Browsing by Author "Mathew, Porunelloor A."
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Item A novel ligand on astrocytes interacts with natural cytotoxicity receptor NKp44 regulating immune response mediated by NK cells(PLOS, 2018-02-15) Bowen, Kelly E.; Mathew, Stephen O.; Borgmann, Kathleen; Ghorpade, Anuja; Mathew, Porunelloor A.NK cells play important role in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. NK cells were shown to be recruited to the CNS following several pathological conditions. NK cells could impact CNS physiology by killing glial cells and by secreting IFN-gamma. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders. There is little data on NK-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. Natural cytotoxicity receptors (NCRs) play a critical role in the cytolytic function of NK cells. Among the NCRs, NKp44 is unique in expression and signal transduction. NKp44 is expressed only upon activation of NK cells and it can mediate both activating and inhibitory signals to NK cells. Here, we have studied the expression and function of natural cytotoxicity receptor NKp44 upon NK-astrocytes interactions in the presence or absence of an HIV peptide (HIV-3S peptide) shown to induce NK cell killing of CD4+ T cells during HIV-infection. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with HIV-3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study showed that NKp44 have a protective effect on astrocytes from NK cell mediated killing during HIV infection and impact astrocyte role in HAND.Item Analysis of expression of immune receptors in pediatric acute lymphoblastic leukemia (ALL)(2016-03-23) Mathew, Stephen O.; Mathew, Porunelloor A.; Bowman, Paul; Powers, SheilaAcute Lymphoblastic leukemia (ALL) is the most common type of cancer in children. It is characterized by overgrowth of the lymphocyte precursor (either B cell or T cell) that is nonfunctioning, and crowds out other immune cells. Current treatment options have a success rate of 80-90%. However, those who relapse have a survival rate of only around 25-40%. Also, side effects of current chemotherapy and radiation treatments have been shown to impact the normal growth and development of children. Research has shown that ALL of the B cell lineage is particularly resistant to killing by Natural Killer (NK) cells. NK cells are part of the innate immune system and specialize in killing tumor and virally infected cells. NK cell activation is dependent on a balance of inhibitory and activating receptors and their ligands expressed on the target cell. Our laboratory has previously cloned three immune receptors, 2B4, CS1 and LLT1, which have been shown to play a role in cancer, however their significance and role in childhood ALL have not been evaluated. In this study, we evaluated the expression of immune receptors 2B4, CS1, LLT1, NKp30 and NKp46 in pediatric ALL subjects both male and female in the age range of 3 – 18 yrs. ALL subjects and healthy subjects were enrolled at Cook Children’s Hospital and UNT Health Science Center, Fort Worth, TX with informed consent/assent according to IRB approval (UNTHSC IRB# 2008-094 & CCMC IRB# 2008-57). The blood samples were collected and peripheral blood mononuclear cells (PBMC) were isolated and analyzed by flow cytometry and real-time qPCR for expression of immune receptors. ALL subjects showed altered expression of immune receptors in the PBMC as compared to healthy subjects. There was an overall decrease in the expression of 2B4, CS1, LLT1 and NKp46 in the B lymphoblasts of ALL subjects as compared to healthy subjects. Expression and functional analysis of these receptors in a larger sample size will provide valuable insights to conduct future mechanistic studies to investigate the role of these immune receptors in ALL resistance and relapse. Funded by Cancer Research Foundation of North Texas (CRFNT).Item Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB(2001-11-01) Chang, Woo-Jin; Alvarez, Rafael; Mathew, Porunelloor A.; Goldfarb, Ronald H.Chang, Woo-Jin, Automodification Reaction of PARP-1 Reversibly Regulates the DNA-Binding of NF-kB, Doctor of Philosophy (Microbiology and Immunology), November, 2001, 92 Pages, 20 figures, 3 schemes, and bibliography. Poly(ADP-ribose) polymerase (PARP-1, E.C. 2.4.2.30) is a constitutively expressed nuclear enzyme. It comprises about 1% of the total nuclear protein and in phylogenetically well conserved in most eukaryotes, with a notable exception in yeast. PARP-1 post transitionally modifies DNA-binding proteins by transferring the ADP-ribose moiety from BNAD+. Although the exact biological function of poly(ADP-ribosyl)ation has not been clearly elucidated, the process is thought to be involved in DNA repair, replication, and gene expression. Previous studies have indicated that PARP-1 participates in eukaryotic gene expression including the genes under the control of nuclear factor-kB (NF-kB). It has been demonstrated that PARP-1 deficient mice are more resistant to lipopolysaccharide-induced endotoxic shock than isogenic wild-type mice due to the inactivation of NP-kB in the mutants. In order to further analyze the interactions between PARP-1, NF-kB, and its consensus DNA in a cell-free system, we co-incubated recombinant PARP-1 protein and the p50-subunit of NF-kB (NF-kB-p50) in the absence of DNA strand-breaks. Electrophoretic mobility shift assays (EMSA) showed that sequence-specific DNA-binding of NF-kB-p50 was dependent on autopoly(ADP-ribosyl)ation of PARP-1. The NF-kB-p50 DNA-binding was inhibitied when PARP-1 was not auto-poly(ADP-ribosyl)ated either in the absence of BNAD+ or in the presence of 3-aminobenzamide, an enzymatic inhibitor of PARP-1. Coimmunoprecipation and immunoblot analysis demonstrated that NF-kB-p50 formed a heterodimer with PARP-1 when PARP-1 was not auto-poly(ADP-ribosyl)ated. In addition, poly(ADP-ribosyl)ation assays showed that NF-kB-p50 protein was not susceptible to poly(ADP-ribosyl)ation under normal incubation conditions. Those in vitro observations described above were confirmed by experiments utilizing HeLa nuclear extracts. EMSA showed that NF-kB DNA-binding was inhibited in 3-AB-pre-treated HeLa cells. To our knowledge, this is the first report demonstrating that auto-poly(ADP-ribosyl)ation reaction by PARP-1 reversibly regulates the function of a transcription factor by inhibiting the formation of heterodimer between PARP-1 and a transcription factor.Item Blocking LLT1-CD161 interaction enhances natural killer cell-mediated lysis of triple-negative breast cancer cells(2018-03-14) Mathew, Stephen O.; Chaudhary, Pankaj; Mathew, Porunelloor A.; Marrufo, Armando M.Purpose: Triple-negative breast cancer (TNBC) accounts for 20 percent of all breast cancer cases and is known to be the most invasive form of breast cancer. TNBC’s absence of estrogen, progesterone, and human epidermal growth factor-2 receptors makes utilizing hormonal treatments ineffective in suppressing tumor growth. TNBC is associated with poorer prognosis and higher incidences of relapse. Therefore, natural killer cell-mediated immunotherapy shows potential as a treatment option for TNBC. Natural killer cells (NK) are innate lymphoid cells that serves its role in the immune system to eradicate infected and tumor cells. NK cell function is regulated through its receptors interacting with activating and inhibitory ligands on target cells. Lectin-like Transcript-1 (LLT1, CLEC2D) is a counter-receptor that interacts with CD161 (NKRP1A) and inhibits NK cell activation. Our study demonstrated that by blocking TNBC’s LLT1 interaction with CD161 with antibodies increases lysis of TNBCs by NK cells. Methods: We have identified the expression and function of LLT1 on TNBC cell lines MDA-MB-231 and MDA-MB-436 by flow cytometry, western blot, immunofluorescent microscopy, and chromium-release assay. LLT1 expression at the cell surface was decreased through gene knockdown with small interference RNA (siRNA) transfection. Primary NK cells were isolated from peripheral blood mononuclear cells from healthy individuals and then were co-incubated with chromium-labeled TNBCs for quantification of specific lysis of TNBCs by NK cells. Results: Our results have demonstrated a higher expression of LLT1 on TNBCs than non-tumorigenic breast cell line MCF10A. We have shown that blocking LLT1 interaction with CD161 with antibodies on TNBCs have increased lysis of TNBCs by primary NK cells. We have also shown that gene knockdown of LLT1 decreases cell surface expression of LLT1 on TNBCs and increases lysis of TNBCs by NK cells. Conclusions: LLT1 expressed on TNBCs is a ligand that interacts with NK receptor CD161 and sends an inhibitory signal to the NK cell thus serving its role for TNBCs to evade immunosurveillance. Respectively, blocking LLT1 with antibodies on TNBCs and decreasing expression of LLT1 by gene knockdown increases susceptibility of TNBCs to NK cell-mediated lysis. Blocking interaction between LLT1 and CD161 with antibodies activates lysis by NK cells and will open a possible new immunotherapeutic strategy for patients diagnosed with TNBC.Item Carisoprodol's Pharmacological Properties at GABAA Receptors, In-vitro and In-silico Studies(2021-05) Claudio, Maria del Carmen; Sumien, Nathalie; Huang, Ren-Qi; Gonzales, Eric B.; Dillon, Glenn H.; Mathew, Porunelloor A.Carisoprodol (CSP) is a centrally-acting prescription muscle relaxant that can directly activate, allosterically modulate and inhibit GABAA receptors. The GABAA receptor is a pentameric chloride ion channel in the cys-loop receptor family. The mechanism of GABAA's inhibitory role in the central nervous system lies in the resulting hyperpolarized state of the cell following chloride ion influx upon ligand binding. GABAA receptors are the target of many different clinically prescribed compounds because of the role they play in regulating the central nervous system. We used pharmacological and computational approaches to investigate the underlying mechanism mediating carisoprodols effects at GABAA receptors, with the ultimate goal of generating a new subunit selective compound related to the structure of carisoprodol and gaining a more thorough understanding of the molecular interaction governing carisoprodol's pharmacoglogical effects. Our evaluation of novel compounds related to the structure of carisoprodol did not yield promising leads, though the potential still remains for development of a novel carisoprodol-related compound with a unique selectivity profile. Probe for a binding site mediating carisoprodols postive modulatory effects and evaluation of additional novel compounds was eventually hindered by time. Our investigaiton into carisoprodol's direct gating effects involved a previously reported single amino acid residue, L415, located at the top of the fourth transmembrane domain (TM4) in the ɑ1 subunit of the GABAA receptor that is critical to carisoprodol's direct gating. Whether the residue is involved in a carisoprodol binding site remained unsolved. In studies probing for a binding site for carisprodol's direct activation of GABAA receptors, promising computaitonal docking data was not able to be validated with elecrophysiology and site-directed mutatgenesis studies, indicating that the residues revealed in docking studies do not form a binding pocket for carisprodol's direct activation effect. Our site directed mutagenesis, electrophysiology and molecular dynamic simulation studies to investigate carisoprodol's inhibitory effects at GABAA receptors revealed a binding site at the Cl- channel pore, in a mechanism similar to picrotoxin, providing a mechanism of action for carisoprodol's inhibitory effects at GABAA receptors.Item Cell surface PCNA is a marker of pancreatic and colon cancer stem cells and inhibits NK cell effector function.(2018-03-14) Mathew, Porunelloor A.; Malaer, JosephPurpose: Cancer stem cells (CSC), a unique subset of tumor cells, possess a stem-cell-like phenotype and are thought to facilitate metastasis by escaping NK cell effector function. There are numerous markers used to identify CSC; of which include surface markers CD44 and CD133, and transcription factors NANOG, SOX2, and Oct-4. NK cells participate in the innate immune response against cancer without prior sensitization. NK cell function depends on a balance of signals transmitted from activating and inhibitory receptors interacting with ligands on the surface of target cells. Cancer cells may evade NK-mediated killing by expressing or secreting ligands for NK cell inhibitory receptors. NKp44 can function as an activating receptor that induces NK cell cytotoxicity or an inhibitory receptor depending on ligand interaction. Proliferating cell nuclear antigen (PCNA) associates with Human Leukocyte Antigen I (HLA I) and forms the inhibitory ligand for NKp44, resulting in the inhibition of NK function. We hypothesize surface PCNA is a marker for CSC in pancreatic and colon cancer. Methods: Pancreatic (Panc-1) and colon (HCT 116) cancer cells were labeled with antibodies against PCNA, CD44, and CD133 and flow cytometry was performed to determine surface expression. Cells were labeled and sorted for cell surface PCNA expression via fluorescence activated cell sorting; NANOG, SOX2, and Oct-4 were analyzed by qRT-PCR from sorted cells. NK receptor-ligand interactions were blocked by incubating cells with anti-PCNA or control antibodies and a chromium release killing assay was performed. Results: In both Panc-1 and HCT 116 cells, a PCNA+CD44+CD133+ population was detected. Furthermore, cell sorting and qRT-PCR confirmed cells with cell surface PCNA have increased expression of CSC transcription factors compared to PCNA- cells. Blocking the interaction of NKp44 and PCNA enhanced the specific lysis of cells by primary NK cells. Conclusions: Cell surface PCNA is associated with co-expression of CD44 and CD133 as well as increased CSC transcription factor expression. Collectively these data demonstrate that surface PCNA is a marker of pancreatic and colon CSC. Additionally, cell surface PCNA on CSC facilitate escape from NK cell killing by interacting with NKp44 and transmission of an inhibitory signal. Our research implicates that blocking NKp44-PCNA interaction may provide novel immunotherapeutic targets for pancreatic and colon cancer stem cells and prevent metastasis.Item Characterization of Markers on Pancreatic and Colon Cancer Stem Cells to Enhance Natural Killer Cell Effector Functions(2021-05) Malaer, Joseph D.; Mathew, Porunelloor A.; Mathew, Stephen O.; Berg, Rance E.; Jones, Harlan P.; Prokai, LaszloDue to advancements in technology and medicine, the average human lifespan in the United States has increased to 79 years since the start of the 20th century. This increase in lifespan is also coupled with an increased risk of developing cancer. While cancer detection and initial treatment has increased the survival of patients, it is not a cure and patients wonder how long they will remain in remission. It is estimated that over 90% of cancer related deaths are due to relapse and metastasis. There is currently a growing body of research that indicates cancer stem cells (CSC) are responsible for relapse and metastasis. CSC are a small subpopulation of cells that retain self-renewal and pluripotency. These cells are also described as being quiescent, which may contribute to their chemotherapy resistance. Additionally, CSC are thought to express ligands to aid in immune evasion mechanisms. Natural Killer (NK) cells are lymphocytes of the innate immune system which function to combat infection and cancer. NK cells, unlike T and B cells, do not require prior sensitization. NK cell function is regulated through activating and inhibitory receptors. Recently, Proliferating Cell Nuclear Antigen (PCNA) was identified as an inhibitory ligand for the natural cytotoxicity receptor NKp44. Lectin-like transcript 1 (LLT1) expression is known to facilitate escape of NK cell effector functions in prostate and breast cancer, and now colorectal cancer. In this study we analyzed the expression of molecules on pancreatic and colon cancer cells that could allow escape from NK cell-mediated immune surveillance. The data presented here demonstrates surface PCNA expressing cells as CSC through co-expression of CSC surface markers CD44 and CD133 as well as overexpression of CSC transcription factors NANOG, SOX-2, and Oct-4. Blocking PCNA or NKp44 alters interferon-ℽ secretion by NK cells when cocultured with pancreatic and colon cancer cells. It is further demonstrated that blocking LLT1 or the PCNA-NKp44 interaction led to an increase in specific lysis of tumor cells. This study suggests that cell surface PCNA could function as a biomarker and an immunotherapeutic target for NK cell mediated killing of pancreatic cancer and colon cancer cells.Item Chemotherapeutic drugs increase CS1 (CD319) expression on multiple myeloma cells enhancing NK cell mediated killing(2019-03-05) Malaer, Joseph; Mathew, Porunelloor A.; Sun, YuanhongBackground: Multiple myeloma (MM) is characterized by malignant plasma cells. The clinical diagnosis includes end-organ damage, such as hypercalcemia and renal insufficiency. CS1, a member of the Signaling Lymphocyte Activation Molecule (SLAM) family of receptors, is expressed on natural killer (NK) cells as an activation receptor. CS1-CS1 binding could activate natural killer cell cytotoxicity. In multiple myeloma cells, CS1 is highly expressed and therefore offers a remarkable target for antibody dependent cell-mediate cytotoxicity (ADCC) by NK cells. Elotuzumab (Empliciti), is a humanized monoclonal antibody against CS1. Clinical trials suggest Empliciti combined with chemotherapeutic drugs has increased efficacy than using Empliciti only. In this study we investigated the mechanism of enhanced killing of MM by NK cells. Hypothesis: Chemotherapeutic drugs up-regulate the CS1 expression on multiple myeloma cell surface and enhance NK cell mediated ADCC. Material and Methods: Multiple myeloma cells, NCI-H929 were treated with vehicle control, lenalidomide and anti-CS1 mAb, doxorubicin and dexamethasone, or dexamethasone and anti-CS1 mAb for 24h and 48h. NCI-H929 cells were labeled with antibody to detect CS1 expression on cell surface by flow cytometry. CS1 mRNA expression in multiple myeloma cells were investigated by RT-qPCR. NK cell mediated killing was determined by chromium release assay. Results: Compare with vertical group, CS1 expression levels are increased in all experiment groups, but only doxorubicin and dexamethasone treatment 24h group show significantly increase, same results in anti-CS1 mAb and lenalidomide treatment 48h group. After treating NCI-H929 cell with dexamethasone and anti-CS1 mAb for 24h and 48h, mRNA expression levels are significantly decreased. No statistically variation is observed among the other groups. Natural killer cell killing abilities are significantly improved in chemotherapeutic drugs combination with anti-CS1 mAb treatment groups, no significant increase in doxorubicin and dexamethasone group. Conclusion: In conclusion, our results suggest chemotherapeutic drugs could increase the CS1 expression on NCI-H929 cell. Combining clinical trial observations and our data, it appears chemotherapeutic drugs may increase the anti-mAb treatment efficacy by up-regulating the CS1 expression level in multiple myeloma cells resulting in enhanced NK cell mediated cytotoxicity.Item Clinical Significance of Annexin A2 in Predicting Poor Prognosis in African American Women with Triple-Negative Breast Cancer(2017-05) Gibbs, Lee D.; Vishwanatha, Jamboor K.; Basha, Riyaz; Lovely, Rehana S.; Mathew, Porunelloor A.; Singh, MeharvanTriple-negative breast cancers (TNBC) are identified by the absence of these three major receptors that drive most breast cancer subtypes. TNBC is the most aggressive breast cancer subtype and studies have shown that the incidence of TNBC is much higher in premenopausal African American (AA) women and woman of African descent in comparison to woman of European descent. TNBC in AA women has been associated with worst overall survival after controlling for socioeconomic factors, treatment latency, and tumor receptor expression. This suggests that the clinical outcome of TNBC in AA women may result more from biological differences than access to adequate healthcare. Utilizing a large archived breast cancer cohort of genome sequencing information and the evaluation of these targets in breast tissue and serum can lead to recognition of reliable biological markers that have tremendous potential to enhance detection, treatment, and prognosis. Our previous studies have shown that Annexin A2 (AnxA2), a 36 kda calcium-dependent phospholipid binding protein, is abundantly expressed in TNBC. We have shown AnxA2 to play multiple roles in TNBC by regulating cellular functions; including plasminogen activation, angiogenesis, proliferation, migration, invasion, and metastasis. AnxA2 is one of the most identified proteins expressed in exosomes (small vesicles that are secreted from tumors as metastatic regulators). We have previously demonstrated exosomal AnxA2 contribution to metastasis of TNBC cells in vivo. The proposed study will determine the correlation of AnxA2 with poor prognoses in AA TNBC patients, and establish the clinical significance of exosomal AnxA2 in contributing to the poor clinical outcomes seen in AA TNBC patients. Three specific aims were addressed in this work. Aim 1- Determine the association of secreted exosomal AnxA2 with TNBC amongst AA patients. Aim 2 - Evaluate AnxA2 expression in TNBC tissue samples amongst a breast cancer patient cohort of various breast subtypes. Aim 3 - Determine the correlation of AnxA2 gene expression with poor pathological, prognostic variables and race/ethnicity in TNBC patients through in silico analysis.Item Combined Chemo/Anti-Angiogenic Cancer Therapy in Lewis Lung Metastases(2002-05-01) Sinha-Datta, Anjuli; Goldfarb, Ronald H.; Agarwal, Neeraj; Mathew, Porunelloor A.Datta, Anjuli. Combined Chemo/Anti-Angiogenic Cancer Therapy in Lewis Lung Metastases. Master of Science (Microbiology and Immunology), May 2002. 41 pp., 17 illustrations, bibliography. The focus of my dissertation studies is an eight amino acid peptide (Å6) derived from the non-receptor binding region of urokinase plasminogen activator (uPA), which partially inhibits the binding of uPA to its receptor (uPAR). Å6 has been synthesized as a potential novel anti-cancer agent and kindly provided by Ångstrom Pharmaceuticals, Inc. (San Diego, CA). We further examined potential therapeutic properties of Å6 in vivo and in vitro. Å6 appeared to directly inhibit the invasion of Lewis lung carcinoma cells through Matrigel by approximately 40-45% compared to control. In addition, Å6 had a morphological effect resulting in thicker tubes on small vessel endothelial tube formation compared to no treatment. Interestingly, doxorubicin had similar effects when added to growing endothelial cells. Moreover, Å6 was administered alone and in combination with a standard clinically used chemotherapeutic agent, doxorubicin, in a Lewis lung carcinoma mouse model to test possible synergy between an anti-angiogenic compound (Å6) and a chemotherapeutic agent. This is the first observation that Å6 has the potential to display a direct anti-metastatic therapeutic effect for established pulmonary metastases in this model. Therefore, we believe that Å6 in combination with doxorubicin has the potential to provide better therapy to cancer patients with tumor metastases than potent chemotherapeutics agents alone, by increasing the dose of non-toxic Å6 and reducing the recommended dose of doxorubicin.Item Copper Tolfenamic Acid as a Novel Survivin Inhibitor for Suppressing Pancreatic Cancer Cell Growth via Downregulating Sp1 and Sp3 Transcription Factors(2018-08) Hurtado, Myrna L.; Basu, Alakananda; Lacko, Andras G.; Mathew, Porunelloor A.; Cheng, Yi-Qiang; Ma, RongDespite medical advancements, PaCa unfortunately still remains a lethal malignancy. Patients are typically diagnosed once the cancer is advanced and metastatic, making them ineligible for surgery. This cancer is usually aggressive at the time of diagnosis, so chemo and radiation offer little benefit. Therefore, PaCa urgently requires more effective and sensitizing agents for treatment. Two targets of interest for PaCa have been transcription factors Sp1 and Sp3. Both Sp1 and Sp3 are involved with regulating cell proliferation, differentiation and apoptosis. Their overexpression has been found to contribute to the progression, advancement, and poor prognosis of many types of cancers, including pancreatic. Survivin, an inhibitor of apoptosis protein, is a gene known to be regulated by both these Sp proteins. Survivin has also been found to be overexpressed in various tumor types and adds to the cancer's resistance to cytotoxic therapies. For these reasons, Sp1, Sp3, and survivin have been targets of interest for PaCa and researchers have becoming interested in finding drugs that inhibit their expression. Tolfenamic Acid (TA) is a generic migraine medicine sold in Europe. The small molecule TA has been gaining popularity for its anti-cancer properties such as inhibition of cell growth and induction of apoptosis in various tumor models. TA has been shown to work by downregulation of Sp proteins and survivin. TA has also been demonstrated to sensitize PaCa cells to radiation treatment. Although the results seen with TA thus far seem promising, its IC50 value is slightly high. Recently, it has been suggested that a copper(II) complex of TA (Cu-TA) can produce a higher therapeutic response; however, its efficacy has yet to be tested in gastro-intestinal cancers. Here, we used human PaCa cell lines (MIA PaCa-2 and Panc1) to evaluate the therapeutic efficacy of Cu-TA in an effort to increase TA's efficacy. This project contains three specific aims. Aim 1: Characterization of Cu-TA and determining its stability and anti-proliferative activity in human pancreatic cancer cell lines. Aim 2: Evaluate the effect of Cu- TA on potential markers associated with PaCa cell growth. Aim 3: Use gene expression analysis to precisely elucidate the underlying mechanisms of Cu-TA's anticancer activity in PaCa cells. Cu-TA was found to have an anti-proliferative effect in PaCa cells and its IC50 value was half that of TA's. Characterization of Cu-TA demonstrated its stability as a compound and its biological stability. Importantly, treatment of Cu-TA on cardiomyocytes did not affect cell viability. This is significant since NSAIDs can potentially cause cardiotoxicity. Cu-TA also downregulates expression of Sp proteins and survivin, molecular markers involved with PaCa growth and progression. Additionally, Cu-TA induces apoptosis and cell cycle arrest in PaCa. Finally, RNA sequencing and subsequent pathway analysis of treatment revealed Cu-TA affects pathways involved with cancer progression and metastasis. Thus, these results demonstrate Cu-TA as a potential anti-cancer agent for PaCa.Item Differential Expression of LLT1, SLAM Receptors CS1 and 2B4 and NCR Receptors NKp46 and NKp30 in Pediatric Acute Lymphoblastic Leukemia (ALL)(MDPI, 2023-02-26) Powers, Sheila B.; Ahmed, Nourhan G.; Jose, Roslin; Brezgiel, Marissa; Aryal, Subhash; Bowman, W. Paul; Mathew, Porunelloor A.; Mathew, Stephen O.Acute lymphoblastic leukemia (ALL) represents the most common pediatric cancer. Most patients (85%) develop B-cell ALL; however, T-cell ALL tends to be more aggressive. We have previously identified 2B4 (SLAMF4), CS1 (SLAMF7) and LLT1 (CLEC2D) that can activate or inhibit NK cells upon the interaction with their ligands. In this study, the expression of 2B4, CS1, LLT1, NKp30 and NKp46 was determined. The expression profiles of these immune receptors were analyzed in the peripheral blood mononuclear cells of B-ALL and T-ALL subjects by single-cell RNA sequencing data obtained from the St. Jude PeCan data portal that showed increased expression of LLT1 in B-ALL and T-ALL subjects. Whole blood was collected from 42 pediatric ALL subjects at diagnosis and post-induction chemotherapy and 20 healthy subjects, and expression was determined at the mRNA and cell surface protein level. A significant increase in cell surface LLT1 expression in T cells, monocytes and NK cells was observed. Increased expression of CS1 and NKp46 was observed on monocytes of ALL subjects at diagnosis. A decrease of LLT1, 2B4, CS1 and NKp46 on T cells of ALL subjects was also observed post-induction chemotherapy. Furthermore, mRNA data showed altered expression of receptors in ALL subjects pre- and post-induction chemotherapy treatment. The results indicate that the differential expression of the receptors/ligand may play a role in the T-cell- and NK-cell-mediated immune surveillance of pediatric ALL.Item Evaluation of NK Cell – Astrocyte Interactions: Potential Role in HIV-Associated Neurocognitive Disorders and HIV- Associated Dementia(2015-05-01) Bowen, Kelly E.; Mathew, Porunelloor A.; Mathew, Stephen O.; Hodge, Lisa M.NK cells play important roles in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. During pathological conditions, NK cells were shown to be recruited to the CNS and could impact CNS physiology by killing glial cells and by secreting IFN-g. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders (HAND). There is little data on NK cell-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. This study aimed to identify NK-associated ligands expressed by human astrocytes that confer this NK-directed cytotoxicity of astrocytes and assay the cytotoxicity differences in presence and absence of HIV 3S peptide. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with 3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study demonstrated that NKp44 has a protective effect on astrocytes from NK cell mediated killing during HIV infection. Astrocytes could also secrete cytokines that affect the expression of NK receptors on NK cells. We evaluated the expression of receptors on NK cells after co-culture with astrocytes. CD38 expression was increased on primary NK cells after incubation with astrocytes. CD38 is expressed on both NK cells and astrocytes and has an important implication in HIV-1 infection. Blocking CD38 signaling in our studies decreased astrocyte lysis, suggesting CD38 signaling has important implications in NK-astrocyte interactions. Future studies providing novel insights into the role of NK cells in the pathogenesis of HAND and other brain disorders might result in the development of NK cell based therapies for brain pathologies.Item Expression and Function of Ligands for Natural Killer Cell Receptors on Triple-negative Breast Cancer Cells(2018-08) Marrufo, Armando M.; Mathew, Porunelloor A.; Mathew, Stephen O.; Jones, Harlan P.; Phillips, Nicole R.Triple-negative breast cancer (TNBC) accounts for 20 percent of all breast cancer cases and is known to be the most invasive form of breast cancer. TNBC's absence of estrogen, progesterone, and human epidermal growth factor-2 receptors makes utilizing hormonal treatments ineffective in suppressing tumor growth. TNBC is associated with poorer prognosis and higher incidences of relapse. Therefore, natural killer cell-mediated immunotherapy shows potential as a treatment option for TNBC. Natural killer cells (NK) are innate lymphoid cells that serves its role in the immune system to eradicate infected and tumor cells. NK cell function is regulated through its receptors interacting with activating and inhibitory ligands on target cells. Lectin-like Transcript-1 (LLT1, CLEC2D) is a ligand that interacts with NKRP1A (CD161) and inhibits NK cell activation. Proliferating Cell Nuclear Antigen (PCNA) is a ligand that interacts with NKp44 and inhibits NK cell activation. We have identified the expression and function of LLT1 and PCNA on TNBC cell lines by flow cytometry, western blot, immunofluorescent microscopy, and chromium-release assay. Our results have demonstrated a higher expression of LLT1 and PCNA on TNBCs than non-tumorigenic breast cell line MCF10A. We have shown that blocking LLT1 interaction with NKRP1A with antibodies and gene knockdown of LLT1, respectively, on TNBCs have increased lysis of TNBCs by primary NK cells. We have also shown that blocking PCNA interaction with NKp44 with antibodies have enhanced killing of TNBCs by NK cells. LLT1 and PCNA expressed on TNBCs sends an inhibitory signal to the NK cell thus serving its role for TNBCs to evade immunosurveillance. Blocking LLT1-NKRP1A or PCNA-NKp44 with antibodies enhances lysis by NK cells and may open a novel immunotherapeutic strategy for patients diagnosed with TNBC.Item Expression of Cancer Stem Cell (CSC) Specific Transcription Factors and Cell Surface PCNA and Their Role in CSC Escape From NK Cell Effector Function(2017-03-14) Horton, Nathan; Mathew, Porunelloor A.; Malaer, JosephPurpose: Natural Killer (NK) cells participate in the innate immune response against cancer and infection without prior sensitization. NK cell function depends on a balance of signals transmitted from activating and inhibitory receptors interacting with ligands on the surface of target cells. Cancer cells may evade NK-mediated killing by expressing or secreting ligands for NK cell inhibitory receptors. The Natural Cytotoxicity Receptor (NCR) family, comprised of NKp30, NKp44, and NKp46, is classically described as a group of activating receptors that induce NK cell activation and cytotoxicity. Notably, NKp44 functions as an activating or inhibitory receptor depending on ligand interaction. Proliferating cell nuclear antigen (PCNA) associates with Human Leukocyte Antigen I (HLA I) and forms the inhibitory ligand for NKp44, resulting in the inhibition of NK function. Cancer stem cells (CSC), a unique subset of tumor cells, possess a stem-cell-like phenotype and are thought to facilitate metastasis by escaping NK cell effector function. Methods: Diffuse B cell lymphoma (DB) cells were labeled and sorted for cell surface PCNA expression via fluorescence activated cell sorting (FACS). Total RNA was isolated, converted to cDNA, and the transcription factors NANOG, SOX2, and Oct-4, which are associated with CSC phenotype, were analyzed by qRT-PCR from sorted PCNA+ and PCNA- cells. NK receptor-ligand interactions were blocked by incubating DB cells with anti-PCNA, anti-NKp44, or control antibodies and a chromium release killing assay was performed. Results: Cell sorting and qRT-PCR confirmed DB cells with cell surface PCNA have increased expression of transcription factors compared to PCNA- cells. Blocking the interaction of NKp44 and PCNA enhanced the killing of DB by NK cells. Conclusions: Cell surface PCNA is associated with increased CSC transcription factor expression. Additionally, cell surface PCNA on CSC may facilitate escape from NK cell killing by interacting with NKp44 and transmission of an inhibitory signal. Characterization of stem cell transcription factors and cell surface PCNA may provide novel immunotherapeutic targets to destroy CSC and thus prevent cancer metastasis.Item Glioblastoma multiforme expresses cell surface PCNA, a potential target for NK cell-mediated immunotherapy.(2022) Cooksey, Luke; Mathew, Porunelloor A.Purpose: Glioblastoma multiforme (GBM) is the most common form of primary brain cancer and carries a dreadful five-year survival rate of 9%. Current treatment options include surgery, chemotherapy, and radiation. Recently, there has been a move to pursue immunotherapy options to improve patient outcomes. These therapies often depend on the identification of molecular markers that are distinctive to the tumor cells. Some markers, such as HER2/Neu and EGFR, are overexpressed on a significant percentage of GBM tumors and are used as targets for immunotherapies. However, to address GBM tumors that do not overexpress HER2/Neu and EGFR, our lab set out to identify novel markers on GBM as future candidates for Natural Killer (NK)cell-mediated immunotherapy. Methods: Previously, our lab demonstrated that Lectin-like Transcript-1 (LLT1), membrane-bound Proliferating Cell Nuclear Antigen (PCNA), NKp44 Ligand (NKp44L) and CS1 (CD319) are targets of NK cell-mediated killing of various cancers. Based on these prior studies, we examined their expression on the well-known GBM cell lines LN-229 and LN-18 by flow cytometry using PE-labeled antibodies specific for each marker. Results: PCNA was the lone marker of our panel identified to be highly expressed on both LN-229 and LN-18 cells. Conclusions: Based on our results, we concluded that cell surface PCNA is an ideal candidate for NK cell-mediated immunotherapy for GBM. Currently, we are evaluating blocking inhibitory signals to NK cells from the PCNA-NKp44 interaction to target GBM for NK-mediated killing. Recent findings demonstrating the ability to transiently open the blood-brain barrier further increase the feasibility of targeting GBM by NK cells with monoclonal antibodies in the future.Item IDENTIFICATION & CHARACTERIZATION OF A MELANOMA SPECIFIC LIGAND FOR NK CELL RECEPTOR 2B4(2014-03) Bowen, Kelly E.; Mathew, Porunelloor A.Purpose (a): Natural Killer (NK) cells are lymphocytes that play a vital role in defense against cancer and infections. NK cell function is regulated by a balance between activating & inhibitory signals transmitted through NK cell surface receptors upon interaction with their ligands. Identification of NK cell receptors and their ligands on tumor cells allow targeted therapy for the specific tumor. CD48 is the only known natural ligand for NK receptor 2B4. Previous studies indicate an in vivo role of 2B4 in B16 melanoma tumor rejection. B16 melanoma cells do not express CD48. We hypothesize that 2B4 interacts with a melanoma specific ligand and that this interaction regulates NK cell function. Methods (b): We generated m2B4-Fc fusion protein, consisting of the extracellular domain of mouse 2B4 and human IgG1 Fc portion. To produce the soluble m2B4-Fc, the p2B4-Fc plasmid was transiently transfected into HEK cells. The purified protein was tested for binding by flow cytometry to mouse CD48 of P815 cells, which express CD48, and for binding to mouse 2B4 melanoma cells, which do not express CD48. The melanoma specific ligand was isolated by immunoprecipitation. Results (c): We show that NK cell receptor 2B4 binds to a melanoma specific ligand on B16 melanoma cells. Conclusions (d): Identification and molecular characterization of the melanoma specific ligand for 2B4 will help in developing new strategies to target melanoma with NK cells.Item Immune and Inflammatory Responses Differ Between the Upper and Lower Respiratory Tract(2001-05-01) Hodge, Lisa M.; Simecka, Jerry; Goldfarb, Ronald H.; Mathew, Porunelloor A.The purpose of these studies was to evaluate the role of upper and lower respiratory immune responses during immunization against respiratory disease antigens, and to characterize which immune responses during immunization against respiratory disease antigens, and to characterize which immune responses contribute to protection in the respiratory tract during infection. After nasal immunization, antigen-specific IgA antibody forming cells dominated throughout the respiratory tract. However, IgG responses were significant in lungs, but not in nasal passages. Furthermore, parental immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions developed within the lungs of mice, but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising immunogenicity. Serum IgE responses were also enhanced in a dose dependent manner by inclusion of CT. During infection, mRNA expression for IL-4 was greater in the nasal passages, while both mRNAs for IL-4 and IFN-y were increased in the lungs. As well, we found increased mycoplasma organisms in the lungs of IFN-y-/- mice, suggesting a protective role for cell-mediated immunity in the lung. In contrast, IL-4-/- mice had greater mycoplasma organisms in the nasal passages, indicating IL-4 responses are crucial for upper respiratory tract protection. Consistent with antigen deposition, nasal inoculation with 10 μl volume of antigen plus CT resulted in significant IgA responses in the nasal passages compared to mice given 24 μl immunizations; however, lower respiratory tract immunizations generated antibody responses in both nasal passages and lungs. In addition, both immunizations resulted in equivalent serum antibody responses. Upper and total respiratory tract immunizations provided protection in the nasal passages when CT was added. However, in the lung, all immunizations resulted in protection against mycoplasma infection, regardless of the inclusion of CT, suggesting a different role for CT as an adjuvant in upper and lower respiratory tract immune protection. In conclusion, we found immune responses generated during immunization and infection are different between the upper and lower respiratory tracts, and the contribution of these responses to clearance of respiratory infection differs.Item Lectin-like transcript 1 as a natural killer cell-mediated immunotherapeutic target for triple negative breast cancer and prostate cancer(OAE Publishing, Inc., 2019-12-17) Sun, Yuanhong; Malaer, Joseph D.; Mathew, Porunelloor A.Breast and prostate cancer are the leading causes of death in females and males, respectively. Triple negative breast cancer (TNBC) does not express the estrogen receptor, progesterone receptor, or human epidermal growth factor receptor 2, resulting in limited treatment options. Androgen deprivation therapy is the standard care for prostate cancer patients; however, metastasis and recurrence are seen in androgen-independent prostate cancer. Both prostate and breast cancer show higher resistance after recurrence and metastasis, which increases the difficulty of treatment. Natural killer (NK) cells play a critical role during innate immunity and tumor recognition and elimination. NK cell function is determined by a delicate balance of inhibitory signals and activation signals received through cell surface receptors. Lectin-like transcript 1 (LLT1, CLEC2D, OCIL) is a ligand of NK cell inhibitory receptor NKRP1A (CD161). Several studies have that reported higher expression of LLT1 is associated with the development of various tumors. Our studies revealed that TNBC and prostate cancer cells express higher levels of LLT1. In the presence of a monoclonal antibody against LLT1, NK cell-mediated killing of TNBC and prostate cancer cells were greatly enhanced. This review highlights the potential that using monoclonal antibodies to block LLT1 - NKRP1A interactions could be an effective immunotherapeutic approach to treat triple negative breast cancer and prostate cancer.Item LLT1-Mediated Immunotherapy for Hepatocellular Carcinoma(2022-08) Allison, Michaela M.; Mathew, Stephen O.; Chaudhary, Pankaj; Mathew, Porunelloor A.; Forster, Michael J.