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Human peripheral blood cells stimulated by phytohemagglutinin (which serve as a model of cancerous cells) and resting cells were incubated in dimethyl sulfoxide solu­tions of various phthalocyanines. In order to diminish the influence of atmospheric ox­ygen the cells were embedded in a polymer (polyvinyl alcohol) film. Fluorescence spectra of the samples were measured over two regions of excitation wavelengths: at 405 nm (predominant absorption of the cell material) and in the regions of strong ab­sorption of phthalocyanines (at about 605 nm and 337 nm). The intrinsic emission of cell material became changed as a result both of cells' stimulation and of incubation of cells in dye solution. In most cases the stimulated cells when stained by dye exhibited higher long wavelength fluorescence intensity than resting cells. This suggests higher efficiency of dye incorporation into cancerous cells than into healthy cells. The ab­sorption spectra of samples were also measured. The spectra of various phthalo- cyanines in incubation solvent, in polymer and in the cells embedded in polymer, were compared. The comparison of properties of the cells stimulated for different time peri­ods enabled to establish the conditions of stimulation creating a population of cells in­corporating a large number of sensitizing molecules.
The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to γ-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as γH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G0phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5–5 Gy) induction of foci containing γH2A.X (1 h after γ-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3+lymphocytes were A+. Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A+PI–) in comparison to non-stimulated PBMCs (38% A+resp. 13.4%). After PHA-stimulation also the amount of γH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to γ-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2–7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3+T lymphocytes by the dose of 4 Gy 65% of cells were A+.
The interleukin-12p40 gene (IL12B) encodes the p40 polypeptide chain, which, together with p19, composes IL-23. A bi-allelic promoter polymorphism (IL12Bpro) located at −2703 bp of the transcription initiation site has been reported to show associations with IL-12p40 production. To elucidate the dependence of IL-12p40 and IL-23 production on IL12Bpro polymorphism in relation to MAPK signal transduction pathways, we examined the effect of JNK and p38 inhibition on the secretion of these cytokines by stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with 1.1 and 1.2/2.2 IL12Bpro genotypes. Stimulation with LPS and C3bgp resulted in approximately equal IL-12p40 production from PBMC with the 1.1 and 1.2/2.2 genotypes. The inhibition of JNK and p38 before stimulation significantly upregulated IL-12p40 production by PBMC with the 1.1 genotype, but did not influence IL-12p40 production from PBMC with the 1.2/2.2 genotype. Cultures of PBMC with the 1.1 genotype produced significantly more IL-12p40 than PBMC with the 1.2/2.2 genotype after stimulation with PHA. Inhibition of p38 kinase upregulated p40 production only in cultures with the 1.1 genotype. Decreased IL-23 production was observed in C3bgp-stimulated cultures after the inhibition of p38 regardless of the genotype of the tested cells. We concluded that IL-12p40 and IL-23 expression, which is mediated by the p38 and JNK intracellular signaling pathways, is influenced by IL12Bpro polymorphism.
AIM. We investigated effects of pulsating electromagnetic field (PEMF-50 Hz, 45 ± 5 mT) on viability and cytokine production by human peripheral blood mononuclear cells (PBMC) from healthy donors and from Crohn’s disease patients (CD). METHODS. The study was performed after activation of cells with phytohaemaglutinin (PHA) and lipopolisaccharide (LPS). Exposure of PBMC cultures to PEMF from both CD patients and from healthy donors decreased cell’s viability of about 10% and 5% (p>0,05) respectively. PEMF influence was most effective after threefold application. Susceptibility of PBMCs to magnetic field exposure differs among the stimulated (PHA, LPS) and not stimulated (NS) cells. Mitogen activated cells during cell division are most susceptible to induction of the cell death as a result of magnetic interaction, contrary PEMF exposure has minimal effect on non-diving PBMCs from CD patients and from controls. Decreased viability of the Crohn derived cells upon magnetic stimulation was accompanied by altered cytokines profile. Exposed and stimulated PBMCs from Crohn patients decreased IFN- proinflammatory and increased IL-10 anti-inflammatory cytokine production. The electromagnetically induced cell death could be an important step for non-invasive PEMF treatment in chronic inflammatory diseases.
Immunosenescence is viewed as a remodeling process with the exhaustion of naïve T cells and filling up of the immunological space with memory cells. In this study some phenotypic changes of CD8+ human cells during in vivo ageing were compared with those observed in long term cultures of lymphocytes derived from cord blood or from peripheral blood from donors of different age. Both in vivo and in vitro a significant decrease of the fraction of CD8+CD28+ cells was observed. Comparing the proportions of other T cell subpopulations (the CD4/CD8+ ratio, CD56, CD57, CD27) made it possible to conclude that replicative senescence in vitro partially reflects in vivo ageing.
The immunoprophylactic properties of Propionibacterium avidum KP-40, a potent stymulator of the macrophage-monocyte system and inducer of endogenous interferon, were tested in newborn calves. A single subcutaneous injection of the immunomodulator was administered either immediately after delivery or to 4-week-old animals. During physiological maturation of the immune system and the disappearance of passive resistance transmitted by colostrum, a gradual reduction of serum γ-globulins and increase in conglutinin occurred in the 2nd month of life in untreated calves. Administration of Propionibacterium avidum caused a significant rise of both γ-globulins and conglutinin. This was accompanied by increased lymphocytosis and a higher number of E-rosetting (T) lymphocytes. It is concluded that administration of Propionibacterium avidum increases the natural resistance of newborn calves and offers an effective prophylaxis against development of spontaneous infections.
The gag gene encoded protein, p24 of bovine leukemia virus (BLV), was cloned and ex­pressed as thioredoxin-6xHis-p24 protein in Escherichia coli. The bacterial cells carrying plasmid pT7THis-p24 expressed the protein of 38 kDa that was detected by immuno­blotting analysis using anti-p24 monoclonal antibodies and sera from BLV infected cattle and sheep. The purified p24 fusion protein was shown to be sensitive and specific for de­tection of BLV antibodies in the infected cattle.
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