Browsing by Author "Donkor, Michael"
Now showing 1 - 9 of 9
- Results Per Page
- Sort Options
Item Assessing Changes to the Lower GI Tract Microbiome in Response to Neglect-related Early Life Stress Exposure(2024-03-21) Choe, Jamie; Donkor, Michael; Zhang, Yan; Gorham, Isabelle; Allen, Michael; Phillips, Nicole; Jones, HarlanGrowing evidence supports exposure to early life stress (ELS) is associated with alterations in the developing immune system and increases the risk for chronic health conditions. It is widely understood that alterations to the gut microbiome can occur from exposure to various environmental factors, including diet and stress. Early life malnutrition is a form of neglect-related ELS that refers to states of both under- and over-nutrition in which a child may have insufficient intake of one or more nutrients due to an imbalanced diet. Malnutrition during childhood is a public health concern with significant health ramifications. Recent research shows the gut microbiome is intimately involved with immune system development—especially during early life when the immune system is being trained. In the present study, we use a modified version of the maternal separation with early weaning (MSEW) model to study the impact of physical neglect and malnutrition on the gut microbiome in mice. Conditions of neglect-related stress were simulated based on scheduled dam-pup separation (physical neglect) and a high carbohydrate early-wean diet (malnutrition). C57BL/6J mice were bred in-house and ELS pups were subjected to: (1) daily dam-pup separation on postnatal days (PD) 2-13 and/or (2) early weaning (EW) to a high carbohydrate diet on PD14-21. All tissues and stool samples were collected on PD21 for analysis. Pups exposed to MSEW or EW alone were assessed separately. 16S rRNA gene sequencing revealed the neglect-related ELS condition, as described under the present model, led to significant shifts in the predominate species in the lower GI tract microbial community. ELS-mediated shifts included increased Bacteroides and Enterococcus and were accompanied by decreased Lachnospiraceae. RTqPCR of bilateral adrenal glands revealed gene expression changes in key enzymes for stress response pathways, namely those implicated in the synthesis of adrenal glucocorticoids. These results demonstrate ELS-mediated dysbiosis can be observed at PD21 under the present model. Our findings at the PD21 timepoint reveal acute changes to the gut microbiome in the context of ELS and characterizes the baseline microbial community in the lower GI tract.Item EXOSOME PROFILING OF BRONCHIAL LAVAGE FLUID IN A MOUSE MODEL OF SURGERY RESECTION OF BREAST CANCER WITH LUNG METASTASIS(2024-03-21) Marikh, Morad; Brown, Ainsley; Hall, Courtney; Donkor, Michael; Garlotte, Isabelle; Subasinghe, Kumudu; Elkassih, Omar; Jones, Harlan; Phillips, NicoleThe lung serves as a primary site for breast cancer metastasis, carrying profound implications for patient prognoses. About 60% of people diagnosed with metastatic breast cancer have lesions in either the lungs or the bones, with triple-negative breast cancer (TNBC) more likely than other types of breast cancers to metastasize to the lungs. Although current targeted chemo-radiotherapy and surgery result in higher survivorship, studies have documented that such curative treatments may also increase risk of lung metastasis. To date, the causal factors that mediate metastasis in the context of cancer treatments remain elusive. Our long-term goal is that a deeper understanding of the mechanisms that mediate relocation of breast tumor cells from its primary origin to its distal site (e.g., lung) will reveal novel complementary diagnostic and preventative treatments to improve TNBC survivorship. Exosomes, serving as tiny extracellular vesicles within tumor cells and other cells (e.g., immune cells) release diverse biomolecules have been implicated in tumor pathogenesis. Specifically, miRNAs as cargo within exosomes are known to regulate cellular function. miRNAs are small RNA molecules that can bind to messenger RNA (mRNA) and inhibit protein synthesis or promote mRNA degradation. This regulatory function allows miRNAs to modulate the expression of multiple genes involved in various cellular processes and their dysregulation has been implicated in various diseases, including cancer. The objective of this study was to determine the expression of miRNA-200b-3p and miRNA-141-5p as known regulators of lung cancer are influenced by surgical removal of a primary breast cancer. We hypothesized that miRNA-200b-3p and miRNA-141-5p mRNA expression is increased in response to surgery. Using an established model of breast cancer metastasis, exosomes were isolated from the bronchiole alveolar lavage fluid (BALF) of tumor bearing mice and mice in which primary tumors were resected compared to tumor-free mice. Results demonstrated that miRNA-200b-3p was present in both tumor-bearing and non-tumor-bearing mice. In contrast, miRNA-141-5p was not expressed in tumor-bearing, non-tumor-bearing mice, and naïve mice determined by quantitative reverse transcriptase polymerase chain reaction (qrtPCR). In conclusion, as we navigate the intricacies of miRNA dynamics in the lung microenvironment, future studies will involve broadening the miRNA panel and refining exosome recovery techniques. This strategic evolution aims to enhance sensitivity, facilitating the detection of elusive, tumor-derived exosome miRNAs. All studies have been approved by UNTHSC IACUC, approval number #2018-0031. Acknowledgement: This research is partially supported by a grant from the Cancer Prevention and Research Institute of Texas (Award#: RP210046) to Dr. Jamboor K. Vishwanatha and National Institute of Cancer Research of the Health under Award 1 P20 CA233355-01 (Vishwanatha, Jones-Project 1).Item NASAL NANO-VACCINE PREVENTS PRIMARY BREAST TUMOR FROM MAKING THE LUNG ITS NEW HOME(2022) Donkor, Michael; Jones, HarlanDespite medical advances in the diagnosis and treatment of cancer, metastatic breast cancers remain a leading cause of death in the U.S. Increasingly, novel immune-based treatments which harness the patient's immune system have promise for improving survivorship in metastatic breast cancer patients. Such therapies take advantage of the immune system's natural defense mechanisms to halt progression of breast cancer. This is mainly through the early activation of innate immune cells such as natural killer cells and the subsequent activation of the adaptive immune responses such as T and B lymphocytes which elicits a tumor-specific cytolytic and humoral antibody response, respectively. Researchers have taken advantage of these immune mechanisms of tumor defense as a complementary approach to current radio-chemo treatments, which have shown to be limited by adverse off-target effects on patients. This is particularly problematic for recurrent highly metastatic lung, brain, and bone disease, where the physiological function is a premium. Ongoing research in our laboratory is focused on using nanotechnology to develop immune-based vaccines to target local immune protection against metastatic lung disease. Because the lung is naturally tolerogenic, making it easy for disseminated tumor cells to grow, the expectation is that boosting immune responses at the lungs before seeding tumors from primary organs would mitigate metastasis and reduce mortality risks. Using an experimental murine breast cancer model of metastasis, we sought to examine the effect of intranasal vaccination to induce local and systemic adaptive immune responses as a first step in conceptualizing an immune-based nano-vaccine. We hypothesized that an intranasal vaccine protocol would induce protective lung mucosal immune protection against secondary lung metastasis. Our results demonstrated that intranasal vaccination provides protection against secondary lung metastasis using murine model of experimental lung metastasis. This protection was due to increased accumulation of both CD4+ and CD8+ T cells in the lungs that produced IFN-gamma as shown by flow cytometry and ELISA techniques. Again, our results show that intranasal vaccination produces higher tumor-specific IgG responses across respiratory tissues. These results provide initial findings suggesting the potential for targeted tumor vaccines to produce a local tumor-specific T-cell and antibody response with the potential to prevent tumor metastasis. Future challenge studies using spontaneous model of lung metastasis will test our working hypothesis that intranasal tumor vaccination protects the lung from tumor development in the presence of a primary breast tumor.Item Nasal Tumor Vaccination Protects against Lung Tumor Development by Induction of Resident Effector and Memory Anti-Tumor Immune Responses(MDPI, 2023-02-26) Donkor, Michael; Choe, Jamie Y.; Reid, Danielle; Quinn, Byron; Pulse, Mark; Ranjan, Amalendu P.; Chaudhary, Pankaj; Jones, Harlan P.Lung metastasis is a leading cause of cancer-related deaths. Here, we show that intranasal delivery of our engineered CpG-coated tumor antigen (Tag)-encapsulated nanoparticles (NPs)-nasal nano-vaccine-significantly reduced lung colonization by intravenous challenge of an extra-pulmonary tumor. Protection against tumor-cell lung colonization was linked to the induction of localized mucosal-associated effector and resident memory T cells as well as increased bronchiolar alveolar lavage-fluid IgA and serum IgG antibody responses. The nasal nano-vaccine-induced T-cell-mediated antitumor mucosal immune response was shown to increase tumor-specific production of IFN-gamma and granzyme B by lung-derived CD8(+) T cells. These findings demonstrate that our engineered nasal nano-vaccine has the potential to be used as a prophylactic approach prior to the seeding of tumors in the lungs, and thereby prevent overt lung metastases from existing extra pulmonary tumors.Item OCIMUM TENUIFLORUM DECREASES THE RATE OF GROWTH AND METASTATIC POTENTIAL OF MURINE 4T1 MAMMARY CARCINOMA CELLS(2021) Donkor, Michael; Jones, HarlanCombination therapies involving chemotherapy and radiation, aimed at reducing metastasis and mortality have faced challenges, including limiting the effectiveness of immunotherapy. Ocimum tenuiflorum (O. tenuiflorum) has been used traditionally in Indian culture for the treatment of disease and proven scientifically to have immunomodulatory effects. The purpose of this study was to determine the anti-tumor effect of the natural plant O. tenuiflorum on 4T1 murine mammary carcinoma cells (4T1). Hypothesis: O. tenuiflorum decreases the rate of growth and metastatic potential of 4T1 tumor cells. 4T1 cells were grown in culture medium and exposed to increasing concentrations of O. tenuiflorum. The metastatic potential was determined using the scratch assay technique. We were also interested in the mechanism by which O. tenuiflorum decreased the rate of growth and metastatic potential of 4T1 cells. We determined the changes in the mRNA expression of IL-4R, previously reported to drive breast cancer metastasis following exposure of 4T1 tumor cells O. tenuiflorum. Results: Exposing 4T1 cells to various concentrations of O. tenuiflorum decreased the rate of growth and metastatic potential of 4T1 tumor cells. Also, O. tenuiflorum downregulated the expression of IL-4R by 4T1 tumor cells with increasing concentration. We conclude that O. tenuiflorum has the potential to be used as adjunct treatment in management of breast cancer. Further studies will investigate in-depth the mechanism of O. tenuiflorum's on 4T1 including apoptosis, migration and other molecular mechanism on tumor evasiveness.Item OCIMUM TENUIFLORUM DECREASES THE RATE OF GROWTH AND METASTATIC POTENTIAL OF MURINE 4T1 MAMMARY CARCINOMA CELLS(2020) Moore, Cayla; Quinn, Bryon; Jones, Harlan; Donkor, MichaelCombination therapies involving chemotherapy and radiation, aimed at reducing metastasis and mortality have faced challenges, including limiting the effectiveness of immunotherapy. Ocimum tenuiflorum (O. tenuiflorum) has been used traditionally in Indian culture for the treatment of disease and proven scientifically to have immunomodulatory effects. The purpose of this study was to determine the anti-tumor effect of the natural plant O. tenuiflorum on 4T1 murine mammary carcinoma cells (4T1). Hypothesis: O. tenuiflorum decreases the rate of growth and metastatic potential of 4T1 tumor cells. Method: 4T1 cells were grown in culture medium and exposed to increasing concentrations of O. tenuiflorum. The metastatic potential was determined using the scratch assay technique. We were also interested in the mechanism by which O. tenuiflorum decreased the rate of growth and metastatic potential of 4T1 cells. Therefore, we determined the changes in the mRNA expression of IL-4R, previously reported to drive breast cancer metastasis following exposure of 4T1 tumor cells to O. tenuiflorum. Results: Exposing 4T1 cells to various concentrations of O. tenuiflorum decreased the rate of growth and metastatic potential of 4T1 tumor cells. Also, O. tenuiflorum downregulated the expression of IL-4R by 4T1 tumor cells with increasing concentration. We conclude that O. tenuiflorum has the potential to be used as adjunct treatment in management of breast cancer. Further studies will investigate in-depth the mechanism of O. tenuiflorum on 4T1 including apoptosis, migration and other molecular mechanism on tumor evasiveness.Item Phosphorylated Annexin A2 at Tyrosine 23 Regulates Exosome Release and Biogenesis in Triple Negative Breast Cancer(2022) Desai, Priyanka P.; Tripathi, Amit K.; Donkor, Michael; Thyagarajan, Srikantha; Jones, Harlan; Van Treuren, Timothy; Lampe, Jana B.; Chaudhary, Panka J.; Vishwanatha, JamboorPurpose: Exosomes are highly involved in the progression of diverse diseases. Targeting exosome biogenesis and release is a potential strategy for the treatment of the disease like cancer which urges an improved understanding of the process. During the exosomes biogenesis, invagination of the plasma membranes forms early endosomes which mature into late endosomes and multivesicular bodies. Annexin A2 (AnxA2), a calcium dependent phospholipid binding protein, is one of the cargo proteins which gets uploading into the exosomes and impart aggressive phenotype in triple negative breast cancer (TNBC). The mechanism how AnxA2 uploads the exosomal cargo into the exosomes and releases exosomes in the tumor microenvironment remains to be unidentified. In this study, we have explored the potential mechanism for exosome biogenesis and release to target it in TNBC, which lacks the targeted based therapies. Methods: Plasmids expressing constitutive phosphomimetic (AnxA2-Y23E) and non-phosphomimetic AnxA2 (AnxA2-Y23F) mutant gene were transfected in MDA-MB-231 cells. Exosomes isolated from AnxA2-Y23E and AnxA2-Y23F mutant cells were analyzed for expression of the exosomal cargo proteins and RNAs by Western blot and RT-PCR. The number of exosomes released were analyzed by Nanotrack analysis (NTA). Mutant cells treated with Rapamycin, mTORC1(Mammalian Target of Rapamycin Complex 1) inhibitor, were analyzed for the cargo and exosomal secretion. Mutant cells were injected in nude mice to generate tumors. Serum exosomes were isolated and analyzed for cargo and number of exosome release by NTA. Results: In this study, we found that phosphorylated Annexin A2 at tyrosine 23 increases exosome secretion. It loads proteins like AnxA2, CD9 (Cluster of Differentiation 9), LC3B, and Tsg101(Tumor susceptibility gene 101), and AnxA2 and mTOR mRNA into the exosomes. Moreover, secretion and loading of cargo into the exosomes is regulated by increased phosphorylation of AnxA2 and reduced downstream mTORC1 activity. Conclusions: Phosphorylation of AnxA2 at tyrosine 23 regulates exosome secretion and cargo loading into the exosomes in TNBC.Item Short Peptides based on the conserved regions of MIEN1 protein exhibit anti-cancer activity by targeting the MIEN1 Signaling Pathway(Elsevier B.V., 2024-01-26) Tripathi, Amit K.; Desai, Priyanka P.; Tyagi, Antariksh; Lampe, Jana B.; Srivastava, Yogesh; Donkor, Michael; Jones, Harlan P.; Dzyuba, Sergei V.; Crossley, Eric; Williams, Noelle S.; Vishwanatha, Jamboor K.Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-kappaB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK SCR and RP-7 SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo.Item T cell alterations in a mouse model of neglect-related early life stress(2023) Choe, Jamie Y.; Donkor, Michael; Jones, Harlan P.Exposure to severe or chronically stressful life events during childhood—referred to as early life stress (ELS)—is associated with negative effects on health across the life course. Neglect is a significant source of ELS during childhood and accounts for over 75% of maltreatment-related child abuse in the United States. Animal models of ELS emulate the nature of childhood neglect through scheduled separation. A major challenge in studying the impact of stress on immune competency has been the difficulty of developing a reliable mouse model. The thymus is responsible for producing self-tolerant T cells and is critical for adaptive immunity. Developing T cells can be identified based on CD4/CD8 expression status which corresponds to defined stages in thymocyte maturation. We developed a novel murine model of neglect-related ELS based on the maternal separation with early weaning (MSEW) paradigm featuring scheduled dam-pup separation and early weaning to a high carbohydrate diet. This study investigates the effect of ELS on surface markers for thymic T cell development and distribution. Pups were produced by in-house breeding and subjected to one of two distinct ELS conditions: (1) daily dam-pup separation with early weaning (MSEW) or (2) the early weaning condition alone (EW) at postnatal day (PD) 14. Control pups were maintained on the dam’s milk to the standard wean date (PD21). Tissues were collected at PD21 after euthanasia. Flow cytometry revealed significant differences in the distribution of PD21 thymocytes between the double-positive CD4+CD8+ (DP) and single-positive (SP) compartments. Our data indicate that neglect-related ELS can disrupt the baseline distribution of developing T cells within defined thymic compartments and suggests ELS exposure may have downstream ramifications on T cell immunity.