Ewa Kozlowska

Serum interleukin (IL-2, IL-10, IL-6, IL-4), TNFα, and INFγ concentrations are elevated in patients with atypical and idiopathic parkinsonism

We investigated serum levels of interleukin (IL)-2, IL-10, IL-6, IL-4, TNFalpha, INFgamma in 7 patients with atypical parkinsonism (AP), 31 idiopathic PD (iPD) patients, 17 idiopathic PD with cardiovascular risk factor (iPD-CVRF) patients, and 20 age-matched controls (healthy, non-parkinsonian patients). Cytokine concentrations were measured using the Becton Dickinson (BD) human Th1/Th2 Cytokine kit II with a flow cytometry system. The concentrations of IL-2, IL-10, IL-4, IL-6, TNFalpha, and INFgamma were detectable in the serum from all groups, including the control. Increased serum IL-2, IL-10, IL-4, IL-6, TNFalpha, and INFgamma concentrations were found in all groups of parkinsonian patients, as compared to the control group. The highest elevations of serum IL-2, IL-4, IL-6, TNFalpha, and INFgamma concentrations were observed in AP patients, as compared to the iPD and iPD-CVRF groups. However, the serum IL-6 concentration was higher in the iPD-CVRF group than in the iPD group. The IL-10 level was significantly higher in all groups of PD patients relative to the control group, but was the lowest in the serum from the AP patients. Moreover, the serum levels of lipid peroxidation products were enhanced 2.1- and 1.5-fold in AP and both iPD groups, respectively. These results argue in favor of the involvement of immunological events in the process of neurodegeneration in AP and PD.

Age-related changes in the occurrence and characteristics of thymic CD4+ CD25+ T cells in mice

Natural regulatory CD4+ CD25+ T cells play an important role in preventing autoimmunity by maintaining self‐tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T‐cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age‐associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4+ CD25+ thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA‐4, CD122, FOXP3), usually used to characterize the phenotype of CD4+ CD25+ T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus‐deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus‐deriving CD4+ CD25+ T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4+ CD25+ thymocytes between young and old animals are discussed in relation to the expression of these surface markers.

Exogenous melatonin modifies the circadian rhythm but does not increase the level of some immune parameters in the chicken

Abstract: The effect of daily injections of the pineal hormone melatonin and naltrexone, an opioid antagonist, on the circadian rhythm and the level of immune parameters (plaque forming cell [PFC] number, serum agglutinin titer, lymphoid gland weight, total white blood cells (WBC) and their fraction number, and serum lysozyme [LZ] content) was examined in White Leghorn cockerels and female BALB/c mice kept in LD 12:12. Animals were immunized ip with sheep red blood cells (SRBC) to stimulate their immune system. Subcutaneous injections of melatonin, naltrexone, or both drugs together were made 2 hr before the end of light, for 4 or 5 days, beginning on the day of immunization. The day following the fifth injection, chickens were sacrificed over a 24 hr period every 4 hr (experiment I) or twice daily, i.e., at the beginning of light and dark phases (experiment II). Mice were killed on the day following the fourth injection at the beginning of light, and splenic PFC number was determined (experiment III). In experiment I, the existence of the diurnal rhythm was evaluated by cosinor analysis. Melatonin injections entrained the circadian rhythm in anti‐SRBC serum agglutinins, but it did not influence circadian rhythmicity in other parameters examined. The circadian rhythm in total WBC number and their fractions was entrained by naltrexone treatment. Melatonin injections did not affect either the diurnal mean of parameters examined or the weight of lymphoid organs. Splenic PFC number in chickens was diminished by both melatonin and naltrexone injections, whereas in mice it was increased by melatonin, and naltrexone antagonized that effect. It is concluded that melatonin in chickens, at least in the dose range used, is not an immunoenhancing agent, as in mice. Possible reasons for this difference are discussed.

Potentiating antitumor effects of a combination therapy with lovastatin and butyrate in the Lewis lung carcinoma model in mice

Lovastatin, the drug used for the treatment of hypercholesterolemia, has previously been reported to exert antitumor activity in experimental murine models. Butyrate and butyric acid derivatives are well known to induce differentiation and apoptosis of tumour cells and also have recently gained acceptance as potential anticancer agents. In this study, we examined the antitumor effects of the combination of lovastatin and butyrate or its prodrug tributyrin in vitro and in vivo against a murine Lewis lung carcinoma (3LL). This combination therapy showed synergistic antitumor activity against 3LL cells in vitro. These effects were at least in part due to apoptosis induction that occurred after 12 hr of incubation with lovastatin and butyrate and was preceded by changes in cell cycle distribution of treated cells and expression of p21, p53 and cyclin D1. Remarkably, a systemic treatment of syngeneic mice inoculated with 3LL cells with both drugs resulted in significant tumour growth retardation.

Thymus-directed immunotoxicity of airborne dust particles from Upper Silesia (Poland) under acute extrapulmonary studies in mice

Industrial air pollutants from Upper Silesia, Poland, contain over 250 polycyclic and heterocyclic aromatic hydrocarbons and heavy metals, including mutagenic and carcinogenic chemicals that have been shown to form DNA adducts. Over 4 million habitants of Silesia are permanently exposed to the industrial pollution by pulmonary and dermal routes and by contaminated food and water. These chemicals, when examined separately in animals models, were proven immunotoxic. We studied the extrapulmonary immunotoxic potential of a typical mixture of Silesian filter-suspended matter from a selected area, over a specific season and time period. Early changes in the immune system were analyzed in BALB/c mice exposed ip to acute doses of 20-330 mg dust mixture/kg body weight (0.06-1.0 LD50). No major changes were noted for weight and the cellularity of spleen, liver and kidneys. However, dramatic decrease in thymus weight index and thymocyte cell count were noted as early as 24-72 h postexposure, which correlated with almost complete depletion of immature, double-positive CD4+CD8+ thymocytes. Changes in spleen were less profound; however, increased depletion of B cells over T cells was noted at high doses of the suspended matter. Exposure to the airborne dust also decreased cytokine production by spleen cells, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Overall, a single exposure to Silesian dust, even at the relatively low 0.06 LD50 dose, affected lymphokine production, suppressed B-cell proliferative response, and depleted thymuses of immature, double-positive CD4+CD8+ cells. A chemical synergism is suspected. To our knowledge, none of the known components of Silesian suspended matter, when examined as a single chemical, was shown to exert such a profound biological effect.

Controlled delivery of BID protein fused with TAT peptide sensitizes cancer cells to apoptosis

BackgroundLow cellular level of BID is critical for viability of numerous cancer cells. Sensitization of cells to anticancer agents by BID overexpression from adenovirus or pcDNA vectors is a proposed strategy for cancer therapy; however it does not provide any stringent control of cellular level of BID. The aim of this work was to examine whether a fusion of BID with TAT cell penetrating peptide (TAT-BID) may be used for controlled sensitization of cancer cells to anticancer agents acting through death receptors (TRAIL) or DNA damage (camptothecin). Prostate cancer PC3 and LNCaP, non-small human lung cancer A549, and cervix carcinoma HeLa cells were used in the study.MethodsUptake of TAT-BID protein by cells was studied by quantitative Western blot analysis of cells extracts. Cells viability was monitored by MTT test. Apoptosis was detected by flow cytometry and cytochrome c release assay.ResultsTAT-BID was delivered to all cancer cells in amounts depending on time, dose and the cell line. Recombinant BID sensitized PC3 cells to TRAIL or, to lesser extent, to camptothecin. Out of remaining cells, TAT-BID sensitized A549, and only slightly HeLa cells to TRAIL. None of the latter cell lines were sensitized to camptothecin. In all cases the mutant not phosphorylable by CK2 (TAT-BIDT59AS76A) was similarly efficient in sensitization as the wild type TAT-BID.ConclusionsTAT-BID may be delivered to cancer cells in controlled manner and efficiently sensitizes PC3 and A549 cells to TRAIL. Therefore, it may be considered as a potential therapeutic agent that enhances the efficacy of TRAIL for the treatment of prostate and non-small human lung cancer.

Identification and Functional Analysis of the erh1+ Gene Encoding Enhancer of Rudimentary Homolog from the Fission Yeast Schizosaccharomyces pombe

The ERH gene encodes a highly conserved small nuclear protein with a unique amino acid sequence and three-dimensional structure but unknown function. The gene is present in animals, plants, and protists but to date has only been found in few fungi. Here we report that ERH homologs are also present in all four species from the genus Schizosaccharomyces, S. pombe, S. octosporus, S. cryophilus, and S. japonicus, which, however, are an exception in this respect among Ascomycota and Basidiomycota. The ERH protein sequence is moderately conserved within the genus (58% identity between S. pombe and S. japonicus), but the intron-rich genes have almost identical intron-exon organizations in all four species. In S. pombe, erh1+ is expressed at a roughly constant level during vegetative growth and adaptation to unfavorable conditions such as nutrient limitation and hyperosmotic stress caused by sorbitol. Erh1p localizes preferentially to the nucleus with the exception of the nucleolus, but is also present in the cytoplasm. Cells lacking erh1+ have an aberrant cell morphology and a comma-like shape when cultured to the stationary phase, and exhibit a delayed recovery from this phase followed by slower growth. Loss of erh1+ in an auxotrophic background results in enhanced arrest in the G1 phase following nutritional stress, and also leads to hypersensitivity to agents inducing hyperosmotic stress (sorbitol), inhibiting DNA replication (hydroxyurea), and destabilizing the plasma membrane (SDS); this hypersensitivity can be abolished by expression of S. pombe erh1+ and, to a lesser extent, S. japonicus erh1+ or human ERH. Erh1p fails to interact with the human Ciz1 and PDIP46/SKAR proteins, known molecular partners of human ERH. Our data suggest that in Schizosaccharomyces sp. erh1+ is non-essential for normal growth and Erh1p could play a role in response to adverse environmental conditions and in cell cycle regulation.

Time schedule-dependent effect of the CK2 inhibitor TBB on PC-3 human prostate cancer cell viability

Inhibitors of CK2 kinase inhibit cell proliferation and induce apoptosis in numerous cancer cell lines. Due to these properties, they are considered potentially useful in anticancer therapy. In this study, we show that the exact effect of the specific CK2 inhibitor TBB on PC-3 human prostate cancer cell viability depends on the time schedule of administration: it was not observed when the treatment was directly followed by the viability assay but it appeared when the treatment and the assay were separated by a 24-h incubation without the inhibitor. Such a pattern was maintained when the TBB treatment was combined with either camptothecin or TRAIL. The time schedule-dependence of cell viability was not reflected by a similar dependence of induction of apoptosis. Despite this, the schedule in which a treatment with the CK2 inhibitor precedes that with an anticancer drug seems to be a good choice for a potential therapy against androgen-refractory prostate cancer.

Different T-cell activation by streptozotocin and Freund's adjuvant in popliteal lymph node (PLN)

A popliteal lymph node (PLN) test was further validated for predictive screening of autoimmunity-inducing drugs. Autoimmune-like T-cell activation of streptozotocin (STZ) was compared with the effect of Freunds complete adjuvant (FCA), injected locally into the foot pad of BALB/c mice. Early cell activation in enlarged PLN was monitored by flow cytometry. Injection of both STZ and FCA markedly increased the absolute PLN cell number as well as specific T-helper (CD4+), T-suppressor/cytotoxic (CD8+), and B (Ig+) subsets. However, quantitative analysis of early T-cell activation revealed important differences between STZ-induced PLN reaction and FCA-related lymphoproliferation. At 72 h, the number of cells stained with anti-early activation marker (EAM+; CD69+) increased over 10 times in STZ-enlarged nodes and only 3 times in the FCA-inflamed nodes. Furthermore, different cytometric profiles were noted for STZ-activated and FCA-activated cells stained with anti-interleukin-2 receptor (IL-2R) (CD25+). The data suggest the applicability of early cytometric screening of enlarged PLN for predictive analysis detection of chemicals inducing an autoimmune-like reaction.

Mutations in the Saccharomyces cerevisiae vacuolar fusion proteins Ccz1, Mon1 and Ypt7 cause defects in cell cycle progression in a num1Δ background

The proteins Ccz1 and Mon1 are known to function together with the Rab-GTPase Ypt7 in membrane fusion reactions at the yeast vacuole. In a genome-wide analysis they have also been found to interact genetically with the nuclear-migration protein Num1. In this study we analyze these synthetic effects and we show that the mutants ccz1Delta num1Delta, mon1Delta num1Delta and ypt7Delta num1Delta exhibit severe defects in cell cycle progression. A large fraction of the mutant cells enter a new cell division cycle without having completed mitotic exit, leading to the accumulation of multinuclear, anuclear and multibudded cells. The double deletion strains display also increased sensitivity to calcium ions. The cell-cycle defects are only weakly observed if deletions of other vacuolar protein sorting genes are combined with num1Delta or if other nuclear-migration genes are deleted together with CCZ1, whereas the calcium sensitivity is characteristic for a large subset of the tested double mutants. Further, the cell-cycle defects of the ccz1Delta num1Delta strain can be partially rescued by overproduction of either the calcium pump Pmc1 or the nuclear-migration factors Kar9 and Bim1. Together, these results indicate that deregulation of the cell cycle in these mutants results from two separate mechanisms, one of which is related to calcium homeostasis.