Analysis of DEFB1 regulatory SNPs in cystic fibrosis patients from North-Eastern Italy.

Cystic fibrosis (CF) transmembrane regulator protein (CFTR) gene is undoubtedly the main genetic factor involved in the modulation of CF phenotype. However, other factors such as human defensins and the genes encoding for these antimicrobial peptides have been hypothesized as possible modifiers influencing airways infection in CF patients, but their role in the pathogenesis of lung disease is still debated. Since DEFB1 gene encoding for human beta-defensin 1 displays features such as antimicrobial or chemotactic activity playing a role in inflammation, it has been considered as a possible candidate CF modifier gene. We analysed three single nucleotide polymorphisms (SNPs) in the 5'-untranslated region of the DEFB1 gene (namely g-52G>A, g-44C>G and g-20G>A) in a group of 62 CF patients from North Eastern Italy, and in 130 healthy controls, with the aim of verifying the possible association of these functional SNPs with the pulmonary phenotype of CF patients. DEFB1 SNPs have been genotyped by using Taqman allele-specific fluorescent probes and a real-time PCR platform. No significant differences were found for allele, genotype and haplotype frequencies of DEFB1 g-52G>A, g-44C>G and g-20G>A SNPs in CF patients stratified for Pseudomonas aeruginosa infection, as well as in patients with a severe and mild clinical phenotype or in patients stratified for CFTR genotypes. DEFB1 allele, genotype and haplotype frequencies of CF patients globally considered were similar to those of healthy controls. Our findings are discordant with respect to another recent study performed on CF patients coming from Southern Italy, probably due to different ethnicity of the patients.

Effects of low-dose parathyroid hormone on bone mass, turnover, and ectopic osteoinduction in a rat model for chronic alcohol abuse.

Parathyroid hormone (PTH) is used clinically in osteoporotic patients to increase bone mass by enhancing bone formation. PTH therapy is not uniformly effective at all skeletal sites and "life-style" factors may modulate the skeletal response to PTH. Alcohol may represent one of these factors. Chronic alcohol abuse is associated with osteoporosis and impaired fracture healing. Therefore, the present study investigated the effects of alcohol on the bone anabolic response to a dose of PTH similar to a human therapeutic dose 1) during normal cancellous and cortical bone growth and turnover, and 2) in a model of demineralized allogeneic bone matrix (DABM)-induced osteoinduction. Three-month-old male Sprague Dawley rats were fed a Lieber-DeCarli liquid diet with 35% of the calories derived from ethanol. The controls were pair-fed an alcohol-free isocaloric diet containing maltose-dextran. Following adaptation to the liquid diets, the rats were implanted subcutaneously with DABM cylinders prepared from cortical bone of rats fed normal chow. The rats were subsequently treated daily with PTH (1 microg/kg/d sc, 5 d/week) or vehicle and measurements on bone and DABM implants performed 6 weeks later. Total bone mass was evaluated on the day of necropsy using DXA. Tibiae were processed for histomorphometry. Bone mass and architecture in tibial diaphysis and DABM implants were evaluated by muCT. PTH treatment increased whole body bone mineral content (BMC) and bone mineral density (BMD). The hormone also increased bone formation and bone area/tissue area in the proximal tibial metaphysis. In contrast, PTH treatment had no effect on periosteal bone formation and minimal effects on DABM-induced osteoinduction. Alcohol consumption decreased whole body BMC. Alcohol also decreased cancellous as well as cortical bone formation and bone mass in tibia and impaired DABM-mediated osteoinduction. There was no interaction between PTH treatment and alcohol consumption for any of the endpoints evaluated. Our results indicate that the bone anabolic response to a therapeutic dose of PTH in the rat is largely confined to cancellous bone. In contrast, alcohol consumption inhibits bone formation at all sites. Furthermore, alcohol inhibits osteoinduction and reduces periosteal and cancellous bone formation, irrespective of therapeutic PTH administration. Based on the animal model, our findings suggest that alcohol consumption could impair the beneficial effects of PTH therapy in osteoporosis.

Impaired osteoinduction in a rat model for chronic alcohol abuse.

Alcohol abuse is a risk factor for bone fractures. Following a fracture, alcoholics have a higher risk for impaired fracture healing. However, the specific alcohol-induced defect(s) in bone healing are not known. Alcohol is a potent inhibitor of bone formation during bone growth and turnover. Thus, the purpose of this study was to determine the effects of alcohol consumption on induction of new bone formation. Demineralized allogeneic bone matrix (DABM) cylinders were used to model osteoinduction in a rat model for chronic alcohol abuse. DABM cylinders, prepared from femurs and tibiae of rats fed a normal diet, were implanted into sexually mature male rats adapted to alcohol (ethanol contributed 35% of caloric intake) or control liquid diets. Food intake in the control rats was restricted to match food intake of alcohol-fed animals. The implants were recovered 6 weeks later and analyzed by histology, muCT and chemical analysis. Histological evaluation revealed a robust osteoinductive response, resulting in mature bone ossicle formation, in DABM implants in rats fed the control diet. Alcohol consumption affected bone mass and architecture of the DABM implants but not volumetric density or mineral composition. Specifically, alcohol consumption resulted in significant decreases in DABM-induced bone volume, bone volume/mg original cylinder weight, connectivity density, trabecular number and thickness, ash weight and % ash weight. There were no changes in mineral (ash) density nor in the relative amounts of calcium, magnesium, iron, selenium and zinc (microg/mg ash), indicating that alcohol consumption did not impair mineralization. Taken together, these results show that alcohol abuse resulted in decreased bone formation within the DABM implant. We conclude that reduced osteoinduction may contribute to impaired bone healing in alcoholics.

Prognostic value of circulating KRAS2 gene mutations in colorectal cancer with distant metastases.

While tissue KRAS2 mutations have been extensively investigated, the role of circulating mutant KRAS2 gene in patients with colorectal carcinoma remains obscure. The aim of the present study was to explore the prognostic significance of circulating KRAS2 gene mutational status in subjects undergoing primary treatment for colorectal cancer. Codon 12 KRAS2 mutations were examined in DNA samples extracted from the serum of 86 patients with colorectal cancer and were compared with the KRAS2 status of their primary tumors. Tissue and serum KRAS2 status was compared with other clinicopathological variables (including CEA and CA 19-9 levels) and with cancer-related survival. KRAS2 mutations were found in tissue samples of 28 patients (33%); serum KRAS2 mutations were detected in 10 of them (36%). Serum KRAS2 status was significantly associated with Dukes' stage D (p=0.001) and with preoperative CA 19-9 levels (p=0.01). At multivariate analysis, cancer-related survival was associated with Dukes' stage (p<0.0001), CEA level (p=0.02), and mutant circulating KRAS2 (p=0.01). All 7 stage D patients with serum KRAS2 mutations died of the disease within 24 months of primary treatment; cancer-related survival was significantly better in 9 stage D patients without serum KRAS2 mutations, with 5 patients (56%) alive after 24 months and 1 patient (13%) alive after 44 months. Residual disease after surgery was evident in all 7 stage D patients with mutant circulating KRAS2, and in 5 out of 9 stage D patients without serum mutations. Serum KRAS2 status may impact substantially on the management of stage D colorectal carcinoma, since it appears to cor-relate with prognosis in this patient subgroup.

On the clinical usefulness of the free-to-total prostate-specific antigen ratio.

The free-to-total prostate-specific antigen ratio (F/T PSA) is associated with the presence of prostate cancer and is thus used as an indicator for suspicion of prostate cancer and as a determinant for biopsy. We reviewed a recent retrospective series of 966 consecutive prostate biopsies where F/T PSA was blindly determined and did not influence biopsy indication. We simulated the association of F/T PSA with biopsy outcome and its impact as a biopsy determinant. When adopting an F/T PSA cutoff of 10%, 13%, 16% or 20% among random sextant biopsies in the 4-10 ng/mL total PSA range, the sensitivity was 15%, 37%, 55% and 72% and the specificity 89%, 80%, 64% and 44%, respectively. Using F/T PSA as a biopsy determinant, from 1.7 to 2.6 cancer biopsies would have been delayed to avoid 10 benign biopsies. As this balance is not acceptable, F/T PSA has no role as a biopsy indicator and its clinical use is questionable.

Mass screening for coeliac disease using antihuman transglutaminase antibody assay.

AIMS: To determine coeliac disease prevalence by an anti-transglutaminase antibody assay in a large paediatric population; to evaluate acceptance of the screening programme, dietary compliance, and long term health effects. METHODS: Cross-sectional survey of 3188 schoolchildren (aged 6-12) and prospective follow up of diagnosed cases. Main outcome measures were: prevalence of coeliac disease defined by intestinal biopsy or positivity to both human tissue transglutaminase and anti-endomysium antibodies in HLA DQ2-8 positive subjects; percentage of children whose families accepted screening; dietary compliance as defined by negativity for anti-transglutaminase antibodies; and presence of clinical or laboratory abnormalities at 24 month follow up. RESULTS: The families of 3188/3665 children gave their consent (87%). Thirty biopsy proven coeliacs were identified (prevalence 1:106). Three other children testing positive for both coeliac related autoantibodies and HLA DQ2-8 but refusing biopsy were considered as having coeliac disease (prevalence 1:96). Of 33 cases, 12 had coeliac related symptoms. The 30 biopsy proven coeliacs followed a gluten-free diet. Of 28 subjects completing 18-24 months follow up, 20 (71.4%) were negative for anti-transglutaminase antibodies, while eight were slightly positive; symptoms resolved in all 12 symptomatic children. CONCLUSIONS: Prevalence of coeliac disease is high in Italian schoolchildren. Two thirds of cases were asymptomatic. Acceptance of the programme was good, as was dietary compliance. Given the high prevalence and possible complications of untreated coeliac disease, the availability of a valid screening method, and evidence of willingness to comply with dietary treatment population mass screening deserves careful consideration.

A reliable screening procedure for coeliac disease in clinical practice.

BACKGROUND: The main autoantigen recognized by the sera of patients with coeliac disease (CD) is tissue transglutaminase (tTG). A human-recombinant form of tTG was used to develop an ELISA to measure anti-tTG serum antibodies for the diagnosis of CD. Preliminary retrospective reports suggest that the human tTG-based ELISA could identify coeliac patients missed by the IgA-anti-endomysium antibody test (AEA). Whether the human recombinant tTG ELISA is sufficiently accurate to become the main diagnostic CD tool in everyday clinical practice is unknown. The objective was to determine, in a prospective study, the sensitivity and specificity of an ELISA test based on the use of human tTG compared with AEA, to analyse the discordant cases for HLA DQ2-8 and for clinical and intestinal biopsy characteristics. METHODS: 1106 patients referred to a gastrointestinal outpatient clinic for symptoms attributable to CD, 52 first-degree relatives of CD patients and 200 healthy controls were tested for both anti-human tTG and AEA antibodies. RESULTS: Out of 1158 subjects, 146 were tested positive for anti-tTG antibodies and 140 were biopsy-proven coeliacs. The AEA test identified 126/1158 coeliacs who also tested positive for anti-tTG antibodies. The 14 patients missed by the AEA test carried the typical HLA-DQ for CD; they had normal levels of total serum IgA and had milder pathology than those with both anti-tTG and AEA positivity (P < 0001). CONCLUSIONS: These results prove that human tTG-based ELISA is an excellent diagnostic tool for CD, for mass screening by both the specialist and the general clinic.

Undiagnosed coeliac disease and risk of autoimmune disorders in subjects with Type I diabetes mellitus.

AIMS/HYPOTHESIS: We tested the hypothesis that silent coeliac disease is more frequent than expected in both patients with Type I (insulin-dependent) diabetes mellitus and their first-degree relatives. We evaluated how the presence of other autoimmune disorders in diabetic patients and their first-degree relatives is related to silent, unrecognized coeliac disease. METHODS: Sera from 491 subjects with Type I diabetes, 824 relatives and 4,000 healthy control subjects were screened for anti-endomysial antibodies and all those subjects who tested positive for anti-endomysial antibodies underwent intestinal biopsy. RESULTS: We found that the prevalence of coeliac disease was 5.7 % among the diabetic patients and 1.9 % among the relatives, values significantly higher than those found among the control subjects (p < 0.0001; p < 0.001). The prevalence of autoimmune disorders in diabetic patients with coeliac disease was significantly higher than in subjects with Type I diabetes alone (p < 0.0001). The prevalence of autoimmune disorders in the relatives with coeliac disease was significantly higher than in those who tested negative for anti-endomysial antibodies (p = 0.01). CONCLUSION/INTERPRETATION: This report provides further confirmation of the high prevalence of undiagnosed coeliac disease among diabetic patients and their relatives. This interesting new finding is the increased presence of other autoimmune diseases in these patients, as well as in their relatives with a delayed diagnosis for coeliac disease. Patients newly diagnosed with coeliac disease showed excellent compliance with the gluten-free diet. This should encourage policymakers to consider introducing an easy-to-use screening programme for diabetic patients and their relatives into everyday clinical practice, in order to prevent coeliac-associated symptoms and the onset of additional, more serious auto-immune disorders.

Development of a novel rapid non-invasive screening test for coeliac disease.

BACKGROUND: Coeliac disease is one of the commonest underdiagnosed diseases in general practice. The autoantigen recognised by the sera of patients with coeliac disease has recently been identified as tissue transglutaminase. AIMS: We evaluated a simple non-invasive immunological dot blot assay for coeliac disease, suitable for use by the general physician in the ambulatory setting. The sensitivity and specificity of this dot blot assay based on recognition of recombinant human transglutaminase were compared with those of antiendomysial antibodies and an enzyme linked immunosorbent assay. METHODS: Serum samples were analysed from 64 healthy controls, 58 first degree relatives of coeliacs, 74 diseased controls, and 70 biopsy confirmed untreated patients with coeliac disease. Dot blot assay and enzyme linked immunosorbent assay were performed using recombinant human transglutaminase as antigen. RESULTS: The dot blot assay, which can be performed in 20 minutes, was positive in all 70 untreated coeliacs (sensitivity 100%). Among the three control groups, there were three false positive tests by dot blot (specificity 98%), all belonging to the group of healthy subjects. The antiendomysial antibodies test missed five untreated coeliac patients (sensitivity 93%) and was negative in all three control groups (specificity 100%). The specificity of the immunosorbent assay was 99% for IgA and 98% for IgG, while sensitivity was 93% for IgA, 47% for IgG, and 100% for IgA and IgG combined. CONCLUSIONS: The dot blot assay is highly accurate in detecting untreated subjects with coeliac disease and can be performed in the general physician's medical office during the course of a routine examination. This innovative test is a practical, reliable alternative to both the immunofluorescent based antiendomysial test and immunosorbent assay for detection of transglutaminase antibodies for the diagnosis of coeliac disease.

Human recombinant tissue transglutaminase ELISA: an innovative diagnostic assay for celiac disease.

OBJECTIVE: Tissue transglutaminase is the autoantigen recognized by the sera of celiac patients. An enzyme-linked immunosorbent assay (ELISA) based on guinea-pig tissue transglutaminase was recently used to measure serum tissue transglutaminase antibodies for the diagnosis of celiac disease. We determine the sensitivity and specificity of an ELISA test based on the use of human recombinant transglutaminase, compared with the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies. METHODS: Serum samples were tested from 65 patients with intestinal biopsy proven celiac disease, from 10 patients with Crohn's disease, and from 150 healthy blood donors. RESULTS: Human transglutaminase ELISA identified 64 of 65 celiac patients, whereas the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies identified 58 of 65 and 60 of 65 subjects, respectively. The three tests showed comparable specificity. CONCLUSIONS: These results proved that the human tissue transglutaminase-based ELISA represents a cost-effective strategy for identifying both symptomatic and atypical forms of celiac disease and could mean that intestinal biopsy need no longer be the gold standard for diagnosing this clinical condition. Furthermore, early identification and treatment of patients with celiac disease in an outpatient setting could have significant implications for reducing long-term morbidity and can produce major savings in future health care costs.

Usefulness of screening program for celiac disease in autoimmune thyroiditis.

We determined the prevalence of celiac disease in subjects with autoimmune thyroiditis compared with sick and healthy subjects. The screening was performed with IgA-class endomysium antibody, by indirect immunofluorescence using human umbilical cord as the antigenic substrate. Six of the 172 patients with autoimmune thyroiditis were found to be anti-endomysium positive (3.4%) and five of these underwent intestinal biopsy, which showed total villous atrophy. By contrast, 3 (0.75%) of 396 patients with nongastroenterologic malignancies and 10 (0.25%) of 4000 blood donors were found to have celiac disease. The prevalence of autoimmune diseases was significantly higher in patients with both celiac disease and autoimmune thyroiditis than in patients with autoimmune thyroiditis alone (P = 0.01). This study confirms that celiac disease is increased among patients with autoimmune thyroiditis. We suggest that these patients may benefit from screening for celiac disease so as to eliminate symptoms and limit the risk of developing other autoimmune disorders.

Molecular detection of codon 12 K-RAS mutations in circulating DNA from serum of colorectal cancer patients.

Point mutations of the K-RAS gene at codon 12 are found in about 40% of cases with colorectal cancer. The diagnostic implications of the detection of these mutations and their clinical utility are still unclear. The aim of this study was to test both the feasibility of the detection of the mutated K-RAS gene in serum and its potential role in colorectal cancer detection and monitoring. Codon 12 K-RAS mutations were examined in DNA extracted from the serum of 35 patients with colorectal cancer and were compared with the K-RAS status in the corresponding primary tumor. Molecular detection was performed by the mutant-enriched PCR (ME-PCR) assay, a sensitive method capable of distinguishing a small quantity of mutated DNA in the presence of abundant wild-type DNA. The occurrence of mutations was compared with clinicopathological parameters as well as CEA and CA19.9 serum levels. We found codon 12 K-RAS mutations in the tissue of 13/35 (37%) patients. Serum mutations were detected in 5/13 (38.5%) patients with mutated K-RAS in the tissue. 26/35 (74%) patients showed an identical K-RAS pattern in tissue and serum. No codon 12 K-RAS alterations were found in serum samples of 22 patients with benign gastrointestinal diseases. Elevated serum CEA levels were detected in 16 patients, four of whom also presented serum RAS mutations. Our results confirm that K-RAS mutations can be found in circulating DNA extracted from serum samples of patients with colorectal cancer and show that there is a correspondence between serum and tissue K-RAS patterns.

Screening for coeliac disease in healthy blood donors at two immuno-transfusion centres in north-east Italy.

BACKGROUND AND AIMS: In the past, the reported prevalence of coeliac disease ranged from 1:1000 to 1:4000, whereas recent studies using serological screening methods have found a significantly higher prevalence. The aim of this study was to investigate the prevalence of coeliac disease in healthy blood donors in a North-eastern region of Italy. SUBJECTS: A total of 4000 healthy blood donors were studied from two immunotransfusion centres. METHODS: Serum IgA-antiendomysium antibodies were detected by indirect immunofluorescence using human umbilical cord vein sections, and positive sera were tested also on monkey oesophagus tissue. Intestinal biopsy was performed in all antiendomysium-positive subjects. RESULTS: Ten out of 4000 sera screened were found to be antiendomysium positive on human umbilical cord vein. All positive patients had flat mucosa on intestinal biopsy. Five subjects had coeliac disease-related clinical features (2 had a history of gastrointestinal symptoms, 1 a family history of IDDM, 1 sideropenic anaemia, and 1 IgA deficiency). One of the ten serum, antiendomysium positive on human umbilical cord vein, was found to be negative when tested on monkey oesophagus. CONCLUSIONS: These data confirm the high prevalence of undiagnosed silent coeliac disease in the healthy adult population. This is the first study where umbilical cord was used for screening coeliac disease in a large population. The human umbilical cord vein indirect immunofluorescence test is more specific for villous atrophy than conventional indirect immunofluorescence test on monkey oesophagus and is a reliable screening test for coeliac disease in an apparently healthy population.

Serologic response to Bartonella henselae in patients with cat scratch disease and in sick and healthy children.

Indirect fluorescent antibody assay (IFA) is the most reliable test for detecting antibody to Bartonella henselae in the diagnosis of cat scratch disease (CSD). Recently, an ELISA test has been proposed, but conflicting results are reported. We compared IgG-IFA and IgG-IgM ELISA methods in CSD patients and in healthy children. We also tested ELISA specificity in a large group of healthy controls and in children with lymphoma-associated lymphadenopathy and with pyogenic lymphadenitis. The ELISA procedure was positive in 69/78 patients with CSD (sensitivity 89.6%), in 5/100 healthy children (specificity 95%), in 2/51 patients with non-Hodgkin's lymphoma or pyogenic lymphadenitis (specificity 96%) and in 27/296 blood donors (specificity 91.6%). In 34 patients with CSD, ELISA IgM and IgG responses decreased significantly between time of diagnosis of the disease and recovery. We found significantly higher IgG-ELISA titres in cat-owners, whether blood donors or healthy children, than in non-cat-owners. The IgG-IFA test gave positive results in 69/78 patients with CSD (sensitivity 89.6%) and in 5/62 healthy controls (specificity 92.5%). The ELISA method is a cheap, sensitive method for determining antibody response to Bartonella henselae infection and is also important for evaluating the clinical course of the disease and the efficacy of antibiotic therapy. The high specificity of ELISA in patients with non-Hodgkin's lymphoma will help the clinician to exclude a potentially life-threatening disease associated with lymphadenopathy.