Singlet oxygen luminescence kinetics in a heterogeneous environment - identification of the photosensitizer localization in small unilamellar vesicles.

In vivo measurement of singlet oxygen luminescence kinetics is affected by the heterogeneity of biological samples. Even though singlet oxygen luminescence detection is technically getting easier, the analysis of signals from biological samples is still far from quantitative real time surveillance as it is aspired by the community. In this paper small unilamellar vesicles (SUVs) are used for modelling the general behaviour of heterogeneous samples. The geometry of the SUVs can be determined independently using dynamic light scattering. Therefore an accurate theoretical description of the generation, deactivation and diffusion of the singlet oxygen is possible. The theoretical model developed here perfectly fits the experimental results. Thus the location of the singlet oxygen generating a photosensitizer molecule can be exactly determined from the kinetics of the singlet oxygen luminescence. The application of the used theoretical approach thus allows for accurate quantitative measurements in SUVs.

Ultraviolet radiation and nanoparticle induced intracellular free radicals generation measured in human keratinocytes by electron paramagnetic resonance spectroscopy.

BACKGROUND: Several nanoparticle-based formulations used in cosmetics and dermatology are exposed to sunlight once applied to the skin. Therefore, it is important to study possible synergistic effects of nanoparticles and ultraviolet radiation. METHODS: Electron paramagnetic resonance spectroscopy (EPR) was used to detect intracellular free radicals induced by ultraviolet B (UVB) radiation and amorphous silica nanoparticle and to evaluate the influence of nanoparticle surface chemistry on particle cytotoxicity toward HaCaT cells. Uncoated titanium dioxide nanoparticles served as positive control. In addition, particle intracellular uptake, viability, and induction of interleukin-6 were measured. RESULTS: We found that photo-activated titanium dioxide particles induced a significant amount of intracellular free radicals. On the contrary, no intracellular free radicals were generated by the investigated silica nanoparticles in the dark as well as under UVB radiation. However, under UVB exposure, the non-functionalized silica nanoparticles altered the release of IL-6. At the same concentrations, the amino-functionalized silica nanoparticles had no influence on UVB-induced IL-6 release. CONCLUSION: EPR spectroscopy is a useful technique to measure nanoparticle-induced intracellular free radicals. Non-toxic concentrations of silica particles enhanced the toxicity of UVB radiation. This synergistic effect was not mediated by particle-generated free radicals and correlated with particle surface charge and intracellular distribution.

Photoinactivation of Escherichia coli (SURE2) without intracellular uptake of the photosensitizer.

AIM: This study was performed to investigate the possibility to photodynamically inactivate Gram-negative bacteria without intracellular uptake of the photosensitizer. The efficiency of the photodynamic growth inhibition of Escherichia coli (SURE2) was proved in a comparative study of a neutral and a cationic photosensitizer. METHODS AND RESULTS: We used confocal laser scanning microscopy (CLSM) to investigate the uptake of the photosensitizer by the bacteria to show that both chlorin e(6) and TMPyP are not accumulated in the cells. Fluorescence lifetime imaging (FLIM) and phototoxicity experiments were used to investigate the photodynamic inactivation of the Gram-negative bacteria. The phototoxicity experiments were carried out using a white light LED-setup to irradiate the bacterial suspensions. The viability of the bacteria was obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay. For the cationic TMPyP, photodynamic inactivation without intracellular uptake was observed, whereas for chlorin e(6) such behaviour was not found. CONCLUSIONS: It was proven that in general, it is possible to photodynamically inactivate Gram-negative bacteria without photosensitizer accumulation in the bacterial cells. This fact is especially interesting, considering that the development of resistances may be prevented, leaving the active components outside the bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: In a world with bacteria that gain the ability to withstand the effects of antibiotics and are able to transmit on these resistances to the next generation, it becomes necessary to develop new approaches to inhibit the growth of multi-resistant bacteria. The photodynamic inactivation of bacteria is based on a three-component system by which the growth of the bacterial cells is inhibited. The well-directed oxidative damage is initiated by visible light of a certain wavelength, which excites a nontoxic photoactive molecule, called photosensitizer. Its reaction with oxygen causes the generation of cytotoxic species like singlet oxygen, which is highly reactive and causes the inactivation of the growth of bacteria.

Cyclodextrin/chlorophyll a complexes as supramolecular photosensitizers.

The interactions between chlorophyll a, and three cyclodextrins, hydroxypropyl-beta-cyclodextrin heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin, were studied in aqueous solutions by means of absorption, emission and circular dichroism spectroscopy. Nanosecond laser flash photolysis and steady-state singlet oxygen generation experiments were performed to clarify the photoactivity of chlorophyll a in these systems. Moreover the photosensitizing activity of these complexes towards human leukemia T-lymphocytes (Jurkat cells) was tested and compared with that of the free sensitizer, chlorophyll a. The results obtained indicate that each cyclodextrin is able to carry the pigment in monomeric form inside of cells producing singlet oxygen.

Bone marrow transplantation for severe aplastic anemia using a phenotypically HLA-identical, SB-compatible unrelated donor.

A three-year-old boy with severe aplastic anemia (HLA-A1,B8(Bw6), Cw7,DR3, MB2, MT2, SB4/A1,B8 (Bw6), Cw7,DR3,MB2,MT2,SB-) received a bone marrow transplant from a phenotypically HLA-identical, SB-compatible female unrelated donor. This donor was selected from eighteen HLA-A1,-B8,-blood donors after extended serotyping, mixed leukocyte culture testing and secondary proliferation assays with primed lymphocyte typing reagents specific for SB. Although patient cells proliferated well as responders in MLR, their stimulatory capability was greatly impaired. Because the patient had inherited the same serological HLA-D haplotype from each parent, it was concluded that a compatible unrelated donor must be homozygous for the same HLA-D antigens as the patient. This HLA-D homozygosity was demonstrated by the lack of MLR responses of both parents to stimulators from the donor. The SB typing results suggested SB compatibility because both the patient and the donor typed as SB4,-. Following bone marrow transplantation, there was rapid hematopoietic engraftment. The patient developed severe diarrhea caused by graft-versus-host disease of the gastrointestinal tract, which necessitated hyperalimentation. He is currently eighteen months posttransplant with full hematopoietic reconstitution and moderate chronic skin graft-versus-host disease.

Serological and cellular definition of a new HLA-DR associated determinant, MC1, and its association with rheumatoid arthritis.

A new serological HLA-DR associated determinant was defined by a cluster of six B cell alloantisera. This determinant, designated as MC1, was strongly associated with DR1 and DR4 and appeared unrelated to MB, MT, and SB. The antigen frequency of MC1 was 47% in Caucasoids and 43% in Blacks. Mixed leukocyte culture cloning yielded an alloreactive T cell clone with PLT specificity associated with MC1. We have also observed that certain alloreactive clones may also acquire expression of MC1 but not DR4 during long-term culture maintenance in the presence of DR4, MC1-positive feeder cells. Disease association studies showed a significantly increased frequency of MC1 in adult rheumatoid arthritis patients (93.5% vs 47.4% in normals; relative risk 16.1). These findings suggest that MC1 represents a novel HLA-D encoded determinant.

HLA types in American black juvenile diabetics strong associations with Dw3 and Dw4.

The distributions of HLA-A, -B, -C and -D antigens in 38 black American insulin-dependent juvenile diabetics were studied. Antigens A1, A2, B8 and Cw3 were slightly increased, but the corrected probability values were not statistically significant. As determined by mixed lymphocyte culture, the frequency of Dw3 was 89% in the juvenile diabetics and that of Dw4 was 42% in comparison with 14 and 8%, respectively, in the controls. The relative risks for juvenile diabetes were 52 for Dw3 (p = 10(-8) and 9 for Dw4 (p = 10(-6). Dw2 was significantly decreased in the diabetics (p equals 0.008). All of these deviations in A, B, C and D locus specificities have been previously reported by others in white juvenile diabetics. Because there are white genes in the American black gene pool and juvenile diabetes is rare in blacks in western Africa, many cases of juvenile diabetes in American blacks could be the result of genes ultimately derived from the white genes. This hypothesis is supported by the similar HLA associations in juvenile diabetes in the black and white ethnic groups.

[Fecal chymotrypsin concentration in childhood. Normal values, specificity, sensitivity]. / Stuhl-Chymotrypsin-Konzentration im Kindesalter. Normalwerte, Spezifität, Sensitivität.

The determination of fecal chymotrypsin is a suitable noninvasive screening test for the evaluation of exocrine pancreatic function. Using a photometric assay we found a specificity of 87.5% and a sensitivity of 80%. Beyond the first year of life the lowest limit of normal values is 72 micrograms chymotrypsin/g stool. The method is reproducible, and a 72-hour stool collection is unnecessary when several fecal chymotrypsin determinations in spot stool samples are performed.

A linkage study of the HLA region using C-band heteromorphisms.

A linkage study was performed to establish the map distance from the HLA region to the centromere on 6p. In this study, seven families were investigated for the segregation of their HLA types and a polymorphic C-band marker on chromosome 6. Four out of 29 children were found to be recombinants. A map distance of 14 cM was determined. Ninety-five percent confidence limits for the recombination frequency were established (0.012 less than or equal to theta less than or equal to 0.263).