Role of the Th1 and Th17 Pathway in Subacute Sclerosing Panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a progressive and fatal disease caused by reactivation of a mutated measles virus in brain tissue. The process of reactivation is yet to be elucidated. In this study, the possible roles of the Th1 (interleukin [IL]-12, interferon [IFN]-γ) and the Th17 axis (IL-23, IL-17, IL-22), particularly of IL-17, in the pathogenesis of SSPE were investigated. Briefly, mononuclear cells from SSPE patients were stimulated using measles virus peptide, and the release of IL-12, IL-23, IL-22, IFN-γ, and IL-17 cytokines was measured using enzyme-linked immunosorbent assay and/or enzyme-linked immunosorbent spot assay (ELISpot). We found that in comparison to the mononuclear cells obtained from healthy donors, cells from SSPE patients exhibited increased levels of IL-12, IL-23, IL-17, IL-22, and IFN-γ cytokines in response to measles virus stimulation. However, the same result was not obtained with cytomegalovirus and phytohemagglutinin. Using flow cytometry, mononuclear cells obtained from SSPE patients and healthy controls were also analyzed for the presence of intracellular IL-17 in response to measles virus stimulation. On stimulation, the number of IL-17-positive cells were found to be higher among mononuclear cells obtained from the patients. In addition, the numbers of IL-17- and IFN-γ-positive cells were significantly increased in SSPE patients. In conclusion, this study demonstrates that both the IL-12/IFN-γ and the IL-23/IL-17/IL-22 pathways are functionally abnormal in SSPE pathogenesis. Targeting these pathways and their specific pro-inflammatory mediator production may provide a new strategy to suppress SSPE development.

Doxorubicin-induced testicular damage is related to PARP-1 signaling molecules in mice.

BACKGROUND: Doxorubicin (DOX), is a chemotherapeutic agent, which evokes oxidative stress and cell apoptosis in testicular tissue. It is known that the activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP), leading to apoptosis induced by DOX. The aim of the present study is to investigate whether PARP pathway has a role in DOX-induced testicular damage and infertility utilizing pharmacological PARP-1 inhibitor, PJ34, and PARP-1 knockout mice model. METHODS: Firstly, we assessed the activation of PARP pathway after DOX-induction at various hours by immunohistochemistry and western blot analysis. Secondly, we evaluated the role of PARP pathway in DOX-induced testicular damage, sperm motility, and fertility with pharmacological inhibition of PARP by using PJ34. Finally, we aimed to correlate a functional relationship between PARP-1 and DOX using PARP-1 knockout mice in DOX-induced testicular damage. Doxorubicin levels in plasma and testis tissue were also assessed. RESULTS: In DOX-induced group; PARP-1, PAR and apoptotic pathway protein expressions, increased significantly. In DOX + PJ34 group; PAR, cytochrome c, and AIF levels decreased significantly. Testicular weights, sperm motility, and mean the number of pups per litter decreased in DOX-induced group after 28 days, however they were similar to the control group in DOX-PJ34 group. In PARP-1 KO group, seminiferous tubule morphology was impaired significantly after 28 days of DOX-administration. CONCLUSIONS: Our study indicates that DOX-induced testicular damage may be related to over-activation of PARP-1. PJ34 application was effective in preventing severe testicular damage caused by DOX-injection and may be considered for fertility protection against DOX-induced testicular damage.

Infectious diseases, autoimmunity and midline defect in a patient with a novel bi-allelic mutation in IL12RB1 gene.

Clinical disease caused by weakly pathogenic mycobacterial species, which is known as Mendelian susceptibility to mycobacterial disease (MSMD), is a rare entity. IFN-γ and IL-17 production are defective due to insufficient response to IL-2 and IL-23 in IL-12Rß1 deficiency; so this also causes tendency to intracellular microorganisms and candidal diseases. Here, we present a patient who suffers IL-12Rß1 deficiency caused by a novel bi-allelic mutation with recurrent salmonellosis, mycobacterial, fungal infections and remained asymptomatic during 13 months of follow-up after hIFN-γ treatment. In addition she had hemolytic anemia and midline defects like cleft lip and palate which have not been reported in a patient with MSMD in the literature prior to this case report. In conclusion, diagnosis of MSMD should be kept in mind in patients with recurrent salmonellosis, mycobacterial and fungal infections especially in countries with a high consanguinity rate.

Genotoxic and cell-transforming effects of titanium dioxide nanoparticles.

The in vitro genotoxic and the soft-agar anchorage independent cell transformation ability of titanium dioxide nanoparticles (nano-TiO2) and its microparticulated form has been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells. Nano-TiO2 of two different sizes (21 and 50 nm) were used in this study. The comet assay, with and without the use of FPG enzyme, the micronucleus assay and the soft-agar colony assay were used. For both the comet assay and the frequency of micronuclei a statistically significant induction of DNA damage, was observed at the highest dose tested (1000 µg/mL). No oxidative DNA damage induction was observed when the comet assay was complemented with the use of FPG enzyme. Furthermore, long-term exposure to nano-TiO2 has also proved to induce cell-transformation promoting cell-anchorage independent growth in soft-agar. Results were similar for the two nano-TiO2 sizes. Negative results were obtained when the microparticulated form of TiO2 was tested, indicating the existence of important differences between the microparticulated and nanoparticulated forms. As a conclusion it should be indicated that the observed genotoxic/tranforming effects were only detected at the higher dose tested (1000 µg/mL) what play down the real risk of environmental exposures to this nanomaterial.

Zinc oxide nanoparticles: genotoxicity, interactions with UV-light and cell-transforming potential.

The in vitro genotoxic and the soft agar anchorage independent cell transformation ability of zinc oxide nanoparticles (NPs) and its bulky forms have been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells, either alone or in combination with UVB-light. The comet assay, with and without the use of FPG and Endo III enzymes, the micronucleus assay and the soft-agar colony assay were used. For the comet assay a statistically significant induction of DNA damage, with and without the enzymes, were observed up of 100µg/mL. ZnO NPs were able to increase significantly the frequency of micronuclei, and similar results were observed in the cell transformation assay where such NPs were able to induce cell-anchorage independent growth. These effects were observed at doses up 100µg/mL. Although UVB-light was able to induce genotoxic damage and cell-anchorage growth, a significant antagonist interaction effect was observed in combination with ZnO NPs. These in vitro results, obtained with the selected cell lines, contribute to increase our genotoxicity database on the ZnO NPs effects as well as to open the discussion about their risk in photo-protection sun screens.

Determination of TiO2, ZrO2, and Al2O3 nanoparticles on genotoxic responses in human peripheral blood lymphocytes and cultured embyronic kidney cells.

In this study a genotoxic evaluation of titanium dioxide (TiO2, 2.3 nm), zirconium oxide (ZrO2, 6 nm), aluminum oxide (Al2O3, 16.7 nm) nanoparticles (NP) and their ionic forms was conducted using human peripheral blood lymphocytes and cultured human embryonic kidney (HEK293) cells by means of a modified alkaline comet assay with/without the formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III) enzymes. Modifications to the comet assay by using lesion-specific endonucleases, such as Endo III and Fpg, detect DNA bases with oxidative damage. Both human peripheral blood lymphocytes and cultured embryonic kidney cells were incubated with TiO2, ZrO2, or Al2O3 NP at concentrations of 1, 10, or 100 µg/ml. Our results showed no significant induction in DNA damage by the comet assay with/without the Endo III and Fpg enzymes at all concentrations of ZrO2 and Al2O3. In the case of TiO2 NP only the highest concentration of 100 µg/ml significantly induced a genotoxic response. Data thus indicate that both ZrO2 and Al2O3 NP were not genotoxic in our system and in the case of TiO2 the lowest-observed-adverse-effect level (LOAEL) for genotoxicity was 100 µg/ml. Evidence indicates that these metallic NP are considered safe in light of the fact that no genotoxicity was noted with ZrO2 and Al2O3 and that the highest TiO2 concentration is not environmentally relevant.

The effect of trans-resveratrol on platelet-neutrophil complex formation and neutrophil burst in hypercholesterolemic rats.

Transresveratrol (t-resveratrol; 3,5,4'-trihydroxy-trans-stilbene) is a polyphenolic compound found in fresh grapes, grape juice and wine, and has been found to reduce the total cholesterol level in hypercholesterolemic rats. The objective of the present study was to assess the effects of t-resveratrol on platelet-neutrophil complex formation and neutrophil reactive oxygen species (ROS) status in control and hypercholesterolemic rats using a modified flow cytometric method. Rats (n=80) were divided into five groups (control, ethanol, resveratrol, hypercholesterolemic and resveratrol-administered hypercholesterolemic groups), comprising 16 animals per group. Serum levels of lipids and H2O2 were determined using commercially available kits, while platelet-neutrophil complex formation and neutrophil ROS status were determined using a modified flow cytometric method. Serum total cholesterol and low-density lipoprotein cholesterol levels were found to be increased and the high-density lipoprotein cholesterol level was found to be decreased in the HC group compared with the control group (P<0.001). Treatment of HC rats with t-resveratrol significantly lowered total cholesterol and low-density lipoprotein cholesterol levels (P<0.001). In the hypercholesterolemic group, levels of serum H2O2 platelet-neutrophil complex formation and neutrophil ROS status were significantly increased (P<0.001). On the other hand, in the resveratrol-administered hypercholesterolemic group, serum H2O2 levels, platelet-neutrophil complex formation and neutrophil ROS status were decreased compared with the hypercholesterolemic group (P<0.001). Serum H2O2 levels, platelet-neutrophil complex and neutrophil ROS status were positively correlated with one another. The present study is the first to demonstrate the protective effect of t-resveratrol against hypercholesterolemia-induced platelet-neutrophil complex formation and neutrophil ROS burst. Further investigations on its plausible role in antihypercholesterolemic treatment are warranted.

Tbx3 represses PTEN and is over-expressed in head and neck squamous cell carcinoma.

BACKGROUND: Despite advances in diagnostic and treatment strategies, head and neck squamous cell cancer (HNSCC) constitutes one of the worst cancer types in terms of prognosis. PTEN is one of the tumour suppressors whose expression and/or activity have been found to be reduced in HNSCC, with rather low rates of mutations within the PTEN gene (6-8%). We reasoned that low expression levels of PTEN might be due to a transcriptional repression governed by an oncogene. Tbx2 and Tbx3, both of which are transcriptional repressors, have been found to be amplified or over-expressed in various cancer types. Thus, we hypothesize that Tbx3 may be over expressed in HNSCC and may repress PTEN, thus leading to cancer formation and/or progression. METHODS: Using immunohistochemistry and quantitative PCR (qPCR), protein and mRNA levels of PTEN and Tbx3 were identified in samples excised from cancerous and adjacent normal tissues from 33 patients who were diagnosed with HNSCC. In addition, HeLa and HEK cell lines were transfected with a Tbx3 expressing plasmid and endogenous PTEN mRNA and protein levels were determined via qPCR and flow cytometry. Transcription assays were performed to demonstrate effects of Tbx3 on PTEN promoter activity. Mann-Whitney, Spearman's Correlation and Wilcoxon signed-rank tests were used to analyze the data. RESULTS: We demonstrate that in HNSCC samples, Tbx3 mRNA levels are increased with respect to their normal tissue counterparts (p<0.001), whereas PTEN mRNA levels are significantly reduced in cancer tissues. Moreover, Tbx3 protein is also increased in HNSCC tissue sections. Over-expression of Tbx3 in HeLa and HEK cell lines causes reduction in endogenous PTEN mRNA and protein levels. In addition, transcription activity assays reveal that Tbx3 is capable of repressing both the basal and induced promoter activity of PTEN. CONCLUSIONS: We show that Tbx3 is up-regulated in tissue samples of HNSCC patients and that Tbx3 represses PTEN transcription. Thus, our data not only reveals a new mechanism that may be important in cancer formation, but also suggests that Tbx3 can be used as a potential biomarker in cancer.

Effect of resveratrol on platelet activation in hypercholesterolemic rats: CD40-CD40L system as a potential target.

Our aim was to investigate whether trans-resveratrol (t-resveratrol), a red wine constituent known for its cardioprotective effects, was able to influence CD40 ligand (CD40L) and its receptor CD40 in platelets of hypercholesterolemic rats. Sixty Wistar rats were divided into 5 groups: control (C), ethanol (E), t-resveratrol (R), hypercholesterolemia (HC), and hypercholesterolemia plus t-resveratrol (HCR). Rats in the C, E, and R groups were fed a normal diet for 80 days. For 20 days before sacrifice, we intraperitoneally (i.p.) administered 0.1 mL ethanol (50% v/v) to the E group, and 0.1 mL t-resveratrol (20 mg·kg(-1)·day(-1)) to the R group. Rats in the HC and HCR groups were fed a 5% cholesterol diet for 80 days. Rats in the HCR group were administered i.p. 0.1 mL t-resveratrol (20 mg·kg(-1)·day(-1)) for 20 days before sacrifice. Serum levels of total cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), very low-density lipoprotein (VLDL-C), and total triglycerides (TG) were assayed with a commercial colorimetric kit. Platelet P-selectin, CD40, and CD40L expression was determined by flow cytometry. sCD40L and IL6 levels were measured by ELISA. In the HC group, we observed a significant increase in serum TC, LDL-C, VLDL-C, TG, sCD40, and IL-6 levels and platelet activation markers compared with levels in the control group. However, t-resveratrol administration to the HC group (HCR group) attenuated the increase in lipids, sCD40, and IL-6 and down-regulated platelet P-selectin, CD40, and CD40L expressions. A positive correlation was found for serum lipids and all the platelet activation markers. Our study showed that the CD40-CD40L dyad is up-regulated in the presence of hypercholesterolemia and that t-resveratrol administration down-regulated the increase.