Madhava C. Reddy

C-Phycocyanin, a selective cyclooxygenase-2 inhibitor, induces apoptosis in lipopolysaccharide-stimulated RAW 264.7 macrophages

C-Phycocyanin (C-PC) is one of the major biliproteins of Spirulina platensis, a blue green algae, with antioxidant and radical scavenging properties. It is also known to exhibit anti-inflammatory and anti-cancer properties. However, the mechanism of action of C-PC is not clearly understood. Previously, we have shown that C-PC selectively inhibits cyclooxygenase-2 (COX-2), an inducible isoform that is upregulated during inflammation and cancer. In view of the reported induction of apoptosis in cancer cells by cyclooxygenase-2 inhibitors, the present study is undertaken to test the effect of C-PC on LPS stimulated RAW 264.7 mouse macrophage cell line. These studies have shown a dose dependent reduction in the growth and multiplication of macrophage cell line by C-PC. This decrease in cell number appears to be mediated by C-PC induced apoptosis as evidenced by flow cytometric and confocal microscopic studies. Cells treated with 20 micro M C-PC showed typical nuclear condensation and 16.6% of cells in sub-G(o)/G(1) phase. These cells also showed DNA fragmentation in a dose dependent manner. The studies on poly(ADP ribose) polymerase (PARP) cleavage showed typical fragmentation pattern in C-PC treated cells. This C-PC induced apoptosis in RAW 264.7 cells appears to be mediated by the release of cytochrome c from mitochondria and independent of Bcl-2 expression. These effects of C-PC on RAW 264.7 cells may be due to reduced PGE(2) levels as a result of COX-2 inhibition.

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome.

Sweeten PAMPs: Role of Sugar Complexed PAMPs in Innate Immunity and Vaccine Biology.

Innate sensors play a critical role in the early innate immune responses to invading pathogens through sensing of diverse biochemical signatures also known as pathogen associated molecular patterns (PAMPs). These biochemical signatures primarily consist of a major family of biomolecules such as proteins, lipids, nitrogen bases, and sugar and its complexes, which are distinct from host molecules and exclusively expressed in pathogens and essential to their survival. The family of sensors known as pattern recognition receptors (PRRs) are germ-line encoded, evolutionarily conserved molecules, and consist of Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin-like receptors (CLRs), and DNA sensors. Sensing of PAMP by PRR initiates the cascade of signaling leading to the activation of transcription factors, such as NF-κB and interferon regulatory factors (IRFs), resulting in a variety of cellular responses, including the production of interferons (IFNs) and pro-inflammatory cytokines. In this review, we discuss sensing of different types of glycosylated PAMPs such as β-glucan (a polymeric sugar) or lipopolysaccharides, nucleic acid, and so on (sugar complex PAMPs) by different families of sensors, its role in pathogenesis, and its application in development of potential vaccine and vaccine adjuvants.

Human HMGB1 directly facilitates interactions between nucleotide excision repair proteins on triplex-directed psoralen interstrand crosslinks.

Psoralen is a chemotherapeutic agent that acts by producing DNA interstrand crosslinks (ICLs), which are especially cytotoxic and mutagenic because their complex chemical nature makes them difficult to repair. Proteins from multiple repair pathways, including nucleotide excision repair (NER), are involved in their removal in mammalian cells, but the exact nature of their repair is poorly understood. We have shown previously that HMGB1, a protein involved in chromatin structure, transcriptional regulation, and inflammation, can bind cooperatively to triplex-directed psoralen ICLs with RPA, and that mammalian cells lacking HMGB1 are hypersensitive to psoralen ICLs. However, whether this effect is mediated by a role for HMGB1 in DNA damage recognition is still unknown. Given HMGB1s ability to bind to damaged DNA and its interaction with the RPA protein, we hypothesized that HMGB1 works together with the NER damage recognition proteins to aid in the removal of ICLs. We show here that HMGB1 is capable of binding to triplex-directed psoralen ICLs with the dedicated NER damage recognition complex XPC-RAD23B, as well as XPA-RPA, and that they form a higher-order complex on these lesions. In addition, we demonstrate that HMGB1 interacts with XPC-RAD23B and XPA in the absence of DNA. These findings directly demonstrate interactions between HMGB1 and the NER damage recognition proteins, and suggest that HMGB1 may affect ICL repair by enhancing the interactions between NER damage recognition factors.

Repair of Genome Destabilizing Lesions

Abstract Reddy, M. C. and Vasquez, K. M. Repair of Genome Destabilizing Lesions. Radiat. Res. 164, 345–356 (2005). Living organisms are constantly exposed to detrimental agents both from the environment (e.g. ionizing radiation, ultraviolet light, natural and synthetic chemicals) and from endogenous metabolic processes (e.g. oxidative and hydrolytic reactions), resulting in modifications of proteins, lipids and DNA. Proteins and lipids are degraded and resynthesized, but the DNA is replicated only during cell division, when DNA damage may result in mutation fixation. Thus the DNA damage generated has the potential to lead to carcinogenesis, cell death, or other genetic disorders in the absence of efficient error-free repair. Because modifications in DNA sequence or structure may be incompatible with its essential role in preservation and transmission of genetic information from generation to generation, exquisitely sensitive DNA repair pathways have evolved to maintain genomic stability and cell viability. This review focuses on the repair and processing of genome destabilizing lesions and helical distortions that differ significantly from the canonical B-form DNA in mammalian cells. In particular, we discuss the introduction and processing of site-specific lesions in mammalian cells with an emphasis on psoralen interstrand crosslinks.

Simultaneous Inhibition of T Helper 2 and T Regulatory Cell Differentiation by Small Molecules Enhances Bacillus Calmette-Guerin Vaccine Efficacy against Tuberculosis

Background: Immunological parameters induced by BCG and the requirement of immunologic responses for optimal vaccine efficacy is incompletely understood. Results: Small-molecule inhibitors of Th2 and Treg cells promote BCG vaccine efficacy. Conclusion: Immunomodulators enhance the capacity of the BCG vaccine to protect against tuberculosis. Significance: Our studies reveal a simple and cost-effective approach to improve BCG vaccine efficacy. Tuberculosis affects nine million individuals and kills almost two million people every year. The only vaccine available, Bacillus Calmette-Guerin (BCG), has been used since its inception in 1921. Although BCG induces host-protective T helper 1 (Th1) cell immune responses, which play a central role in host protection, its efficacy is unsatisfactory, suggesting that additional methods to enhance protective immune responses are needed. Recently we have shown that simultaneous inhibition of Th2 cells and Tregs by using the pharmacological inhibitors suplatast tosylate and D4476, respectively, dramatically enhances Mycobacterium tuberculosis clearance and induces superior Th1 responses. Here we show that treatment with these two drugs during BCG vaccination dramatically improves vaccine efficacy. Furthermore, we demonstrate that these drugs induce a shift in the development of T cell memory, favoring central memory T (Tcm) cell responses over effector memory T (Tem) cell responses. Collectively, our findings provide evidence that simultaneous inhibition of Th2 cells and Tregs during BCG vaccination promotes vaccine efficacy.

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.

The Need for Physiologically Relevant Peroxisome Proliferator-Activated Receptor-gamma (PPAR-γ) Ligands

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a nuclear transcription factor which is involved in the differentiation of fibroblasts to adipocytes in vitro. PPAR-γ also plays a pivotal role in inflammation and macrophage activation. Furthermore, type 2 diabetes mellitus (T2DM), a condition in which an individuals ability to respond to insulin is lowered, is treated by drugs called thiazolidinediones (TZDs) that are known to activated PPAR-γ, thus augmenting insulin signaling and glucose uptake by adipose tissue. Unfortunately, these otherwise effective drugs are responsible for side effects such as obesity and cardiovascular diseases. The ligand-binding ability of PPAR-γ is different from other nuclear receptors since it can bind to a wide variety of ligands. Although a number of compounds have been shown to activate PPAR-γ, knowledge of its endogenous ligands and their physiological functions is lacking. The known ligands were either ambiguous or found to produce ill effects in vivo. In this review we discuss the structure and functions of PPAR-γ, ligands discovered so far, and focus on the importance of identification of physiologically relevant endogenous ligands.