Dakshayani Lomada

Thymus Medulla Formation and Central Tolerance Are Restored in IKKα−/− Mice That Express an IKKα Transgene in Keratin 5+ Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-κB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IκB kinase (IKK)α, a required intermediate in the noncanonical NF-κB signaling pathway. Introduction of an IKKα transgene driven by a K5 promoter restores the K5+K14+ mTEC subset in IKKα−/− mice. Unexpectedly, the K5-IKKα transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKα transgene ameliorates autoimmune symptoms in IKKα−/− mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-κB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.

Ovarian Cancer Biology and Immunotherapy

Ovarian cancer is the most lethal malignancy of the female reproductive system and the fifth leading cause of cancer death in women. In the year 2012 alone, United States had 22,280 new ovarian cancer cases and 15,500 deaths were reported. About 7%–10% of ovarian cancers result from an inherited tendency to develop the disease. Ovarian cancer has the ability to escape the immune system because of its pathological interactions between cancer cells and host immune cells in the tumor microenvironment create an immunosuppressive network that promotes tumor growth, protects the tumor from immune system. The levels of immune suppressive elements like regulatory T cells, plasmacytoid dendritic cells and cytokines such as IL-10, IL-6, TNF-α, and TGF-β are elevated in the tumor microenvironment. Vascular endothelial growth factor is known to have an immune suppressing role besides its angiogenic role in the tumor microenvironment. Ovarian cancer is associated with high mortality partly due to difficulties in early diagnosis and development of metastases. These problems may overcome by developing accurate mouse models that should mimic the complexity of human ovarian cancer. Such animal models are better suited to understand pathophysiology, metastases, and also for preclinical testing of targeted molecular therapeutics. Immunotherapy is an area of active investigation and off late many clinical trials is ongoing to prevent disease progression. The main aim of dendritic cells vaccination is to stimulate tumor specific effector T cells that can reduce tumor size and induce immunological memory to prevent tumor relapse.

γδ T cell mediated immune responses in Disease and Therapy

The role of γδ T cells in immunotherapy has gained specific importance in the recent years because of their prominent function involving directly or indirectly in the rehabilitation of the diseases. γδ T cells represent a minor population of T cells that express a distinct T cell receptor (TCR) composed of γδ chains instead of αβ chains. Unlike αβ T cells, γδ T cells display a restricted TCR repertoire and recognize mostly unknown non-peptide antigens. γδ T cells act as a link between innate and adaptive immunity, because they lack precise major histocompatibility complex (MHC) restriction and seize the ability to recognize ligands that are generated during affliction. Skin epidermal γδ T cells recognize antigen expressed by damaged or stressed keratinocytes and play an indispensable role in tissue homeostasis and repair through secretion of distinct growth factors. γδ T cell based immunotherapy strategies possess great prominence in the treatment because of the property of their MHC-independent cytotoxicity, copious amount of cytokine release, and a immediate response in infections. Understanding the role of γδ T cells in pathogenic infections, wound healing, autoimmune diseases, and cancer might provide knowledge for the successful treatment of these diseases using γδ T cell based immunotherapy. Enhancing the human Vγ9Vδ2 T cells functions by administration of aminobisphosphonates like zoledronate, pamidronate, and bromohydrin pyrophosphate along with cytokines and monoclonal antibodies shows a hopeful approach for treatment of tumors and infections. The current review summarizes the role of γδ T cells in various human diseases and immunotherapeutic approaches using γδ T cells.

Stat3 Signaling Promotes Survival And Maintenance Of Medullary Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) are essential for establishing central tolerance by expressing a diverse array of self-peptides that delete autoreactive thymocytes and/or divert thymocytes into the regulatory T cell lineage. Activation of the NFκB signaling pathway in mTEC precursors is indispensable for mTEC maturation and proliferation resulting in proper medullary region formation. Here we show that the Stat3-mediated signaling pathway also plays a key role in mTEC development and homeostasis. Expression of a constitutively active Stat3 transgene targeted to the mTEC compartment increases mTEC cellularity and bypasses the requirement for signals from positively selected thymocytes to drive medullary region formation. Conversely, conditional deletion of Stat3 disrupts medullary region architecture and reduces the number of mTECs. Stat3 signaling does not affect mTEC proliferation, but rather promotes survival of immature MHCIIloCD80lo mTEC precursors. In contrast to striking alterations in the mTEC compartment, neither enforced expression nor deletion of Stat3 affects cTEC cellularity or organization. These results demonstrate that in addition to the NFkB pathway, Stat3-mediated signals play an essential role in regulating mTEC cellularity and medullary region homeostasis.

Ti–O based nanomaterials ameliorate experimental autoimmune encephalomyelitis and collagen-induced arthritis

Multiple sclerosis (MS) and rheumatoid arthritis (RA) are the most common chronic autoimmune inflammatory diseases that affect the central nervous system and joints respectively. Treatment of autoimmune diseases usually concentrates on alleviating symptoms. High-mobility group box 1 protein (HMGB1) cytokine had been reported to play a key role in autoimmune disorders as HMGB1 levels correlate with active inflammation and neutralizing HMGB1 can rescue various autoimmune diseases. Nano-size titania (TiO2) is an exceptional multi-functional material that showed several practical applications ranging from pigments in paints, UV light absorbent in sunscreen lotion to coatings on non-fogging surfaces, biomedicine and agriculture. However, the in vivo role of Ti–O based nanomaterials in autoimmune disease models has not been examined. This study was designed to investigate the role of Ti–O based nanomaterials such as H2Ti3O7 nanotubes (TNT) and anatase TiO2 fine particles (TFP) in well established animal models experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA). We showed for the first time that the administration of Ti–O based nanomaterials attenuated clinical signs of pathophysiology and correlated with the reduction of the pro-inflammatory cytokine HMGB1. The clinical signs, histology and HMGB1 secretion data showed the therapeutic role of TNT and TFP in EAE and TNT in CIA. Thus, TNT and TFP have potential applications in specific treatment of MS/RA and this may provide an effective novel therapeutic approach for other autoimmune diseases.

Isolation and in vitro evaluation of bacteriophages against MDR-bacterial isolates from septic wound infections

Multi-drug resistance has become a major problem for the treatment of pathogenic bacterial infections. The use of bacteriophages is an attractive approach to overcome the problem of drug resistance in several pathogens that cause fatal diseases. Our study aimed to isolate multi drug resistant bacteria from patients with septic wounds and then isolate and apply bacteriophages in vitro as alternative therapeutic agents. Pus samples were aseptically collected from Rajiv Gandhi Institute of Medical Science (RIMS), Kadapa, A.P., and samples were analyzed by gram staining, evaluating morphological characteristics, and biochemical methods. MDR-bacterial strains were collected using the Kirby-Bauer disk diffusion method against a variety of antibiotics. Bacteriophages were collected and tested in vitro for lytic activity against MDR-bacterial isolates. Analysis of the pus swab samples revealed that the most of the isolates detected had Pseudomonas aeruginosa as the predominant bacterium, followed by Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli. Our results suggested that gram-negative bacteria were more predominant than gram-positive bacteria in septic wounds; most of these isolates were resistant to ampicillin, amoxicillin, penicillin, vancomycin and tetracycline. All the gram-positive isolates (100%) were multi-drug resistant, whereas 86% of the gram-negative isolates had a drug resistant nature. Further bacteriophages isolated from sewage demonstrated perfect lytic activity against the multi-drug resistant bacteria causing septic wounds. In vitro analysis of the isolated bacteriophages demonstrated perfect lysis against the corresponding MDR-bacteria, and these isolated phages may be promising as a first choice for prophylaxis against wound sepsis, Moreover, phage therapy does not enhance multi-drug resistance in bacteria and could work simultaneously on a wide variety of MDR-bacteria when used in a bacteriophage cocktail. Hence, our results suggest that these bacteriophages could be potential therapeutic options for treating septic wounds caused by P. aeruginosa, S. aureus, K. pneumoniae and E. coli.

Molecular docking and simulation of Curcumin with Geranylgeranyl Transferase1 (GGTase1) and Farnesyl Transferase (FTase)

Protein prenylation is a posttranslational modification that is indispensable for translocation of membrane GTPases like Ras, Rho, Ras etc. Proteins of Ras family undergo farnesylation by FTase while Rho family goes through geranylgeranylation by GGTase1. There is only an infinitesimal difference in signal recognition between FTase and GGTase1. FTase inhibitors mostly end up selecting the cells with mutated Ras proteins that have acquired affinity towards GGTase1 in cancer microcosms. Therefore, it is of interest to identify GGTase1 and FTase dual inhibitors using the docking tool AutoDock Vina. Docking data show that curcumin (from turmeric) has higher binding affinity to GGTase1 than that of established peptidomimetic GGTase1 inhibitors (GGTI) such as GGTI-297, GGTI-298, CHEMBL525185. Curcumin also interacts with FTase with binding energy comparable to co-crystalized compound 2-[3-(3-ethyl-1-methyl-2-oxo-azepan-3-yl)-phenoxy]-4-[1-amino-1-(1-methyl-1h-imidizol-5-yl)-ethyl]-benzonitrile (BNE). The docked complex was further simulated for 10 ns using molecular dynamics simulation for stability. Thus, the molecular basis for curcumin binding to GGTase1 and FTase is reported.

Role of STAT3 in Colorectal Cancer Development

Colorectal cancer is the cancer of the colon, located at the lower part of the digestive system. Although the role of STAT3 in cancer is known, its role particularly in colon cancer is largely unknown. STAT3 is a cytoplasmic transcription factor that involves extracellular signaling to the nucleus regulating fundamental functions, like cell proliferation, apoptosis, differentiation, and angiogenesis. STAT3 is a key regulator; abrogated activation leads to several diseases, including cancer. Aberrant interleukin (IL)-6-mediated JAK/STAT3 signaling pathway is closely related to the advancement of several human solid tumors including colorectal cancer. With other upstream regulators, IL-6/JAK signaling can activate STAT3, and its role appears to be critical in various types of cancer. STAT3 has been traditionally recognized as an oncogene; more recently the dual role of STAT3 in cancer, either tumor inductive or suppressive, has been appreciated. This chapter describes the potential role of STAT3 in colon cancer based on in vitro, in vivo, and patient studies. Furthermore, we will discuss the mechanism of action and roles of the IL-6/JAK/STAT3 pathway in colorectal cancer and exploit current therapeutic strategies, to treat colorectal cancer. Understanding the complexity of STAT3 function in colorectal cancer has the potential to elucidate important molecular aspects of colorectal cancer with significant therapeutic implications.

In vitro and in vivo evaluation of anti-cancer activity: Shape-dependent properties of TiO2 nanostructures

Cancer is a complex and widespread disease, and it is going to be the first cause of death in the world. Chemotherapy has been used to treat cancer, but it is detrimental to immune cells and known to induce numerous side effects. Therefore it is imperative to develop new drugs for the treatment of cancer without any side effects and toxicity. TiO2 nanomaterials are human safe, cost effective, chemically stable and have numerous biomedical applications. Spherical TiO2 fine particles (TFP), TiO2 nanosquares (TNS) and TiO2 nanotubes (TNT) were developed and evaluated for anti-cancer activity in vitro and in vivo. Our data suggest that these nanostructured materials significantly inhibited proliferation of breast cancer MDAMB 231 cells in in vitro shape dependent manner. In addition, we found that TiO2 nanostructures inhibited the migration and colony formation of breast cancer MDAMB231 cells. More importantly, we found that TNS/TNT/TFP had anti-angiogenic effect in CAM assay and TNT had comparable anti-angiogenic effect with the positive control staurosporine. Additional qRT-PCR data suggest that TiO2 nanostructures induced the upregulation of tumor suppressor genes p53, MDA7, TRAIL and transcription factor STAT3, which suggests the probable mechanism for the anticancer activity of TiO2 nanostructures. Finally, analysis of TEM confirms the dispersion and interaction of nanostructures in the cells. Thus these materials could be potential therapeutic targets for the treatment of cancer.

Abstract 4873: IKKα bridges central tolerance to innate immunity and inflammation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA IKKα has emerged as a tumor suppressor in squamous cell carcinomas (SCCs) of skin, head and neck, and lung. Our recent studies indicate that inflammation is involved in lung SCCs in kinase-dead knock-in IkkαKA/KA mice. However, how IKKα prevents inflammation has not been fully understood yet. In the present studies, we showed that IkkαKA/KA mice developed reduced thymic medullary regions, systemic inflammation, and severe skin damage, indicating that IKKα plays an essential role linking central tolerance to innate immunity and inflammation. In line with previous studies of the role of NF-κB pathway in thymic medulla formation and the establishment of central tolerance, we found that NF-κB pathway was inactivated in the presence of kinase inactive form of IKKα in thymic epithelial cells. Depleting the lymphocytes, thymus, T cells, macrophages, and/or reintroducing transgenic IKKα into the skin or thymus rescued the severe skin phenotypes. Transferring IkkαKA/KA T cells, but not wild-type T cells, reconstituted the severe skin phenotypes and SCCs in the lungs and forestomach of IkkαKA/KA/Rag-/- mice. We found that the self-reactive T cells provided TGFβ signaling, which enhanced the expression of monocyte chemoattractant protein-1 (MCP-1) in keratinocytes. As a result, increased level of MCP-1 led to robust recruitment of macrophages to the skin, resulting in skin inflammation and hyperproliferation. This study reveals an important IKKα/TGFβ/MCP-1 signaling axis that orchestrates central tolerance and inflammation in the maintenance of tissue homeostasis. Citation Format: Feng Zhu, Zhisong Chen, Jami Willette-Brown, Dakshayani Lomada, Sean R. Davis, Timothy Back, Teizo Yoshimura, Zhonghe Sun, Xiaolin Wu, Robert Wiltrout, Ellen Richie, Ulrich Siebenlist, Giorgio Trichieri, Yinling Hu. IKKα bridges central tolerance to innate immunity and inflammation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4873. doi:10.1158/1538-7445.AM2014-4873