Molecular Biology and Genetics

The advancement in combinatory antiretroviral therapy (cART) has granted people with HIV-1 an improved lifespan by decreasing the likelihood of AIDS-defining illnesses. People diagnosed early in their infection and undergo cART can keep the virus suppressed and live as long as their HIV-negative peers. As of 2015, more than 50% of people living with HIV in the United States are aged 50 and older. With improved life expectancy, individuals living with long-term HIV infection exhibit many clinical characteristics commonly observed in aging such as: cardiovascular disease, lung disease, certain cancers, HIV-Associated Neurocognitive Disorders (HAND), and liver disease (including hepatitis B and hepatitis C), among others. Regarding neurocognitive disorders, HIV/AIDS patients seem to have learning deficits and working memory impairment such as easy forgetfulness and slowness in action, difficulties in concentration, planning, and multitasking in the condition of having a relatively uneventful and well-controlled clinical course with low HIV viral titers. Neuropsychological studies have disclosed cognitive impairment in a substantial (15–50%) proportion of patients, including learning and working memory deficits which may affect their quality of life, adherence to treatment and ultimately result in increased comorbidity. Studies have described cAMP responsive-element binding (CREB)-1 protein - involved in mitochondrial biogenesis, long-term memory and synaptic plasticity - as a key player in protecting neurons and preventing neurodegeneration. However, loss of CREB protein expression and phosphorylation leads to the development of neurocognitive impairments such as learning deficit and working memory alteration. CREB performs its functions by regulating several genes such, PGC-1 and BDNF being the few (key regulators of mitochondrial bioenergetics, synaptic plasticity and long-term memory, respectively). In here, we have shown that exposure of neuronal cells and animals to HIV-1 gp120 protein decreases expression level of phosphorylated CREB and inhibits its function. Therefore, it will lead to altered neuronal communication and mitochondrial functions. Using pharmacological reagent – rolipram (activates cAMP by inhibiting PDE-4), we were able to reverse the effect of Gp120. Rolipram treatment restored CREB expression and functions altered by gp120. During my graduate years, using in vitro and in vivo studies, I was able to determine the mechanisms used by gp120 leading to the loss of; i – energy metabolism; ii – synaptic plasticity; and iii – long-term memory. We partially determined the relation between gp120, CREB and downstream targets of CREB (PGC-1α and/or BDNF). This approach allowed me to further understand the relation between gp120 and mitochondria as well as between gp120 and neuronal communication. Overall, our study has marked a milestone with regards to understanding the role of gp120 and learning deficiency. Our study mechanistically unravels for the first time the…

Microbiology and Immunology

COPD is characterized by an abnormal regulatory T cell (Treg) response with a shift towards a Th1 and Th17 cell responses. However, it is unclear if the function of Treg cells is impaired by smoking and in COPD. In addition, the miRNA profile of Treg cells in COPD is unknown and whether miRNA deregulation contributes to COPD immunopathogenesis. We set the objective to study Treg cell function isolated from peripheral blood of patients with COPD versus controls and to compare their miRNA profiles. We also were interested in exploring the function of some of the differentially expressed Treg cell miRNAs. We assessed the Treg cell function by observing their suppressive activity on autologous effector T cells and analyzed their miRNA expression initially by microarray analysis then conducted real time RT-PCR validation for selected miRNAs. In Silico target gene analysis for the validated miRNAs suggested that miR-199-5p is particularly relevant to Treg cell physiology so its function was investigated further using CCD-986Sk and MOLT-4 cells. We found no difference in Treg cell function between COPD and controls but we were able to identify 6 and 96 miRNAs that were differentially expressed in COPD versus control Treg cells. We confirmed that miR-199a-5p was repressed by approximately 4 fold in Treg cells of COPD patients compared to cells in healthy smokers. Importantly, miR-199a-5p had significant overrepresentation of its target genes in the Treg cell transcriptome, with many targets associated with the TGF-b activation pathway. We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan® arrays of the Human TGF-b Pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favor of a Th1 and Th17 response.

Temple University – Theses

Molecular Biology and Genetics

Opioid receptors are members of the superfamily of seven transmembrane G protein-coupled receptors (GPCRs) which share several structural and functional characteristics. There are 3 subtypes of opioid receptors, designated μ (MOR), δ (DOR), and κ (KOR) opioid receptors, have been found in the immune, nervous, gastrointestinal and other tissues. We have attempted to clarify the nature of MOR-induced signal transduction pathways in leukocytes. We found that the activation of MOR leads to a significant induction of ERK phosphorylation in peripheral blood mononuclear cells from normal donors using the MOR-selective agonist DAMGO. We are also interested in determining the role of this signaling pathway in the regulation of the immune response. Recent experiments using selective inhibitors suggests that the activation of ERK involves a pathway composed of Raf, Ras, and MEK1/2 kinases, but is independent of PI3 kinase. After treatment of multiple protein kinase inhibitors we found the PKC inhibitor Go-6983 and PLC inhibitor U73122 could also inhibit ERK phosphorylation in MOR stable line (HEK-MOR). According to the results from the Go-6983 and H-89 inhibitor treatment experiments, we found PKCμ/PKD1, a family member of Protein Kinase D, may be involved in MOR-induced ERK phosphorylation. We also found PKCμ/PKD1 S916 phosphorylation after MOR activation and the PKCμ specific inhibitor CID755673 inhibited the MOR-mediated ERK activation. ERK phosphorylation activated several transcription factors in human monocytes, the activation of transcription factors has been proved to induce miRNA expression. We have initiated a series of experiments to study the regulation of miRNA expression by MOR in human monocytes. We found miR-21, miR-155, miR-29a, miR-20b expression were significantly up-regulated following morphine treatment, and morphine-induced miR-21 expression is down-regulated following pretreatment with the ERK inhibitor U0126 and PKD inhibitor CID755673 in human primary monocytes. The results suggest that morphine-induced MOR activation results in up-regulation of miRNA expression human monocytes and this may regulate monocyte and/or macrophage function thought PKCμ/Ras/Raf/ERK signaling pathway.

Temple University – Theses

Molecular Biology and Genetics

The isocitrate dehydrogenase (IDH) family of enzymes is central to cellular metabolism, catalyzing the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and production of NADPH. Recently, cancer-associated heterozygous somatic mutations in family members IDH1 and IDH2 were discovered and present predominantly in glioblastoma multiforme and acute myeloid leukemia. The major consequence of these point mutations is the biochemical production of the ‘oncometabolite’ (R)-2-hydroxyglutarate [(R)-2-HG] from α-KG. Recent studies indicate that unalike mutations of IDH2 produce different intracellular levels of (R)-2-HG, suggesting that IDH2 mutations may differentially influence tumorigenesis. Contrasting clinical studies find IDH mutations to be associated either with increased metastatic potential, or higher survival and enhanced chemosensitivity probabilities. Nevertheless, these studies failed to indicate specifically which of the various IDH mutations were found in each of the patients’ tumors. This raises important questions as to whether specific IDH mutations contribute differently to tumorigenesis. In this study, specific IDH2 mutations were evaluated and compared for their chemosensitivity, tumorigenic activity, and production of (R)-2-HG- all notable determinants for their potential use as tumor biomarkers. Three individual clinically relevant IDH2 mutations (IDH2-R172K, -R172M, and -R140Q) or IDH2-WT were expressed in human U87MG glioblastoma cells. We observed distinct changes in cell morphology, proliferation, migration, invasion, anchorage-independent growth and response to chemotherapeutic agents. Differences in base-line activation of various stress pathways were also observed, lending a plausible explanation to the differing phenotypic outcomes. Interestingly, the variable levels of endogenous (R)-2-HG produced by the cell panel inversely correlated with their respective growth rates, implicating (R)-2-HG as a negative regulator of tumor growth. Indeed, treatment of tumor cell lines, expressing IDH2-WT, with exogenous (R)-2-HG induced a decrease in cell proliferation in a dose-dependent manner. When tested in vivo, treatment of tumor-bearing mice with (R)-2-HG significantly reduced tumor volume. These in vivo results complement the results of soft-agar colony forming assays, demonstrating again that (R)-2-HG inhibits tumor growth. In contrast, immortalized cells subjected to long-term (R)-2-HG treatment showed enhanced cell proliferation. Their response to (R)-2-HG, however, could be switched from growth promotion to that of growth inhibition through expression of oncogenic Ras. Thus, these findings demonstrate conclusively that IDH2 mutations are not alike and that oncometabolite (R)-2-HG plays dual roles in tumorigenesis.

Temple University – Theses

Microbiology and Immunology

Cannabinoids are known to have anti-inflammatory and immunomodulatory properties. Cannabinoid receptor 2 (CB2) is expressed mainly on leukocytes and is the receptor implicated in mediating many of the effects of cannabinoids on immune processes. The capacity of delta-9-tetrahydrocannabinol (delta-9-THC) and of two CB2-selective agonists to inhibit the murine Mixed Lymphocyte Reaction (MLR), an in vitro correlate of graft rejection following skin and organ transplantation was tested. Both CB2-selective agonists and delta-9-THC significantly suppressed the MLR in a dose dependent fashion. The inhibition was via CB2, as suppression could be blocked by pretreatment with a CB2- selective antagonist, but not by a CB1 antagonist, and none of the compounds suppressed the MLR when splenocytes from CB2 deficient mice were used. The CB2 agonists were shown to act directly on T-cells, as exposure of CD3+ cells to these compounds completely inhibited their action in a reconstituted MLR and proliferation of purified T-cells by anti-CD3 and anti-CD28 antibodies was inhibited. Treatment of both CD4+ and CD8+ T-cells with a CB2-selective agonist inhibited the MLR, though significantly less than when both cell types were treated. T-cell function was decreased by CB2 agonists, as an ELISA of MLR culture supernatants revealed IL-2 release was significantly reduced in the cannabinoid treated cells. Further, treatment with O-1966 dose- dependently decreased levels of the active nuclear forms of the transcription factors NF- kappa-B and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures, generated using a PCR T-cell activation array, showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. An increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures and pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-dependent decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. The ability of O-1966 treatment to block rejection of skin grafts in vivo was also tested. Mice received skin grafts from a histoincompatible donor, and the time to graft rejection was analyzed. Compared to mice that received the vehicle, mice that received O-1966 treatment had significantly prolonged graft survival and increased Tregs in the spleen. The spleen cells from O-1966-treated mice had reduced proliferation in an MLR and an increased percentage of Tregs. Together, these data support the potential of this class of compounds as useful therapies to prolong graft survival in transplant patients and possibly as a new class of…

Physiology

Several studies have reported that administration of cannabinoid receptor agonists in inflammatory/autoimmune and CNS injury models resulted in significant attenuation of clinical disease. The beneficial effects correlated with the observed reduction of inflammatory mediators and peripheral immune cell infiltration into the site of inflammation. Previous studies from our laboratories demonstrated that administration of cannabinoid type 2 receptor agonist attenuated disease score and improved recovery in two murine models of neuroinflammation; spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. The goal of the current investigation was to evaluate the mechanisms through which administration of selective cannabinoid-2 receptor (CB2R) agonists modify inflammatory responses and help to improve function in SCI and EAE. In SCI, an acute neuroinflammatory disorder, administration of CB2R agonist at 1 h and 24 h following contusion injury to the cord resulted in improved recovery of motor function and bladder function (the ability to spontaneously void) compared to control animals. Evaluation of inflammatory mediators at 48h demonstrated a dramatic reduction in the expression of the chemokines CXCL9, 10, 11 and cytokines IL-23 and its receptor in CB2R agonist-treated cords. There was also a reduction in the expression of toll-like receptors (TLR1, TLR4, TLR6, and TLR7), which correlated with a decreased number of immunoreactive microglia. Interestingly, at seven days post injury, CB2R agonist-treated injured cords showed a significant reduction in both hematopoietic and myeloid cell infiltration. In EAE, a chronic neuroinflammatory disorder, our laboratories demonstrated previously that administration of a CB2R agonist led to lower disease scores and improved recovery. In this study, we observed reduced numbers of infiltrating hematopoietic and myeloid cells into the spinal cord and brain of CB2 agonist-treated mice. This reduction was observed at the peak of disease (day 17) and the effect was maintained at the chronic stage of disease (day 30). Evaluation of molecules associated with cell migration showed decreased levels of the adhesion molecule VCAM-1 and matrix metalloproteinases MMP-2 and 9 at peak of EAE in treated mice. The decrease in VCAM-1 correlates with our previous observation of decreased leukocyte rolling and adhesion to brain microvasculature. However, the reduction in MMP-2/9 expression suggests that CB2R agonists may also affect leukocyte transmigration into the perivascular space and further infiltration into the CNS parenchyma. This process requires both chemokine cues and the gelatinases MMP2/9. Animals deficient in these MMPs show leukocyte accumulation in the perivascular space and are resistant to EAE. There are no reports in the literature on possible CB2R agonist effects on gelatinases in myeloid cells. Although both MMP-2 and -9 are produced by antigen-presenting cells and act on similar substrates, MMP-9 appears to play…

Molecular Biology and Genetics

Opioid receptor modulation of pro-inflammatory cytokine production is vital for host defense and the inflammatory response. Previous results have shown the mu-opioid receptor (MOR) selective agonist, DAMGO, has the capacity to increase the expression of the pro-inflammatory chemokines, CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 in peripheral blood mononuclear cells (PBMCs). We have shown that MOR activation is able to induce the expression of TGF-β, and TGF-β appears to be required for induction of CCL5 following MOR activation. This work suggests a novel role for TGF-β in the inflammatory response. NF-κB is a transcription factor that plays a pivotal role in inflammation and the immune response. We have found that NF-kB inhibitors can prevent the MOR-induced activation of CCL2 and CCL5, and that the NF-kB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to μ-opioid receptor activation. In vivo, DAMGO administration can induce binding of p. 65 to the enhancer region of the CCL2 promoter. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation and, is essential for NF-kB activity as well as CCL2 expression and transcriptional activity. In conclusion, these data suggest a pro-inflammatory role for MOR which involves NF-κB activation and PKCζ as well as a novel role for TGF-β as a regulator of pro-inflammatory chemokines.

Temple University – Theses

Molecular Biology and Genetics

Opioid receptor-mediated regulation of chemokine receptors is vital for the host immune response, development, and neurological function. Previous studies have demonstrated that the kappa opioid receptor (KOR) activation results in decreased infectivity of human immunodeficiency virus 1 (HIV-1) in human peripheral blood mononuclear cells (PBMCs). We have found this effect is due to down-regulation of the major HIV-1 co-receptors, CCR5 and CXCR4. Using molecular techniques, CCR5 and CXCR4 mRNA levels drop dramatically following KOR activation. To dissect the mechanism involved, we used transcription factor binding arrays and compared control cell extracts to KOR activated cell extracts. We determined that the interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) could be involved in the KOR-mediated repression of CCR5 and CXCR4 transcription and protein expression. Using chemical inhibitors and small interfering RNA (siRNA) molecules, we determined that JAK2, STAT3, and IRF2 are critical members of this signal transduction pathway. The understanding of these particular mechanisms should prove to be beneficial for the development of potential pharmacological agents targeted at HIV-1 binding and infection since virus infection requires expression of the co-receptors CXCR4 and CCR5. Understanding the molecular basis for KOR-induced inhibition of co-receptor expression may provide a basis for the development of KOR agonist-based therapeutics to treat individuals infected with HIV.

Temple University – Theses