A. Rossolini

Pathogenic Mechanisms of Helicobacter pylori: Production of Cytotoxin

Bacteria associated with mucosal infections of the digestive system generally produce toxins, especially when they cause inflammatory lesions. Illnesses due to thermotolerant campylobacters, to enterohemorrhagic Escherichia coli, and to Clostridium difficileare only some examples. It would be surprising if Helicobacter pylori (HP) did not produce any toxic substances. The difficulty consists in attributing a pathogenic meaning to the toxin, since the range is quite wide of clinical and histological presentation of gastroduodenal inflammatory diseases linked to the presence in the stomach of H. pylori organisms [1]. Johnson and Lior [2] firstly reported the production of heat-labile cytotoxin by 80.6% of 36 HP strains they tested. However, most of our knowledge of the cytotoxigenicity of HP is from Leunk et al. [3] whose work has inspired us in part. They found that about 55% of 201 HP strains isolated in four different parts of the world produced a substance which caused intracellular vacuolization in cells of several lines in vitro, not only in lines generally employed in toxigenicity tests, like Chinese hamster ovary (CHO) cells, Vero cells, and Y-1 cells, but also in human tumoral cells like HeLa, KATO III, and HEp-2, as well as in human embryonic intestinal cells which were the most responsive. They also inferred that the toxin was proteinaceous in nature being heat labile (destroyed at 70 °C for 30 min), protease sensitive, and ammonium-sulfate precipitable. Its molecular weight ought to be higher than 100 kDa since cytotoxic activity could be found only in the retentate of a concentrated broth culture filtrate (CBCF) passed through 100 kDa molecular weight limit ultrafiltration membrane.

MOLECULAR EPIDEMIOLOGY OF NASOPHARYNGEAL CORYNEBACTERIA IN HEALTHY ADULTS FROM AN AREA WHERE DIPHTHERIA VACCINATION HAS BEEN EXTENSIVELY PRACTICED

In addition to conventional biochemical tests, a DNA probe specific for Corynebacterium diphtheriae was used to characterize 53 cystinase-positive and urease-negative corynebacteria strains isolated from pharyngeal and nasal swabs obtained from 515 healthy adults living in an urban area of central Italy. No Corynebacterium diphtheriae strain was found. Six “atypical” strains were isolated, which could not be classified in any of the species so far defined in the Corynebacterium genus. These strains appeared to be biochemically close to Corynebacterium pseudodiphtheriticum and genetically close to Corynebacterium diphtheriae, since their DNAs strongly hybridized, under relatively low stringency conditions, with a Corynebacterium diphtheriae-specific probe and since insertion sequences which are usually found in Corynebacterium diphtheriae genomes were also found to be present in their genomes. No one of these six strains was either toxigenic or susceptible to lysogenization by β-corynephage carrying the tox gene. Therefore, they do not seem to have any epidemiological relevance as possible hosts for β-phages.

Specific antibody patterns over a two-year period after rubella immunization with RA 27/3 live attenuated vaccine.

Screening for rubella antibodies was carried out on 1557 schoolgirls aged 9-20. Of seronegative subjects 70% (442/631) were immunized with RA 27/3 rubella vaccine and some of the vaccinees underwent a serological and clinical follow-up over a two year period. Adverse reactions occurred in 27% of vaccinees, usually 1-2 weeks after immunization; late reactions were never observed. The vaccine-induced seroconversion rate evaluated at 4-5 weeks after immunization was 99.7%. Both one and two years after immunization the seropositivity rate of vaccinees was 100%. The maximum geometric mean antibody titre (GMT) was observed at 4-5 weeks after vaccination and a significant GMT decrease was evident on both the following annual controls. Specific antibody patterns in vaccinees were highly variable and in a small number of subjects a remarkable antibody titre decrease was noticed.

Antibodies to Vacuolating Toxin of Helicobacter pylori in Dyspeptic Patients

Certain Helicobacter pylori (HP) strains are capable of inducing a cytopathic effect in mammalian cells in vitro consisting in the formation of intracytoplasmic vacuoles [1, 2]. In vitro cells exposed to broth culture filtrate (BCF) of vacuolating HP (VHP) strains die more rapidly than do cells exposed to BCF of non-VHP ones and to uninoculated broth. In addition, VHP causes a strong reduction of the proliferation index of Epstein-Bass Virus (EBV) -transformed B lymphocytes [2].

Immunity to diphtheria, six to 15 years after a basic three-dose immunization schedule

The results of a study of the immunity to diphtheria of 283 girls (9-18 years of age) vaccinated at the age of two years with three doses of vaccine, are reported. The rabbit skin test was used to determine the titre of serum diphtheria antitoxin. 55.8% of the subjects were found to be protected (titre greater than or equal to 0.1 IU/ml), 38.9% were only relatively immune (titre greater than or equal to 0.01- less than 0.01 IU/ml), and 5.3% were unprotected (titre less than 0.01 IU/ml). The antitoxin titres showed a tendency to decrease with time. Even so, 6-15 years after vaccination, the percentages of protected and partially protected subjects were still high (95%).

Rubella in teenagers: epidemiology and prophylaxis in Siena, Italy

Six hundred and fifty-three teenagers (aged 11-13 year) living in Siena and its surroundings (Tuscany, Italy) were the sample for serological screening intended to ascertain immunity to rubella. It was found that 324 of the teenagers (49.62%) lacked antibodies and, hence, were unprotected against the infection. Out of the 324 girls, 196 (around 3/5) were vaccinated using live vaccine. Post-vaccinal complications, with clinical signs of rubella infection, were recorded in almost one third of the vaccinees. Virus isolation from the blood was, in every case, not possible after either 10 or 30 days from vaccination. The serological findings, expressed in hemagglutination inhibition antibodies, could be summarized in the following way: (i) antibodies at low titre were found in only eight out of 184 girls (4.35%) ten days after vaccination; (ii) serological conversion was recorded in 187 out of 188 girls (99.47%) 30 days after vaccination; (iii) the titres were moderately high but much lower than those recorded for the natural infection. The results are discussed in the context of their implications for the strategies of rubella vaccination as far as the safety and the effectiveness of the vaccine are concerned, with emphasis on the duration of the protective immunity.

Antibiotic susceptibility of 206Haemophilus influenzae isolates collected from children in central Italy

Susceptibility of 206H. influenzae isolates was evaluated by disk diffusion method for 11 antimicrobial agents. No isolates were found to be resistant to third-generation cephalosporins, amoxi-cillin+clavulanic acid, gentamicin and ciprofloxacin. Four untypable isolates (1.9%) were β-lactamase producing ampicillin-resistant; one of these was also resistant to chloramphenicol. The rate of resistance against rifampin was 0.5 percent.

The 130-kDa Vacuolating Cytotoxin-Associated Protein Is a Component of Cytotoxic Helicobacter pylori Organisms

Helicobacter pylori (HP) is the agent of type B gastritis and a cofactor in the development of peptic ulcer and probably also of gastric cancer. Certain HP strains produce a toxin which induces intracellular vacuolization on mammalian cells in vitro. Proteins associated with vacuolating activity (VA) have molecular weights of 130, 95, and 80 kDa and can be detected broth and in concentrated broth culture filtrates (CBCF) of cytotoxic strains [1].

Affinity and Ion Exchange Chromatography in the Purification and Characterization of Helicobacter pylori Cytotoxin-Associated Proteins

The study of the virulence characteristics of Helicobacter pylori (HP) seeks to contribute to explaining why only few persons infected with HP develop peptic ulcers. Cytotoxicity plays a crucial role in the pathogenetic mechanisms of most infections of the digestive system. Some HP isolates produce one or more substances which induce intracytoplasmic vacuolization in mammahan cells in culture. Several studies have shown that vacuolating activity or cytotoxicity of these isolates is associated with proteins of apparent molecular weights of 130, 95, and 82 kDa in SDS-PAGE [3,4]. This association is based mainly on the presence of these proteins in concentrated broth culture filtrates of cytotoxic strains and their absence in those of noncytotoxic ones. In addition, by immunoblotting with human sera a serological recognition of these proteins is more prevalent among subjects infected with cytotoxic strain than in persons colonized by non-cytotoxic HP [1]. Sera from patients infected with HP are also able to neutralize the vacuolization suggesting that cytotoxin is produced in vivo [5].

Serum Antibodies to the Vacuolating Toxin Produced by Helicobacter pylori

Cytotoxin is one of the candidate factors of virulence of Helicobacter pylori (HP) [1]. Toxigenicity occurs frequently in strains isolated from patients with peptic ulceration [2], and there is serological evidence that cytotoxin production also occurs in vivo. In fact, in immunoblotting tests we saw that all the 31 individuals infected by cytotoxic (CT+) HP strains tested and 14 of 20 patients (70%) infected by noncytotoxic (CT−) organisms had serum immunoglobulin G (IgG) which recognized HP cytotoxin-associated proteins (CAP). IgA to CAP were hardly detected, mostly in patients infected by CT+ strains, while IgM were not detected at all (N. Figura et al., in preparation).