pH-Dependent Changes in the Mechanisms of Transport of Chlorine e6 and Its Derivatives in the Blood.

We studied the effects of medium pH on steady-state distribution of chlorine e6 and its derivatives between the main transport proteins of human blood plasma. The decrease in medium pH from weakly alkaline (pH 7.4) to acid (pH 5.0) was followed by an increase in relative affinity of chlorines to lipoproteins and reduced their affinity to serum albumin. pH-Dependent changes in the parameters of distribution of photosensitizers between the plasma and blood cells was revealed. We discussed the role of charge and polarity degree of photosensitizer molecule in the mechanism of binding to serum albumin. A possible role of changes in hydrogen ion activity in the processes of selective accumulation of chlorines by tumor cells is discussed.

[Evaluation of Photo- and Cytotoxicity of Chlorin e6 Ester Derivatives and Their Liposomal Forms].

Photophysical characteristics and photosensitizing activity of the chlorin e6 dimethyl and trimethyl ester derivatives in various solution and their liposomal forms were studied. It was shown that in lipid vesicles chlorin e6 ester derivatives are predominantly in the monomeric state and possess optimal photophysical properties and high photochemical activity. The rate of redistribution of the chlorin e6 dimethyl ester from lipid vesicle to cells was higher as compared with that one of the chlorin e6 trimethyl ester. The increase of the serum concentration in the incubation medium has a different effect on processes of accumulation of the liposomal forms of the chlorin e6 dimethyl and trimethyl ester derivatives by the cells. Cell culture studies showed that application of liposomal forms of the chlorin e6 dimethyl and trimethyl ester derivatives significantly decreases their cytotoxicity but keeps high cytotoxic effect of photodynamic activity of the chlorin e6 ester derivatives.

[Distribution of porphyrin sensitizers among protein and cellular blood elements]. / Raspredeleniie porfirinovykh sensibilizatorov mezhdu belkovymi i kletochnymi élementami krovi.

The distribution of porphyrin pigments between plasma proteins and blood cells was studied. It was shown that the relative fraction of sensitizer bound by blood cells changed significantly depending on the physicochemical features of pigment molecules. This parameter strongly correlates with porphyrin polarity. Polar watersoluble tetraphenylporphin derivatives, chlorine e6 and hematoporphyrin are bound by plasma proteins only. The decrease in pigment polarity by substitution of polar side groups results in a drastic increase of pigment affinity to blood cells. The binding of extremely apolar pigments by cells in blood occurs for a long period of time, probably as a result of a low rate of pigment redistribution between serum proteins and cellular membrane. The data obtained show that blood cells may be involved into the control of pigment transport and distribution in organism during photodynamic therapy. The parameters of porphyrin distribution between plasma proteins and cells in blood are of certain importance when the pharmacokinetic behavior of various sensitizers is compared.

Effect of FLT3 ligand and granulocyte colony-stimulating factor on expansion and mobilization of facilitating cells and hematopoietic stem cells in mice: kinetics and repopulating potential.

We have previously identified a cellular population in murine bone marrow that facilitates engraftment of highly purified hematopoietic stem cells (HSC) across major histocompatibility complex (MHC) barriers without causing graft-versus-host disease. Here we investigated the effect of flt3 ligand (FL) and granulocyte colony-stimulating factor (G-CSF) on the mobilization of facilitating cells (FC) and HSC into peripheral blood (PB). Mice were injected with FL alone (day 1 to 10), G-CSF alone (day 4 to 10), or both in combination. The number of FC (CD8(+)/alpha betaTCR-/gamma deltaTCR-) and HSC (lineage-/Sca-1(+)/c-kit+) was assessed daily by flow cytometry. Lethally irradiated allogeneic mice were reconstituted with PB mononuclear cells (PBMC). FL and G-CSF showed a highly significant synergy on the mobilization of FC and HSC. The peak efficiency for mobilization of FC (21-fold increase) and HSC (200-fold increase) was reached on day 10. Our data further suggest that the proliferation of FC and HSC induced by FL in addition to the mobilizing effect mediated by G-CSF might be responsible for the observed synergy of both growth factors. Finally, the engraftment potential of PBMC mobilized with FL and G-CSF or FL alone was superior to PBMC obtained from animals treated with G-CSF alone. Experiments comparing the engraftment potential of day 7 and day 10 mobilized PBMC indicate that day 10, during which both FC and HSC reached their maximum, might be the ideal time point for the collection of both populations.

FLT3 ligand induces the generation of functionally active dendritic cells in mice.

FLT3 ligand (FL) is a recently described hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic progenitors. We have investigated the effect of FL on murine hematopoiesis and dendritic cell (DC) generation and accumulation in lymphoid tissues and liver in vivo and in vitro evaluating the morphologic, phenotypic, and functional characteristics of these DC. We have observed extramedullary hematopoiesis in the mouse spleen with all lineages of hematopoietic cells represented after the administration of FL. Injection of FL results in a time-dependent and reversible accumulation of DC in the spleen, bone marrow, lymph nodes, and liver. Both flow cytometry and immunohistochemistry revealed a significant accumulation of DC in these tissues. Results of mixed leukocyte reaction suggested that these cells, isolated from murine bone marrow or spleen, were active as antigen presenting cells. Furthermore, cultivation of splenic and marrow cells with GM-CSF and IL-4 gave rise to large numbers of functionally active mature DC. Thus, the results of this study suggest that FL is a promising growth factor that stimulates the generation of large number of DC and may be a useful cytokine for the immunotherapy of cancer.

Bone marrow-derived dendritic cells serve as potent adjuvants for peptide-based antitumor vaccines.

Dendritic cells (DCs) are considered the most effective antigen-presenting cells (APCs) for primary immune responses. Since presentation of antigens to the immune system by appropriate professional APCs is critical to elicit a strong immune reaction and DCs seem to be quantitatively and functionally defective in the tumor host, DCs hold great promise to improve cancer vaccines. Even though they are found in lymphoid organs, skin and mucosa, the difficulty of generating large numbers of DCs has been a major limitation for their use in vaccine studies. A simple method for obtaining DCs from mouse bone marrow cells cultured in the presence of GM-CSF + interleukin 4 is now available. In four different tumor models, mice injected with DCs grown in GM-CSF plus interleukin 4 and prepulsed with a cytotoxic T lymphocyte-recognized tumor peptide epitope developed a specific cytotoxic T lymphocyte response and were protected against a subsequent tumor challenge with tumor cells expressing the relevant tumor antigen. Moreover, treatment of day 5-14 tumors with peptide-pulsed DCs resulted in sustained tumor regression in five different tumor models. These results suggest that presentation of tumor antigens to the immune system by professional APCs is a promising method to circumvent tumor-mediated immunosuppression and is the basis for ongoing clinical trials of cancer immunotherapy with tumor peptide-pulsed DCs.

Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.

Dendritic cells, the most potent 'professional' antigen-presenting cells, hold promise for improving the immunotherapy of cancer. In three different well-characterized tumour models, naive mice injected with bone marrow-derived dendritic cells prepulsed with tumour-associated peptides previously characterized as being recognized by class I major histocompatibility complex-restricted cytotoxic T lymphocytes, developed a specific T-lymphocyte response and were protected against a subsequent lethal tumour challenge. Moreover, in the C3 sarcoma and the 3LL lung carcinoma murine models, treatment of animals bearing established macroscopic tumours (up to 1 cm2 in size) with tumour peptide-pulsed dendritic cells resulted in sustained tumour regression and tumour-free status in more than 80% of cases. These results support the clinical use of tumour peptide-pulsed dendritic cells as components in developing effective cancer vaccines and therapies.

Photodynamic activity of chlorin e6 and chlorin e6 ethylenediamide in vitro and in vivo.

Several parameters of chlorin e6 and its derivative chlorin e6 ethylenediamide have been investigated as these compound are potential sensitizers for photodynamic therapy. A study carried out to compare the cellular uptake of the pigments indicates that chlorin e6 ethylenediamide possesses an enhanced affinity for tumour cells and cellular membranes. Comparison of the uptake in induced sarcoma shows that chlorin e6 ethylenediamide is a much better tumour localizer than chlorin e6. The efficiency of phototherapy with chlorin e6 ethylenediamide is higher than that with chlorin e6. These data show the influence of the substitution of the carboxyl groups in chlorin e6 by ester and amide groups on the photosensitizing properties of the pigments.

[Photodamage of gram-positive and gram-negative bacterial cells in the presence of chlorin e6 derivatives]. / Fotopovrezhdenie grampolozhitel'nykh i gramotritsatel'nykh bakterial'nykh kletok v prisutstvii proizvodnykh khlorina e6.

The kinetics of photoinduced damage sensitized by chlorine e6 and its derivatives was studied in Escherichia coli and Bacillus subtilis. The effectiveness of E. coli photoinactivation in the presence of chlorines was 100-200 times lower as compared with that of B. subtilis. The structural organisation of bacterial cell walls apparently played an essential role in the penetration of tetrapyrrole pigments into the cell and in their binding.

[Chlorine e6-sensitized photoinhibition of thrombocyte aggregation. Participation of active oxygen forms]. / Sensibilizirovannoe khlorinom e6 fotoingibirovanie agregatsii trombotsitov. Uchastie aktivnykh form kisloroda.

It has been shown that photosensitized action of chlorin e6 (Che6) on platelet-rich plasma leads to platelet aggregation inhibition. Che6-sensitized photoinactivation of platelets was intensified in the presence of singlet oxygen interceptor. The aggregation rate of light-irradiated platelets in the presence of Che6 in D2O buffer was higher than in H2O buffer. The participation of active oxygen forms in the determination of platelet function has been considered.

[The chlorine e6-photosensitized inhibition of human thrombocyte aggregation]. / Fotosensibilizirovannoe khlorinom e6 ingibirovanie agregatsii trombotsitov cheloveka.

ADP-induced aggregation of platelets in the presence of chlorin E6 under the action of visible light depending on the pigment concentration and the time of light action was studied. It was shown that the photosensitized effect of chlorin E6 on the platelet-enriched plasma results in platelet aggregation inhibition.

[The effect of the bone marrow fibroblasts from patients with malignant diseases of the blood system on the colony-forming capacity of granulomonocytic precursor cells]. / Vliianie fibroblastov kostnogo mozga bol'nykh zlokachestvennymi zabolevaniiami sistemy krovi na kolonieobrazuiushchuiu sposobnost' granulomonotsitarnykh kletok-predshestvennikov.

The inhibiting activity of bone marrow fibroblasts and their ability to sustain survival of granulocytic cell precursors have been studied in patients with chronic myelocytic leukemia, chronic lymphoproliferative disorders, acute leukemia, myelodysplastic syndrome. Fibroblast conditioned medium inhibits proliferation of granulomonocytic precursors stimulated by leucocyte feeder layer or leucocyte conditioned medium. The effect depends both on the presence of monocytes and the specificity of the disease. The inhibitory effect is related with long-ranged factors. Bone marrow fibroblasts are able to sustain survival of hemopoietic precursors via humoral and cell-cell mechanisms. The regulatory role of bone marrow fibroblasts in hemopoietic disorders is discussed.

[The distribution of chlorine e6 and its derivatives in HeLa cells studied using luminescence microscopy]. / Issledovanie raspredelenie khlorina e6 i ego proizvodnykh v kletkakh HeLa metodom liuminestsentnoi mikroskopii.

The influence of the chemical structure of porphyrin pigments on their accumulation and localization in HeLa cells has been examined by the scanning fluorescence microphotometry. It has been found that the replacement of carboxyl groups of chlorine e6 for methyl and amino groups has no influence on the pigment distributions in cells. All the pigments are bound by cell membrane structures. The chemical modification of chlorine e6 structure is essential for the ability of pigment to be accumulated by cells that can be used to increase the efficiency of cancer phototherapy. The charge and hydrophobic properties of pigment molecules are of great importance for accumulating porphyrin sensitizers by cells.

[Damage photosensitized by chlorine e6 in bacteria with various defects of the DNA repair system]. / Fotosensibillizirovannoe khlorinom e6 povrezhdenie bakterii s razlichnymi defektami sistem reparatsii DNK.

The goal of the work was to study the sensitivity of isogenic Escherichia coli cells differing in their ability to mediate DNA repair steps to the action of visible light sensitized by chlorine e6. Cells incapable of excision repair as well as those deficient in post-replicative recombination DNA repair were found to be much more sensitive to the combined action of visible light and chlorine e6 as compared to cells whose genes responsible for DNA repair were not damaged. The results indicate that visible light damages bacterial DNA in the presence of chlorine e6.

[Photosensitization by chlorine and porphyrin causing structural damage of erythrocytes]. / Fotosensibilizirovannye khlorinami i porfirinami structurnye povrezhdeniia éritrotsitov.

Kinetics of hemolysis of human erythrocytes photosensitized by porphyrin and chlorin derivatives was investigated by small angle light scattering method. The compounds used were arranged in the following order of photosensitizing activity decrease: ethylendiamide of chlorine e6, chlorine e6 dimethylester chlorine e6 haematoporphyrin diacetate tetracarboxyphenylporphyn. The substances having a greater number of binding sites with liposomes (chlorine e6 dimethylester) or with albumin (ethylendiamide of chlorine e6) showed the greatest photohemolytic activity. The photohemolytic activity of porphyrins was suggested to depend on the number of the pigment molecules bound by erythrocytes membranes.