Natale Figura

Detection in an enzyme immunoassay of an immune response to a recombinant fragment of the 128 kilodalton protein (CagA) ofHelicobacter pylori

The possibility of using a recombinant fragment of the CagA (128 kDa protein) for the diagnosis ofHelicobacter pylori infection was evaluated. Following cloning of the gene coding for the CagA, a recombinant fragment of it was expressed inEscherichia coli, purified and used in Western blot and an EIA to screen sera from 82 patients with gastroduodenal disease who underwent endoscopic examination. In Western blot, good correlation was found between the serological data obtained with the recombinant antigen and those obtained using non-purified extracts ofHelicobacter pylori. The EIA using the antigen showed a sensitivity of 96.2 % and a specificity of 96.6 % compared with Western blot. These data indicate that the recombinant protein is a reliable antigen for detection of infections withHelicobacter pylori strains that are associated with disease. The EIA assay described may be used in follow-up of the progression of the illness and the results of therapy.

The presence of bacteria species in semen and sperm quality

PurposeTo verify the prevalence of semen bacterial contamination and whether the contamination could decrease sperm quality.MethodsSpermiogram, semen culture, and sperm transmission electron microscopy (TEM) analysis were performed. TEM data were elaborated using a mathematical formula that calculates a fertility index (FI)—able to define patients as fertile or infertile—and the percentage of sperm apoptosis, immaturity and necrosis. We aligned the amino acid sequence of beta-tubulin with protein of the most frequent species isolated from semen.ResultsPatients were divided according to the contaminating species; in each group, we observed fertile individuals, in whom the semen quality was similar to that of controls and infertile men whose sperm quality was significantly decreased, in terms of motility, FI, apoptosis and necrosis. Partial homology between β-tubulin and bacterial proteins was observed.ConclusionSperm bacterial contamination is quite frequent and could contribute to the deterioration of the sperm quality of infertile men.

Antimicrobial activity against Helicobacter pylori strains and antioxidant properties of blackberry leaves (Rubus ulmifolius) and isolated compounds

Rubus spp. (Rosaceae) provide extracts used in traditional medicine as antimicrobial, anticonvulsant, muscle relaxant and radical scavenging agents. Resistance to antibiotics used to treat Helicobacter pylori infection as well as their poor availability in developing countries prompted us to test the antimicrobial activity of Rubus ulmifolius leaves and isolated polyphenols against two H. pylori strains with different virulence (CagA+ strain 10K and CagA(-) strain G21). The antioxidant activity (TEAC values) of the tested compounds ranged from 4.88 (gallic acid) to 1.60 (kaempferol), whilst the leaf extract gave a value of 0.12. All the isolated polyphenols as well as the leaf extract showed antibacterial activity against both of the H. pylori strains. The minimum bactericidal concentrations (MBCs) of the extract for H. pylori strains G21 and 10K, respectively, were 1200 microg/mL and 1500 microg/mL after 24h of exposure and 134 microg/mL and 270 microg/mL after 48 h exposure. Ellagic acid showed very low MBC values towards both of the H. pylori strains after 48 h (2 microg/mL and 10 microg/mL for strains G21 and 10K, respectively) and kaempferol toward G21 strain (MBC=6 microg/mL). A relationship between antimicrobial activity and antioxidant capacity was found only for H. pylori strain G21 CagA(-) strain.

Helicobacter pylori infection and infertility

Objective To determine (1) the prevalence of Helicobacter pylori infection in male and female patients with reproductive disorders and controls; (2) the presence of anti-H. pylori antibodies in samples of follicular fluid, vaginal secretions and sperm; and (3) the existence of a structural homology between a major spermatozoa protein, tubulin, and H. pylori proteins. Patients and methods Serum samples from 167 patients with infertility and 837 age- and gender-matched controls (blood donors) were examined by enzyme-linked immunosorbent assay (ELISA) and Western blotting to determine the seropositivity for H. pylori infection. The presence of anti-H. pylori antibodies in samples of follicular fluid, vaginal secretions and sperm was determined using the same techniques. The possible cross-reactivity with spermatozoa of anti-H. pylori hyperimmune sera and human antibodies was studied by immunofluorescence. The N-acid homology of human tubulin with the principal H. pylori proteins was assayed by the WU-blastp program available on the Internet. Results The prevalence of infection was significantly higher in patients than controls (49.1%v. 33.5%, P < 0.001). Follicular fluids from infected patients contained specific antibodies in all cases, sperm samples in about 50% of cases, and vaginal secretions in a minority of cases. Sera to H. pylori whole antigens and VacA reacted with the tails and the pericentriolar area of human spermatozoa (which are rich in tubulin); sera to urease and heat-shock protein (Hsp) did not. Follicular fluids with anti-H. pylori antibodies immune reacted with spermatozoa. A linear homology was found between β-tubulin and three H. pylori proteins, flagellin, VacA and CagA. Conclusions H. pylori infection may increase the risk of developing reproductive disorders or worsen the clinical expression of this syndrome.

Effect of trans-resveratrol on induced oxidative stress in human sperm and in rat germinal cells

Resveratrol is a phytoalexin with antioxidant properties. We evaluated resveratrol toxicity in swim-up selected human sperm and in rat spermatocytes and spermatids separated by the STAPUT method. Resveratrol antioxidant activity was tested against lipid peroxidation (LPO) induced by tert-butyl hydroperoxide. LPO was evaluated using the C11-BODIPY(581/591) probe and transmission electron microscopy in samples incubated with and without resveratrol. LD50 for human sperm and rat spermatids was 50 μM; spermatocytes were more sensitive to resveratrol cytotoxicity. Sperm motility increased progressively at 30 μM, 15 μM and 6 μM. 15 μM resveratrol acts against LPO, preserving sperm chromatin and plasma membranes. LPO were more marked in spermatocytes than in spermatids and the effect of resveratrol was more evident in spermatocytes. In this study, the scavenger properties of resveratrol were demonstrated in vitro in human sperm and rat germ cells, thus resveratrol could be added to the media used in assisted reproduction techniques and cryopreservation when oxidative stress is exacerbated.

Extragastric Manifestations of Helicobacter pylori Infection: H. pylori and Extragastric Diseases

The possible role of Helicobacter pylori as a trigger for some extragastric diseases has been largely investigated in the last year. There are, in fact, several studies concerning cardiovascular diseases, neurological disorders, diabetes mellitus, ear and eyes diseases, immunological and hematological disorders, liver and bile tract diseases, gynecological and respiratory tract pathologies. Among them, idiopathic sideropenic anemia and idiopathic thrombocytopenic purpura still remain the extragastric diseases showing the most convincing results. Concerning ischemic heart disease, there are new interesting data playing in favor of the association, even though there are still some open issues to be clarified. For the other diseases, more studies are needed to clarify the reality of the proposed association.

Most Helicobacter pylori-Infected Patients Have Specific Antibodies, and Some Also Have H. pylori Antigens and Genomic Material in Bile: Is It a Risk Factor for Gallstone Formation?

Bile may contain a 130-kDa protein endowed withaminopeptidase activity and the ability to promotecholesterol crystallisation. As > 90% of H. pyloristrains have a similar peptidase activity, and half the isolates express a 110- to 140-kDa antigen, theCagA protein, we investigated a possible associationbetween H. pylori infection and gallstones, and thepresence in bile samples of factors related to H. pylori that could increase cholesterolcrystallization. The prevalence of H. pylori infectionwas 82.1% in 112 patients with gallstones and 80.3% in112 controls (NS). Fifteen bile samples out of 23specimens from infected patients (65.2%) containedanti-CagA antibodies. A ~60-kDa antigen only reactingwith an anti-CagA antibody was found in five bilesamples (21.7%) from 23 infected patients. One bilesample (4.1%) contained ureA and cagA genes of H.pylori. The homology of CagA with the N-terminalsequence of aminopeptidase N was very low. We concludedthat the presence of specific antibody to H. pylori in most bile samples tested and of an H. pyloriputative antigen in a discrete number of cases mayrepresent factors that increase the risk of gallstoneformation.

Inhibition of Helicobacter pylori urease by omeprazole

Objectives To investigate the effect of the proton pump inhibitor omeprazole on the function of purified urease enzyme of Helicobacter pylori and to examine the in vitro activities of omeprazole against urease-positive and -negative Helicobacter strains. Methods Urease was purified chromatographically from H. pylori. The effect of omeprazole on urease enzyme activity was assayed spectrophotometrically after incubation with urea substrate, phenol red indicator and 3mmol/l phosphate buffer. Minimal inhibitory concentrations of omeprazole against H. pylori and other Helicobacter species were determined using an agar dilution assay. Results Omeprazole was found to inhibit the enzyme activity of purified H. pylori urease in a dose-dependent manner. In vitro growth of both urease-positive and mutant urease-negative H. pyloristrains was equally inhibited by omeprazole. Conclusions Whilst omeprazole competitively inhibits the urease enzyme in vitro, urease enzyme inhibition does not account for the in vitro susceptibility of H. pylori to proton pump inhibitors. Urease inhibition by omeprazole in vivo, however, could increase the effectiveness of antibiotics. It may also affect the reliability of the urease-based tests used to assess H. pylori status.

Helicobacter pylori exotoxins and gastroduodenal diseases associated with cytotoxic strain infection.

This paper describes the characteristics of exotoxins produced by Helicobacter pylori, and in particular the vacuolating toxin (VacA) and the cytotoxin‐associated protein (CagA). The possible association between infection by strains of certain phenotypic or genomic types and the seriousness of gastroduodenal diseases is discussed. Helicobacter pylori induces various morphological changes in cells in vitro, but only infection by strains which induce cytovacuolation has been studied at present. In its native form, VacA is a protein aggregate made of subunits with a mass of 95 kDa. In vitro it stimulates a cellular v‐ type ATPase present on the endosomes and creates an acidic environment inside the vacuoles. It also alters in vitro a K(+)‐dependent phosphatase activity and could impair the flux of sodium through the cells. Purified VacA causes ulceration in mice; experimental infection in mice with strains which also express the CagA protein causes gastric erosions, vacuolation and epithelial and stromal polymorphonuclear (PMN) cell infiltration. In vivo vacuolation can be observed in gastric cells from patients infected with type I (VacA‐CagA positive) H. pylori. CagA is a protein of 128‐140 kDa molecular weight, noncytotoxic and highly immunogenic. It is coexpressed in approximately 70% of cytotoxin‐producing strains. In CagA positive strain infection, increased levels of interleukin‐8 (IL‐8) are secreted by the colonized gastric mucosa. Patients infected by cytotoxic strains and/or patients with anti‐CagA antibodies are more likely to have active gastritis, and are more likely to develop peptic ulcer or gastric cancer. The different outcomes of infection could be determined by host factors, diet, or by the age at which infection is acquired.

Prevalence of Helicobacter pylori infection in male patients with osteoporosis and controls.

Cytokines that regulate bone turnover (tumor necrosis factor-α, interleukin-6, etc.) may influence the pathogenesis of skeleton disorders, such as osteoporosis. Since Helicobacter pylori infection increases the systemic levels of inflammatory cytokines, we investigated the possibility that this infection increases the risk of developing osteoporosis and affects the bone metabolism in a group of male patients with osteoporosis. We examined 80 osteoporotic male patients and 160 controls for serum antibodies to H. pylori and the CagA protein and determined, in patients alone, the most important biochemical and instrumental parameters of the disease. Fifty-one patients (63.7%) and 107 controls (66.8%) were seropositive for H. pylori infection (nonsignificant); 30 infected patients (58.8%) and 43 infected controls (40.1%) were positive for anti-CagA antibodies (P = 0.028; OR = 2.13). Levels of estradiol in infected CagA-positive patients were significantly lower than in infected CagA-negative patients (28.5 [SD = 10.18] vs. 39.5 [SD = 14.50] pg/ml; P = 0.002) and uninfected patients (35.2 [SD = 12.7] pg/ml; P = 0.028). Levels of urinary cross-laps(a marker of bone resorption) were increased in patients infected by CagA-positive strains compared to patients infected by CagA-negative strains (282.9 [SD = 103.8] vs. 210.5 [SD = 150.1] μ g/mmol; P = 0.048) and uninfected patients (204.3 [SD = 130.1] μ g/mmol; P = 0.016). Differences among uninfected and infected patients, independent of CagA status, were observed for other markers of bone turnover, but they did not reach statistical significance. Infection by CagA-positive H. pylori strains is more prevalent in men with osteoporosis, who show reduced systemic levels of estrogens and increased bone turnover. H. pylori infection by strains expressing CagA may therefore be considered a risk factor for osteoporisis in men.