The in vitro Photoinactivation of Helicobacter pylori by a Novel LED-Based Device.

The rise of antibiotic resistance is the main cause for the failure of conventional antibiotic therapy of Helicobacter pylori infection, which is often associated with severe gastric diseases, including gastric cancer. In the last years, alternative non-pharmacological approaches have been considered in the treatment of H. pylori infection. Among these, antimicrobial PhotoDynamic Therapy (aPDT), a light-based treatment able to photoinactivate a wide range of bacteria, viruses, fungal and protozoan parasites, could represent a promising therapeutic strategy. In the case of H. pylori, aPDT can exploit photoactive endogenous porphyrins, such as protoporphyrin IX and coproporphyrin I and III, to induce photokilling, without any other exogenous photosensitizers. With the aim of developing an ingestible LED-based robotic pill for minimally invasive intragastric treatment of H. pylori infection, it is crucial to determine the best illumination parameters to activate the endogenous photosensitizers. In this study the photokilling effect on H. pylori has been evaluated by using a novel LED-based device, designed for testing the appropriate LEDs for the pill and suitable to perform in vitro irradiation experiments. Exposure to visible light induced bacterial photokilling most effectively at 405 nm and 460 nm. Sub-lethal light dose at 405 nm caused morphological changes on bacterial surface indicating the cell wall as one of the main targets of photodamage. For the first time endogenous photosensitizing molecules other than porphyrins, such as flavins, have been suggested to be involved in the 460 nm H. pylori photoinactivation.

Spectroscopic characterization and fluorescence imaging of Helicobacter pylori endogenous porphyrins.

Conventional antimicrobial strategies have become increasingly ineffective due to the rapid emergence of antibiotic resistance among pathogenic bacteria. In order to overcome this problem, antimicrobial PhotoDynamic Therapy (PDT) is considered a promising alternative therapy. PDT has a broad spectrum of action and low mutagenic potential. It is particularly effective when microorganisms present endogenous photosensitizing pigments. Helicobacter pylori (Hp), a pathogen notoriously responsible of severe gastric infections (chronic gastritis, peptic ulcer, MALT lymphoma and gastric adenocarcinoma), produces and accumulates the photosensitizers protoporphyrin IX and coproporphyrin, thus it might be a suitable target of antimicrobial PDT. With the aim to design and develop an ingestible LED-based robotic pill for intragastric phototherapy, so that irradiation can be performed in situ without the use of invasive endoscopic light, photophysical studies on the Hp endogenous photosensitizers were carried out. These studies represent an important prerequisite in order to select the most effective irradiation conditions for Hp eradication. The photophysical characterization of Hp porphyrins, including their spectroscopic features in terms of absorption, steady-state and time-resolved fluorescence, was performed on bacterial extracts as well as within planktonic and biofilm growing Hp cells.

Main photophysical properties of oxyblepharismin.

Oxyblepharismin is the photo-oxidized form of blepharismin, the chromophore responsible for the photophobic response of heterotrich ciliate Blepharisma japonicum, and represents a nice model for the study of photo-transduction. In this work, we focused on the photophysical characterization of OxyBP, in view of highlighting the main features related to excitation and emission. By a combined experimental and computational approach we identified the main features of absorption and fluorescence emission of the molecule in solvents of different properties, identifying the nature of transitions as well as the possible heterogeneity at ground/excited state. The thorough photophysical characterization of OxyBP is meant to provide the starting point for the elucidation of the photo-transduction pathway in vivo.

Kinetics of nuclear phosphorylation (γ-H2AX) in human lymphocytes treated in vitro with UVB, bleomycin and mitomycin C.

After double-strand break induction, formation of γ-H2AX foci due to phosphorylation at Ser-139 of histone H2AX represents an early event of the DNA damage response (DDR). γ-H2AX foci are then rapidly dephosphorylated as signal for the subsequent recruitment of effector proteins. The induction and disappearance of the foci can be, therefore, used to monitor the functioning of the DDR machinery in a cell population exposed to genotoxic stress. Here, we investigated the time-course of γ-H2AX in unstimulated or cultured peripheral lymphocytes in vitro treated with UVB, bleomycin and mitomycin C (MMC). Once the mutagen exposure was performed, cells were harvested at different interval times from 0.5 to 5h. The results show that (i) in 20-h stimulated peripheral lymphocytes, UVB irradiation caused extensive and dose-dependent increases in nuclear phosphorylation, and disappearance of γ-H2AX foci progressed, proportionally to the UV fluence, with increasing the harvesting time; (ii) UVB-exposed G0 cells cultured for 20-h post-irradiation displayed low amounts of DNA phosphorylation, depicting a time-course in which the maximum effect was reached at 0.5h and dephosphorylation started after 1h; (iii) treatment of unstimulated lymphocytes with bleomycin sulphate induced an increase in nuclear phosphorylation of several folds higher than that of untreated cells, depicting kinetics comparable to those observed for UVB-exposed G1 cells; (iv) in stimulated cells, MMC caused a severe and dose-dependent high degree of H2AX phosphorylation together with a very slower kinetic of dephosphorylation with respect to the other experimental treatments. This study confirms the feasibility of the γ-H2AX focus assay as a genotoxic end-point and supports the view that the proposed type of analysis should be introduced in biomonitoring studies of human populations. This could also represent a feasible and useful tool in the screening and diagnosis of precancerous states or very early stages of other diseases.

Biological weighting function of the UV-B-induced impairment of phototaxis in the freshwater ciliate Ophryoglena flava.

We determined the biological weighting function (BWF) of the effect of UV radiation on phototaxis of the freshwater, histophagous ciliate Ophryoglena flava. Dose-effect curves were measured by exposing the cells to 12 different irradiation regimens obtained with two different levels of UV-B radiation and by using six filters with cutoff wavelengths ranging from 280 to 335 nm. The results show that there are significant damages to phototaxis at the doses used and that the effect increases when the cutoff is shifted toward short wavelengths. The data were used to calculate the BWF of phototaxis impairment by applying a nonlinear fit procedure. The BWF thus obtained decays exponentially with increasing wavelength in agreement with similar findings reported in the literature for other systems.