In vitro studies of different irradiation conditions for Photodynamic inactivation of Helicobacter pylori.

Helicobacter pylori (HP) infections are considered to be the main cause for chronic gastritis and gastric ulcers, whereby more than half of the world's population is nowadays infected. The increased use of antibiotics is leading to an enhanced resistance. Photodynamic inactivation of bacteria seems to be a potential alternative for antibiotic therapies. In our study we used the photosensitizer Chlorin e6 (Ce6) in combination with red light-emitting diodes to inactivate HP in vitro. Ce6 uptake is determined by spectroscopy. Furthermore diverse experiments of different concentrations in the range of 0-100 µM of the photosensitizer and exposure times up to 300 s are carried out in order to find optimal irradiation parameters (wavelength: 660 nm, power density: 9 mW/cm(2), absorbed dose: up to 2.7 J/cm(2)). The data show a significant reduction after already a few seconds of illumination, even with a low Ce6 concentration in the sub-µM-region. At a concentration of 100 µM a nearly total inactivation (6-log10-reduction) of HP was achieved within 60s of irradiation.

Pharmacokinetic of ALA and h-ALA induced porphyrins in the models Mycobacterium phlei and Mycobacterium smegmatis.

Photodynamic inactivation (PDI) of bacterial strains presents an attractive potential alternative to antibiotic therapies. Success is dependent on the effective accumulation in bacterial cells of photochemical substances called photosensitizers, which are usually porphyrins. It is also important to know the distribution of the photosensitizer in bacteria at the microscopic level. The present results examine the accumulation of photosensitizers by Mycobacterium phlei and Mycobacterium smegmatis, which serve as models for the important pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. The kinetics of porphyrin synthesis after treatment with the precursors ALA and h-ALA were studied. The goal was to describe the biosynthesis and the pharmacokinetics of sensitizers in both bacterial strains using fluorescence microscopy and spectroscopy. We could show that both Mycobacterium strains enrich porphyrins after ALA and h-ALA administration detected by fluorescence peaks at about 620nm. By HPLC analyses the major porphyrin could be identified as coproporphyrin. In the future we will apply the new knowledge in in vitro and in vivo experiments to strains of M. tuberculosis, M. leprae and M. bovis and examine cell destruction by PDI.

Autofluorescence detection of tumors in the human lung--spectroscopical measurements in situ, in an in vivo model and in vitro.

To detect bronchial carcinoma by autofluorescence, we measured the spectra of tumor and normal tissue in situ, in an in vivo model and in vitro by fiber optic spectrometer and two-dimensional resolved microspectroscopy. The in situ measurements were performed in bronchi of nine patients with squamous cell carcinoma during regular bronchoscopy with autofluorescence assistance. The fluorescence was monitored with a fiber optical spectrometer under blue light excitation (lambda=405nm). In an in vivo model, the resected lobe of a lung was perfused under physiological conditions. Tumorous and normal tissues were examined spectroscopically during perfusion and after blood removal and substitution with formol. In another setup the wavelength dependency of autofluorescence was examined on resected parts of physiological bronchi and central bronchial carcinomas. Under the variation of the excitation from 385 to 465nm the autofluorescence response was monitored with a fiber optic spectrometer. For investigation of the origin of autofluorescence, two-dimensional resolved spectroscopy was performed with the SpectraCube system on several sections of tumor and normal tissues All measurements, performed in vivo, in the in vivo model and in vitro agreed, that the main difference of the autofluorescence between tumor and normal bronchus tissue is the intensity of the fluorescences' main peak at 505nm. The signal on tumor tissue is in all cases significantly lower than that of normal tissue. The shape of the autofluorescence peaks is in healthy and carcinoma tissue approximately the same with two characteristic minima at 540 and 580nm. After the preparation with formaldehyde those minima disappeared from the spectra. A comparison with the absorption spectra of hemoglobin showed, that the variation of the spectra may be due to the blood content in the tissue. Two-dimensional spatially resolved spectroscopy showed, that the lower intensity of fluorescence in tumor tissue is due to the irregular and low-concentrated formation of fluorescent structures, which seen to be the elastic structures of bronchial tissue.