Giuseppe Fanciulli

Generation of high-quality protein extracts from formalin-fixed, paraffin-embedded tissues

A wealth of information on proteins involved in many aspects of disease is encased within formalin‐fixed paraffin‐embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this “hidden treasure” is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full‐length proteins. Here, we describe an optimised method for extraction of full‐length proteins from FFPE tissues. This method builds on the classical “antigen retrieval” technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3 μg and 86.8 μg of proteins were obtained per 80 mm2 tissue slice of formalin‐fixed paraffin‐embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS‐PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC‐nanoESI‐Q‐TOF MS of FFPE proteins resolved by SDS‐PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported.

Risk Factors Associated with Helicobacter pylori Infection among Children in a Defined Geographic Area

Factors influencing the pattern of Helicobacter pylori infection among children living in adjacent urban and rural areas of northern Sardinia, Italy, were compared. The seroprevalence of H. pylori infection was 22% (625 of 2810 children) in the study population and was significantly higher among children in rural areas (37%) than in urban areas (13%) (odds ratio [OR], 3.8; 95% confidence interval [CI], 3.2-4.7; P<.005). This difference was consistent within each age group. In rural areas, children who had dogs were at greatest risk for H. pylori infection (OR, 1.8; 95% CI, 1.3-2.6; P<.05). No association was seen between H. pylori seropositivity and a history of breast-feeding. Urban children attending day care centers had a higher prevalence of infection (17%) than did those who never attended (12%) (OR, 1.5; 95% CI, 1.1-2.0; P<.05). The epidemiology of H. pylori infection is complex; even within the same geographic area, different factors influence acquisition of H. pylori infection.

Natural history of gastro-entero-pancreatic and thoracic neuroendocrine tumors. Data from a large prospective and retrospective Italian Epidemiological study: THE NET MANAGEMENT STUDY

Background: The few epidemiological data available in literature on neuroendocrine tumors (NET) are mainly based on Registry databases, missing therefore details on their clinical and natural history. Aim: To investigate epidemiology, clinical presentation, and natural history of NET. Design and setting: A large national retrospective survey was conducted in 13 Italian referral centers. Among 1203 NET, 820 originating in the thorax (T-NET), in the gastro-entero-pancreatic tract (GEP-NET) or metastatic NET of unknown primary origin (U-NET) were enrolled in the study. Results: 93% had a sporadic and 7% a multiple endocrine neoplasia type 1 (MEN1)-associated tumor; 63% were GEP-NET, 33% T-NET, 4% U-NET. Pancreas and lung were the commonest primary sites. Poorly differentiated carcinomas were <10%, all sporadic. The incidence of NET had a linear increase from 1990 to 2007 in all the centers. The mean age at diagnosis was 60.0±16.4 yr, significantly anticipated in MEN1 patients (47.7±16.5 yr). Association with cigarette smoking and other non-NET cancer were more prevalent than in the general Italian population. The first symptoms of the disease were related to tumor burden in 46%, endocrine syndrome in 23%, while the diagnosis was fortuity in 29%. Insulin (37%) and serotonin (35%) were the most common hormonal hyper-secretions. An advanced tumor stage was found in 42%, more frequently in the gut and thymus. No differences in the overall survival was observed between T-NET and GEP-NET and between sporadic and MEN1 -associated tumors at 10 yr from diagnosis, while survival probability was dramatically reduced in U-NET. Conclusions: The data obtained from this study furnish relevant information on epidemiology, natural history, and clinico-pathological features of NET, not available from the few published Register studies.

Prevalence of silent celiac disease in patients with autoimmune thyroiditis from Northern Sardinia

Celiac disease (CD) is frequently associated with other autoimmune diseases such as Type 1 diabetes mellitus, autoimmune thyroiditis (AT), and Addison’s disease. The frequency of these associations varies with the populations studied. We conducted this study to ascertain the prevalence of CD in patients with AT from Sardinia, an area with a very high prevalence of CD. To this aim, 297 consecutive patients with AT (as defined by elevated antithyroid antibody levels and a positive ultrasound scan) were studied. Immunoglobulin A and G-class antigliadin antibodies were assayed in serum; if either or both were positive, antiendomysium antibodies were determined. If two markers were positive, serum ferritin, folate, and vitamin B12 levels were measured and jejunal biopsy was suggested. Thirteen out of the 14 patients who showed at least two positive markers consented to jejunal biopsy and all of them showed histological features of CD. The prevalence of CD in AT patients was 4-fold greater than that observed in the general population (4.37 vs 1.06%, p<0.0001). Ferritin was low in 6 and vitamin B12 in 2 out of 13 patients; serum folates were normal in all patients. Molecular typing of HLA class II alleles showed an increased frequency of the extended haplotype DRB1*0301/DQA1*0501/DQB1* 0201. None of our patients had a history of gastrointestinal symptoms. We confirm the increased prevalence of silent CD in patients with AT. Patients with AT ought to be regarded as a highrisk group for CD and should be screened routinely for it; if negative, screening tests should be repeated at regular intervals.

Proteomic analysis of formalin-fixed, paraffin-embedded lung neuroendocrine tumor samples from hospital archives

Hospital tissue repositories host an invaluable supply of diseased samples with matched retrospective clinical information. In this work, a recently optimized method for extracting full-length proteins from formalin-fixed, paraffin-embedded (FFPE) tissues was evaluated on lung neuroendocrine tumor (LNET) samples collected from hospital repositories. LNETs comprise a heterogeneous spectrum of diseases, for which subtype-specific diagnostic markers are lacking. Six archival samples diagnosed as typical carcinoid (TC) or small cell lung carcinoma (SCLC) were subjected to a full-length protein extraction followed by a GeLC-MS/MS analysis, enabling the identification of over 300 distinct proteins per tumor subtype. All identified proteins were categorized through DAVID software, revealing a differential distribution of functional classes, such as those involved in RNA processing, response to oxidative stress and ion homeostasis. Moreover, using spectral counting for protein abundance estimation and beta-binomial test as statistical filter, a list of 28 differentially expressed proteins was generated and submitted to pathway analysis by means of Ingenuity Pathway Analysis software. Differential expression of chromogranin-A (more expressed in TCs) and stathmin (more expressed in SCLCs) was consistently confirmed by immunohistochemistry. Therefore, FFPE hospital archival samples can be successfully subjected to proteomic investigations aimed to biomarker discovery following a GeLC-MS/MS label-free approach.

Gastrointestinal Symptoms and Helicobacter pylori Infection in School-Age Children Residing in Porto Torres, Sardinia, Italy

Background:  Helicobacter pylori infection is typically acquired in childhood, and following the acute event, it is thought that most infections remain asymptomatic. H. pylori has been suggested to protect against diarrhea in childhood.

Is there anything to the reported association between Helicobacter pylori infection and autoimmune thyroiditis

Higher serological prevalence rates of Helicobacter pylori (Hp) infection have been reported in patients with autoimmune thyroiditis (AT), and it has been suggested that monoclonal antibodies against Cag-A positive Hp strains can cross-react with follicular cells of the thyroid gland. We studied the prevalence of AT and thyroid functional status in patients who underwent gastroscopy for dyspeptic symptoms. Patients were tested for TSH, free thyroid hormones, and antithyroglobulin and antithyroperoxidase antibodies (ATPO). Hp positivity was determined using urea breath test (UBT). Serum samples from 302 patients (59.9% women) were evaluated. One hundred ninety-one subjects (63.2%) were Hp-negative, and 111 of 302 (36.8%) were Hp-positive. Forty-three of 191 Hp-negative patients (22.5%; 95% CI, 17.1–29.0%) had an increase of either antibody, compared to 30 of 111 (27.0%; 95% CI, 19.6–36.0%) Hp-positive patients (P = 0.40). Similar results were obtained using positivity for both antibodies (7.3 vs. 7.2%; P = 1) or for ATPO (18.8 vs. 21.6%; P = 0.54). The prevalences of hypothyroidism (4.7 vs. 5.5%) or hyperthyroidism (5.8 vs. 5.5%) were also similar (P = 0.95). Hormonal levels were not different in the two groups (P > 0.22 in all cases). The previously reported association between AT and Hp infection was not observed in our study. Infection by Hp does not appear to increase the risk of AT in individuals with dyspeptic symptoms, and screening for this condition in patients with a positive UBT is not indicated.

Multiple endocrine neoplasia syndrome type 1: institution, management, and data analysis of a nationwide multicenter patient database

ObjectiveThe aim of this study was to integrate European epidemiological data on patients with multiple endocrine neoplasia type 1 by creating an Italian registry of this syndrome, including clinical and genetic characteristics and therapeutic management.MethodsClinical, familial and genetic data of patients with multiple endocrine neoplasia type 1, diagnosed, treated, and followed-up for a mean time of 11.3 years, in 14 Italian referral endocrinological centers, were collected, over a 3-year course (2011–2013), to build a national electronic database.ResultsThe Italian multiple endocrine neoplasia type 1 database includes 475 patients (271 women and 204 men), of whom 383 patients (80.6%) were classified as familial cases (from 136 different pedigrees), and 92 (19.4%) patients were sporadic cases. A MEN1 mutation was identified in 92.6% of familial cases and in 48.9% of sporadic cases. Four hundred thirty-six patients were symptomatic, presenting primary hyperparathyroidism, gastroenteropancreatic neuroendocrine tumors and pituitary tumors in 93, 53, and 41% of cases, respectively. Thirty-nine subjects, belonging to affected pedigrees positive for a MEN1 mutation, were asymptomatic at clinical and biochemical screening. Age at diagnosis of multiple endocrine neoplasia type 1 probands was similar for both familial and simplex cases (mean age 47.2 ± 15.3 years). In familial cases, diagnosis of multiple endocrine neoplasia type 1 in relatives of affected probands was made more than 10 years in advance (mean age at diagnosis 36.5 ± 17.6 years).ConclusionsThe analysis of Italian registry of multiple endocrine neoplasia type 1 patients revealed that clinical features of Italian multiple endocrine neoplasia type 1 patients are similar to those of other western countries, and confirmed that the genetic test allowed multiple endocrine neoplasia type 1 diagnosis 10 years earlier than biochemical or clinical diagnosis.

Application of 2D-DIGE to formalin-fixed diseased tissue samples from hospital repositories: results from four case studies

In the recent past, the potential suitability of fixed samples to 2D‐DIGE studies has been demonstrated on model tissues, but not on “real‐world” archival tissues. Therefore, this study was aimed to assess the quality of the results delivered by 2D‐DIGE on samples retrieved from hospital tissue repositories.

Successful treatment of retrograde ejaculation with the α 1 -adrenergic agonist methoxamine: case study

We treated two patients affected by retrograde ejaculation (RE) with the pure α1-adrenergic agonist methoxamine; the drug was self-administered intramuscularly by the patients 30 min prior to intercourse or masturbation. A previous trial with oral imipramine had been ineffective in both patients. Sperm count increased substantially, particularly in the first patient who had insulin-dependent diabetes and was seeking fertility. In this patient, total ejaculated sperm increased from 22 millions to 488 and 419.5 millions on two different occasions, with good motility; two clinical pregnancies were obtained in the partner of this patient after 3 and 4 months of treatment, respectively. The second patient did not desire fertility. In both patients, no side effects were seen except for slight piloerection; blood pressure values increased slightly, and heart rate was unchanged. We conclude that self-administered methoxamine can be a useful, noninvasive and inexpensive treatment of RE, when oral agents are ineffective.