Giuditta F. Schiavano

Activity of Brassica oleracea Leaf Juice on Foodborne Pathogenic Bacteria

Many vegetables of the Cruciferae family have been found to possess antimicrobial properties against several microorganisms of clinical importance. In this study, we reported the antibacterial effect of Brassica oleracea juice on several food-borne pathogens. The juice was found to be effective in inhibiting the growth of Salmonella Enteritidis, verotoxigenic Escherichia coli O157:H7, E. coli HB producing thermolabile toxin, nontoxigenic E. coli, and Listeria monocytogenes, but not Enterococcus faecalis. All cauliflower cultivars tested suppressed bacterial growth in a dose-dependent manner after 5 h of treatments, and the reduction in the number of viable cells ranged from 1 log with a 10% juice concentration to more than 3 log with a 20% juice concentration. The foodborne bacteria tested were also markedly reduced by isothiocyanates, natural components abundant in the genus Brassica, indicating that glucosinolate-derived isothiocyanates can play a major role in the antimicrobial activity of cauliflower. The antimicrobial effect of juice was reduced in presence of cysteine, suggesting that one mechanism of action of the juice involves blocking bacterial sulfhydryl groups.

Swimming pools and fungi: An environmental epidemiology survey in Italian indoor swimming facilities

Abstract A growing number of people attend swimming facilities for recreational activities, rehabilitative treatments, or sport. Filamentous fungi and yeast can be isolated from contaminated air, water and surfaces and may represent a biological risk for employees and users. Here we investigated the occurrence of mycotic species, in a sample of Italian swimming pools (n = 10). Detection and identification of isolated species were achieved by cultural and morphological methods. Results revealed moderate mycotic titres and a high biodiversity. Penicillium spp., Aspergillus spp., Cladosporium spp. and Alternaria sp., were constantly detected in air and surfaces sampled by the swimming area, while pathogenic yeast Candida albicans was never detected. Fusarium spp. was the most common taxon isolated from surfaces. For one facility, we typed the genotypic profiles and studied, by genetic typing, the spatial and temporal distribution of isolates. Phylogenetic relationships between species were analysed by alignment of small ribosomal subunit RNA sequences.

Heterodimer-Loaded Erythrocytes as Bioreactors for Slow Delivery of the Antiviral Drug Azidothymidine and the Antimycobacterial Drug Ethambutol

Disseminated infection with Mycobacterium avium complex (MAC) remains the most common serious bacterial infection in patients with advanced AIDS. The organisms that make up this complex are found ubiquitously in the environment, yet rarely cause disseminated disease in nonimmunocompromised human patients; on the contrary, up to 50% of patients with AIDS may ultimately develop the pathology. Hence, therapeutic strategies able to inhibit HIV and Mycobacterium replication are needed. Because of the rapid plasma elimination and toxicity of the most commonly used drugs, daily multiple-drug therapies must often be continued throughout life, frequently causing major side effects and, as a consequence, poor patient compliance. Therefore, alternative strategies that reduce the toxicity of the drugs and allow prolonged application intervals are sorely needed. Since erythrocytes (RBCs) can behave as bioreactors able to convert impermeant prodrugs to membrane-releasable active drugs, new compounds (AZTpEMB, AZTpEMBpAZT, and AZTp2EMB) consisting of both an antiretroviral and an antimicrobial drug were designed and synthesized. Among these, only AZTp2EMB was hydrolyzed by erythrocyte enzymes and could be encapsulated inside RBCs. AZTp2EMB-loaded RBCs slowly released AZT and EMB in culture medium, reducing its concentration by one-half about every 48 hr of incubation at 37 degrees C. Moreover, when AZTp2EMB-loaded erythrocytes were incubated for 6 days in the presence of human macrophages infected with Mycobacterium avium (M. avium) a marked bactericidal effect (>1 log) was observed. Thus, AZTp2EMB-loaded erythrocytes could be used as endogenous bioreactors for AZT and EMB delivery in the treatment of HIV and M. avium infection.

Drug Delivery through Phagocytosis of Red Blood Cells

Human red blood cells, once in circulation, survive for 120 days and are then removed through an immunomediated mechanism by resident macrophages. We have taken advantage of these unique properties to develop a new drug delivery system. In fact, a number of conventional and new drugs can be encapsulated into human erythrocytes by several procedures. Once re-infused in the same donor the circulating red cells behave as a slow drug delivery system. Alternatively, drug-loaded red cells can be modified to increase their phagocytosis by inducing band 3 clustering and opsonization by autologous IgGs and complement (up to C3b). Macrophages recognize and phagocytose such drug-loaded red cells through the Fc and C3b receptors. This drug targeting system permits the administration of membrane-impermeable molecules to macrophages and allows to optimize treatment of human infections by a number of pathogens with a tropism for macrophages. Furthermore, as reviewed in this paper, modified red cells can be conveniently used to deliver peptides, modified proteins, antisense nucleotides, or small organic molecules. The red cell-based drug targeting system has been proved to be effective and safe in preclinical studies. The administration of drugs by autologous red cells has been proved to be effective in humans with more than 600 administrations.

Inhibition of murine AIDS by pro-glutathione (GSH) molecules.

Antioxidant molecules can be used both to replenish the depletion of reduced glutathione (GSH) occurring during HIV infection, and to inhibit HIV replication. The purpose of this work was to assess the efficacy of two pro-GSH molecules able to cross the cell membrane more easily than GSH. We used an experimental animal model consisting of C57BL/6 mice infected with the LP-BM5 viral complex; the treatments were based on the intramuscular administration of I-152, a pro-drug of N-acetylcysteine and S-acetyl-beta-mercaptoethylamine, and S-acetylglutathione, an acetylated GSH derivative. The results show that I-152, at a concentration of 10.7 times lower than GSH, caused a reduction in lymph node and spleen weights of about 55% when compared to infected animals and an inhibition of about 66% in spleen and lymph node virus content. S-acetylglutathione, at half the concentration of GSH, caused a reduction in lymph node weight of about 17% and in spleen and lymph node virus content of about 70% and 30%, respectively. These results show that the administration of pro-GSH molecules may favorably substitute for the use of GSH as such.

Inhibition of Breast Cancer Cell Proliferation and In Vitro Tumorigenesis by a New Red Apple Cultivar

Purpose The aim of this study was to evaluate the antiproliferative activity in breast cancer cells and the inhibition of tumorigenesis in pre-neoplastic cells of a new apple cultivar with reddish pulp, called the Pelingo apple. Methods The antiproliferative activity was evaluated in MCF-7 and MDA-MB-231 human breast cancer cells. The inhibition of tumorigenesis was performed in JB6 promotion-sensitive (P+) cells. Results Results showed that Pelingo apple juice is characterized by a very high polyphenol content and strongly inhibited breast cancer cell proliferation. Its antiproliferative activity was found to be higher than the other five apple juices tested. Pelingo juice induced cell accumulation in the G2/M phase of the cell cycle and autophagy through overexpression of p21, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) activity and an increase in lipidated microtubule-associated protein-1 light chain-3 beta (LC3B). Remarkably, Pelingo juice inhibited the 12-o-tetra-decanoyl-phorbol-13-acetate (TPA)-induced tumorigenesis of JB6 P+ cells, suppressing colony formation in semi-solid medium and TPA-induced ERK1/2 phosphorylation. Conclusions Our data indicate that the Pelingo apple is rich in food components that can markedly inhibit in vitro tumorigenesis and growth of human breast cancer cells and could provide natural bioactive non-nutrient compounds, with potential chemopreventive activity.

The potency of acyclovir can be markedly different in different cell types

Acyclovir is an acyclic guanine analog with a considerable activity against herpes simplex viruses. We studied the antiherpetic activity of acyclovir in macrophages and fibroblast cell lines. Utilising a plaque reduction assay we found that acyclovir potently inhibited the HSV-1 replication in macrophages (EC50) = 0.0025 microM) compared to Vero (EC50 = 8.5 microM) and MRC-5 (EC50 = 3.3 microM) cells. The cytotoxicity of acyclovir was not detected at concentrations < or = 20 microM, thus the selective index in macrophages was >8000. This marked difference in antiherpetic activity between macrophages and fibroblasts was not observed with Foscarnet and PMEA. We suggest that this potent antiviral effect of acyclovir is mainly due to a proficient phosphorylation of the drug and/or a favourable dGTP/acyclovir triphosphate ratio in macrophage cells.

New treatment protocol including lympholytic and antiretroviral drugs to inhibit murine AIDS

Summary: Highly active antiretroviral therapy (HAART), although very efficient in reducing viral load to undetectable levels within 2 weeks, does not eradicate HIV‐1 infection and after the suspension of therapy, HIV RNA rebounds to pretherapy levels. This limited efficacy is mainly due to the existence of viral reservoirs such as CD4+ T cells, macrophages, and dendritic cells in which the virus can remain latent. Elimination of these latent reservoirs would be a possible solution to this problem and various efforts are now being directed to this end. With this goal in mind, we investigated a lympholytic drug with known activity against lymphoproliferative malignancies, 2‐fluoro‐ara‐AMP (fludarabine). The murine model of AIDS was used to evaluate the efficacy of alternating administration of fludarabine and azidothymidine (AZT). The aim of this experiment was to eliminate infected cells with fludarabine and protect noninfected cells with AZT. LP‐BM5‐infected mice were treated with two different therapeutic protocols: one group was treated with two alternating 3‐week cycles of fludarabine and AZT (treatment A), whereas the other was treated with three alternating 2‐week cycles of fludarabine and AZT (treatment B); both treatments lasted 12 weeks and the animals in the two groups received the same amount of drug. At different times of infection, disease‐related findings (i.e., splenomegaly, lymphadenopathy, hypergammaglobulinemia, T‐cell and B‐cell spleen cell proliferative index, and phenotypes of peripheral blood lymphocytes) were analyzed and the content of proviral DNA in the lymph nodes was quantified. The results obtained show that treatment B was more effective in inhibiting disease progression than treatment A. In fact, all parameters investigated were almost within control values. These results were also confirmed by the quantification of proviral DNA content in the lymph nodes, which after 12 weeks of treatment A declined by ˜50%, whereas treatment B decreased proviral DNA content by ˜85% with respect to infected/untreated mice. The data obtained suggest that a therapeutic protocol including three cycles rather than two of a lympholytic drug and antiretroviral drugs is more advantageous. The efficacy of the treatment could likely increase if other drugs were used in addition to AZT and more cycles of fludarabine were added. This approach appears to be of potential interest in an HIV‐1 eradication protocol.

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli).

The toxicity of H2O2 in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H2O2 (i.e. mode one killing, which is produced by concentrations of H2O2 lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H2O2 higher than 10 mM). Although the data obtained do not clarify the molecular basis of H2O2 toxicity and/or do not explain the specific function of superoxide ions in H2O2-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H2O2 lethality. A mechanism of H2O2 toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O2-. generating system. This enzyme should be active at low concentrations of H2O2 (less than 5 mM) and high concentrations of the oxidant (greater than 5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H2O2 concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H2O2 in the range 5-10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H2O2 (greater than 10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe2+ since superoxide ions may also reduce trivalent iron to the divalent form.

Molecular detection of Pseudomonas aeruginosa in recreational water.

The aim of this study was the development of a new molecular assay for Pseudomonas aeruginosa identification in recreational water. The method includes bacterial cell concentration through membrane filtration, a short (6 h) culture-enrichment step, DNA extraction and its amplification through a Real-Time PCR assay. The performance of the molecular approach was evaluated on 44 samples of swimming pool water and compared with the reference method UNI EN ISO 16266:2008. Positivity rates of 6% and 74% in pool and inlet water, respectively, with the standard culture method, and of 23% and 74% with the molecular method were found. Statistical analysis indicated “substantial agreement” (Cohens Kappa index: 0.6831) between the two approaches. RAPD typing of P. aeruginosa isolates showed identical fingerprint profiles, indicating their epidemiological correlation. The developed protocol showed very high specificity and a detection limit of 10 genomic units. This technique has the potential to screen large numbers of environmental samples, and could be proposed as part of a self-monitoring plan for recreational facilities, improving surveillance and early warning systems.