dy10. Methods: Recombinant HMW-GS-1Dy10 was deamidated using human recombinant tissue transglutaminase. MALDI-TOF was performed to compare the level of deamidation of glutamine residues between material before and after treatment. Enzyme-linked immunosorbent assays were developed. Sera from patients with untreated CD and gastrointestinal disease controls were tested and receiver operator characteristics were used to calculate cutoffs. Results: MALDI-TOF revealed a number of fragments matching known HMW-GS-1Dy10 sequences within both the deamidated and non-deamidated material. Evidence of deamidation of glutamine residues was found only within the human transglutaminase-treated material. Patients with untreated CD had significantly increased levels of serum antibodies to HMW-GS-1Dy10 compared to controls. Undeamidated HMW-GS-1Dy10 IgA antibodies had sensitivities and specificities of 72.5 and 78.26%, respectively. Deamidated HMW-GS-1Dy10 IgA antibodies had sensitivities and specificities of 76.8 and 65.2%. Undeamidated HMW-GS-1Dy10 IgG antibodies had sensitivities and specificities of 75.3 and 68.1%. Deamidated HMW-GS-1Dy10 IgG antibodies had sensitivities and specificities of 36.2 and 92.8%. Conclusion: Patients with untreated CD have raised antibody levels to HMW-GS-1Dy10, indicating the participation of these proteins in the adaptive immune response to gluten. Discrimination between CD patients and controls is not enhanced by deamidation of HMW-GS-1Dy10. Thus antibodies to these proteins are not useful markers for CD detection.

INTRODUCTION: Whipple's disease is a rare chronic infection caused by Tropheryma whipplei. Although most patients respond to antibiotics, in some of them the start of the treatment is followed by recurrence of inflammation. Since polymerase chain reaction is negative for Tropheryma whipplei, this reinflammation cannot be a relapse of Whipple's disease itself. Very recently, it has been recognised as a complication of Whipple's disease and defined immune reconstitution inflammatory syndrome (IRIS). Our aim is to study the prevalence and the clinical features of IRIS in Italian patients with Whipple's disease. METHODS: Evidence of IRIS was retrospectively revaluated in the clinical notes of 22 patients with Whipple's disease. Patients with no evidence of IRIS served as controls for the clinical findings. RESULTS: Recurrence of arthralgia and/or fever allowed a diagnosis of IRIS in 5/22 patients. One patient died. Previous immunosuppressive therapy was found in all patients with IRIS but only in 7/17 controls (Fisher test, p=0.039). Age at diagnosis and diagnostic delay were higher in patients with IRIS compared to controls. However, statistical significance was not reached. CONCLUSIONS: IRIS is a frequent complication of Whipple's disease and it can be fatal. The risk of IRIS is greatly increased in patients previously treated with immunosuppressive therapy.

BACKGROUND HLA-DQB1*02 homozygosity was shown to be more common in patients with complicated rather than uncomplicated celiac disease (CD). GOALS To study HLA-DQA1 and DQB1 profile in adult patients with different forms of CD, including patients with complicated and potential CD, the most affected and the most preserved histologic end of the pathologic celiac spectrum. STUDY HLA-DQA1 and DQB1 molecular typing was performed in 218 adult CD patients (169 with uncomplicated CD, 27 with complicated CD, and 22 with potential CD) and 224 healthy stem cell donors. HLA-DQA1 and DQB1 gene polymorphism was analyzed using polymerase chain reaction sequence-specific primers and/or reverse polymerase chain reaction sequence-specific oligonucleotides techniques. RESULTS As expected, the frequency of HLA-DQB1*02 allele, DQB1*02 homozygosity, and DQB1*0302 gene were statistically different in the 4 groups. However, multivariate analysis demonstrated that patients with potential CD have a higher frequency of both HLA-DQB1*0302 and HLA-DQB1*0603 alleles and a reduced frequency of DQB1*02 homozygosity compared with patients with uncomplicated and complicated CD. CONCLUSIONS The increased frequency of DQB1*0302 and the reduced frequency of DQB1*02 homozygosity in potential CD is consistent with the idea that different clinical/pathologic evolutions might be related to different immunogeneses. This could be clinically relevant in the future.

Tissues exposed to ischemia and reperfusion develop an inflammatory response. We investigate the morphological and immunological changes occurring in the mucosa of a jejunal loop transplanted in the oropharynx of a man undergoing circular pharyngolaryngectomy. Jejunal biopsies were collected during the transplantation procedures (cold and warm ischemia, reperfusion), during the 7 post-operative days through an exteriorized jejunal segment for flap monitoring, and 45 days after transplantation through an upper endoscopy. Matrix metalloproteinase (MMP)-3 and MMP-12 increase was accompanied by a parallel rise in apoptotic enterocytes, and by a concomitant reduction of surface area to volume ratio and enterocyte height. Goblet cell hyperplasia is coupled with Paneth cell disappearance at the crypt base. CD8-positive intraepithelial lymphocytes initially decrease, then they increase in accordance with the peak of enterocyte apoptosis. We identified alterations in lymphocyte infiltration, mucosal architecture and epithelial cell turnover, which may give a window to mechanisms of small bowel ischemia-reperfusion in humans.

In the past few years, the number of celiac disease diagnoses not confirmed at the Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, a tertiary referral centre, was particularly high. Therefore, a decision was made to investigate the reasons why these diagnoses were wrong and by whom they had been made. The clinical histories of all celiac patients referred to the centre were re-evaluated. Between December 1998 and January 2007, 614 patients who were diagnosed at other institutions and presumed to be affected by celiac disease attended the tertiary referral outpatient clinic. The histological and serological results allowed for confirmation the diagnosis in 434 patients. In the remaining 180 patients, the initial diagnosis of celiac disease could not be confirmed; therefore, the patients were re-investigated. After re-evaluation, the diagnosis of celiac disease was confirmed in only 61 of these 180 cases. The reasons for incorrect initial diagnosis were analyzed. A mere 80% correct diagnosis rate is a very disappointing result. Although it should be obvious that celiac disease must be investigated with duodenal biopsies and celiac antibody testing, this well-known strategy is not always followed, probably resulting in an incorrect diagnosis.

Matrix metalloproteinases (MMPs) are a family of endopeptidases playing a key role in tissue remodelling in both physiological and pathological conditions. Since little information is available about their role in celiac disease (CD), our aims were to quantify their expression/activity and to investigate their relation to proinflammatory cytokines in this condition. Duodenal biopsies from untreated, treated celiac patients and controls were used to quantify the expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-12, MMP-14, their inhibitor TIMP-1, IFN-gamma and TNF-alpha by using real-time reverse transcription-polymerase chain reaction and the gelatin/casein/elastin activities by gel zymography, and to isolate lamina propria mononuclear cells (LPMCs). These cells and myofibroblasts isolated from jejunal specimens were incubated in the absence or presence of IFN-gamma and TNF-alpha. MMP-1 and MMP-12 mRNA levels were significantly increased in active CD compared to treated (P<0.01 and P<0.0005, respectively) and normal mucosa (P<0.01 and P<0.0005, respectively), and this was paralleled by an upregulation of caseinolytic and elastolytic activities. Furthermore, MMP-12 levels significantly (P<0.05) correlated with those of IFN-gamma and the degree of villous flattening. MMP-2 turned out to be significantly (P<0.05) reduced in untreated and treated celiacs compared to controls. In active CD, transcripts of TIMP-1 were higher than in treated and controls (P<0.005 and P<0.05, respectively), such as those of IFN-gamma (P<0.05), whereas TNF-alpha levels were suppressed (P=0.0001). In physiological condition, myofibroblasts represent the main source of MMP-2, whereas LPMCs produce almost all MMPs only after cytokine stimulation. Conversely, cells isolated from active patients constitutively express MMPs without any increase after cytokine stimulation, while those from treated patients are in a resting condition. In conclusion, our results show the presence of a peculiar MMP pattern in active CD strongly dominated by MMP-12, correlating either with IFN-gamma or the degree of mucosal damage.

Whipple's disease is a chronic multisystemic infection, due to Tropheryma whipplei, a bacterium ubiquitously present in the environment. Although it is very rare, its clinical features are non-specific and can affect several different districts. Whipple's disease is therefore a condition that should always be kept in mind by doctors working in several branches of medicine, such as internal medicine, gastroenterology, rheumatology, neurology, and cardiology. The condition is fatal if not promptly recognized and treated, but the best treatment is still not completely defined, especially in relapsing disease, neurological manifestations, and in cases of immunoreconstitution after initiation of antibiotic treatment.

OBJECTIVES:Capsule endoscopy (CE) allows for the assessment of the small bowel in numerous intestinal diseases, including celiac disease (CD). The main advantage of CE is the complete visualization of the intestinal mucosal surface. The objective of this study was to investigate whether CE can predict the severity of CD and detect complications.METHODS:We retrospectively studied the medical files of 9 patients with symptomatic CD, 11 patients with refractory celiac disease type I (RCDI) and 18 patients with refractory celiac disease type II (RCDII), and 45 patients without CD who were investigated both CE and upper endoscopy or enteroscopy. The type of CD was diagnosed on the basis of a centralized histological review, flow cytometry analysis of intraepithelial lymphocytes, and the analysis of T-cell receptor rearrangement by multiplex polymerase chain reaction.RESULTS:A total of 47 CEs (10, 11, and 26 CEs in the symptomatic CD, RCDI, and RCDII groups, respectively) from the 38 celiac patients and 47 CEs from the 45 nonceliac patients were retrospectively reviewed. Villous atrophy, numerous, or distally located ulcers were more frequent in celiac patients than in controls. Among celiac patients, CE was of acceptable quality in 96% of cases and was complete in 62% of cases. The concordance of CE with histology for villous atrophy was better than that of optic endoscopy (κ coefficient =0.45 vs. 0.24, P<0.001). Extensive mucosal damage on CE was associated with low serum albumin (P=0.003) and the RCDII form (P=0.02). Three cases of overt lymphoma were detected by CE during the follow-up.CONCLUSIONS:CE findings have a satisfactory concordance with histology and nutritional status in patients with symptomatic or refractory CD. Moreover, CE may predict the type of RCD and allows for the early detection of overt lymphoma.Am J Gastroenterol advance online publication, 11 September 2012; doi:10.1038/ajg.2012.199.

In refractory celiac disease (RCD), intestinal epithelial damage persists despite a gluten-free diet. Characteristic for RCD type II (RCD II) is the presence of aberrant surface TCR-CD3(-) intraepithelial lymphocytes (IELs) that can progressively replace normal IELs and eventually give rise to overt lymphoma. Therefore, RCD II is considered a malignant condition that forms an intermediate stage between celiac disease (CD) and overt lymphoma. We demonstrate in this study that surface TCR-CD3(-) IEL lines isolated from three RCD II patients preferentially lyse epithelial cell lines. FACS analysis revealed that DNAM-1 was strongly expressed on the three RCD cell lines, whereas other activating NK cell receptors were not expressed on all three RCD cell lines. Consistent with this finding, cytotoxicity of the RCD cell lines was mediated mainly by DNAM-1 with only a minor role for other activating NK cell receptors. Furthermore, enterocytes isolated from duodenal biopsies expressed DNAM-1 ligands and were lysed by the RCD cell lines ex vivo. Although DNAM-1 on CD8(+) T cells and NK cells is known to mediate lysis of tumor cells, this study provides, to our knowledge, the first evidence that (pre)malignant cells themselves can acquire the ability to lyse epithelial cells via DNAM-1. This study confirms previous work on epithelial lysis by RCD cell lines and identifies a novel mechanism that potentially contributes to the gluten-independent tissue damage in RCD II and RCD-associated lymphoma.

Recently, several European centers of lymphoma diagnosis and research developed various polymerase chain reaction (PCR) methods for clonality analysis in suspect T-cell and B-cell proliferations (Biomed-2 Concerted Action). They have mainly been applied to frozen material of systemic B-cell and T-cell malignancies. Thus far, only limited data exist with regard to cutaneous T-cell lymphoma (CTCL) and paraffin-embedded material. Thus, we applied the Biomed-2 T-cell receptor (TCR) gamma and TCRbeta PCR as well as an in-house TCRgamma PCR to a collection of 107 archival skin samples (84 CTCL, 3 systemic TCL and 20 controls). As a result, the Biomed-2 TCRgamma PCR revealed 81% clonality, the in-house TCRgamma method revealed 86% clonality, and the Biomed-2 TCRbeta revealed 78% clonality in CTCL samples generating at least the 300 bp fragment in the Biomed-2 control PCR. We found clonal TCRbeta rearrangements in 5 of 17 CTCL samples that were polyclonal in the Biomed-2 TCRgamma PCR. By combining all Biomed-2 assays, one or more clonal rearrangements were detected in 87% of CTCL and in all 3 systemic TCLs. By combining all TCR PCR assays applied here, clonality was shown in 90% of the CTCL cases. In conclusion, we showed that the Biomed-2 TCR PCR worked well with DNA from paraffin-embedded tissue, revealing a high-clonality detection rate in CTCL, and thus should be highly recommended for routine molecular analysis. In addition, the performance of our in-house TCRgamma assay verifies our previously published findings on clonally expanded T-cells in CTCL.

Monoclonal expansions of immunoglobulin (Ig) and T-cell receptor (TCR) genes are, respectively, important markers for B- and T-cell malignancies. We used BIOMED-2 protocols to detect clonality of these genes in B-(BCL) and T-cell lymphoma (TCL) in southern Taiwan. Heteroduplex analysis was used after PCR reactions. Ig/TCR clonality rates were 95%(22 in 23 cases) for BCL and 76%(19 in 25 cases) for TCL. These results reveal overall satisfactory detection rates for both BCL and TCL. None of the four natural killer (NK)/T-cell lymphomas detected showed TCR gene clonality, which suggested that BIOMED-2 protocols distinguished the NK/T-cell lymphoma from TCL. In addition, three of the five tested precursor T-cell lymphoblastic lymphomas showed no TCR gene clonality, probably because the immature T-cells in this type of TCL had not undergone TCR gene rearrangements. We conclude that BIOMED-2 protocols are suitable for detecting Ig and TCR clonalities in B- and T-cell malignancies in southern Taiwan.

A rare case of primary intestinal T-cell lymphoma (ITL) of an 8-year-old boy is reported. Medium- to large-sized tumor cells were betaF1+, CD3+, CD8+. TIA-1+, but CD4-, CD5-, CD30-, CD56-, CD20-, CD79a-, TdT-, consistent with an intraepithelial lymphocyte (IEL) origin. They showed monoclonal rearrangement of the T-cell receptor gamma-chain and no evidence of EBV infection. No clinical, histologic, laboratory, or genetic evidence of celiac disease was detected. In adults, ITL is often associated with enteropathy and has a very poor outcome. Our patient remains in first remission 30 months after finishing the acute lymphoblastic leukemia protocol COALL-07-03 high risk standard.

OBJECTIVE An aberrant immunophenotype and monoclonality of intraepithelial lymphocytes (IELs) are frequently found in refractory coeliac disease (RCD). However, the utility of continual monitoring of IEL immunophenotype and clonality in the surveillance of RCD remains to be studied. DESIGN The diagnostic and follow-up biopsies from 33 patients with CD, 7 with suspected RCD, 41 with RCD and 20 with enteropathy-associated T cell lymphoma (EATL)(including 11 evolved from RCD) were investigated by CD3epsilon/CD8 double immunohistochemistry and PCR-based clonality analysis of the rearranged T cell receptor (TCR) genes. RESULTS An aberrant immunophenotype (CD3epsilon(+)CD8(-) IELs > or =40%) and monoclonality were detected occasionally in CD biopsies, either transiently in patients with CD not compliant with a gluten-free diet or in those who subsequently developed suspected RCD, RCD or EATL. In contrast, the aberrant immunophenotype and monoclonality were found in 30 of 41 (73%) and 24 of 37 (65%) biopsies, respectively, at the time of RCD diagnosis. Among the patients with RCD who did not show these abnormalities in their diagnostic biopsies, 8 of 10 (80%) and 5 of 11 (45%) cases gained an aberrant immunophenotype and monoclonality, respectively, during follow-up. Irrespective of whether detected in diagnostic or follow-up biopsies, persistence of both abnormalities was characteristic of RCD. Importantly, the presence of concurrent persistent monoclonality and aberrant immunophenotype, especially > or =80% CD3epsilon(+)CD8(-) IELs, was a strong predictor of EATL development in patients with RCD (p=0.001). CONCLUSIONS Continual monitoring of both immunophenotype and clonality of IELs is more important than snapshot analysis for RCD diagnosis and follow-up, and could provide a useful tool for surveillance of patients at risk of EATL.

OBJECTIVE To establish a sensitive and effective method for detection of immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangement,and to explore its role in diagnosis and differential diagnosis of lymphoproliferative disorders. METHODS Fifty-eight lymphoid tissue samples from 54 patients with lymphoproliferations were evaluated by the novel BIOMED-2 multiplex polymerase chain reaction (PCR) for antigen receptor genes rearrangement. RESULTS Multiplex PCR demonstrated monoclonal Ig/TCR gene rearrangements in 22 of 25 (88.0%) B-cell malignancies and 8 of 15 (53.3%) T-cell malignancies. Among 17 benign lymphoproliferations confirmed histopathologically, polyclonal rearrangements were detected in 14 cases (82.4%). In total, the clonality analysis and the final clinico-histopathological diagnosis were concordant in 77.2%. Combination detection of Iglambda and TCR delta gene rearrangements did not increase the detection rate of monoclonal rearrangement of Ig/TCR, but might help to the detection of Iglambda+ or TCR delta+ lymphomas. CONCLUSION The novel BIOMED-2 multiplex PCR strategy is a rapid, reliable and sensitive approach to detecting clonality in suspected lymphoproliferations, especially in atypical cases.

OBJECTIVE To evaluate the practical values of PCR detectable T-cell receptor (TCR) gene rearrangement in paraffin embedded tissue samples in the diagnosis of T-cell malignancies using BIOMED-2 PCR multiplex tubes TCRgamma(A+B). METHODS Traditional phenol-chloroform method was used to extract DNA from 55 cases of archival paraffin embedded tissues samples of T-cell malignancies and the DNA quality was evaluated by PCR-based amplification of housekeeping gene beta-globin. The selected BIOMED-2 PCR multiplex tubes TCRgamma(A+B) were used to detect TCR gene rearrangement and comparison with the results of universal TCR primers (T(VG)/T(JX)) was performed. RESULTS Positive detection rates by the BIOMED-2 multiplex tubes TCRgamma(A+B) and the universal primers (T(VG)/T(JX)) were 76.4% and 60.0%, respectively. There were not statistical difference between the methods (P > 0.05). CONCLUSION BIOMED-2 multiplex tubes TCRgamma(A+B) is suitable for detection of clonal rearrangements of TCR genes in current archival paraffin embedded tissue samples of T-cell malignancies.

BACKGROUND & AIMS Refractory celiac disease (RCD) occurs when both symptoms and intestinal damage persist or recur despite strict adherence to a gluten-free diet. In RCD, the immunophenotype of intraepithelial lymphocytes may be normal and polyclonal (RCD I) or abnormal and monoclonal (RCD II). The aim is to describe the clinical characteristics, treatment, and long-term outcome in a large single-center cohort of patients with RCD. METHODS We compared the clinical characteristics and outcome in 57 patients with RCD: 42 with RCD I and 15 with RCD II. RESULTS Fifteen of 57 patients died during follow-up (n=8 with RCD I and n=7 with RCD II), each within the first 2 years after RCD diagnosis. The overall 5-year cumulative survival is 70%, 80%, and 45% for the entire cohort, RCD I, and RCD II, respectively. The refractory state itself and enteropathy-associated T-cell lymphoma (EATL) were the most common causes of death, respectively. A new staging system is proposed based on the cumulative effect of 5 prognostic factors investigated at the time of the refractory state diagnosis: for patients in stages I, II, and III, the 5-year cumulative survival rate was 96%, 71%, and 19%, respectively (P<.0001). CONCLUSIONS RCD is associated with high mortality with RCD II having an especially poor prognosis because of the development of EATL. A new staging model is proposed that may improve the precision of prognosis in patients with RCD.

INTRODUCTION: Refractory sprue is characterised by distinctive morphologic alterations and the emergence of clonal intraepithelial lymphocytes. Aim: In this case report the authors emphasize the importance of histopathology in the diagnosis of refractory sprue. METHODS: The sequential biopsies from this patient have been investigated with routine histology, immunohistochemistry and molecular genetics for T-cell clonality analysis. RESULTS: The severely cachectic patient presenting with malabsorption syndrome has been diagnosed with celiac disease through a duodenal biopsy, and the CD8 negativity of the intraepithelial lymphocytes suggested the possible diagnosis of refractory sprue. Azathioprine and glucocorticoid therapy was administered due to the failed jejunal feeding and gluten-free diet, resulting in clinically complete, morphologically partial remission. Intestinal T-cell lymphoma developed in the ileocecal region within two years after the first clinical presentation. DISCUSSION: Refractory sprue and the enteropathy-type T-cell lymphoma constitute a disease spectrum. The reported case shows how a simple method can provide crucial information in the diagnosis of refractory sprue.