Life Sciences Commons^™

Development Of Antigen-Specific Memory Cd8+ T Cells Following Live-Attenuated Chimeric West Nile Virus Vaccination, Heidi Smith, Thomas Monath, Pamela Pazoles, Alan Rothman, Diane Casey, Masanori Terajima, Francis Ennis, Farshad Guirakhoo, Sharone Green

Alan Rothman

ChimeriVax-WN02 is a novel live-attenuated West Nile virus (WNV) vaccine containing modified WNV premembrane (prM) and envelope (E) sequences inserted into the yellow fever 17D vaccine genome. We investigated the induction and evolution of CD8(+) T cell responses to a WNV envelope epitope, which is a dominant target in naturally infected HLA-A*02-positive individuals. WNV epitope-specific CD8(+) T cells were detected by HLA tetramer staining in 22 of 23 donors tested, with peak frequencies occurring between days 14 and 28. WNV epitope-specific T cells evolved from an effector phenotype to a long-lived memory phenotype. In the majority of donors, CD8(+) T …

Cross-Reactive Memory Cd8(+) T Cells Alter The Immune Response To Heterologous Secondary Dengue Virus Infections In Mice In A Sequence-Specific Manner, Coreen Beaumier, Anuja Mathew, Hema Bashyam, Alan Rothman

Alan Rothman

Dengue virus is the causative agent of dengue fever and the more-severe dengue hemorrhagic fever (DHF). Human studies suggest that the increased risk of DHF during secondary infection is due to immunopathology partially mediated by cross-reactive memory T cells from the primary infection. To model T cell responses to sequential infections, we immunized mice with different sequences of dengue virus serotypes and measured the frequency of peptide-specific T cells after infection. The acute response after heterologous secondary infections was enhanced compared with the acute or memory response after primary infection. Also, the hierarchy of epitope-specific responses was influenced by the …

Cytokine Gene Expression And Protein Production In Peripheral Blood Mononuclear Cells Of Children With Acute Dengue Virus Infections, Susan Gagnon, Masuko Mori, Ichiro Kurane, Sharone Green, David Vaughn, Siripen Kalayanarooj, Saroj Suntayakorn, Francis Ennis, Alan Rothman

Alan Rothman

Plasma leakage in dengue hemorrhagic fever (DHF) is associated with elevated plasma levels of cytokines. To define further the contribution of immune activation to DHF and the source of cytokines, we analyzed the production of cytokines in peripheral blood mononuclear cells (PBMC) obtained from children with dengue, using RT-PCR and immunostaining. Tumor necrosis factor-alpha (TNF-alpha) and TNF-beta expression was detected in all samples by PCR and in < 50% of samples by immunostaining. Interferon-gamma (IFN-gamma) expression was detected in < 50% of samples by either method. Interleukin-2 (IL-2) and IL-4 expression was detected in a few samples by immunostaining but was not …

Bystander Target Cell Lysis And Cytokine Production By Dengue Virus-Specific Human Cd4(+) Cytotoxic T-Lymphocyte Clones, Susan Gagnon, Francis Ennis, Alan Rothman

Alan Rothman

Dengue hemorrhagic fever, the severe form of dengue virus infection, is believed to be an immunopathological response to a secondary infection with a heterologous serotype of dengue virus. Dengue virus capsid protein-specific CD4(+) cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells. After activation by anti-CD3 or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Lysis of HepG2 cells suggests a potential role for CD4(+) CTL in the liver involvement observed during dengue virus infection. Three CD4(+) CTL clones …

Dengue Virus-Reactive Cd8+ T Cells Display Quantitative And Qualitative Differences In Their Response To Variant Epitopes Of Heterologous Viral Serotypes, Hema Bashyam, Sharone Green, Alan Rothman

Alan Rothman

Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the …

Development Of Antigen-Specific Memory Cd8+ T Cells Following Live-Attenuated Chimeric West Nile Virus Vaccination, Heidi Smith, Thomas Monath, Pamela Pazoles, Alan Rothman, Diane Casey, Masanori Terajima, Francis Ennis, Farshad Guirakhoo, Sharone Green

Sharone Green

ChimeriVax-WN02 is a novel live-attenuated West Nile virus (WNV) vaccine containing modified WNV premembrane (prM) and envelope (E) sequences inserted into the yellow fever 17D vaccine genome. We investigated the induction and evolution of CD8(+) T cell responses to a WNV envelope epitope, which is a dominant target in naturally infected HLA-A*02-positive individuals. WNV epitope-specific CD8(+) T cells were detected by HLA tetramer staining in 22 of 23 donors tested, with peak frequencies occurring between days 14 and 28. WNV epitope-specific T cells evolved from an effector phenotype to a long-lived memory phenotype. In the majority of donors, CD8(+) T …

Dengue Virus-Reactive Cd8+ T Cells Display Quantitative And Qualitative Differences In Their Response To Variant Epitopes Of Heterologous Viral Serotypes, Hema Bashyam, Sharone Green, Alan Rothman

Sharone Green

Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the …

Cytokine Gene Expression And Protein Production In Peripheral Blood Mononuclear Cells Of Children With Acute Dengue Virus Infections, Susan Gagnon, Masuko Mori, Ichiro Kurane, Sharone Green, David Vaughn, Siripen Kalayanarooj, Saroj Suntayakorn, Francis Ennis, Alan Rothman

Sharone Green

Plasma leakage in dengue hemorrhagic fever (DHF) is associated with elevated plasma levels of cytokines. To define further the contribution of immune activation to DHF and the source of cytokines, we analyzed the production of cytokines in peripheral blood mononuclear cells (PBMC) obtained from children with dengue, using RT-PCR and immunostaining. Tumor necrosis factor-alpha (TNF-alpha) and TNF-beta expression was detected in all samples by PCR and in < 50% of samples by immunostaining. Interferon-gamma (IFN-gamma) expression was detected in < 50% of samples by either method. Interleukin-2 (IL-2) and IL-4 expression was detected in a few samples by immunostaining but was not …

Fatty Acid And Endotoxin Activate Inflammasomes In Mouse Hepatocytes That Release Danger Signals To Stimulate Immune Cells, Timea Csak, Michal Ganz, Justin Pespisa, Karen Kodys, Angela Dolganiuc, Gyongyi Szabo

Gyongyi Szabo

The pathogenesis of nonalcoholic steatohepatitis (NASH) and inflammasome activation involves sequential hits. The inflammasome, which cleaves pro-interleukin-1beta (pro-IL-1beta) into secreted IL-1beta, is induced by endogenous and exogenous danger signals. Lipopolysaccharide (LPS), a toll-like receptor 4 ligand, plays a role in NASH and also activates the inflammasome. In this study, we hypothesized that the inflammasome is activated in NASH by multiple hits involving endogenous and exogenous danger signals. Using mouse models of methionine choline-deficient (MCD) diet-induced NASH and high-fat diet-induced NASH, we found up-regulation of the inflammasome [including NACHT, LRR, and PYD domains-containing protein 3 (NALP3; cryopyrin), apoptosis-associated speck-like CARD-domain containing …

Aberrations In Post-Trauma Monocyte (Mo) Subpopulation: Role In Septic Shock Syndrome, Carol Miller-Graziano, Gyongyi Szabo, Karen Kodys, Katherine Griffey

Gyongyi Szabo

Appearance of increased proportions of monocytes bearing the 72kd(FcRI) receptor for IgG correlated to aberrant monocyte (MO) functions, depressed immune functions, and poor clinical outcome. The trauma patients' FcRI+ MO subpopulation produced the majority of their elevated IL-6, TNF alpha, TGF beta, and PGE2. IgG stimulation of patients' MO through FcRI not only stimulated TNF alpha, IL-6, and PGE2 levels, but also greatly augmented the levels of these monokines produced after subsequent bacterial challenge. Post-trauma increased IL-6 levels can lead to polyclonal B-cell activation and high levels of circulating, nonspecific IgG as seen in trauma patients. This nonspecific IgG triggers …

Effect Of Ethanol On Inflammatory Responses. Implications For Pancreatitis, Gyongyi Szabo, Pranoti Mandrekar, Shilpa Oak, Julia Mayerle

Gyongyi Szabo

BACKGROUND/AIMS: Alcohol use alters inflammatory cell responses. While alcohol has direct effects on pancreatic acinar cells, activation of inflammatory cells is a major component of the pathology of alcoholic pancreatitis.

METHODS: The effects of acute or chronic alcohol exposure were evaluated in human monocytes on the production of TNFalpha or IL-10 production, pro-inflammatory gene and nuclear factor-kappaB (NF-kappaB) activation.

RESULTS: Moderate, acute alcohol consumption or equivalent doses of alcohol in vitro had anti-inflammatory effects on monocyte activation via inhibition of pro-inflammatory genes and NF-kappaB activation, inhibition of TNFalpha production and augmentation of the anti-inflammatory cytokine, IL-10. In contrast, acute alcohol …

Induction And Regulation Of Monocyte Procoagulant Activity, Gyongyi Szabo, Carol Miller-Graziano

Gyongyi Szabo

Monocyte (MO) procoagulant activity (PCA) is induced by various stimuli including allogeneic stimulation, immunocomplexes, and bacterial products. Antigen-antibody complex stimulation therefore represents a pathway for MO PCA induction. Activation of MO PCA has been demonstrated in immunocomplex disease and could represent a major pathology in transplanted immunocomplex disease patients. Stimulation of monocytes via their FcRI receptor has been demonstrated to induce TNF and PGE2. This report demonstrates that stimulation of the high-density FcRI receptor-bearing (FcRI+) MO by resetting with anti-Rh coated erythrocytes also induces significant PCA levels (P less than 0.001). Muramyl dipeptide (MDP), a Gram-positive bacterial cell wall analogue, …

The Opposite Effects Of Acute And Chronic Alcohol On Lipopolysaccharide-Induced Inflammation Are Linked To Irak-M In Human Monocytes, Pranoti Mandrekar, Shashi Bala, Donna Catalano, Karen Kodys, Gyongyi Szabo

Gyongyi Szabo

Impaired host defense after alcohol use is linked to altered cytokine production, however, acute and chronic alcohol differently modulate monocyte/macrophage activation. We hypothesized that in human monocytes, acute alcohol induces hyporesponsiveness to LPS, resulting in decreased TNF-alpha, whereas chronic alcohol increases TNF-alpha by sensitization to LPS. We found that acute alcohol increased IL-1R-associated kinase-monocyte (IRAK-M), a negative regulator of IRAK-1, in human monocytes. This was associated with decreased IkappaB alpha kinase activity, NFkappaB DNA binding, and NFkappaB-driven reporter activity after LPS stimulation. In contrast, chronic alcohol decreased IRAK-M expression but increased IRAK-1 and IKK kinase activities, NFkappaB DNA binding, and …

Increased Lipopolysaccharide Sensitivity In Alcoholic Fatty Livers Is Independent Of Leptin Deficiency And Toll-Like Receptor 4 (Tlr4) Or Tlr2 Mrna Expression, Laszlo Romics, Pranoti Mandrekar, Karen Kodys, Arumugam Velayudham, Yvonne Drechsler, Angela Dolganiuc, Gyongyi Szabo

Gyongyi Szabo

BACKGROUND: Both alcoholic (AFL) and nonalcoholic (NAFL) fatty livers show increased sensitivity to endotoxin-induced injury. Lipopolysaccharide (LPS) is recognized by toll-like receptor 4 (TLR4), whereas lipopeptide triggers TLR2 to induce common downstream activation of nuclear factor (NF)-kappaB and pro-inflammatory pathways that are activated in AFL and NAFL. METHODS: Serum alanine aminotransferase (ALT), tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6 levels; hepatic NF-kappaB activity; and expression of TLR2, TLR4, inducible nitric oxide synthase (iNOS), and heme oxygenase (HO)-1 mRNAs were investigated in lean and leptin-deficient ob/ob mice after LPS challenge in combination with acute or chronic alcohol feeding. RESULTS: Increased LPS …

Elevated Tumor Necrosis Factor Alpha Production Concomitant To Elevated Prostaglandin E2 Production By Trauma Patients' Monocytes, Thomas Takayama, Carol Miller-Graziano, Gyongyi Szabo

Gyongyi Szabo

The level of tumor necrosis factor alpha (TNF alpha), a monokine implicated in mediating septic shock, is elevated in the blood of some patients with sepsis. Monocytes from 11 trauma patients and 11 burn patients were suboptimally stimulated with interferon gamma and muramyl dipeptide, an analogue of bacterial wall products. The patients with sepsis showed significantly greater total TNF alpha levels (secreted in combination with cell-associated) 3 days before septic episodes, as compared with normal controls (32.38 to 2231.76 ng/10(6) monocytes per milliliter, median = 121.03 ng/10(6) monocytes per milliliter; normal control: 0.00 to 18.20 ng/10(6) monocytes per milliliter, median …

Regulation Of Human Monocyte Functions By Acute Ethanol Treatment: Decreased Tumor Necrosis Factor-Alpha, Interleukin-1 Beta And Elevated Interleukin-10, And Transforming Growth Factor-Beta Production, Gyongyi Szabo, Pranoti Mandrekar, Linda Girouard, Donna Catalano

Gyongyi Szabo

We and others have previously shown that even acute ethanol exposure has the capacity to modulate immune functions, particularly monocyte functions. Herein, we tested the hypothesis that acute ethanol treatment inhibits inflammatory, while increasing inhibitory cytokine production in human blood monocytes that, in turn, could contribute to the overall immune abnormalities seen after alcohol use. Our data show that in vitro treatment of blood monocytes with a physiologically relevant dose of alcohol (25 mM) results in significantly decreased induction of tumor necrosis factor-alpha (TNF alpha) and interleukin (IL)-1 beta by bacterial stimulation of either Gram-positive [staphylococcal enterotoxin B (SEB), 1 …

Ethanol-Mediated Regulation Of Transcription Factors In Immunocompetent Cells, Gyongyi Szabo, Pranoti Mandrekar

Gyongyi Szabo

The immunomodulatory effects of acute and chronic alcohol use are characterized by impaired antigen-specific immune activation and by increased susceptibility to infections due to alterations in innate immune responses and inflammatory mediator production. The central feature of cellular responses to inflammatory and stress signals is the activation of the nuclear regulatory kappa B/Rel family of transcriptional factors via various surface receptor systems in immunocompetent cells. Activation of NF-kappa B, however, is regulated at multiple levels including I-kappa B degradation, nuclear translocation, and by interaction of NF-kappa B/Rel with other transcription factors. Data from our and other laboratories demonstrate that acute …

Modulation Of Non-Alcoholic Steatohepatitis By Pattern Recognition Receptors In Mice: The Role Of Toll-Like Receptors 2 And 4, Gyongyi Szabo, Arumugam Velayudham, Laszlo Romics, Pranoti Mandrekar

Gyongyi Szabo

Toll-like receptors (TLR) recognize pathogen-derived molecules and induce downstream activation of inflammatory pathways. Fatty liver has been shown to result in increased sensitivity to lipopolysaccharide (LPS), a TLR4 ligand. In this study, we investigated the roles of TLR2 and TLR4 in liver damage and on cytokine induction in a methionine-choline deficient (MCD) diet-induced model of nonalcoholic steatohepatitis. We found that mice with nonalcoholic fatty liver had increased liver injury and inflammatory cytokine induction after challenge with a TLR4 but not with a TLR2 ligand. TLR2 deficient mice were not protected against the development of steatohepatitis after MCD diet feeding. On …

The Critical Role Of Toll-Like Receptor (Tlr) 4 In Alcoholic Liver Disease Is Independent Of The Common Tlr Adapter Myd88, Istvan Hritz, Pranoti Mandrekar, Arumugam Velayudham, Donna Catalano, Angela Dolganiuc, Karen Kodys, Evelyn Kurt-Jones, Gyongyi Szabo

Gyongyi Szabo

The Toll-like receptor 4 (TLR4) that recognizes endotoxin, a trigger of inflammation in alcoholic liver disease (ALD), activates two signaling pathways utilizing different adapter molecules: the common TLR adapter, myeloid differentiation factor 88 (MyD88), or Toll/interleukin immune-response-domain-containing adaptor inducing interferon (IFN)-beta. The MyD88 pathway induces proinflammatory cytokine activation, a critical mediator of ALD. Here we evaluated the role of MyD88 in alcohol-induced liver injury in wild-type, TLR2-deficient, TLR4-deficient, or MyD88-deficient (knockout [KO]) mice after administration of the Lieber-De-Carli diet (4.5% volume/volume ethanol) or an isocaloric liquid control diet for 5 weeks. Alcohol feeding resulted in a significant increase in serum …

Antigen Presentation By The Cd4 Positive Monocyte Subset, Gyongyi Szabo, Carol Miller-Graziano, Karen Kodys

Gyongyi Szabo

Although CD4 antigen is expressed on monocytes (MO), its functional role is uncharacterized. In this study, isolated human MO were separated into CD4+ and CD4- MO subsets and assessed for presentation of tetanus toxoid. The CD4- MO subset had decreased antigen presenting cell (APC) capacity as well as increased PGE2 production when compared to the CD4+ MO subset. Addition of a cyclo-oxygenase inhibitor (Indomethacin) did not restore the CD4- MO subset's APC capacity to that of the similarly treated CD4+ MO subset, eliminating differential PGE2 production as the primary cause of differential APC capacity. Production of monokines such as IL-1 …

Differential Tumor Necrosis Factor Production By Human Monocyte Subsets, Gyongyi Szabo, Carol Miller-Graziano, Jia-Yan Wu, Thomas Takayama, Karen Kodys

Gyongyi Szabo

The human monocyte (M phi subset rosetting with anti RH-coated human erythrocytes via high-affinity, 72 kD receptors (FcRI+), contains the PGE2-producing immunosuppressive subpopulation, while the non-rosetting M phi subset (FcRI-) is the major plasminogen activator-producing and antigen-presenting M phi. This study gives additional evidence for the functional disparity of the FcRI- and FcRI+ M phi subsets. We are demonstrating that the normal human M phi subset isolated by rosetting via the FcRI receptor (FcRI+) produces greater quantities of tumor necrosis factor (TNF) than the non-rosetting (FcRI-) M phi. TNF production by the FcRI+ M phi subset is greater than that …

Inhibition Of Superantigen-Induced T Cell Proliferation And Monocyte Il-1 Beta, Tnf-Alpha, And Il-6 Production By Acute Ethanol Treatment, Gyongyi Szabo, Pranoti Mandrekar, Donna Catalano

Gyongyi Szabo

Alcohol use has been shown to decrease monocyte antigen presentation capacity and inflammatory cytokine production, thereby increasing susceptibility to infections. Here, we demonstrate that in vitro acute treatment of normal monocytes with pharmacological doses of ethanol can decrease superantigen [Staphylococcus enterotoxins B (SEB) and A (SEA)]-induced T cell proliferation. Furthermore, ethanol treatment (25-100 mM) significantly inhibited SEA- or SEB-induced production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 in monocytes. Ethanol-induced down-regulation of monocyte TNF-alpha, IL-1 beta, and IL-6 occurred at both the protein and mRNA levels. Additional data suggest that ethanol can decrease IL-1 beta mRNA …

Tlr2- And Tlr4-Mediated Signals Determine Attenuation Or Augmentation Of Inflammation By Acute Alcohol In Monocytes, Shilpa Oak, Pranoti Mandrekar, Donna Catalano, Karen Kodys, Gyongyi Szabo

Gyongyi Szabo

Most pathogens express ligands for multiple TLRs that share common downstream signaling. In this study, we investigated the effects of acute alcohol on inflammatory pathways induced by TLR2 or TLR4 ligands and their combination. In human monocytes, alcohol attenuated TLR4- but not TLR2-induced TNF-alpha protein and mRNA levels and NF-kappaB activation. In contrast, acute alcohol augmented TNF-alpha production when both TLR2 and TLR4 ligands were present. IL-1R-associated kinase (IRAK)-1 activity was reduced by alcohol in TLR4, but it was augmented in TLR2- plus TLR4-stimulated cells. IRAK-monocyte, an inhibitor of IRAK-1, was induced in TLR4, but it was reduced in TLR2- …