Francesco Dubini

Degranulation of Paneth Cells via Toll-Like Receptor 9

The release of antimicrobial peptides and growth factors by Paneth cells is thought to play an important role in protecting the small intestine, but the mechanisms involved have remained obscure. Immunohistochemistry and immunofluorescence showed that Paneth cells express Toll-like receptor 9 (TLR9) in the granules. Injection of mice with oligonucleotides containing CpG sequence (CpG-ODNs) led to a down-modulation of TLR9 and a striking decrease in the number of large secretory granules, consistent with degranulation. Moreover CpG-ODN treatment increased resistance to oral challenge with virulent Salmonella typhimurium. Moreover, our findings demonstrate a sentinel role for Paneth cells through TLR9.

Helicobacter pylori: ureA, cagA and vacA expression during conversion to the coccoid form.

As viability of coccoid forms of Helicobacter pylori can only be verified by demonstrating the integrity of the DNA and active protein synthesis, we analysed the expression of ureA, cagA, vacA genes after prolonged incubation in a liquid medium. Exponentially growing and ageing phase cultures were used. Our results showed that, although the coccoid forms had decreased DNA and RNA levels after 31 days, they were not degraded and still expressed the urease, cytotoxic island and vacuolating toxin genes. Coccoid forms are therefore viable and may act as a transmissible agent that plays a crucial role in disease relapses after antibiotic therapy.

Helicobacter pylori: cultivability and antibiotic susceptibility of coccoid forms

Helicobacter pylori is an actively dividing helical bacterium that changes to coccoid morphology as the culture ages. It has been suggested that the coccoid forms may be involved in transmission of infection and in relapses following antimicrobial therapy. The aim of this investigation was to determine the survival and susceptibility of the coccoid forms to amoxycillin, erythromycin, gentamicin and metronidazole. Colony counts and microscopic examination were performed after 1-4 weeks of culture. At 2 and 4 weeks, identical cultures were treated with the antibiotics for 24 h. Our results showed that 4-week cultures of coccoid forms were cultivable after antibiotic treatment.

A new furoxan NO-donor rabeprazole derivative and related compounds.

The design of hybrid molecules by combining appropriate pharmacophoric groups with NO-releasing moieties is a promising approach to the production of new drugs with interesting potential for treating a variety of diseases. Our research group has been active in this field and we have designed several such products. These compounds include NO-donor nonsteroidal antiinflammatory drugs (NSAIDs) 5] and NO-donor H2-receptor antagonists. We pursued this line because nitric oxide (NO.) has protective effects on the gastric mucosa through a number of mechanisms, such as promotion of mucus secretion, increased mucosal blood flow and decreased adherence of neutrophils to the gastric vascular endothelium. In confirmation of this idea, we herein describe the new hybrid 12, obtained by joining the 4-alkoxy-3-phenylsulfonylfuroxan substructure to rabeprazole (1; Scheme 1). It is known that furoxans are able to release NO at physiological pH, in the presence of thiol cofactors. The mechanism of this release appears to be complex and may contributed to the dramatic T1 relaxivity changes. Gd±DTPA alone did not provide any meaningful enhancement of the signal, even when a 600-fold excess was used in the experiment. In conclusion, we have developed new types of imaging probes that have the potential to be used for in vivo imaging of blood coagulation. The specific peptide derived from 2AP labeled with various reporter groups can be covalently attached to fibrin by blood coagulation factor FX13 through transglutamination. Theoretically, the same approach could be applied to other transglutaminases, which are widely found in generic tissue stabilization and also contribute to a variety of diseases, such as cancer, neurodegenerative diseases, and celiac disease. 18]

Antimicrobial Susceptibility Testing of Helicobacter pylori Determined by Microdilution Method Using a New Medium

Antibiotic susceptibility testing of Helicobacter pylori isolates was performed by broth microdilution method with MegaCellTM RPMI-1640 Medium (SIGMA). Fifty five clinical isolates of H. pylori were tested against metronidazole, tinidazole, amoxicillin, and clarithromycin. The results were compared to those obtained by standard agar dilution method. The microdilution method performed with new medium, showed excellent correlation with agar dilution results, with 100% agreement for metronidazole, 96.3% for amoxicillin, 90.7% for clarithromycin, and 92.8% for tinidazole. MICs determined by proposed method were highly reproducible: replicate results were variable within one-two-fold dilution by using different inocula and different batches of medium.

Anti-Helicobacter pylori agents endowed with H2-antagonist properties

New anti-Helicobacter pylori (H. pylori) agents endowed with H2-antagonists properties were obtained by combining the lamtidine derived pharmacophoric group with the antibiotic calvatic acid. All the compounds were tested for their irreversible H2-antagonist properties and for their ability to inhibit 20 H. pylori strains, two of them metronidazole resistant. The most active derivative (compound 4) displayed antimicrobial activity similar to metronidazole.

Susceptibility of Helicobacter pylori and Pharmacokinetic Properties in Mice of New 5-Nitroimidazole Derivatives Devoid of Mutagenic Activity in the Ames Test

The minimum inhibitory concentrations of metronidazole and four new 5-nitroimidazole derivatives (EU 11100, EU 11102, EU 11103, EU 11104), obtained by the reaction of 1-methyl-5-nitroimidazolyl-2-carboxyaldehyde and terbutyl-phenol, were determined against 25 clinical isolates of Helicobacter pylori. Three of them (EU 11100, EU 11103, EU 11104) exhibited an antibacterial activity higher than that of metronidazole. The last one, the molecule EU 11102, was less active than metronidazole. In mice, after a single equimolar oral administration, the molecules EU 11100 and 11103 were poorly absorbed and poorly excreted in urine. The molecular EU 11104 was well adsorbed and its urinary recovery was slightly lower than that of metronidazole. The substance EU 11102 was not demonstrable in blood and urine. In the Salmonella/microsome mutagenicity test only the molecule EU 11100 showed an increase of mutation frequency in S. typhimurium TA 100.

‘In vitro’ development of metronidazole, erythromycin, amoxicillin and gentamicin resistance in Helicobacter pylori

Serial passage of 37 Helicobacter pylori clinical isolates on increasing concentrations of metronidazole rapidly produced five strains with MICs up to 512 fold higher than those for the original strains. For these five metronidazole-resistant strains the MICs of erythromycin, gentamicin and amoxicillin were unchanged. When they were submitted to the same technique for these last antimicrobial agents, only one strain developed high level resistance to erythromycin and gentamicin having MIC values respectively up to 32 and 64-fold increased. Finally, no amoxicillin-resistant Helicobacter pylori could be obtained.

Antifungal activity of two benzofuran-imidazoles in different experimental conditions.

A number of experiments was performed in order to analyze the in vitro activity of two new benzofuran-imidazoles, IM/B/4-62 and IM/B/4-66. Studies included the determination of minimum inhibitory concentrations (MICs) on three culture media, at different pH values and at different inoculum sizes. Furthermore, the killing activity and induced resistance were determined. In all the experiments econazole, clotrimazole and bifonazole were the reference compounds. The best MIC values of the two new imidazoles were observed on modified Sabourauds medium, at neutral pH and an inoculum size of 10(4) cells/ml. The two substances showed killing activity and no resistance was observed. On the whole, the more favorable results were obtained with the compound IM/B/4-66.