Alfredo Di Leo

EGF, TGF-a, and EGF-R in Human Colorectal Adenocarcinoma

: The over-expression of epidermal growth factor receptor (EGF-R), EGF and transforming growth factor alpha (TGFalpha) could be a mechanism for colorectal tumor cells to escape normal growth controls. Our aims were: (i) to evaluate EGF, TGFalpha, and EGF-R concentrations in neoplastic tissue and surrounding mucosa from 40 patients with colorectal adenocarcinoma, and (ii) to assess the expression of these growth factors and their receptor in relation to the tumor site. EGF, TGFalpha, and EGF-R were detected in either colorectal neoplastic tissue or surrounding mucosa. Significantly increased levels of EGF and EGF-R were present in neoplastic samples compared to surrounding mucosa. Furthermore, a significant increase in TGFalpha and in EGF levels was observed in the left-sided surrounding mucosa and left-sided neoplastic tissue, respectively. EGF-R stimulation by its ligands may play an important role in colorectal neoplastic tissue. Moreover, the higher content of growth factors in the left-side than the right-side colon suggests different growth properties in the proximal and distal colon.

Clarithromycin-Resistant Genotypes and Eradication of Helicobacter pylori

Context Point mutations in the peptidyltransferase region of the 23S ribosomal RNA gene may be responsible for Helicobacter pylori clarithromycin resistance. Contribution This study related mutations to eradication rates in 156 adults treated with clarithromycin regimens for H. pylori infection. Eradication was successful in 14 of 15 patients with either A2142G or A2142C point mutations but in only 11 of 23 patients with the A2143G point mutation. Cautions This was a post hoc subgroup study of selected participants in a multicenter randomized trial. Implications The A2143G point mutation may be associated with a low eradication rate of H. pylori infection. The Editors Helicobacter pylori infection plays a major role in peptic ulcer disease, low-grade mucosa-associated lymphoid tissue lymphoma, and gastric cancer (1), and its eradication dramatically affects the natural history of both peptic ulcer and gastric lymphoma (2). European and U.S. guidelines advised the use of triple therapies (proton-pump inhibitor, clarithromycin plus amoxicillin, or metronidazole) for 7 to 14 days to cure this infection (3, 4). However, H. pylori resistance against clarithromycin is increasing worldwide, reducing the success rate of standard triple therapies to mean values as low as 18% to 44% (5-7). Novel culture-free polymerase chain reaction (PCR)based assays have allowed the detection of the genetic mutations that are involved in the mechanisms of clarithromycin resistance (8, 9). In detail, A2143G and A2142G transitions are the most prevalent point mutations in Europe and the United States (10, 11), while the A2144G mutation is more frequent in Asia (12, 13). Although such genetic mutations have been associated with different degrees of bacterial resistance in vitro, data are still conflicting (7, 14). Moreover, no study has assessed the role of these different mutations on H. pylori treatment outcome. In a recent multicenter study, a novel sequential regimen, consisting of a simple dual therapy given for the first 5 days followed by a triple therapy for the remaining 5 days, achieved a very high cure rate as compared with standard triple therapy (92% vs. 74%) (15). Whether such a high cure rate may depend on increased efficacy of the sequential regimen against the clarithromycin-resistant strains is unknown. We wanted to evaluate the role of different point mutations in the success of eradication therapy and to compare the efficacy of standard triple therapy and the sequential regimen for these mutations. Methods Study Design To assess the role of primary clarithromycin resistance in therapeutic outcome, we designed a post hoc subgroup analysis of a previous study involving 8 Italian centers (15). In detail, we selected patients from those who were previously enrolled by our 2 centers to participate in a multicenter study between January and December 2001 (Figure). Demographic and clinical characteristics of patients enrolled in our substudy were similar to those of patients enrolled in the original multicenter study. Briefly, in the original study, Zullo and colleagues (15) allocated patients who were never treated for H. pylori infection, according to a computer-generated randomization list drawn in each center, to receive standard 7-day treatment (20 mg of rabeprazole, 500 mg of clarithromycin, and 1 g of amoxicillin twice daily) or 10-day sequential therapy (20 mg of rabeprazole plus 1 g of amoxicillin twice daily for the first 5 days followed by 20 mg of rabeprazole, 500 mg of clarithromycin, and 500 mg of tinidazole twice daily for the remaining 5 days). To assess H. pylori status, the investigators performed upper endoscopy with several gastric biopsies for histology (Giemsa staining), a rapid urease test, and a standard 13C-urea breath test at entry and at 4 to 6 weeks after therapy. Investigators considered infections to be eradicated when all 3 test results were negative and considered treatment to have failed when at least 1 test result was positive. The local ethics committee approved the protocol, and all participants gave written informed consent. Figure. Flow chart showing data of patients recruited in this substudy from the previous multicenter study. For our current study, we selected 75 of 192 patients who were treated with standard triple therapy and 81 of 185 patients who were treated with the sequential regimen in the 2 participating centers. We recruited patients consecutively from the randomization lists of the previous study, independent of the eradication status. The final study sample included 58 of 140 patients whose infections were eradicated and 17 of 52 patients whose infections were not eradicated after standard triple therapy and 76 of 177 patients whose infections were eradicated and 5 of 8 whose infections were not eradicated after the sequential regimen. Clarithromycin Resistance Assessment We assessed the 3 point mutations (A2142C, A2142G, and A2143G) of clarithromycin resistance by using a validated real-time PCR, as reported elsewhere (16). Briefly, we extracted the DNA by using NucleoSpin Tissue (Macherey-Nagel GmbH & Co., Dren, Germany), according to the manufacturers instructions, applied on paraffin-embedded sections. We applied the same procedure to homogeneous bacterial cultures of H. pylori (positive and negative controls), for which clarithromycin resistance had been previously assessed with Etest (AB BIODISK, Solna, Sweden). We estimated final DNA concentrations by ultraviolet absorbance at 260 nm. Preparation of the Probes and Primers We designed TaqMan minor groove binder (MGB) probes and primers to hybridize with wild-type and mutant DNA by using the Primer Express program and Custom TaqMan SNP Genotyping Assay service (Applied Biosystems, Foster City, California) that synthesized the primers and probes for each mutation. Genotyping Assay The assay reagents for the genotyping single nucleotide mutation from the Assays-by-Design service (Applied Biosystems) consisted of a 40X mix of unlabeled PCR primers and TaqMan MGB probes (FAM and VIC fluorochrome dyelabeled). These assays were designed for the genotyping of specific mutations. Each assay enables scoring of both genotypes in a single well. Since a recent study showed that the conjugation of MGB to oligonucleotides stabilizes nucleic acid duplexes, causing a dramatic increase in oligonucleotide melting temperature (17, 18), we used an attachment of the MGB, which enables the use of shorter fluorogenic probes, thus resulting in improved mismatch discrimination. Our probes were distinguished by being labeled with different fluorescent reporter dyes (FAM dye and VIC dye). A substantial increase in FAM or VIC dye fluorescence indicated homozygosity for the FAM- or VIC-specific allele, while an increase in both signals indicated heterozygosity (19). Real-Time PCR Assay and Allelic Discrimination We performed the real-time PCR procedure according to the method of Wada and colleagues (20). We enclosed positive and negative controls in each assay. We analyzed fluorescence of hybridized probes by multicomponent graphics, where we examined dye-labeled (FAM and VIC), background, and passive control (ROX fluorochrome dye-labeled) fluorescence and expressed them as normalized reporter signal (Rn). We clustered all samples by using the maximum likelihood algorithm based on the ratio of normalized reporter dye signal. The result of the analysis yields 3 major clusters corresponding to the 3 genotypic constituents: wild-type homozygous, mutated-type homozygous, and heterozygous. Characterization of Positive and Negative Controls by Amplification and Sequencing of the Hp23S Fragment We obtained the Hp23S fragment by PCR amplification of H. pylori extracted DNA from homogeneous bacterial cultures (strains with and without clarithromycin resistance, previously assessed by Etest) by using primer Hp23-F (5-CCACAGCGAT GTG GTCTCAG-3) and Hp23-R (5-CTCCATAAGAGCCAAAGCCC-3) according to conventional PCR assay (21). Before sequencing, we purified the PCR products by using the Wizard PCR preps (Promega, Madison, Wisconsin). We performed the sequencing reaction with the same primers for PCR, as described by Sanger and colleagues (22), by using the Dye Terminator 3.1 Ready Reaction Kit (Applied Biosystems) as indicated by the manufacturer. We performed sequencing on the 2 strands of each PCR product with the automated ABI Prism 377 DNA Sequencer (Applied Biosystems) and aligned the resulting nucleotide sequence by using the Sequence Navigator software package (Applied Biosystems). Statistical Analysis We determined sample size before the start of the study on the basis of the available data in the literature. In detail, an eradication rate ranging from 18% to 44% was reported after standard triple therapy in patients with primary clarithromycin-resistant strains (5-7), whereas the sequential regimen eradicated the infection in 79% of such patients (15). Assuming a high eradication rate for the triple therapy (45%) and a relatively poor success rate for the sequential regimen (70%) in patients with primary clarithromycin-resistant strains, we calculated that at least 68 patients per group were needed to detect a statistically significant difference with 0.8 power and an level of 0.05 (2-sided). After the study was completed, we realized that our sample size estimate provided the necessary number of clarithromycin-resistant patients and should have been inflated, on the basis of a presumed overall rate of clarithromycin resistance, to provide an estimate of total sample size. We compared eradication rates by H. pylori clarithromycin-resistant strain mutation (A2142C, A2142G, and A2143G) by using the Fisher exact test or chi-square test, as appropriate. We determined point mutation groupings after reviewing eradication rates by individual mutation. We compared clinical characteristics among the different groups by using the Student t-test for unpaired da

The influence of Lactobacillus brevis on ornithine decarboxylase activity and polyamine profiles in Helicobacter pylori-infected gastric mucosa.

Background.  Functional probiotics may prevent Helicobacter pylori infection, and some evidence suggests that they also possess antitumor properties. Lactobacillus brevis (CD2) is a functional Lactobacillus strain with peculiar biochemical features, essentially related to the activity of arginine deiminase. This enzyme catalyzes the catabolism of arginine and affects the biosynthesis of polyamines (putrescine, spermidine, and spermine). Polyamines are polycations found in high concentrations in both normal and neoplastic cells. Our aims were: 1, to assess whether oral administration of L. brevis (CD2) affects H. pylori survival in the human gastric mucosa; 2, to evaluate the effects of L. brevis (CD2) on polyamine biosynthesis in gastric biopsies from H. pylori‐positive patients.

Early Menopause Is Associated With Lack of Response to Antiviral Therapy in Women With Chronic Hepatitis C

BACKGROUND & AIMS Chronic hepatitis C (CHC) and liver fibrosis progress more rapidly in men and menopausal women than in women of reproductive age. We investigated the associations among menopause, sustained virologic response (SVR), and liver damage in patients with CHC. METHODS We performed a prospective study of 1000 consecutive, treatment-naïve patients 18 years of age and older with compensated liver disease from CHC. Liver biopsy samples were analyzed (for fibrosis, inflammation, and steatosis) before patients received standard antiviral therapy. From women (n = 442), we collected data on the presence, type, and timing of menopause; associated hormone and metabolic features; serum levels of interleukin-6; and hepatic tumor necrosis factor (TNF)-α. RESULTS Postmenopausal women achieved SVRs less frequently than women of reproductive age (46.0% vs 67.5%; P < .0001) but as frequently as men (51.1%; P = .283). By multivariate regression analysis, independent significant predictors for women to not achieve an SVR were early menopause (odds ratio [OR], 8.055; 95% confidence interval [CI], 1.834-25.350), levels of γ-glutamyl transpeptidase (OR, 2.165; 95% CI, 1.364-3.436), infection with hepatitis C virus genotype 1 or 4 (OR, 3.861; 95% CI, 2.433-6.134), and cholesterol levels (OR, 0.985; 95% CI, 0.971-0.998). Early menopause was the only independent factor that predicted lack of an SVR among women with genotype 1 hepatitis C virus infection (OR, 3.933; 95% CI, 1.274-12.142). Baseline levels of liver inflammation, fibrosis, steatosis, serum interleukin-6 (P = .04), and hepatic TNF-α (P = .007) were significantly higher among postmenopausal women than women of reproductive age. CONCLUSIONS Among women with CHC, early menopause was associated with a low likelihood of SVR, probably because of inflammatory factors that change at menopause.

Epidemiology of hepatitis C Virus infection in an area of Southern Italy

BACKGROUND/AIMS Hepatitis C virus (HCV) has been recognized as a major cause of liver disease, but little is known about its diffusion at population level. To estimate the prevalence and incidence of HCV infection and to explore potential risk factors at population level, an epidemiologic study was carried out. METHODS A cohort was built up in 1985, on a random sample of the population of Castellana, a small town in southern Italy (Bari province), and followed up until 1993. HCV ELISA II and RIBA HCV 2.0 were used as screening and confirmatory tests, respectively. RESULTS The overall anti-HCV prevalence was 26.0% (511/1969) at enrollment. The HCV infection incidence rate was 34.2x100,000 person-years (3 cases/8766 persons-years). A secular trend (referent born before 1930; born 1930-39 Odds Ratio (OR) 0.72, 95% Confidence Interval (95% CI) 0.56-0.94; born 1940-49, OR 0.33, 95% CI 0.25-0.44; born 1950 or after, OR 0.15, 95% CI 0.09-0.23) and geographical pattern (referent born outside Bari province; born in Bari province, OR 1.71, 95% CI 0.93-3.16; born in Castellana G, OR 2.29, 95% CI 1.29-4.05) were found by logistic regression analysis after controlling for several confounding factors. CONCLUSIONS The high prevalence, moderate incidence, and marked decrease in HCV infection in the cohort of birth in a population without known risk factors suggest that an epidemiological transition has been operating at population level since the 1950s.

Regenerating Rat Liver: Correlations Between Estrogen Receptor Localization and Deoxyribonucleic Acid Synthesis

Estrogen receptor activity was quantitated in the cytosol and nucleus of normal rat liver and in regenerating rat liver at several time intervals after 75% hepatectomy. Cytosolic estradiol binding in regenerating liver decreases at 12, 24, and 48 h after hepatectomy and at 48 h is 30% of that in normal rat liver. Nuclear estrogen binding 48 h after surgery is elevated fivefold over normal values. No alterations in affinity of the receptor for estrogen have been observed. Specificity studies indicate that the estrogen receptors from both normal and regenerating liver were similar and are highly specific for estrogens. These changes in cellular distribution of receptors parallel increases in nuclear deoxyribonucleic acid synthesis and mitotic indices in the liver.

Circulating cytokines and gastrin levels in asymptomatic subjects infected by Helicobacter pylori (H. pylori).

The pathophysiology of hypergastrinemia in H. pylori infection has been largely investigated and different reports clearly show that the infected antrum has a marked inflammatory response with a suggestive local production of cytokines. Notwithstanding, a few data are available on the circulating levels of cytokines and gastrin in the asymptomatic people carrying H. pylori infection. Thus, aim of the study was to evaluate circulating proinflammatory cytokines [Interleukin (IL)-8, Interleukin (IL)-10, Interferon (IFN)-γ, and Tumor Necrosis Factor (TNF)-α] and gastrin levels in H. pylori positive asymptomatic subjects vs. H. pylori negative ones. To this end, thirty healthy volunteers with no digestive symptoms or systemic disease were enrolled and H. pylori infection was identified by a 13C-urea breath test. Plasma levels of gastrin were determined using the RIA kit whereas IL-8, TNF-α, IL-10, and IFN-γ levels in serum were measured with a solid-phase ELISA. Fifteen infected people showed significantly higher gastrin and TNF-α levels than uninfected subjects. On the contrary, IL-8 levels were significantly higher in the uninfected subjects than in H. pylori positive ones (P < 0.0422). IFN-γ and IL-10 circulating levels were not affected by H. pylori presence, being not significantly different in the two groups.

Reproductive status is associated with the severity of fibrosis in women with hepatitis C.

Introduction Chronic hepatitis C is the main cause of death in patients with end-stage liver disease. Prognosis depends on the increase of fibrosis, whose progression is twice as rapid in men as in women. Aim of the study was to evaluate the effects of reproductive stage on fibrosis severity in women and to compare these findings with age-matched men. Materials and Methods A retrospective study of 710 consecutive patients with biopsy-proven chronic hepatitis C was conducted, using data from a clinical database of two tertiary Italian care centers. Four age-matched groups of men served as controls. Data about demographics, biochemistry, liver biopsy and ultrasonography were analyzed. Contributing factors were assessed by multivariate logistic regression analysis. Results Liver fibrosis was more advanced in the early menopausal than in the fully reproductive (P<0.0001) or premenopausal (P = 0.042) group. Late menopausal women had higher liver fibrosis compared with the other groups (fully reproductive, P<0.0001; premenopausal, P = <0.0001; early menopausal, P = 0.052). Multivariate analyses showed that male sex was independently associated with more severe fibrosis in the groups corresponding to premenopausal (P = 0.048) and early menopausal (P = 0.004) but not late menopausal pairs. In women, estradiol/testosterone ratio decreased markedly in early (vs. reproductive age: P = 0.002 and vs. premenopausal: P<0.0001) and late menopause (vs. reproductive age: P = 0.001; vs. premenopausal: P<0.0001). In men age-matched with menopausal women, estradiol/testosterone ratio instead increased (reproductive age group vs. early: P = 0.002 and vs. late M: P = 0.001). Conclusions The severity of fibrosis in women worsens in parallel with increasing estrogen deprivation and estradiol/testosterone ratio decrease. Our data provide evidence why fibrosis progression is discontinuous in women and more linear and severe in men, in whom aging-associated estradiol/testosterone ratio increase occurs too late to noticeably influence the inflammatory process leading to fibrosis.

Estrogens, phytoestrogens and colorectal neoproliferative lesions

Epidemiological and experimental studies suggest a protective role of estrogens against colorectal cancer. This effect seems to be mediated by their binding to estrogen receptor beta (ER-β), one of the two estrogen receptors with high affinity for these hormones. Very recently, the demonstration of an involvement of ER-β in the development of adenomatous polyps of the colon has also been documented, suggesting the use of selective ER-β agonists in primary colorectal cancer prevention. Phytoestrogens are plant-derived compounds that structurally and functionally act as estrogen-agonists in mammals. They are characterized by a higher binding affinity to ER-β as compared to estrogen receptor alpha (ER-α), the other estrogen receptor subtype. These biological characteristics explain why the administration of phytoestrogens does not produce the classical side effects associated to estrogen administration (cerebro- and cardio-vascular accidents, higher incidence of endometrial and breast cancer) and makes these substances ideal candidates for the prevention of colorectal cancer.

Serum fructosamine and colorectal adenomas.

The relationship of glucose in the blood with colorectal adenoma or cancer is not clear. Fructosamine, equivalent to total serum glycated proteins, is a marker of blood glucose levels in the previous 3 weeks. We evaluated in a case–control study the association between fructosamine and colorectal adenoma, a precursor of colorectal cancer. Cases were subjects with the first occurrence of one or more histologically confirmed colorectal adenomatous polyps removed after a complete colonoscopy (153 cases), and controls were subjects with normal colonoscopy performed in the same endoscopy units during the same period (84 controls). Serum fructosamine was measured by a colorimetric method. Unconditional multiple logistic regression was used for statistical analysis. We found that in non-diabetic subjects the risk of colorectal adenoma increased with the level of fructosamine, and the odds ratio of colorectal adenoma in subjects with fructosamine levels higher than the median (270μg/100 ml), in comparison with subjects with fructosamine lower than the median, was 2.3 (95% CI: 1.1–4.8). The risk of colorectal adenoma increased also with increasing levels of serum triglycerides and cholesterol, and decreased with increasing levels of fasting serum insulin. The results of this study show that the risk of colorectal adenoma increases with the level of fructosamine, an indicator of the level of glucose in the blood more sensitive to foods with a high glycemic index.