Microbiology Commons^™

Hypoxia Inducible Factor 1 Alpha (Hif-1a): A Major Regulator Of Placental Development, Renee E. Albers

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Hypoxia-inducible factor-1 alpha (HIF-1a) is a critical component of the cellular oxygen-sensing machinery and is essential for placental formation and embryonic survival. In this study, we promoted prolonged expression of HIF-1a, by using a form that is insensitive to oxygen, denoted as CA-HIF-1a. In order to have continual placental specific expression of the CA-HIF-1a, lentiviral infection was performed on embryos at the blastocyst stage of development and transferred into pseudo-pregnant mothers. Placental analysis was performed via in situ hybridization on embryonic day 14.5 (E14.5) to determine the effects of CA-HIF-1a prolonged expression. Data indicate that prolonged activity of CA-HIF-1a restricted …

Type I Interferon Activation Of Natural Killer (Nk) Cells By Cytomegalovirus (Cmv) And Their Interaction With Dendritic (Dc) And Nkt Cells, Howaida A. Oulad Abdelati

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In our current study the roles of natural killer (NK) cells in regulation of the acute phase of murine cytomegalovirus (MCMV) infection were demonstrated. NK cells utilize perforin and gamma interferon to kill MCMV infected cells. Activation of NK cells is controlled by the balance between inhibitory and activation receptors and modulated by cytokines produced from the infected cells. Type I interferons are produced mainly by plasmacytoid dendritic cells (PDCs). Type I interferons have a substantial role in enhancing NK cells cytotoxic activity and NK cells proliferation and maintain cell survival through STAT mediated signaling pathway. IL-12 produced by dendritic …

Amp-Activated Protein Kinase Knockdown In Labyrinthine Trophoblast Cells Results In Altered Morphology And Function, Erica Ashton Kayleigh Carey

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The placenta is a transient organ that develops upon initiation of pregnancy and is essential for fetal development and survival. The rodent placenta consists of three layers with predominantly analogous cell types in the human placenta. The labyrinth layer, which lies closest to the fetus, facilitates nutrient and waste exchange between mother and baby. Abnormalities of the placenta may occur as a result of cellular stress and have been associated with the pregnancy-associated disorders, intrauterine growth restriction and placental insufficiency. AMP-activated protein kinase (AMPK) is a master sensor of cellular stress and changes in AMPK expression or activation could be …

The Effect Of Hsv-1 Infection On Differentiated And Polarized U937 Cells, Allolo Dreiwish Aldreiwish

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Herpes simplex virus type 1 (HSV-1) challenges the host immune system through several mechanisms (Frey, et al., 2009). In vitro, U937 cells (human macrophage-like precursor cell line) are not susceptible to HSV-1 infection when they are not differentiating (Lopez-Guerrero and Alonso, 1997). Differentiation of these cells resistance can abrogates their resistance to HSV-1 (Tenney and Morahan, 1991). In this study, we examined the effect of HSV-1 infection on differentiated and polarized U937 cells. U937 cells are differentiated to M0 cells.

Then, M0 cells are polarized to distinct phenotypes, M1 or M2. M1 are proinflammatory macrophages while M2 are anti-inflammatory cells. …

Establishment Of A Quiscence Herpes Simplex Type 1 Infection In L929 Fibroblasts And Neuro-2a Cells By A Nucleoside Analogue Acyclovir, Noura Shaklawoon

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Herpes simplex type 1 (HSV-1) quiescent infection was established in L929 cells, murine fibroblasts, and in Neuro-2A (N2A) cells, mouse neuroblastoma, by treating them with the nucleoside analogue acyclovir (ACV) 24 hours before infection. Subsequent release of virus from the non-productive state was accomplished by treating the cells with the histone deacetylase inhibitor trichostatin A (TSA). Treatment of both L929 and N2A cell lines with ACV 24 hours before infection induced protection from HSV-1 cytopathic effect. A quiescent state was confirmed by absence of virus plaques when supernatant fluids from ACV-treated HSV-1 infected cultures were titered on Vero cell monolayers. …

N-Glycosylation, Localization And Trafficking Of Endogenous Nkcc1 In Cos7 Cells, Richa Singh

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The Na+K+2Cl^- co-transporter-1 (NKCC1) effects the electroneutral movement of Na+, K+ and 2Cl^- ions across plasma membranes. NKCC1 is considered a widely distributed, highly N-glycosylated ion transporter involved in the regulation of the intracellular chloride concentration ([Cl^-]i), cell volume and salt secretion. NKCC1 locates in the plasma membrane of all cells studied so far, particularly on the basolateral side of most polarized cells. However, the majority of immunoreactive NKCC1 distributes in intracellular compartments of unknown nature whereas a small proportion of the transporter appears to be located in the plasma membrane. Here, we identified and characterized …

Novel Approaches For The Eradication Of Hiv Latently Infected Cells, Sally Al Ali

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The development of a suitable experimental cell model to study HIV latency in primary cells could have a massive effect on the current approaches to eradicate virus in latently infected cells. The main proposal of this paper is to develop an in vitro HIV cell model that represents HIV latency in vivo, then to create a more effective viral vector in order to target HIV reservoirs. For this goal, a directed evolution method is suggested to be used in order to mutate the AAV cap gene to generate a recombinant AAV vector that is capable of infecting primary resting CD4+ …

Conversion Of The U937 Monocyte Into "Macrophage-Like" Populations Exhibiting M1 Or M2 Characteristics, Bradley M. Sharp

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The U937 cell line is an oncogenic human monocyte cell line. These monocytes have the potential of differentiating into either macrophages or dendritic cells (Lawrence et al., 2011). This differentiation pattern depends on the characteristics of the tissue microenvironment (Kigerl et al., 2009). PMA (Phorbol 12-Myristate 13-Acetate) is a phorbol ester capable of transforming monocytic cells toward the macrophage pathway. Upon treatment with PMA, U937 cells under-go a series of morphological and functional changes. Traditionally monocytic cell lines are used as a model of macrophage function, because current human macrophage cell lines require a T-cell conditioned growth medium and contact …

Hpai H5n1: A Global Pandemic Concern, With Implications For Pandemic Preperation And Public Health Policy, Lauren M. Koontz

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Research on highly pathogenic avian influenza A (HPAI) H5N1 has gained much attention in recent years due to its devastating impact within the bird population. While transmission to humans is rare, infections are fatal in more than half of the cases. One of the main concerns among the scientific community is the ability for this virus to mutate in a way much like seasonal influeanza that would allow it to be transmitted efficiently among the human population. Two researchers were able to separately create mutant isolates of H5N1 influenza A virus that could be transmitted via the respiratory route between …

Establishment Of A Quiescent Infection Of Hsv-1 In L929 Fibroblasts Using A Mitotic Inhibitor And Ifn-Γ, Neelam V. Shinde

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The goal for this study was to determine if a quiescent infection of HSV-1 could be induced in murine fibroblasts L929 by treating them with the anti-mitotic agent 5-fluoro 2'deoxy uridine (FUDR) alone and with interferon-γ. Since neurons are post-mitotic and exhibit a lower metabolic rate than other cells, fibroblasts were treated with FUDR to induce a post-mitotic state. The cell cycle arrest of fibroblasts would decrease the thymidylate metabolism and impair HSV-1 replication. An evaluation of cytopathic effects of FUDR was used to determine the optimal concentration which arrests cell growth and inhibits viral replication. Image J program developed …

Q-Ve-Oph, A Control Caspase Inhibitor For Analyzing Neuronal Death, Rebecca L. Bricker

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Sarin is a neurotoxin that has been used in terrorist attacks in Japan and is a potential bioterrorist weapon. It induces seizures by affecting the regulation of neurotransmitters in the brain. Seizures are directly correlated to neuronal damage. Two types of neuronal damage that can occur are apoptosis and necrosis. One of the main regions of the brain where neuronal death occurs is the hippocampus, which is involved in memory. Victims showed chronic decline of memory loss 3 and 7 years after the Tokyo's terrorist attack incident. There are treatments available that can break down sarin or can block the …

The Effects Of Enteropathogenic And Commensal Escherichia Coli On Tight Junction Permeability, Hilary Kaye Allen

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The intestinal mucosa maintains a barrier between materials from the external environment and the internal environment of the host. Disruption of the gut wall integrity is involved in the development of various intestinal diseases, such as Irritable Bowel Syndrome (IBS) and Crohn's disease. The intestinal mucosa is lined with epithelial cells that are connected by tight junctions, the intercellular junctions that form a selectively permeable barrier between paracellular pathways. Enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), can disrupt the tight junctions of epithelial cells by altering the cellular cytoskeleton or by directly affecting tight junction proteins. Commensal Escherichia coli …

Mhc Class I Expression In Murine Fibroblast And Keratinocyte Cell Lines During The First Twenty-Four Hours Of Infection With Herpes Simplex Virus-1 (Hsv-1), Prasanthi Kumchala

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The hypothesis of this study is: HSV-1 infection of murine fibroblasts and keratinocytes inhibits expression of MHC class I molecules during first 24 hours of infection. IFN-γ pretreatment of fibroblasts protected the cells from virus-induced inhibition of MHC class I expression, but did not protect keratinocytes. Herpesviruses are known for their ability to establish persistent infections. Herpesviruses exert many different ways to suppress host defense mechanisms. One such way is by down regulating expression of the major histocompatibility complex I (MHC I) molecules in infected cells. Epidermal cells such as keratinocytes are the major sites for herpes simplex virus type …

The Effects Of Hsv-1 Challenge On Polarized Murine Macrophages: An In Vitro Model Using The J774a.1 Murine Macrophage Cell Line, Adam Craig Reichard

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In our current study we examined the effects of HSV-1 challenge on J774A.1 macrophages polarized to either a proinflammatory (M1) or anti-inflammatory (M2) phenotype. Polarized J774A.1 macrophages were characterized using CD14-CD86 and SOCS1-SOCS3 expression levels. SOCS proteins are a family of proteins that are capable of inhibiting cytokine-signaling pathways. HSV-1 up regulates expression of SOCS1 protein levels in infected cells, inhibiting the ability of infected cells to produce proinflammatory products (Nowoslawski Akhtar and Benveniste, 2011). This study shows that signals within the microenvironment play a greater role in macrophage polarization, and SOCS1-SOCS3 expression levels, than does HSV-1 challenge. M1 macrophages …

Suppressor Of Cytokine Signaling (Socs) 1 & 3 Expression In Hsv-1- Infected And Interferon-Γ-Treated Neuro-2a Cells, Melinda Jones

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This study examined the effects of HSV-1 infection and IFN-γ treatment on Neuro-2A cells. HSV-1 induces expression of SOCS1 and SOCS3 in infected cells, inhibiting the ability of these cells to produce the pro inflammatory, antiviral cytokine IFN-γ (Nowoslawski and Benveniste, 2011). SOCS1 and SOCS3 levels were determined in IFN-γ-treated cells, virus-infected cells, and cells that were both IFN-γ-treated and virus-infected. Results were compared with untreated, uninfected control cells. Flow cytometry data analysis showed a slight decrease in SOCS1 and SOCS3 protein levels in cells treated with IFN-γ for 6 hours compared to control cells. A significant decrease in SOCS1 …

The Construction Of A Plasmid For Detecting The Pathway Of Arginine Metabolism In Human Macrophages: A Real-Time Assessment Of Macrophage Polarity, Benjamin A. Holmes

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Macrophages are "first responders", innate immune system cells which quickly arrive to a site of infection and injury, consuming cell debris and foreign matter and recruiting other immune system cells to the area. While historically they have been thought to react uniformly to all challenges, the discovery of toll-like receptors has shown that macrophages actually work closely with the adaptive immune system in fine-tuning the immune response. Furthermore, it has recently been discovered that macrophages can become polarized to one of two subtypes-M1 or M2.

M1 macrophages are efficient producers of reactive oxygen species, nitrogen intermediates, and inflammatory cytokines. They …

Utilization Of A Custom-Designed Microbiota Array To Determine The Distal Gut Microbiota Of Healthy Human Adults, Richard Thomas Agans

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The human microbiota is an essential component of human health and disease. It is involved in metabolism of dietary components and is at the forefront of the intestinal immune response. Classical techniques applied to study intestinal microbiota illustrated higher presences of aerobic and facultative-anaerobic bacteria, however; the levels of obligate anaerobes had been underrepresented. Modern technologies based on DNA and RNA analysis have circumvented previous challenges allowing researchers to gain more extensive insight into the complex intestinal environment. In this work, a recently developed Microbiota Array was used to assess intestinal microbiota of 10 healthy adults (age 22-61, ave. 34.3yrs). …

Hsv-1 Infection In Keratinocyte Cell Lines Treated With Mitotic Inhibitors, Asma A. Abbas

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The hypothesis for this research was: Herpes simplex virus type 1 (HSV-1) infection of murine keratinocyte cell lines (HEL-30 and PAM-212) treated with mitotic inhibitors leads to silencing of virus replication. At 0.1 (Multiplicity of infection) MOI, PAM-212 keratinocytes showed some cell lysis and viral plaques were seen in HEL-30 keratinocyte cultures. PAM-212 keratinocytes, infected at 0.01 MOI, were more susceptible to the lytic effect of HSV-1 than were HEL-30 cells. In this study, 5-fluoro-2'-deoxyuridine (FUDR) treatment of both keratinocyte cell lines permitted an increase survival of HSV-1- infected keratinocytes especially for PAM-212 cell line which was further examined for …

Distribution Of Cellular Interferon Beta (Ifn-Β) In Murine Fibroblast Cell Lines Upon Infection Of Hsv-1, Rachael E. Curtis

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The hypothesis for this study was: IFN-β expression differs between murine fibroblasts (A2R1 and L929) of different mouse strains upon infection of HSV-1. Fluorescent microscopy was used to examine localization of IFN-β in the different murine fibroblast and keratinocyte cell lines after 6 hours of infection with herpes simplex virus type-1 (HSV-1). Because keratinocytes cell lines HEL-30 and PAM-212 grew in clusters, staining patterns in individual cells could not be determined. A notable difference in localization of IFN-β immune staining was seen between the two cell lines when infected with HSV-1. The A2R1 cell line showed perinuclear (PN) localization, while …

Targeted Knockdown Of Amp-Activated Protein Kinase Alpha 1 And Alpha 2 Catalytic Subunits, Larissa J. Tangeman

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AMP-activated protein kinase (AMPK) regulates cellular metabolism and promotes ATP production when energy is depleted. Evidence suggests that AMPK may be involved in oxygen sensing by carotid body cells, which are responsible for regulating the breathing rate to maintain proper blood oxygen levels. There are two isoforms of the catalytic alpha subunit, AMPKalpha1 and AMPKalpha2, which could be involved in oxygen sensing. Here, the production of a short hairpin RNA (shRNA) targeting both catalytic isoforms of AMPK in human, mouse, and rat is described. The shRNA causes significant knockdown of both isoforms of AMPK alpha in mouse and human cells …

Strategies For Long-Term Renal Allograft Survival In Iga Nephropathy Patients, Erin E. Lintz

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IgA nephropathy is one of the most common kidney diseases worldwide. Most IgA nephropathy patients will undergo a kidney transplant as a treatment. Treatment currently includes the use of immunosuppressants which are necessary to prevent graft rejection but present harmful side-effects when taken long-term. This review focuses on strategies which have the ability to promote tolerance of both donor and recipient cells within a transplant recipient. Such strategies allow development of mixed chimerism and thereby alleviate the need for long-term immunosuppressant usage in allograft recipients. These strategies could be applied to IgA nephropathy patients to allow kidney allograft acceptance without …

Amino Acid Substitutions Created In Reverse Transcriptase And Their Influence On Hiv-1 Mutation Frequencies, Amani Saud Alhejely

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As research has shown, a high mutation rate occurs during the reverse transcription RT process because HIV reverse transcriptase fails to correct erroneously incorporated nucleotides during the reverse transcription process. Based on various research articles, a series of amino acid substitutions created in RT were collected to find out their influence on virus mutation frequencies. Two tables were used; one for amino acid substitutions created in RT that increased the HIV-1 mutation rate and another for amino acid substitutions created in RT that decreased the HIV-1 mutation rate. The tables show which amino acid substitutions created in RT decreased or …

Developing A Quantitative Pcr Assay For Detecting Viral Vector Shedding From Animals, Swathee Chinnasamy

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Replication deficient viruses have been used widely for replacing, repairing, and deleting target genes. These recombinant viruses are tested on research animals or patients in clinical trials. Although viral vectors distribute in the body, they are also disseminated into the environment through secretion and excretion processes. By studying the extent of shedding, a proper risk assessment can be performed and appropriate biocontainment can be achieved. Adenoviral and lentiviral vectors were produced from commercially available kits. The transgene present in both vector systems was the lacZ reporter gene encoding for β-galactosidase. Primers and probes were designed for the encapsidation region of …

The Im-9 Cell Line: A Model For Evaluating Tcdd-Induced Modulation Of The Polymorphic Human Hs1,2 Enhancer Within The 3' Immunoglobulin Heavy Chain Regulatory Region, Ruth C. Chambers-Turner

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2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a disrupter, of B-cell differentiation, induces binding of the aryl hydrocarbon receptor (AhR) nuclear complex to dioxin responsive elements (DRE) within the mouse immunoglobulin heavy chain regulatory region (3'IgHRR), and produces a marked inhibition of 3'IgHRR activation, IgH expression, and antibody secretion in a well-characterized mouse B-cell line (CH12.LX). The mouse 3'IgHRR consists of at least four enhancers (hs3a; hs1,2; hs3b; hs4), and is highly homologous with the three enhancers (hs3; hs1,2; hs4) of the human 3'IgHRR. A polymorphism of the human hs1,2 enhancer (resulting in varying numbers of tandem repeats containing a DRE and κB site) has …

Rev Interacts With Tubulin Heterodimers To Cause Cell Cycle Defects, Poornima Kotha Lakshmi Narayan

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Rev is a regulatory protein that plays an important role in the replication of HIV virus by post-transcriptionally promoting expression of viral proteins late in infection. Rev expression also slows cell growth, leads to an accumulation of cells in G2/M specifically before the spindle checkpoint, and can produce changes in ploidy. Because Rev is capable of depolymerizing microtubules (MTs) in vitro, possibly by a mechanism shared with Kinesin-13 proteins, themselves potent cellular MT depolymerases, I tested the hypothesis that these cellular defects were due to an interaction between Rev and tubulin.

To this end, Rev and select Rev mutants defective …

Host Defense Mechanisms Against Mycobacterium Tuberculosis, Laurin Nicole Holloway

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Mycobacterium tuberculosis is one of the world's most infectious diseases. Approximately 2 million people die each year from Mycobacterium tuberculosis infection and one-third of the world's population remains infected. For decades research has focused on uncovering the tactics used by Mycobacterium tuberculosis to evade host immune responses and defense mechanisms used to fight tuberculosis infection. The following review focuses on the host defense mechanisms used to combat Mycobacterium tuberculosis with an emphasis placed on the roles that Toll-like receptors (TLRs), T cells (gamma delta, CD4, CD8), and macrophages play in mounting the innate and adaptive immune responses necessary to eradicate …

Viral Vectors In The Research Laboratory: Just How Safe Are They?, Dawn P. Wooley, Kimberly Kay Morris, Robert Mcrae, John C. Trefry

Neuroscience, Cell Biology & Physiology Faculty Publications

No abstract provided.

The Response Of Vascular Dermal Enodethial Cells To House Dust Mite Extracts, Aaron Mathew Newman

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House dust mites are microscopic arthropods that can trigger moderate allergic symptoms such as sneezing, watery eyes, itching, and wheezing in sensitized individuals. People with more serious allergies to house dust mites can develop allergic diseases like atopic dermatitis and asthma. The effects of house dust mites on allergy sufferers make house dust mites and the study of their effects on the human body of great medical and economic importance. A majority of the research that has been done on house dust mite's effects on humans has dealt with the lungs and relevant disease like asthma. Little work has been …

Human Immunodeficiency Virus-1 Productively Infects Mature Terminally Differentiated Eosinophils In Hiv/Aids Patients, Jude N. Atem

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Eosinophils express membrane CD4 protein and can bind HIV-1 glycoprotein (gp) 120. Therefore, eosinophils could serve as host cells for HIV-1 infection in vivo, especially in the late phase of the infection. In culture, HIV-1 infects eosinophil precursors and primary eosinophils. Additionally HIV-1 proviral DNA sequences have been detected in the eosinophils of some HIV-1 positive patients. Since elevated levels of eosinophils occur during HIV-1 infection and parasitic infections, it implies that co-infection of parasites and HIV-1 could cause a much greater increase in the level of potential HIV-1 susceptible eosinophils. Therefore HIV-1 infection of eosinophils could partly explain the …

The Role Of Phospholipase D (Pld) And Grb2 In Chemotaxis, Katie J. Knapek

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Phospholipase D (PLD) is an enzyme that hydrolyzes phosphatidylcholine yielding choline and phosphatidic acid. PLD is activated by mitogens (lead to cell division) and motogens (leading to cell migration). PLD is known to contribute to cellular proliferation and deregulated expression of PLD has been implicated in several human cancers. PLD has been found to play a role in leukocyte chemotaxis and adhesion as studied through the formation of chemokine gradients. We have established a model of cell migration comprising three cell lines: macrophages RAW 264.7 and LR-5 (for innate defense), and fibroblast COS-7 cells (for wound healing). COS-7 cells respond …