Ishani Das

Zinc-Oxide Nanoparticles Exhibit Genotoxic, Clastogenic, Cytotoxic and Actin Depolymerization Effects by Inducing Oxidative Stress Responses in Macrophages and Adult Mice

Zinc oxide nanoparticles (ZnO-NPs) have wide biological applications, which have raised serious concerns about their impact on the health and environment. Although, various studies have shown ZnO-NP toxicity on different cells underin vitroconditions, sufficient information is lacking regarding toxicity and underlying mechanisms underin vivoconditions. In this work, we investigated genotoxic, clastogenic, and cytotoxic effects of ZnO-NPs on macrophages and in adult mice. ZnO-NP-treated mice showed signs of toxicity such as loss in body weight, passive behavior and reduced survival. Further mechanistic studies revealed that administration of higher dose caused severe DNA damage in peripheral blood and bone marrow cells as evident by the formation of COMET tail, micronuclei, chromosomal fragmentation, and phosphorylation of H2A histone family member X. Moreover, ZnO-NPs inhibited DNA repair mechanism by downregulating the expression offen-1andpolBproteins. Histopathological examinations showed severe inflammation and damage to liver, lungs, and kidneys. Cell viability and wound healing assays revealed that ZnO-NPs killed macrophages in a dose-dependent manner, caused severe wounds and inhibited cellular migration by irreversible actin depolymerization and degradation. Reduction in the viability of macrophages was due to the arrest of the cell cycle at the G0/G1 phase, inhibition of superoxide dismutase and catalase and eventually reactive oxygen species. Furthermore, treatment with an antioxidant drug N-acetyl cysteine significantly reduced the ZnO-NP induced genotoxicity bothin vitroandin vivo Altogether, this study gives detailed pathological insights of ZnO-NP that impair cellular functions, thus will enable to arbitrate their biological applications.

Biocompatible chitosan nanoparticles as an efficient delivery vehicle for Mycobacterium tuberculosis lipids to induce potent cytokines and antibody response through activation of γδ T cells in mice

The activation of cell-mediated and humoral immune responses to Mycobacterium tuberculosis (Mtb) is critical for protection against the pathogen and nanoparticle-mediated delivery of antigens is a more potent way to induce different immune responses. Herein, we show that mice immunized with Mtb lipid-bound chitosan nanoparticles (NPs) induce secretion of prominent type-1 T-helper (Th-1) and type-2 T-helper (Th-2) cytokines in lymph node and spleen cells, and also induces significantly higher levels of IgG, IgG1, IgG2 and IgM in comparison to control mice. Furthermore, significantly enhanced γδ-T-cell activation was observed in lymph node cells isolated from mice immunized with Mtb lipid-coated chitosan NPs as compared to mice immunized with chitosan NPs alone or Mtb lipid liposomes. In comparison to CD8+ cells, significantly higher numbers of CD4+ cells were present in both the lymph node and spleen cells isolated from mice immunized with Mtb lipid-coated chitosan NPs. In conclusion, this study represents a promising new strategy for the efficient delivery of Mtb lipids using chitosan NPs to trigger an enhanced cell-mediated and antibody response against Mtb lipids.

Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages

Mycobacterium tuberculosis is known to modulate the host immune responses to facilitate its persistence inside the host cells. One of the key mechanisms includes repression of class-II transactivator (CIITA) and MHC-II expression in infected macrophages. However, the precise mechanism of CIITA and MHC-II down-regulation is not well studied. M. tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a known potent virulence and antigenic determinant. The M. tuberculosis genome encodes 23 such ESAT-6 family proteins. We herein report that M. tuberculosis and M. bovis bacillus Calmette–Guérin infection down-regulated the expression of CIITA/MHC-II by inducing hypermethylation in histone H3 lysine 9 (H3K9me2/3). Further, we showed that M. tuberculosis ESAT-6 family protein EsxL, encoded by Rv1198, is responsible for the down-regulation of CIITA/MHC-II by inducing H3K9me2/3. We further report that M. tuberculosis esxL induced the expression of nitric-oxide synthase, NO production, and p38 MAPK pathway, which in turn was responsible for the increased H3K9me2/3 in CIITA via up-regulation of euchromatic histone-lysine N-methyltransferase 2 (G9a). In contrast, inhibition of nitric-oxide synthase, p38 MAPK, and G9a abrogated H3K9me2/3, resulting in increased CIITA expression. A chromatin immunoprecipitation assay confirmed that hypermethylation at the promoter IV region of CIITA is mainly responsible for CIITA down-regulation and subsequent antigen presentation. We found that co-culture of macrophages infected with esxL-expressing M. smegmatis and mouse splenocytes led to down-regulation of IL-2, a key cytokine involved in T-cell proliferation. In summary, we demonstrate that M. tuberculosis EsxL inhibits antigen presentation by enhancing H3K9me2/3 at the CIITA promoter, thereby repressing its expression through NO and p38 MAPK activation.

Mycobacterium tuberculosis LprE enhances bacterial persistence by inhibiting cathelicidin and autophagy in macrophages

Mycobacterium tuberculosis(Mtb) lipoproteins are known to facilitate bacterial survival by manipulating the host immune responses. Here, we have characterized a novel Mtb lipoprotein LprE(LprEMtb), and demonstrated its role in mycobacterial survival. LprEMtb acts by down-regulating the expression of cathelicidin, Cyp27B1, VDR and p38-MAPK via TLR-2 signaling pathway. Deletion of lprEMtb resulted in induction of cathelicidin and decreased survival in the host. Interestingly, LprEMtb was also found to inhibit autophagy mechanism to dampen host immune response. Episomal expression of LprEMtb in non-pathogenic Mycobacterium smegmatis(Msm) increased bacillary persistence by down-regulating the expression of cathelicidin and autophagy, while deletion of LprEMtb orthologue in Msm, had no effect on cathelicidin and autophagy expression. Moreover, LprEMtb blocked phago-lysosome fusion by suppressing the expression of EEA1, Rab7 and LAMP-1 endosomal markers by down-regulating IL-12 and IL-22 cytokines. Our results indicate that LprEMtb plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Protection of Bio-Cultural Property in the Cradle of Traditional Knowledge

Human communities have always generated, refined and passed on knowledge from generation to generation. Such “traditional” knowledge” is often an important part of their cultural identities. Traditional knowledge encompasses the beliefs, knowledge, practices, innovations, arts, spirituality, and other forms of cultural experience and expression that belong to indigenous communities worldwide. Since the adoption of the Universal Declaration of Human Rights in 1948, intellectual property has been considered a fundamental human right of all people. Only recently, however, has the need to protect, preserve and provide for the fair use of indigenous intellectual property ‘traditional knowledge’ entered the domestic and international debate on intellectual property rights. Non-indigenous groups, such as corporations, have been exploiting the traditional knowledge that has been developed by indigenous beings over centuries. However, the international community has sought to recognize and protect such traditional knowledge. Many developing countries, holders of traditional knowledge, and campaigning organizations are pressing in a multitude of fora for traditional knowledge to be better protected. Such pressure has led, for example, to the creation of an Intergovernmental Committee on Intellectual Property and Genetic Resources, Traditional Knowledge and Folklore in WIPO. Such knowledge and folklore is also being discussed within the framework of the CBD and in other international organizations such as UNCTAD, WHO, FAO and UNESCO. In addition, the Doha WTO Ministerial Declaration highlighted the need for further work in the TRIPS Council on protecting traditional knowledge.