Microbiology Commons^™

Virus Production And Cell Viability Of Hsv-1-Infected Murine Keratinocytes (Hel-30) Co-Cultured With Murine Macrophages (Raw 264.7), Barry Graffagna

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Keratinocytes are the most abundant type of cell in the outer layer of skin, the epidermis, and provide barrier against pathogens from invading. However, Herpes Simplex Virus Type 1 (HSV-1) targets these keratinocytes for infection, and later infects neurons to establish lifelong latency. The keratinocytes stimulate the innate immune system to engage and to destroy the virus. Among the cells of the innate immune system to respond to the viral invasion is the macrophage. In this study, RAW 264.7 macrophage and HEL-30 keratinocyte monolayers were challenged in vitro with HSV-1 at a multiplicity of infection (MOI) of 0.1 to investigate …

Indian Research Output In Immunology And Microbiology 2012-2016: A Scientometric Study, Jyoti Rana Ph.D. Scholar, Rakesh Kumar Bhatt Associate Professor

Library Philosophy and Practice (e-journal)

Background: The study examines India’s research productivity in immunology and microbiology during 2012-2016, depending on various parameters, including India’s annual average research growth rate, institutional output profile of institutions and profiles of some of the most productive authors.

Aim: The focus of this study is to analyze performance of India’s research output in immunology and microbiology, the quality and productivity of major institutions participating in research in microbiology and immunology and the productivity and quality of leading authors in research in immunology and microbiology.

Methods: The study in the area of immunology and microbiology using 5 years publications data from …

The Effect Of Gram-Positive Staphylococcus Aureus Cell Wall Components Lipoteichoic Acid And Peptidoglycan On Cytokine Production, Cytoskeletal Arrangement, And Cell Viability On Raw 264.7 Murine Macrophages, Gabrielle String

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In this study, gram positive Staphylococcus aureus cell wall components such as lipoteichoic acid (LTA) and peptidoglycan (PGN) were used to study the potential inflammatory and anti-inflammatory cytokine response, cytoskeletal arrangement and cell viability on RAW264.7 murine macrophages over 24 hours. The effect of S.aureus LTA and PGN (5 µg/mL) on RAW 264.7 macrophages was evaluated every six hours for twenty-four hours. Inflammatory cytokine (TNF-a) production peaked at 6 hours before decreasing over time. Anti-inflammatory cytokine (IL-10) production peaked between 12 and 18 hours. During the first twenty-four hours, cytotoxicity of treated macrophages, as defined as the release of lactate …

Characterization Of A Putative Hemolysin Expressed By Sneathia Amnii, A Preterm Birth-Associated Pathogen, Lizette Carrasco, Kimberly Jefferson

Undergraduate Research Posters

The gram-negative bacteria Sneathia amnii is a poorly-characterized commensal of the female urogenital tract frequently associated with adverse clinical outcomes such as bacterial vaginosis (BV), amnionitis, and preterm labor. To investigate its potential role in virulence, we sought to identify and characterize virulence determinants produced by S. amnii in an effort to better understand the pathogenesis of infectious preterm birth. Through sequencing of the Sn35 genome (type strain of S. amnii), we identified two genes with amino acid sequence similarity and structural similarity to the filamentous hemagglutinin (FHA) protein of Bordetella pertussis and its Type Vb transporter. Because S. amnii …

The Effect Of Gram-Positive Staphylococcus Aureus Cell Wall Components Lipoteichoic Acid And Peptidoglycan On Cytokine Production, Cytoskeletal Arrangement, And Cell Viability On Raw 264.7 Murine Macrophages, Gabrielle String

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In this study, gram positive Staphylococcus aureus cell wall components such as lipoteichoic acid (LTA) and peptidoglycan (PGN) were used to study the potential inflammatory and anti-inflammatory cytokine response, cytoskeletal arrangement and cell viability on RAW264.7 murine macrophages over 24 hours. The effect of S.aureus LTA and PGN (5 [mu]g/mL) on RAW 264.7 macrophages was evaluated every six hours for twenty-four hours. Inflammatory cytokine (TNF-a) production peaked at 6 hours before decreasing over time. Anti-inflammatory cytokine (IL-10) production peaked between 12 and 18 hours. During the first twenty-four hours, cytotoxicity of treated macrophages, as defined as the release of lactate …

The Impact Of Socs1 And Socs3 Peptide Mimetics On Rho And Cdc42 Proteins Expression, F-Actin Cytoskeleton Rearrangements, And Cytokines Production Of Uninfected And Hsv-1 Infected M1 And M2 Raw 264.7 Murine Macrophages, Maha A. Elwardany

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The immune system plays an essential role in the pathogenesis of HSV-1 during the lytic phase of the disease, orchestrating the inflammatory response, retaining the virus in its latent phase and preventing the recurrence of HSV-1 infection. Macrophages display a vital role in the innate and adaptive immune responses during multiple phases of HSV-1 infection. Polarized macrophages are categorized into two distinct classes with diverse functions. The classically activated M1 can engulf and destroy the microbial agents, produce proinflammatory cytokines, and participate in the pathogenesis of many inflammatory diseases. The alternatively activated M2 induces anti-inflammatory mediators and stimulates tissue remodeling …

Effect Of Exposure Of Raw264.7 Macrophages To Salmonella Typhimurium Components On Cell Viability, Cytoskeleton Re-Arrangement And Cytokine Secretion, Khalid Abdullah Alyahya

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Innate immune system plays an important role in individual's protection against pathogens and in activation of adaptive immune system. Utilizing RAW 264.7 murine macrophages as an innate immune response representative in this study, we analyzed the effect of invasive pathogen's components (e.g. flagellin) on the arrangement of macrophage's cytoskeleton, on viability of immune cells and on secretion of pro-inflammatory and anti-inflammatory cytokines and on fluorescence intensity of cytoskeleton after rearrangement. Additionally, we studied the similarity and differences between bacterial (Salmonella typhimurium) and synthetic TLR4 agonist (synthetic lipid-A) on viability, fluorescence intensity, cytokine secretion, and cytoskeleton rearrangements. Similarly, we studied the …

The Pmrhfijklm Operon In Yersinia Pseudotuberculosis Enhances Resistance To Ccl28 And Promotes Phagocytic Engulfment By Neutrophils, Lauren Elizabeth Johnson

Theses and Dissertations

Yersinia pseudotuberculosis is a foodborne pathogen that is the ancestral strain to Yersinia pestis, the causative agent of Plague. Y. pseudotuberculosis invades a host through the intestinal epithelium. The bacteria resist mucosal innate immune defenses including antimicrobial chemokines and phagocytic cells, and replicate in local lymph nodes. They cause Tuberculosis-like symptoms, including necrosis of local tissue and granuloma formation. Like all bacteria, Y. pseudotuberculosis has a net negative charge, which contributes to its susceptibility to some cationic antimicrobial peptides. Y. pseudotuberculosis is able to reduce this negative charge by adding 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A portion of lipopolysaccharide. The …

Changes In Cytoskeleton Proteins In Hsv-1 Infection Of J774a.1 Macrophage Phenotype, Riham Abbas Subahi

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The cell cytoskeleton, a unique intracellular matrix found in all eukaryotes, is composed of three main protein structures, microtubules, microlaments and intermediate laments. The cytoskeleton maintains cell shape and internal structures providing mechanical support that facilitates intracellular transport (Parker et al., 2014). Many viruses such as the herpesviruses use the cytoskeleton system of the cell for infectivity (Henry Sum, M. S. 2015). HSV-1 utilizes the cell cytoskeleton in many steps of its life cycle from entry through assembly to egress (Lyman et al., 2008). During infection, HSV-1 viral proteins cause drastic changes and rearrangement of the cellular actin and cell …

Identification Of Protein-Protein Interactions Of Amyotrophic Lateral Sclerosis Associated Protein Tdp-43, Hanoor Sharma

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by progressive degeneration of upper and lower motor neurons in the brain and spinal cord. Multiple mutations are found in some of the proteins associated with ALS, including superoxide dismutase (SOD1), fused in sarcoma (FUS) and trans-activation response DNA-binding protein (TDP-43). TDP-43 is a DNA and RNA binding protein, well conserved, and ubiquitously expressed in all tissues. TDP-43 resides in the nucleus and sometimes shuttles between nucleus and cytoplasm. Mutations in TDP-43 leads to mislocalization of TDP-43 to the cytosol where it was ubiqutinated and hyperphosphsorylated, ultimately leading to neuronal cell …

The Expression Of Aryl Hydrocarbon Receptor In Raw 264.7 Macrophages In The Presence Of Socs1 Peptide And Socs3 Peptide Mimetic And Cells Infected With Hsv-1, Maher Salem Alwethaynani

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Macrophages play a crucial role for our immune system and protect our body from infection. Suppressor of cytokine signaling (SOCS) proteins negatively regulate cytokine receptor and TLRs. The aryl hydrocarbon receptor (AhR) also performs an important role in immunity. This study investigated the changes in expression of AhR in RAW 264.7 macrophage cells after the addition of SOCS1 and SOCS3 peptide mimetics and also examined AhR expression in RAW 264.7 macrophage cells before and after the addition of HSV-1 RAW 264.7 murine macrophage cell lines which are from male BALB/c mice were used in this study. The addition of the …

Cell Viability, Cytoskeleton Organization And Cytokines Secretion Of Raw 264.7 Macrophages Exposed To Gram-Negative Bacterial Components, Ali Awadh Alshehri

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Macrophages play an important role in innate immunity by controlling cellular responses. In this study, the effects of gram-negative bacterial components (Flagellin, lipoprotein, lipopolysaccharides (LPS), outer membrane proteins-A (OMP-A) and peptidoglycan) were determined on cell viability, morphology, cytoskeletal filament and cytokines secretion of murine RAW 264.7 macrophages at 24 hours. The effect of LPS, flagellin and peptidoglycan from gram negative bacteria on viability murine RAW 264.7 macrophages were evaluated using different concentrations (1, 5 and 10 µg/ml). Cells stimulated with LPS displayed ~ 2-fold decrease (P=0.001) in cell viability compared to control cells at 24 hours whereas cells stimulated with …

Species Differences In The Effects Of Tcdd On A Transcriptional Regulatory Region Within The Ig Heavy Chain Gene, Zahra J. Alfaheeda

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The immunoglobulin heavy chain gene (Igh) is regulated by numerous regulatory elements including the 3'Igh regulatory region (3'IghRR). Several transcription factors are involved in modulating the 3'IghRR including NF-B, AP-1, and the aryl hydrocarbon receptor (AhR). The AhR is a ligand-activated transcription factor that mediates the transcription of genes involved in the metabolism of environmental toxicants such as TCDD. TCDD binds AhR and regulates immunoglobulin (Ig) expression in B cells of animal models. This modulation appears to be directly mediated by binding of the AhR to dioxin response elements (DRE) within the 3'IghRR. Structural differences have been found between the …

Selective Involvement Of The Checkpoint Regulator Vista In Suppression Of B-Cell, But Not T-Cell, Responsiveness By Monocytic Myeloid-Derived Suppressor Cells From Mice Infected With An Immunodeficiency-Causing Retrovirus, Kathy A. Green, Li Wang, Randolph J. Noelle, William R. Green

Dartmouth Scholarship

Inhibition of T-cell responses in tumor microenvironments by myeloid-derived suppressor cells (MDSCs) is widely accepted. We demonstrated augmentation of monocytic MDSCs whose suppression of not only T-cell, but also B-cell, responsiveness paralleled the immunodeficiency during LP-BM5 retrovirus infection. MDSCs inhibited T cells by inducible nitric oxide synthase (iNOS)/nitric oxide (NO), but uniquely, inhibition of B cells was ~50% dependent each on iNOS/NO and the MDSC-expressed negative-checkpoint regulator VISTA. Blockade with a combination of iNOS/NO and VISTA caused additive or synergistic abrogation of MDSC-mediated suppression of B-cell responsiveness.

An Examination Of Eisenia Fetida Coelomic Fluid For Antimycobacterial Activity, Christopher William Meiers

All Graduate Theses, Dissertations, and Other Capstone Projects

Antibiotic resistance is a growing problem with real world consequences to human health and no known solution. The genus Mycobacterium contains several bacteria that can cause serious illnesses including tuberculosis and Johne’s disease. Some of these pathogens also have resistance to several antibiotics. This project sought to find antimycobacterial activity related to the phagocytic coelomocytes or soluble substances in the coelomic fluid of the earthworm Eisenia fetida. The phagocytic activity was determined by observing the adherence of phagocytes to Mycobacterium smegmatis (strain mc2155) bacteria. The antimycobacterial ability of the coelomic fluid was assessed by comparison of viable plate counts after …

Mcl1 Enhances The Survival Of Cd8+ Memory T Cells After Viral Infection, Jingang Gui, Zhuting Hu, Ching-Yi Tsai, Tian Ma, Yan Song, Amanda Morales, Li-Hao Huang, Ethan Dmitrovsky, Ruth Craig, Edward Usherwood

Dartmouth Scholarship

Viral infection results in the generation of massive numbers of activated effector CD8+ T cells that recognize viral components. Most of these are short-lived effector T cells (SLECs) that die after clearance of the virus. However, a small proportion of this population survives and forms antigen-specific memory precursor effector cells (MPECs), which ultimately develop into memory cells. These can participate in a recall response upon reexposure to antigen even at protracted times postinfection. Here, antiapoptotic myeloid cell leukemia 1 (MCL1) was found to prolong survival upon T cell stimulation, and mice expressing human MCL1 as a transgene exhibited a skewing …

The Impact Of Hsv-1 Infection On Cell Viability, Morphology, And Cd Markers Expression By Unpolarized And Cytokine-Polarized J774a.1 Mouse Macrophages, Sarah Saad Alsharif

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Macrophages play an important role in the immune system, particularly in neutralizing pathogens via phagocytosis and the production of multiple cytokines and chemokines that control infection after exposure to specific stimuli. Macrophages exhibit two different phenotypes, M1 and M2. This study evaluated the role of Suppressors of cytokine signaling (SOCS)1 and SOCS3 on Herpes Simplex Virus (HSV-1) infection of polarized macrophages, cell viability, cell morphology, and the expression of cell surface CD markers. I hypothesized that J774A.1 murine macrophages cells in the naive state (M0), and M1 and M2 phenotypes would display differences in CD markers CD80,CD163, and CD200R, cell …

The Use Of Antibody-Coated Latex Beads To Determine Single Positive And Double Positive Mouse Spleen Cells Expressing Cd5 And/Or Cd19 Glycoproteins, Abdulrazzag Abdulaziz Othman

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Flow cytometry is the standard method used to diagnose, stage, and monitor patients' response to the treatment given by counting the numbers of CD5, CD19 and CD5+ CD19+ B lymphocytes. In this study, a comparison was done between numbers of single CD5+, single CD19+ and dual CD5+ CD19+ mouse spleen B lymphocytes using flow cytometry and antibody-latex beads. The bead method involved antibody-coated latex bead and yielded results similar to those of flow cytometry. For cells exhibiting both markers (CD5+ CD19+), the bead method used antibody-coated beads of two different colors yielded similar results to those of flow cytometry results. …

Intrinsic Innate Immunity Fails To Control Herpes Simplex Virus And Vesicular Stomatitis Virus Replication In Sensory Neurons And Fibroblasts, Pamela C. Rosato, David A. Leib

Dartmouth Scholarship

Herpes simplex virus 1 (HSV-1) establishes lifelong latent infections in the sensory neurons of the trigeminal ganglia (TG), wherein it retains the capacity to reactivate. The interferon (IFN)-driven antiviral response is critical for the control of HSV-1 acute replication. We therefore sought to further investigate this response in TG neurons cultured from adult mice deficient in a variety of IFN signaling components. Parallel experiments were also performed in fibroblasts isolated concurrently. We showed that HSV-1 replication was comparable in wild-type (WT) and IFN signaling-deficient neurons and fibroblasts. Unexpectedly, a similar pattern was observed for the IFN-sensitive vesicular stomatitis virus (VSV). …

Socs1/Socs3 Expression And Virus Replication Of Denv2 And Hsv-1 In Cytokine-Polarized Subsets Of Raw 264.7 Macrophages, Nagarjuna Reddy Cheemarla

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Cytokine activation of macrophages leads to "macrophage polarizatio" The M1 subset is polarized by exposure to lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) and M2 polarized cells develop after exposure to interleukin-4 (IL-4) or IL-13. In this study, the RAW 264.7 murine macrophage cell line was used to study the effect herpes simplex virus-Type I (HSV-1) and Dengue virus serotype-2 (DENV2) infections have on cultures of unpolarized (M0) or polarized subsets. The macrophage subsets were characterized using cluster of differentiation markers CD14 and CD86. Uninfected M1 macrophages showed up regulation in expression of the co-stimulatory factor B7.2 (CD86) compared to expression by …

Comparison Between Flow Cytometry And Bead Method In Counting Cd4 And Cd8 T Lymphocytes In Mouse Spleen Cells Suspension, Abdulrahman Abdalla Allabidi

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The flow cytometry is gold standard method to count CD4, CD8 human T lymphocytes, and calculate CD8 to CD4 ratio in HIV patients peripheral blood to stage them and monitor their response to treatment. In this study, a comparison was made by counting CD4and CD8 mouse spleen T lymphocytes by flow cytometry, and a method involving antibody coated latex beads. The bead method yielded results comparable to those obtained by flow cytometry. These results indicate that antibody coated beads are suitable to determine CD4 and CD8 + T lymphocytes counts in situation where flow cytometry is not readily available.

Expression Of The Alpha, Beta, And Gamma Subunits Of The Interleukin-2 Receptor By Human Vascular Smooth Muscle Cells, Sultan Mohammed Alhayyani

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Interleukin 2 (IL-2) is a member of the cytokine family and contributes to the proliferation, survival, and death of lymphocytes [1]. The interleukin-2 receptor (IL-2) is a tripartite receptor commonly expressed on the surfaces of many lymphoid cells and is composed of three non-covalently associated subunits, alpha (CD25), beta (CD122), and gamma (CD132) [2]. Our laboratory has previously described IL-2 receptor Beta expression by vasculature smooth muscle cells (VSMC) in mice and humans [3]. The current work expands our previous observations by assessing the expression of the alpha and gamma subunits of the IL-2R by VSMCs. Analysis of IL-2R expression …

Glut 4 And Insulin Resistance, Salmin Ali

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Five types of glucose transporters stimulate the transport of glucose from the bloodstream into cells such as muscle cells and adipocytes in response to insulin signaling or exercise. Skeletal muscle and adipose tissue are the main sites for glucose uptake and dispose in insulin action dependent pathway. In non-insulin stimulated condition, the GLUT4 storage vesicles stay away from the plasma membrane, so there is no GLUT4 translocation or GLUT4 mediated glucose uptake. However, in insulin stimulated state, insulin initiates insulin signaling cascade which results in rapid GLUT4 translocation and increased glucose uptake at the cell surface. Failure of the translocation …

Characterizing Dsrna Production In Virus-Infected Fish Cells, Amal Brek Aloufi

Theses and Dissertations (Comprehensive)

Viral dsRNA is produced by almost all viruses sometime during their replicative cycle. These viral nucleic acids are potent inducers of both innate and adaptive immune responses, and are therefore considered important immuno-modulators. Previous studies have shown that viruses produce dsRNA when replicating in mammalian cells; however, to date no one has demonstrated viral dsRNA production in virus infected fish cells. Therefore, the goal of this study is to investigate dsRNA production by fish viruses in fish cells, verifying production and performing initial characterization of the dsRNA molecules being produced. Three different rainbow trout cell lines were used in this …

Divergent Antibody Subclass And Specificity Profiles But Not Protective Hla-B Alleles Are Associated With Variable Antibody Effector Function Among Hiv-1 Controllers, Jennifer I. Lai, Anna F. Licht, Anne-Sophie Dugast, Todd Suscovich, Ickwon Choi, Chris Bailey-Kellogg, Galit Alter, Margaret E. Ackerman

Dartmouth Scholarship

Understanding the coordination between humoral and cellular immune responses may be the key to developing protective vaccines, and because genetic studies of long-term HIV-1 nonprogressors have associated specific HLA-B alleles with spontaneous control of viral replication, this subject group presents an opportunity to investigate relationships between arms of the adaptive immune system. Given evidence suggesting that cellular immunity may play a role in viral suppression, we sought to determine whether and how the humoral immune response might vary among controllers. Significantly, Fc-mediated antibody effector functions have likewise been associated with durable viral control. In this study, we compared the effector …

Use Of Irf-3 And/Or Irf-7 Knockout Mice To Study Viral Pathogenesis: Lessons From A Murine Retrovirus-Induced Aids Model, Megan A. O'Connor, William R. Green

Dartmouth Scholarship

Interferon regulatory factor (IRF) regulation of the type I interferon response has not been extensively explored in murine retroviral infections. IRF-3(-/-) and select IRF-3/7(-/-) mice were resistant to LP-BM5-induced pathogenesis. However, further analyses strongly suggested that resistance could be attributed to strain 129-specific contamination of the known retrovirus resistance gene Fv1. Therefore, caution should be taken when interpreting phenotypes observed in these knockout mice, as strain 129-derived genetic polymorphisms may explain observed differences.

Nuclear Bmp2 And The Immune Response, Daniel S. Olsen

Theses and Dissertations

Nuclear bone morphogenetic protein 2 (nBMP2) is a nuclear variant of the secreted growth factor BMP2. Experiments in nBmp2NLStm mutant mice, which lack nBMP2 in the nucleus, have shown that nBMP2 affects intracellular calcium transport in skeletal muscle and hippocampal neurons. The objective of this study was to determine whether nBMP2 affects the immune system, since activation of lymphocytes and other immune cells depends on intracellular calcium transport. We found that spleens in nBmp2NLStm mutant mice were 24% smaller than in wild type mice. The white pulp of the spleen contains many immune cells, particularly B and T lymphocytes and …

Cd4 And Cd8 T Cells Directly Recognize Murine Gammaherpesvirus 68-Immortalized Cells And Prevent Tumor Outgrowth, Xiaozhan Liang, Rebecca L. Crepeau, Weijun Zhang, Samuel H. Speck, Edward J. Usherwood

Dartmouth Scholarship

There has been extensive research regarding T cell recognition of Epstein-Barr virus-transformed cells; however, less is known regarding the recognition of B cells immortalized by gamma-2 herpesviruses. Here we show that B cells immortalized by murine gammaherpesvirus 68 (MHV-68, γHV-68) can be controlled by either CD4 or CD8 T cells in vivo. We present evidence for the direct recognition of infected B cells by CD4 and CD8 T cells. These data will help in the development of immunotherapeutic approaches combating gamma-2 herpesvirus-related disease.

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+ …

Corneal Replication Is An Interferon Response-Independent Bottleneck For Virulence Of Herpes Simplex Virus 1 In The Absence Of Virion Host Shutoff, Tracy J. Pasieka, Vineet D. Menachery, Pamela C. Rosato, David A. Leib

Dartmouth Scholarship

Herpes simplex viruses lacking the virion host shutoff function (Δvhs) are avirulent and hypersensitive to type I and type II interferon (IFN). In this study, we demonstrate that even in the absence of IFN responses in AG129 (IFN-αβγR^−/−) mice, Δvhs remains highly attenuated via corneal infection but is fully virulent via intracranial infection. The data demonstrate that the interferon-independent inherent replication defect of Δvhs has a significant impact upon peripheral replication and neuroinvasion.