Evaluation of cytotoxic, genotoxic and inflammatory responses of nanoparticles from photocopiers in three human cell lines

Background: Photocopiers emit nanoparticles with complex chemical composition. Short-term exposures to modest nanoparticle concentrations triggered upper airway inflammation and oxidative stress in healthy human volunteers in a recent study. To further understand the toxicological properties of copier-emitted nanoparticles, we studied in-vitro their ability to induce cytotoxicity, pro-inflammatory cytokine release, DNA damage, and apoptosis in relevant human cell lines. Methods: Three cell types were used: THP-1, primary human nasal- and small airway epithelial cells. Following collection in a large volume photocopy center, nanoparticles were extracted, dispersed and characterized in the cell culture medium. Cells were doped at 30, 100 and 300 μg/mL administered doses for up to 24 hrs. Estimated dose delivered to cells, was ~10% and 22% of the administered dose at 6 and 24 hrs, respectively. Gene expression analysis of key biomarkers was performed using real time quantitative PCR (RT-qPCR) in THP-1 cells at 5 μg nanoparticles/mL for 6-hr exposure for confirmation purposes. Results: Multiple cytokines, GM-CSF, IL-1β, IL-6, IL-8, IFNγ, MCP-1, TNF-α and VEGF, were significantly elevated in THP-1 cells in a dose-dependent manner. Gene expression analysis confirmed up-regulation of the TNF-α gene in THP-1 cells, consistent with cytokine findings. In both primary epithelial cells, cytokines IL-8, VEGF, EGF, IL-1α, TNF-α, IL-6 and GM-CSF were significantly elevated. Apoptosis was induced in all cell lines in a dose-dependent manner, consistent with the significant up-regulation of key apoptosis-regulating genes P53 and Casp8 in THP-1 cells. No significant DNA damage was found at any concentration with the comet assay. Up-regulation of key DNA damage and repair genes, Ku70 and Rad51, were also observed in THP-1 cells, albeit not statistically significant. Significant up-regulation of the key gene HO1 for oxidative stress, implicates oxidative stress induced by nanoparticles. Conclusions: Copier-emitted nanoparticles induced the release of pro-inflammatory cytokines, apoptosis and modest cytotoxicity but no DNA damage in all three-human cell lines. Taken together with gene expression data in THP-1 cells, we conclude that these nanoparticles are directly responsible for inflammation observed in human volunteers. Further toxicological evaluations of these nanoparticles, including across different toner formulations, are warranted

Recent insight into enzymatic degradation of plastics prevalent in the environment: A mini - review

Plastic pollution has been prevalent in the world ever since its use in majority of the areas including packaging, electronic industries, building and construction, healthcare, transportation etcetera. This has lead to a constantly increasing burden on the environment which has attracted many environmentalists and scientists towards working on different ways to cope up with this threat. Many studies have been done to find out ways to naturally degrade plastics using hidden capabilities of microbes that can use these plastics as their sole carbon source. Enzymatic degradation of plastics has been thought of serving this purpose since the revelation of microbial enzymes that can act on plastic in their natural environment and because of it being a much quicker and efficient way as compared to others. This paper gives a brief review on the degradation of plastics using enzymes from various sources and future prospects related to this area of research

Polarographic Study of L-Tryptophan Complexes of Zn(II), Ni(II) & Co(II)

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An approach to control relapse of inflammatory lesions after discontinuation of primary therapy.

Long-term treatment with the fungal metabolite drug FTY720 (Fingolimod) was shown to be highly effective in controlling viral immunopathological lesions. However, in this report we show that the anti-inflammatory effect of FTY720 in herpes simplex virus-1 (HSV-1) induced ocular inflammation is lost upon the discontinuation of treatment and lesions rapidly recurred. The lesions that developed after FTY720 treatment withdrawal involved mainly Th17 cells rather than Th1 cells explained in part by differential expression of surface CD103, an integrin that permits migration of effector cells to inflammatory sites. The expression of IL-6, a proinflammatory cytokine involved in the generation of Th17 cells, was found to be increased in FTY treated mice as compared to controls and this effect could be abrogated upon administration of neutralizing antibody to IL-6. Furthermore, IL-17RKO mice failed to show the recurrence of stromal keratitis (SK) lesions upon FTY720 withdrawal. These results indicate that approaches such as neutralization of proinflammatory cytokines might be considered along with FTY720 treatment if interruption of drug therapy becomes necessary

Efficacious bioconversion of waste walnut shells to xylotetrose and xylopentose by free xylanase (Xy) and MOF immobilized xylanase (Xy-Cu-BTC)

This study uses a cost effective and efficient method for production of higher DP (degree of polymerization) Xylooligosaccharides (XOS) from xylan extracted from the waste walnut shells. Copper based metal organic framework (Cu-BTC MOF) was prepared for immobilization of free xylanase (Xy) enzyme by green synthesis method. Both free and immobilized xylanase (Xy-Cu-BTC) were able to cause the bioconversion of xylan (87.4% yield) into XOS. Predominant production of xylotetrose (X4) and xylopentose (X5) was observed for both the methods. Percentage XOS conversion for free enzyme (Xy) was found to be 4.1% X4 and 60.57% X5 whereas these values increased in case of immobilized system where 11.8% X4 and 64.2% X5 were produced. Xylose production was minute in case of immobilized xylanase 0.88% which makes it a better method for XOS production free from xylose interference. Xy-Cu-BTC MOF can hence be used as an attractive alternative for pure XOS production

FTY720 ligation to S1P receptors promotes Tregs and Th17 cell differentiation.

<p>DO11.10 Rag2−/− mice were treated with 0.3 mg/kg body wt of FTY720 for 7 days. Splenocytes were isolated either from FTY720 treated or control animals and their TCR was stimulated under Th1, Th17 or regulatory T cell polarizing conditions in vitro for 5 days. After 5 days of incubation, cells were stained for measuring the production of cytokines and transcription factor Foxp3 by intra cellular/nuclear staining. (A) FACS plots showing the frequencies of IL-17 producing Th17 cells in FTY720 pre-treated and control groups. (B) Bar graphs for the cumulative data on the frequencies of Th17 cells in two groups. (C) Representative FACS plots showing the frequencies of IFN-γ producing Th1 cells in FTY720 pre -treated and control groups. (D) Bar graphs depict the cumulative data of IFN-γ producing cells from different replicates. (E) FACS plots depicting the frequencies of CD25 and transcription factor, Foxp3 staining in differentiated regulatory T cells in FTY720 pre-treated and control groups. (F) Bar graphs represent the cumulative frequencies of Foxp3⁺ cells in different groups. (G) Histograms show the MFI of CD25 and Foxp3 expression in polarized Tregs in control (faint lines) and FTY720 pre treated (thick lines) groups (H) Bar graphs show the cumulative data of the MFI for CD25 and Foxp3 in control and FTY720 pre-treated group. Data represent means ± SEMs of at least two independent experiments. Statistical significance was calculated by one-way ANOVA with *p≤0.05, **p≤0.01, and ***p≤0.001.</p

FTY720 treatment delays viral clearance from infected corneas and enhance IL-6 expression.

<p>C57BL/6 mice were infected with 1×10⁴ PFU of HSV-1. One group served as infected control and the other was treated with FTY720 (0.3 mg/kg body weight) starting from 24 h pi. (A) Corneal swabs were collected from treated and control groups at various days pi. and virus titers were determined using standard plaque assays using Vero cells. 4–5 mice per group for each time point was used to quantify the viral titers. (B) IL-6 concentration measured by sandwich ELISA in the corneal homogenate samples of FTY720 treated and control groups at different time points are shown. Pooled corneas from each group were used for the ELISA quantification. Bar graphs represent the levels of IL-6 in corneal samples of different groups of mice. Statistical levels of significance were analyzed by a Student t test. Data represent means ± SEM (C). Bar graphs represent the levels of IL-6 in DLNs of different groups of mice. Statistical levels of significance were analyzed by a Student t test. Data represent means ± SEM.</p

An alternative approach for supportive supervision and skill measurements of health workers for integrated management of neonatal and childhood illnesses program in 10 districts of Haryana

Context: “Integrated Management of Neonatal and Childhood Illnesses” (IMNCI) needs regular supportive supervision (SS). Aims: The aim of this study was to find suitable SS model for implementing IMNCI. Settings and Design: This was a prospective interventional study in 10 high-focus districts of Haryana. Subjects and Methods: Two methods of SS were used: (a) visit to subcenters and home visits (model 1) and (b) organization of IMNCI clinics/camps at primary health center (PHC) and community health center (CHC) (model 2). Skill scores were measured at different time points. Routine IMNCI data from study block and randomly selected control block of each district were retrieved for 4 months before and after the training and supervision. Statistical Analysis Used: Change in percentage mean skill score difference and percentage difference in median number of children were assessed in two areas. Results: Mean skill scores increased significantly from 2.1 (pretest) to 7.0 (posttest 1). Supportive supervisory visits sustained and improved skill scores. While model 2 of SS could positively involve health system officials, model 1 was not well received. Outcome indicator in terms of number of children assessed showed a significant improvement in intervention areas. Conclusions: SS in IMNCI clinics/camps at PHC/CHC level and innovative skill scoring method is a promising approach

Inhibition of Th17 generation prevents recurrence of lesions in HSV-1 infected mice.

<p>C57BL/6 and IL-17 RKO mice were infected with 1×10⁴ PFU of HSV-1. First group of mice were left untreated which served as controls. Second group of mice received FTY720 starting from 24 hrs pi. until 14 days pi. Third group of mice received FTY720 until day 10 pi. and later FTY720 treatment was discontinued. Fourth group of mice received FTY720 until day 10 pi and this group was also treated with IL-6 neutralizing antibody every alternate day until the day 14 pi. Fifth group of mice received only IL-6 neutralizing antibody treatment every alternate day until the day 14 pi. and the disease progression was observed. (A) SK progression was measured in treated and control groups at different time points till day 15 pi. (B) SK lesion scores on day 15 pi. Statistical significance was calculated by one-way ANOVA (*p≤0.05, ** p≤0.01, and ***p≤0.001). (C) Angiogenesis scores were measured in anti-IL-6 Ab treated and control groups on day 15 pi. (n = 5 to 6 mice per group). (D) The comparative analysis of disease progression in WT and IL-17 RKO mice that were given FTY720 treatment or where treatment was withdrawn. SK lesion scores were measured on day 11 and 14 pi. in different groups of mice i.e., infected controls, infected control mice treated with FTY720 (0.3 mg/kg body wt) from day 1–10, IL-17RKO mice and IL-17 RKO mice treated from day 1–10 with FTY720 (0.3 mg/kg body wt) from day 1–10 pi. Per group five mice were used and the experiments were repeated twice. Statistical significance was calculated by one-way ANOVA (*p≤0.05, **p≤0.01, and ***p≤0.001). (E) FACS plots representing the frequencies of IL-17 producing cells isolated from draining lymph nodes of various groups and stimulated in the presence of PMA/Ionomycin. (F) Cumulative data for the production of Th17 in different groups of mice is represented by bar graphs.</p

Continued therapy with FTY720 reduces the severity of SK lesions while discontinued treatment leads to relapse.

<p>C57BL/6 mice infected with 1×10⁴ PFU of HSV-1 were treated with FTY720 with various concentrations (0.03, 0.3 and 3.0 mg/Kg body weight) starting from 24 h pi. (A–B) FTY720 treatment depletes T cells from the periphery. (A) FACS plots showing the frequencies of CD4⁺ and CD8⁺ T cells from peripheral blood of FTY720 treated and control mice gated on total live cell population. (B) Bar graphs represents the frequencies of CD4⁺ and CD8⁺ T cells in blood isolated from FTY720 treated and control mice. (C) SK disease progression was measured in FTY720 and control groups at different time points till day 15 pi. (n = 5 to 6 mice per group). D–E. SK lesion (D) and angiogenesis (E) scores in different groups of mice on 15 dpi is shown. One-way ANOVA with Bonferroni’s multiple comparison test was used to calculate significance between control and FTY720 treated groups. (F) Bar graph depicts disease incidence (SK score >2.0) in various groups of mice measured on day 15 pi. Data represents mean ± SEM of at least two independent experiments. (G) FACS plots represent the frequencies of CD4⁺ T cell infiltration in various groups of mice. Numbers in the plots represent the cumulative data with SEM in indicated groups of mice. (H) Bar graphs showing the number of CD45⁺ CD11b⁺ double positive cells (macrophages/monocytes/dendritic cells) and CD45⁺CD11b⁺Gr1⁺ neutrophils in the corneas of different groups. Data represent means ± SEMs of at least two independent experiments. Statistical significance was calculated by one-way ANOVA (*p≤0.05, **p≤0.01, and ***p≤0.001).</p