Genotoxic and cytotoxic properties of PM2.5 collected over the year in Wroclaw (Poland).

In the ambient is >2000 chemical substances, some of them are absorbed on the surface of the particulate matter and may causes many health problems. Air pollution is responsible for >3.2 million premature deaths which classifies it as a second place environmental risk factor. Especially dangerous for health are polycyclic aromatic hydrocarbons and their derivatives which shows mutagenic and carcinogenic properties. Air pollutions were also classified by International Agency for Research on Cancer to group which carcinogenic properties on human were proved by available knowledge. Air pollutions, are one of the biggest problem in Polish cities. The article presents results of mutagenicity, genotoxicity and cytotoxicity researches conducted on a particulate matter fraction 2.5 µm collected during all year long in Wroclaw agglomeration (Poland). The material was collected on filters using high-flow air aspirator and extracted using dichloromethane. Additionally it was fractionated into 4 parts containing: all pollutants, only polycyclic aromatic hydrocarbons, nitro derivatives of PAHs and dinitro derivatives of PAHS. Dry residue of this fraction was dissolving in DMSO and tested using biological methods. Biological methods include mutagenicity properties which are investigated by Salmonella assay (Ames assay). Other biological method was comet assay and 4 parameter cytotoxicity test PAN-I assay. Results of the conducted experiments show differences in mutagenic, genotoxic and cytotoxic properties between seasons of collection and between volumes of dust pollutions fractions. The worst properties shows particles collected in autumn and winter season Results showed also some correlations in results obtained during different methods and properties. Due to the limited possibilities of testing all chemical compounds present in the PM2.5 fraction, it is recommended to carry out tests based on a set of genotoxic and cytotoxic tests, which is confirmed by the conducted research.

Synthesis and quantitative structure-activity relationship analysis of 2-(aryl or heteroaryl)quinolin-4-amines, a new class of anti-HIV-1 agents.

Thirty-eight 2-(aryl or heteroaryl)quinolin-4-amines, N,N-disubstituted, N-monosubstituted, and without a substituent at the amino group have been synthesized with use of novel chemistries developed by us recently. Some of these derivatives show anti-HIV-1 activity at a concentration level of 1 microM and low cell toxicity in vitro. The most active and least toxic compounds are derivatives of 2-(3-pyridyl)quinoline. The results of the quantitative structure-activity relationship analyses, including several classical, linear regression correlations and a Free-Wilson approach of de novo model, provide guidelines for the design of new active compounds of this class.

The study of cell-mediated immune response in recurrent vulvovaginal candidiasis.

The aim of this work was to examine in vitro the ability of cells from patients with recurrent vulvovaginal candidiasis (RVVC) to cell-mediated immune response. Peripheral blood mononuclear cells (PBMC) and whole blood cells (WBC) of 37 RVVC patients in acute infection and 14 in remission were examined for the ability to proliferation and cytokines production (IFN, TNF, IL-6). As a control, a group of 25 healthy women were examined. The cells were stimulated with Candida antigen (HKCA), LPS and PHA. To indicate the level of cytokines, the following cell-lines were used: A549 for IFN, WEHI 164 for TNF and 7TD1 for IL-6. The proliferation/death of cells was determined by colorimetric test using MTT. Distinct suppression of cell-mediated immune response (CMI) was shown in all patients comparing to the control. Greatest suppression was found in the acute phase of the disease. The ability of cells to proliferate and produce IFN increases only in remission. The data seem to suggest that in this phase of disease, the ability of cell-mediated immune response is restored. It was also indicated that IFN may take part in protection against Candida infection.

The occurrence of glycine in bacterial lipopolysaccharides.

The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [14C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100 degrees C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.

Structural and serological characterization of Hafnia alvei lipopolysaccharide core region.

The structures and serological activities of core oligosaccharide of Hafnia alvei strains have been investigated. Methylation analysis, NMR spectroscopy and various specific degradation procedures were the principal methods used. It is concluded that, core hexasaccharides are identical in the lipopolysaccharides tested and are built of two glucose, three heptose and one 2-keto-3-deoxyoctulosonic acid residues. The antiserum raised against the ATCC13337 oligosaccharide core-tetanus toxoid conjugate cross-reacted strongly with all lipopolysaccharides used as antigens in ELISA test, suggesting that this core region is the common structure in the Hafnia genus.

Investigation on extracellular slime from Pseudomonas aeruginosa as interferon inducer in mice.

The investigation concerns interferon (IFN) production in the sera and spleens of 129/Ao/Boy mice induced with Pseudomonas aeruginosa slime extract. Interferon was present in the serum as early as 2 h after i.v. and i.p. injection of the immunogenic dose of the slime - 100 micrograms/mouse. Likewise, in the spleen the same dose induced interferon production at the second hour after its administration. In the spleen interferon was synthetized longer, even up to 7 days. On the other hand, it disappeared from the serum after 24 h. In vitro investigations on interferon induction in peritoneal cells and spleen revealed that after slime extract stimulation, only non-adherent cells are capable of IFN production; while adherent cells are not. Interferon synthesis in peritoneal cells in vitro was much enchanced if for the experiments, cells isolated 2 - 4 h after i.v. administration of mice with Ps. aeruginosa slime extract, were used. Besides, the stimulatory effect of the extract on interferon production was well marked in the Newcastle virus-induced peritoneal cells. For comparison, interferon obtained after induction with slime extract in vivo (in the serum) and in vitro (in peritoneal cells) was tested for its properties. The interferons although both acidstable, displayed significant differences. IFN obtained in vitro from peritoneal cells culture appeared thermolabile and susceptible for neutralization with gamma-globulin of rabbit serum against interferon from Newcastle virus-induced L929 cells (anti-MuIFN alpha/beta). IFN from serum was thermostabile, undergoing only slight neutralization with anti-MuIFN alpha/beta globulin.

Humoral response in mice immunized with outer membrane proteins of Shigella flexneri.

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Sh. flexneri induced in the animals a synthesis of specific antibodies. Their level determined by ELISA test was found to be relatively low in the sera of animals immunized with a single dose (10 micrograms) of OMP; it was markedly higher in mice immunized with two doses of OMP, and very high after three fold immunization. The specific antibodies maintained in the animals for 8-16 weeks after immunization. Anti-OMP sera given to normal mice by intraperitoneal route protected them not only against challenge with homologous Shigella but also against Proteus and Escherichia.

Humoral response in mice immunized with outer membrane proteins of Hafnia alvei. Protective activity of anti-OMP-antibodies.

Intraperitoneal immunization of mice with outer membrane proteins (OMP) of Hafnia alvei induced in the animals a synthesis of specific antibodies. The antibody levels, determined by ELISA test, were found to be relatively low in the sera of mice immunized with a single dose (5 micrograms) of OMP and after a second immunization. However, they were higher in mice immunized with three doses of OMP. The antibodies were present in circulation for a relatively short time after immunization. Serum containing anti-OMP antibodies given intraperitoneally to normal mice protected them only against challenge with a homologous Hafnia strain.

Studies on immunity in mice immunized with outer membrane proteins of Hafnia.

Nonspecific protection induced in mice after administration outer membrane proteins of Hafnia alvei against infection with homologous and heterologous bacteria was transferred into other mice with lymphocytes isolated from spleens of mice immunized with outer membrane proteins. It was also found that mice sensitized with outer membrane proteins derived from H. alvei or with living bacteria induced in animals delayed hypersensitivity (DTH) in homologous and heterologous systems. The observed type of hypersensitivity was transferable to normal mice by lymphocytes obtained from donor animals which were previously sensitized with OMP. The experiments revealed that immunity induced with outer membrane proteins of Hafnia alvei is cell-mediated.

Effect of outer membrane proteins from Shigella on humoral immunity induced in mice by SRBC.

Shigella flexneri outer membrane proteins (OMP) which had been earlier found to exert immunomodulatory effect on cell mediated immune response were also found to act as immunomodulator of the humoral immune response. Effects of OMP were investigated in the experiments in vitro and in vivo, where the level of humoral immune response, measured as the number of plaque-forming cells (PFC) to SRBC in the spleen was evaluated. We demonstrate that small doses of OMP (1-5 micrograms) stimulate, whereas higher doses (10-50 micrograms) suppress the humoral immunity.

Some biological properties of outer membrane proteins of Hafnia.

Outer membrane proteins (OMP) isolated from four antigenically different strains of Hafnia alvei were tested for the toxicity, pyrogenicity, ability to induce Shwartzman reaction as well as for their influence on the leukocyte system. LD50 doses for the studied preparations determined on inbred mice were 18, 20, 28 and 34 mg/kg. These differences in the toxicity of the preparations were reflected in manifestation of Shwartzman reaction; more toxic preparations induced strongest necrohemorrhagic changes at the site of injection. The OMP preparations injected intravenously to rabbits caused moderate increase of body temperature. They induced changes in the number of leukocytes in the animals comparable with those of other preparates of bacterial origin.

Fraction of spleen cells responsible for suppression of cellular immune response to SRBC in mice treated with outer membrane proteins of Shigella.

Effect of splenocytes isolated from mice immunized with suppressive dose of OMP from Shigella on delayed hypersensitivity, induced in mice with sheep red blood cells was investigated. Only the population of T lymphocytes was found to suppress the delayed hypersensitivity, as measured by the footpad reaction. The results suggest that OMP of Shigella are able to induce in the spleens of animals active T cells which are responsible for the suppression of cellular response induced by SRBC.

Effect of outer membrane proteins (OMP) of Shigella on interleukin 2 (IL-2) production by spleen cells of mice.

The kinetics of interleukin (IL-2) release from mouse spleen cells incubated with different doses of outer membrane proteins (OMP) from Shigella was investigated. OMP induced very low activity of IL-2 after 2 and 4 h, and only a slightly higher level of the cytokine was detected after 6 h. However, IL-2 activity increased markedly after 20 and 24 h of incubation, and doses of 5 and 10 micrograms of OMP were found to be the most effective. Spleen cells cultured with OMP for 48 h contained reduced concentration of IL-2.

Studies on nonspecific protection induced with OMP of Shigella flexneri and other genera of Enterobacteriaceae.

Outer membrane proteins (OMP) isolated from Shigella flexneri, Escherichia coli, Proteus vulgaris and Salmonella typhimurium were tested for their protective activity. Each OMP preparate given to mice intraperitoneally in a single injection (5 micrograms/per mouse) protected the animals not only in homologous but also in varying intensity in heterologous systems. Evidence was obtained that this nonspecific protection is cell mediated.

Immunogenic and protective properties of outer membrane proteins of Hafnia alvei.

Outer membrane proteins (OMP) extracted from antigenically distinct or related strains of Hafnia alvei containing defined composition of major proteins proved to be immunogenic. Intraperitoneal immunization of mice with a single dose of such preparations protected the animals against homologous and heterologous Hafnia strains. The OMP preparations were also found to induce protection with varying intensity against Escherichia, Proteus, Shigella and Salmonella.

Phagocytic and bactericidal properties of macrophages of mice immunized with outer membrane proteins (OMP) of Shigella.

Macrophages obtained from peritoneal exudates of mice immunized with the single doses (1 and 5 micrograms) of OMP were shown to have stronger phagocytic as well as bactericidal properties in relation to Shigella flexneri bacilli than nonactivated macrophages. Macrophages from the animals immunized with 10, 20 and 30 micrograms OMP doses showed phagocytic and bactericidal properties similar to those of nonactivated macrophages while the immunization with the dose 50 micrograms resulted in their suppression. Likewise, activity of macrophages from mice immunized twice or three times with various doses of OMP did not differ much from that obtained after immunization with single OMP doses. On the other hand, immunization of mice with a sublethal dose of live Shigella flexneri did not activate either phagocytic or bactericidal properties of macrophages. Besides, phagocytic and bactericidal activity of macrophages of mice immunized with OMP of Shigella was determined in relation to Salmonella typhimurium. The doses 1 and 5 micrograms of OMP resulted in slight activation of macrophages which manifested itself by a little increase in their phagocytic and bactericidal ability. When used in the dose 10 micrograms, OMP remained without any effect on the above activity of macrophages. Only the dose 50 micrograms, slightly suppressed their phagocytic properties.

Induction of tumor necrosis factor and interleukin 6 by outer membrane proteins of Shigella in spleen cells and macrophages of mice.

The ability of outer membrane proteins (OMP) of Shigella flexneri to induce tumor necrosis factor (TNF) and interleukin 6 (IL-6) production by spleen cells and macrophages of mice was investigated. Treatment of spleen cells with OMP resulted in the release of only traces of TNF activity. In contrast, macrophages treated with OMP produced moderate levels of TNF. OMP was also found to be an inducer of IL-6. Both spleen cells and macrophages, treated with OMP, were found to produce substantial levels of this cytokine. The effect of OMP on the release of TNF and IL-6 was dose and time dependent, maximal production being reached at 10 micrograms of OMP after 20 h. The ability of OMP to induce production of these cytokines may explain part of the previously described immunomodulatory effects of this preparation on the immune system.

Effect of outer membrane proteins of Shigella on delayed hypersensitivity in mice to sheep red blood cells.

Small doses of outer membrane proteins (OMP) of Shigella flexneri injected intraperitoneally into mice 1 to 3 days before or 3 days after sensitization of animals with sheep erythrocytes were found to increase delayed hypersensitivity as measured by the footpad reaction. In contrast, administration of higher doses of OMP resulted in suppression of hypersensitivity response. Cell transfer experiments showed that the spleen cells from sensitized and OMP treated mice transferred stimulating and suppressing activity to normal recipients. Suppression of hypersensitivity was also observed when recipients were injected with OMP 24 h before they were infused with spleen cells obtained from donor mice sensitized with sheep erythrocytes.

Some biological properties of outer membrane proteins of Shigella.

Outer membrane proteins (OMP) derived from two antigenically different representatives of Shigella (Sh. flexneri 3a and Sh. sonnei phase I) were tested for the toxicity, pyrogenicity, ability to induce Shwartzman reaction as well as for their influence on the leukocyte system. LD50 dose determined on mice was 28 mg/kg for OMP of Sh. flexneri and 23 mg/kg for OMP of Sh. sonnei. Both preparations injected intravenously to rabbits caused moderate increase of body temperature, expressed by the value 1.8 degrees C. Intravenous administration of protein preparations to rabbits, induced at first leukopenia and then transient leukocytosis. When injected subcutaneously in the dose of 500 micrograms and after 24 h intravenously in the dose 100 micrograms, they produced slight hemorrhagic changes at the site of administration.

Phagocytosis and intracellular killing of Pseudomonas aeruginosa by peritoneal macrophages of mice.

The poorly mucoid P. aeruginosa 87 strain was better phagocytized by peritoneal macrophages from normal mice than highly mucoid P. aeruginosa 219. However, the macrophages killed significantly greater number of mucoid bacterial cells of P. 219 strain. The immunization of mice with 10, 50 or 100 micrograms of slime-extract P. 87 or P. 219 did not significantly enhance the phagocytic and bactericidal properties of macrophages. The vaccination of animals with viable bacterial cells of P. 219 strain, using several schemes of treatment, slightly augmented both activities of macrophages but the differences between them and control group were statistically not significant. However, the ingestion and killing of the bacteria were suppressed when macrophages were harvested from mice treated with 200 or 400 micrograms of slime-extract P. 219.