Marco Greco

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.

Radioimmunoassay to detect antitransglutaminase autoantibodies is the most sensitive and specific screening method for celiac disease

OBJECTIVE:The aim of this study was to establish the most sensitive and specific screening method for celiac disease. We tested three methods based on different principles, which all detect autoantibodies against the same antigen (tissue transglutaminase).METHODS:Sixty-two celiac children at the first biopsy (group 1), 78 celiac children on a gluten-free diet (group 2), 14 celiac children on a gluten-challenge (group 3), and 56 controls with a normal duodenal mucosa (group 4) were studied. The methods used were: 1) radioimmunoprecipitation assay using recombinant tissue transglutaminase (RIA); 2) commercial enzyme immunoassay using guinea pig tissue transglutaminase (ELISA); and 3) indirect immunofluorescence method for detection of antiendomysium antibodies (IF-EMA).RESULTS:RIA antitransglutaminase autoantibodies were detected in 100% of group 1, 43.6% of group 2, 100% of group 3, and none of the control subjects. ELISA antitransglutaminase autoantibodies were detected in 90.3% of group 1, 9% of group 2, 78.6% of group 3, and in none of the control subjects. IF-EMA were detected in 95.2% of group 1, 11.5% of group 2, 92.3% of group 3, and 1.8% of the controls.CONCLUSIONS:Our results demonstrate a very high sensitivity and specificity of the RIA method to detect antitransglutaminase autoantibodies in comparison to ELISA and IF-EMA assays. We can explain this finding with the use of human recombinant antigen and the increased capacity of the RIA method to detect low titers of autoantibodies. If our data are confirmed by studies on larger series, tissue transglutaminase RIA could be proposed as the best screening method for celiac patients.

Expression of protein tyrosine phosphatase alpha (RPTPα) in human breast cancer correlates with low tumor grade, and inhibits tumor cell growth in vitro and in vivo

Tyrosine phosphorylation is controlled by a balance of tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Whereas the contribution of PTKs to breast tumorigenesis is the subject of intense scrutiny, the potential role of PTPs is poorly known. RPTPα is implicated in the activation of Src family kinases, and regulation of integrin signaling, cell adhesion, and growth factor responsiveness. To explore its potential contribution to human neoplasia, we surveyed RPTPα protein levels in primary human breast cancer. We found RPTPα levels to vary widely among tumors, with 29% of cases manifesting significant overexpression. High RPTPα protein levels correlated significantly with low tumor grade and positive estrogen receptor status. Expression of RPTPα in breast carcinoma cells led to growth inhibition, associated with increased accumulation in G0 and G1, and delayed tumor growth and metastasis. To our knowledge, this is the first example of a study correlating expression level of a specific bona fide PTP with neoplastic disease status in humans.

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

Abstract. 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.

Computed tomography of the small bowel in adult celiac disease: the jejunoileal fold pattern reversal

Abstract. The aim of this retrospective study was to establish whether the distinctive intestinal fold pattern of celiac disease (CD), known by barium studies as jejunoileal fold pattern reversal (JFPR) may be recognized at CT. The number of intestinal folds per 2.5 cm, seen at CT, were counted in the jejunum and in the ileum of 22 adult patients with CD and compared with the folds of 30 consecutive subjects in whom an intestinal disease had been excluded. The results were submitted to statistical analysis by Students t-test. In the control group the number of folds per 2.5 cm were 4.88 (SD ± 0.78) in the jejunum and 2.84 ( ± 0.62) in the ileum; in the CD group the number of folds were 2.42 ( ± 1.61) in the jejunum and 5.11 ( ± 1.24) in the ileum. There was a statistically significant difference in the number of jejunal and ileal folds between the CD patients and the control group (in both cases p < 0.001). The JFPR was seen in 15 patients with CD (68.2 %) but in none of the controls. Our study shows that JFPR is not a normal finding and can be demonstrated by CT in the majority of patients with CD.

Nuclear medicine techniques for the study of breast cancer.

The diagnosis of breast cancer is based on the utilization of physical examination, mammography and/or ultrasonography, and fine needle aspiration biopsy (FNAB) or core biopsy in accordance with the palpability and characteristics of the lesion, the age of the patient, and the density of the mammary gland [1, 2]. Even though clinical examination (inspection and palpation) is not a very sensitive test and its specificity is low, in particular for small lumps (less than 1 cm), it should be stressed that it remains the first and fundamental approach in the diagnosis of palpable breast cancer. However, mammography has been completely transformed during the past two decades into a sophisticated technological method, with greatly improved image quality; it not only allows the recognition of very small, frequently non-palpable lesions, but also has become the method of choice for identifying breast carcinomas. The increasing use of screening mammography has resulted in an increase in the rate of detection of non-palpable lesions, and consequently in an increased demand for needle localizations and biopsies. In fact, in some cases the presence of microcalcifications at mammography is the only sign of breast cancer. It is possible to localize the position of this type of lesion with various methods: generally, a needle is positioned under mammographic control, leaving either a coloured substance or a hook wire that provides a guide for the surgeon. In this way, the surgeon is able to remove the portion of the mammary gland that includes the lesion; the specimen should be submitted to radiography for confirmation of the complete removal of microcalcifications [3, 4]. It is important to point out that ultrasonography has very good ability to differentiate between cystic and solid masses, but its sensitivity for the detection of small carcinomas is not high. Its ideal use is in young women with full glandular breasts, owing to their intrinsic radiopacity, while it can also be used for guidance in obtaining aspiration material for cytology [5]. Cytology entails the microscopic examination of cells obtained from nipple secretion, cystic fluids or fine needle biopsy of solid nodules, guided by ultrasonography or mammography in the case of non-palpable lesions.

Breast cancer staging using technetium-99m sestamibi and indium-111 pentetreotide single-photon emission tomography

We evaluated the clinical usefulness of single-photon emission tomography (SPET) with technetium-99m sestamibi and indium-111 pentetreotide in breast cancer staging. Fifteen patients with clinical and/or mammographic findings suggesting T1-2N0-1 breast cancer were studied. SPET images were acquired 20 min after99mTc-sestamibi injection and 4 and 24 h after111In-pentetreotide injection. Patients underwent surgery the day after the later111In-pentetreotide acquisition. Pathological examination showed 16 tumours in the 15 patients, with one bilateral carcinoma. The mean tumour diameter was 18.7 mm. Metastatic axillary involvement was found in 6/16 tumours, with a mean of five metastatic nodes per axilla. Both tracers correctly identified 15/16 primary tumours and five of the six cases of metastatic axillary node involvement. No difference between the tracers was observed in breast cancer staging.99mTc-sestamibi seems to be the better tracer in terms of physical characteristics, execution time and cost-effectiveness. Our data suggest the future possibility of using nuclear medicine imaging to avoid axillary dissection in patients with T1 breast cancer.

Nuclear medicine advances in breast cancer imaging.

Primary breast cancer imaging can be done by various means. Mammography is the most widely used technique because of its excellent diagnostic performance, patient compliance, and cost-effectiveness ratio. Other radiological techniques (such as ultrasonography) are indicated in particular circumstances, while some (such as digital mammography and magnetic resonance imaging) seem very promising but are still under evaluation. The recent technological progress in nuclear medicine has resulted in the availability of two diagnostic procedures that have been validated by extensive international clinical experience: scintimammography with Ses-ta-MIBI and positron emission tomography (PET) with fluorodeoxyglucose (FDG). The general advantage of nuclear medicine imaging is that tumor-seeking radiopharmaceuticals accumulate in cancer lesions, which makes scintimammography and PET fundamentally different from the radiological techniques that image the tumor mainly on the basis of morphological alterations. Scintimammography is indicated for the study of breast lesions in patients in whom mammography is non-diagnostic or difficult to interpret; it may be useful also to assess and even predict the response to primary chemotherapy. FDG-PET is increasingly used in oncology and is particularly useful in breast cancer as it gives more accurate information than scintimammography in the evaluation of patients with ambiguous mammographies and in discriminating between viable tumor, fibrotic scar or necrosis following surgery, chemo- or radiotherapy. The FDG uptake in the tumor correlates with the histological grade and potential aggressiveness of breast cancer, which may have prognostic implications. In addition to its usefulness in the study of breast lesions, FDG-PET shows great efficacy in detecting lymph node involvement prior to surgery. Whole-body PET provides information on soft tissue and bone metastases in a single scanning session, and has an important clinical role in detecting recurrent metastatic disease. On the basis of the above-mentioned evidence, nuclear medicine techniques, integrated with radiological techniques, offer an interesting opportunity to improve the diagnostic imaging yield in breast cancer, which will eventually lead to better patient management. This paper reports on the latest developments in this field.

Angiosarcoma of the breast and vascular endothelial growth factor receptor

Background Breast angiosarcoma is rare and often associated with previous breast cancer treatment. The present study aimed to define long-term outcomes in relation to common prognostic factors. The expression of vascular endothelial growth factor receptor was also evaluated, as it may be a potential target for anti-angiogenic therapy. Patients and methods We retrospectively assessed outcomes in relation to age, association with previous breast-conserving treatment for breast cancer, tumor size, and grade in 19 patients without metastases at diagnosis. Vascular endothelial growth factor receptor was also assessed. Results Median follow-up was 33 months (range, 1–121). There were 6 local recurrences and 6 deaths for disease progression. Five-year disease-free survival and overall survival were 53% (95% CI, 20–86%) and 49% (95% CI, 14–84%), respectively. No factor significantly affected survival. Vascular endothelial growth factor receptor was positive in 50% of cases and was more frequent in better differentiated cancer. Conclusions The association of vascular endothelial growth factor receptor with G1/G2 tumors requires further investigations. Our findings suggest that anti-angiogenic treatment in vascular endothelial growth factor receptor-positive cases be considered as a novel therapeutic modality in this rare and aggressive disease. Although information is still incomplete, we propose a multimodal therapeutic approach including surgery, radiotherapy and chemotherapy. Free full text available at www.tumorionline.it

Angiotensin-converting enzyme activity in stools of healthy subjects and patients with celiac disease

Angiotensin-converting enzyme (ACE) is a dipeptidylcarboxypeptidase that occurs in three types of cells: endothelial, epithelial, and neuroepithelial. ACE activity is present in plasma, urine, and vascular endothelium. High levels of ACE are found in the brush border of human small bowel. The aim of this study was to evaluate ACE activity in human stools and to find a correlation with the intestinal loss of epithelial cells. Fifteen healthy subjects (HS) (8 males, 7 females; age range 6–56 years), 20 patients with celiac disease (CD) (11 males, 9 females; age range 15–53 years), and 18 patients with CD in remission after a gluten-free diet (CD-GFD) (8 males, 10 females; age range 14–54 years) were enrolled in the study. The fecal ACE activity was measured in all groups. Fecal samples were kept at −20°C for a subsequent test. In HS, fecal ACE activity was 21.03±16.17 nmol/min/100 g (mean ±sd). In patients with CD with subtotal mucosa atrophy, ACE activity was significantly higher (113 ± 88.94) than in HS and CD on GFD (36.65±23.9). We have demonstrated ACE activity in human stools. ACE activity in stools seems to derive from the microvilli of the intestinal mucosa, thus suggesting the potential usefulness of ACE determination as an index of enterocyte damage.