Mythily Srinivasan

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https://hdl.handle.net/1805/17678

Targeting Neuroinflammation Using Novel Glucorticoid-Induced Leucine Zipper Analogs

Unresolved inflammation and altered immune signals contribute to the pathology of many chronic diseases. Dr. Srinivasan's research includes the design and development of immunotherapeutic peptides using motif-mediated protein-protein interactions as drug targets.

Recently, she investigated approaches to suppress neuroinflammation in Alzheimer's disease, the most common cause of dementia that is projected to affect over seven million individuals in the United States by 2025. These studies resulted from earlier observations that the blockade of T cell co-receptor CD28 by CD80 competitive antagonist peptides unregulated glucocorticoid-induced leucine zipper (GILZ)-an anti-inflammatory molecule that directly inhibits the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-kB).

Adopting data from the GILZ and NF-kB interaction complex, Dr. Srinivasan developed modified analogs of the p65 binding motif of GIlZ. Efficacy studies, in collaboration with Dr. Debomoy Lahiri, Professor of Neuroscience, and Dr. Deborah Hickman of the IU School of Medicine, suggested that the novel therapeutics exhibited suppressive potential in cellular and animal models of Alzheimer's disease.

Together with the Indiana University Research and Technology Corporation, Dr. Srinivasan initiated a startup venture, Provaidya LLC, to develop the patented GILZ analogs as investigational new drugs and received phase I funding from the National Institutes of Aging.

Dr. Srinivasan's work to develop peptide therapeutics to suppress neuroinflammation is another example of how IUPUI faculty are TRANSLATING RESEARCH INTO PRACTICE.

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Now showing 1 - 10 of 11

Assessment of Salivary Adipokines Resistin, Visfatin, and Ghrelin as Type 2 Diabetes Mellitus Biomarkers
(Hindawi Publishing Corporation, 2018-02-01) Srinivasan, Mythily; Meadows, Melinda L.; Maxwell, Lisa; Oral Pathology, Medicine and Radiology, School of Dentistry
Type 2 diabetes mellitus (T2DM) is emerging as a metabolic epidemic worldwide. Pathologically, dysregulation of many biological pathways precedes hyperglycemia and the clinical diagnosis of T2DM. Changing trajectories along the process of T2DM development necessitates frequent measurement of biomarkers for early identification of at-risk individuals and successful prevention. Increase in circulating inflammatory adipokines has been suggested as predictive of T2DM. Human saliva is an easily accessible biospecimen amenable for painless frequent collection and possesses nearly 50% of serum proteome. In this study, we measured the adipokines resistin, visfatin, TNF-α, and ghrelin as markers for T2DM in unstimulated whole saliva (UWS) using specific assay kits. Resistin and visfatin concentrations were significantly higher in T2DM saliva. Although the concentration of acylated or unacylated ghrelin was lower in diabetic saliva, the decrease was not significant. Since resistin and visfatin are biomarkers integral to T2DM pathology, their salivary assessments may receive clinical acceptance.
Salivary epithelial cells as model to study immune response against cutaneous pathogens
(Wiley, 2014-02) Negrini, Thais C.; Arthur, Rodrigo A.; Waeiss, Robert A.; Carlosa, Iracilda Z.; Srinivasan, Mythily; Oral Pathology, Medicine and Radiology, School of Dentistry
The human skin not only provides passive protection as a physical barrier against external injury, but also mediates active surveillance via epidermal cell surface receptors that recognize and respond to potential invaders. Primary keratinocytes and immortalized cell lines, the commonly used sources to investigate immune responses of cutaneous epithelium are often difficult to obtain and/or potentially exhibit changes in cellular genetic make-up. Here we investigated the possibility of using salivary epithelial cells (SEC) to evaluate the host response to cutaneous microbes. Elevated secretion of IFN-γ and IL-12 was observed in the SEC stimulated with Staphylococcus aureus, a transient pathogen of the skin, as mono species biofilm as compared to SEC stimulated with a commensal microbe, the Staphylococcus epidermidis. Co-culture of the SEC with both microbes as dual species biofilm elicited maximum cytokine response. Stimulation with S. aureus alone but not with S. epidermidis alone induced maximum toll-like receptor-2 (TLR-2) expression in the SEC. Exposure to dual species biofilm induced a sustained upregulation of TLR-2 in the SEC for up to an hour. The data support novel application of the SEC as efficient biospecimen that may be used to investigate personalized response to cutaneous microflora.
Glucocorticoid-Induced Leucine Zipper in Central Nervous System Health and Disease
(Springer, 2016) Srinivasan, Mythily; Lahiri, Debomoy K.; Department of Oral Pathology, Medicine and Radiology, School of Dentistry
The central nervous system (CNS) is a large network of intercommunicating cells that function to maintain tissue health and homeostasis. Considerable evidence suggests that glucocorticoids exert both neuroprotective and neurodegenerative effects on the CNS. Glucocorticoids act by binding two related receptors in the cytoplasm, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). The glucocorticoid receptor complex mediates cellular responses by transactivating target genes and by protein: protein interactions. The paradoxical effects of glucocorticoids on neuronal survival and death have been attributed to the concentration and the ratio of mineralocorticoid to glucocorticoid receptor activation. Glucocorticoid-induced leucine zipper (GILZ) is a recently identified protein transcriptionally upregulated by glucocorticoids. Constitutively, expressed in many tissues including brain, GILZ mediates many of the actions of glucocorticoids. It mimics the anti-inflammatory and anti-proliferative effects of glucocorticoids but exerts differential effects on stem cell differentiation and lineage development. Recent experimental data on the effects of GILZ following induced stress or trauma suggest potential roles in CNS diseases. Here, we provide a short overview of the role of GILZ in CNS health and discuss three potential rationales for the role of GILZ in Alzheimer’s disease pathogenesis.
Epithelial expression of keratinocytes growth factor in oral precancer lesions
(2016) Jimson, Sudha; Murali, S.; Zunt, Susan L.; Goldblatt, Lawrence I.; Srinivasan, Mythily; Department of Oral Pathology, Medicine and Radiology, School of Dentistry
Background: Keratinocyte growth factor (KGF) is a potent epithelial mitogen that acts by binding the KGF receptors (KGFRs) expressed on epithelial cells and regulates proliferation and differentiation. The objective of this study was to investigate the expression of KGF in the epithelium in oral precancer. Materials and Methods: Archival tissues of oral submucous fibrosis (SMF) and leukoplakia were assessed for epithelial KGF expression by immunohistochemistry and real-time quantitative polymerase chain reaction. Results: KGF was predominantly expressed in the basal and parabasal cells in the epithelium of SMF tissues. KGF transcript in the epithelial cells increased with increasing severity of epithelial dysplasia in oral leukoplakia. Conclusion: Although widely reported as a product secreted by the mesenchymal cells, our data suggest that the KGF is also expressed in oral epithelial cells much like the expression in ovarian epithelial cells. Based on the localization of KGF in cells at the epithelial mesenchymal junction and that of the reported presence of KGFR in oral keratinocytes, a potential mechanism involving paracrine and autocrine interactions of KGF and KGFR in early stages of oral precancer is postulated.
Distinct Salivary Biomarker Profile in Chronic Periodontitis
(Office of the Vice Chancellor for Research, 2012-04-13) PRAKASAM, S.; SRINIVASAN, M.
Background: Saliva has potential to diagnose chronic periodontitis (CP). Changes in tissue-expression of pattern-recognition-receptors (PRRs), which recognize periodontal-pathogens, correlate with CP. It follows that PRRs-expression in nucleated-cells (NCs) shed in saliva and soluble-PRRs may differentiate CP from health. Additionally, cytokines in gingival cervical fluid (GCF) correlate with worsening CP, which may be reflected in saliva. One significant test for biomarkers is changes in response to treatment. Objectives: Comparison of CP salivary-biomarkers profile with health and to study treatment effects of scaling and root planning (SRP). Methods: Unstimulated whole saliva (UWS) collection and recording of routine clinical periodontal parameters was done for two groups (n=16): healthy (H) (minimal clinical loss of attachment (CAL) and clinical inflammation) and CP (≥30% sites with ≥4mm CAL). UWS was collected at 3 different time points: before, 1-week and 6-weeks after SRP from the CP group. NCs and clarified saliva (CS) were separated from UWS. Messenger RNA was extracted from NCs and TLR-2 expression was quantitated through real-time-PCR. CS depleted of immunoglobulin and amylase to prevent large molecule interferences and diluted to 1 μg/ml of salivary-protein in PBS, normalize for variations in liquid volume, was used to quantify biomarkers through ELISA. Statistical significance between H- and CP-groups biomarkers was determined through Mann-Whitney ‘U' test and one tailed paired ‘t' test. Results: Statistically significant differences were noted for clinical profiles of H- and CP-groups and for changes after SRP within CP-group. Salivary sTLR-2, IL-17 and IL-10, were significantly higher, and sCD14, IL-6, IL-4 and TLR-2 mRNA were significantly lower in H compared to CP. In CP, salivary sTLR-2 and IL10 increased significantly at 1- and 6-weeks after SRP, whilst IL-4 decreased significantly at 6-weeks. Conclusions: Salivary biomarkers profiles are distinct between health and CP as well as before and after SRP treatment. sTLR-2, IL-10 and IL-4 may serve as short-term biomarkers for monitoring response to SRP. sCD14, TLR2-mRNA and other cytokines need exploration as long-term response biomarkers. Depletion of amylase and immunoglobulin, and normalization for total salivary protein may be important in biomarkers quantification.
Enhancement of Cancer Immunotherapy Using Immune Modulating Peptides
(Office of the Vice Chancellor for Research, 2013-04-05) Chang, Hua-Chen; Han, Ling; Lewis, David; Tung, Chun-Yu; Srinivasan, Mythily; Robertson, Michael J.; Yeh, Wu-Kuang
Immune Peptide Therapeutics (IPT) LLC, an Indiana-based small business and its research partner Indiana University previously identified a novel property of lunasin as a distinct class of immune modulating agent that enhances anti-tumor immunity, which may promote disease-free survival by limiting tumor progression, and thus prolong lives of cancer patients. Lunasin, a synthetic 43-amino acid peptide, was originally isolated from soybeans. Our studies have demonstrated that lunasin exerts robust synergistic effects with cytokines on augmenting IFNγ and granzyme B expression by Natural Killer (NK) cells, which is associated with increased tumoricidal activity of NK cells. In addition, this combination regimen is capable of rescuing IFNγ production ex vivo by NK cells from chemotherapy-treated Non-Hodgkin’s Lymphoma (NHL) patients who are immunocompromised with acquired immune deficiency. The long-term goal is to develop an efficacious immunotherapy which will impact the treatment and improve the clinical outcomes for NHL patients. The dose-response study indicates the optimum concentration of lunasin is at the range of μM, which would undermine its use in clinical studies. To enhance the medicinal value lunasin must be optimized for in vitro and in vivo efficacy. The objective is to develop a second generation of lunasin, which will increase its potency to improve the performance. In this study we have implemented several strategies to design and modify the prototype. The newly developed peptide called IPT.103 has 15 amino acids that are in the D-isoform configuration. Activity of IPT.103 has been tested in vitro with EC50 of 0.78 μM as compared to 4.54 μM for lunasin. IPT.103 also has in vivo activity on enhancing the serum levels of IFNγ production using a mouse model. Taken together, we have developed a peptide derivative (IPT.103) that deviates from its parental type lunasin to increase intellectual merit for commercialization as well as support clinical application.
GILZ-mimics as novel therapeutic agents for progressive multiple sclerosis
(Office of the Vice Chancellor for Research, 2013-04-05) Srinivasan, M.; Dunker, K. A.; Lahiri, Debomoy K.; Pollok, Karen E.
Multiple sclerosis (MS), a leading cause of neurological disability is an inflammatory demyelinating disease of the central nervous system (CNS). The clinical course of MS is highly variable ranging from isolated neurologic episodes to frequently relapsing or progressive disease. Currently there are no effective treatments for progressive MS. The long-term goal of this project is to evaluate a novel therapeutic strategy for progressive MS. Under physiological conditions signaling via the transcription factor, nuclear factor-kappa B (NF-κB) and glucocorticoid (GC) stimulation pathways regulate the immuno-inflammatory responses of the CNS resident glial cells. While NF-κB induces transcriptional activation, signaling via GC receptor functions to suppress immune responses. Persistent activation of NF-κB in the glial cells precipitates neuronal degeneration and axonal loss characteristic of progressive MS. Interactome analysis between the GC and NF-κB pathways suggested a novel strategy to inhibit NF-κB. Glucocorticoid-induced leucine zipper (GILZ) is a GC inducible protein that binds p65, the functionally critical subunit of NF-κB, and prevent transactivation of pathological mediators. The sites of interaction are localized to the proline rich region of the GILZ protein and the p65 transactivation domain. A 23 residue GILZ peptide prevented nuclear translocation of p65 and suppressed disease in an animal model of MS. Structurally GILZ peptide adopted polyproline type II (PPII) helical conformation, a favorable feature for drug development. The objective of this study is to optimize the lead peptide and develop drug like analogs. Specific features of the GILZ-p65 interactions were adapted in the design of over 25 GILZ analogs such that each exhibit optimum PPII helix, bind p65 transactivation domain and potentially accommodate modified residues that enhance the binding specificity with the p65. The analogs were ranked after passing through the Lipinski filter to determine the drug like properties. The top ranked analogs will be evaluated for functional efficacy.
Literature-Based Discovery of Salivary Biomarkers for Type 2 Diabetes Mellitus
(Libertas Academia, 2015-02) Srinivasan, Mythily; Blackburn, Corinne; Mohamed, Mohamed; Sivagami, A. V.; Blum, Janice; Department of Oral Pathology, Medicine and Radiology, Indiana University School of Dentistry
The alarming increase in type 2 diabetes mellitus (T2DM) underscores the need for efficient screening and preventive strategies. Select protein biomarker profiles emerge over time during T2DM development. Periodic evaluation of these markers will increase the predictive ability of diabetes risk scores. Noninvasive methods for frequent measurements of biomarkers are increasingly being investigated. Application of salivary diagnostics has gained importance with the establishment of significant similarities between the salivary and serum proteomes. The objective of this study is to identify T2DM-specific salivary biomarkers by literature-based discovery. A serial interrogation of the PubMed database was performed using MeSH terms of specific T2DM pathological processes in primary and secondary iterations to compile cohorts of T2DM-specific serum markers. Subsequent search consisted of mining for the identified serum markers in human saliva. More than 60% of T2DM-associated serum proteins have been measured in saliva. Nearly half of these proteins have been reported in diabetic saliva. Measurements of salivary lipids and oxidative stress markers that can exhibit correlated saliva plasma ratio could constitute reliable factors for T2DM risk assessment. We conclude that a high percentage of T2DM-associated serum proteins can be measured in saliva, which offers an attractive and economical strategy for T2DM screening.
Soluble toll like receptor 2 (TLR-2) is increased in saliva of children with dental caries
(2014-08) Zhao, Alyssa; Blackburn, Corinne; Chin, Judith; Srinivasan, Mythily
Background Dental caries is the most common microbial disease affecting mankind. Caries risk assessment methods, identification of biomarkers and vaccine development strategies are being emphasized to control the incidence of the largely preventable disease. Pattern recognition receptors such as the toll like receptors (TLR) have been implicated as modulators of host-microbial interactions. Soluble TLR-2 and its co-receptor, CD14 identified in saliva can bind the cell wall components of cariogenic bacteria and modulate the disease process. The objective of this study is to determine the potential of salivary sTLR-2 and sCD14 as biomarkers of caries activity and indirect measures of the cariogenic bacterial burden. Methods Unstimulated whole saliva was collected from twenty caries free and twenty caries active children between the ages of 5 and 13 years. The concentration of sCD14 and sTLR-2 together with that of the cytokine IL-8 reported to be increased in dental caries was assessed by the enzyme linked immunosorbent assay. Results While the level of sCD14 and that of IL-8 was equivocal between the two groups, the sTLR-2 concentration in caries active saliva was significantly higher than that in caries free saliva. Conclusions The sTLR-2 in saliva could serve as a potential biomarker for caries activity.
Modulating NK-mediated Immunity by Lunakine
(Office of the Vice Chancellor for Research, 2014-04-11) Chang, Hua-Chen; Tung, Chun-Yu; Lewis, David; Han, Ling; Srinivasan, Mythily; Robertson, Michael J.; Yeh, Wu-Kuang
Despite the plethora of immune modulating agents available in cancer treatment, their effectiveness relies on a functional immune system. However, the adverse side effects by chemotherapy impede the therapeutic benefits from immunotherapy. It remains a major challenge to prevent relapse for cancer patients who have already undergone rigorous chemotherapy. Lunasin, a 43-amino acid peptide, was originally isolated from soybeans. Our team has recently discovered a novel function of lunasin as an immune modulating agent that exerts robust synergistic effects imposed by several therapeutic cytokines. Such synergism strongly augments IFNγ and granzyme B expression by Natural Killer (NK) cells, which is associated with increased tumoricidal activity. The combination regimen with lunasin and cytokine is capable of restoring NK activation from lymphoma patients with chemotherapy-induced immune dysfunction. Our results support the potential application of lunasin to improve the therapeutic effects of existing cytokine treatment that has been used to eliminate residual tumors cells from lymphoma patients after chemotherapy. We designate lunakine as new formulation by combing lunasin and selected cytokine (filed for US Patent Cooperation Treat). In working with Indiana University and Technology Corporation (IURTC), we have started a startup company, Immune Peptide Therapeutics (IPT), LLC. Our mission is to develop a more efficacious immunotherapy that prevents relapse and confers progression-free survival for cancer patients. With the support from FORCES, our team has successfully developed a second generation of lunasin called IPT.103 that deviates from its parental type. Activity of IPT.103 has been tested in vitro with EC50 of 0.78 μM as compared to 4.54 μM for lunasin, indicating an improved potency to induce IFNγ production by NK cells. The newly developed peptide IPT.103 is expected to strengthen the intellectual property (IP) position for commercialization. We are currently working on tumor models for preclinical assessment of IPT’s regimens in immunotherapy for lymphoma.

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