Patric Lundberg | Eastern Virginia Medical School (original) (raw)
Papers by Patric Lundberg
Typescript. Thesis (Ph. D.)--University of Wisconsin--Madison, 1998. Includes bibliographical ref... more Typescript. Thesis (Ph. D.)--University of Wisconsin--Madison, 1998. Includes bibliographical references.
Journal of neuroimmunology, Jan 15, 2017
Rapid eye movement (REM) sleep is rapidly and persistently suppressed during vesicular stomatitis... more Rapid eye movement (REM) sleep is rapidly and persistently suppressed during vesicular stomatitis virus (VSV) encephalitis in C57Bl/6J (B6) mice. REM sleep suppression was associated with a complex global brain chemokine/cytokine response with bimodal kinetics although regionally distinct cytokine profiles were readily identified. Cytokine mRNA was translated either immediately or suppressed until the pathogen was cleared from the CNS. Innate signaling pathway (TLRs, RIG-I) activation occurred rapidly and sequentially prior to VSV neuroinvasion suggesting that antiviral states are quickly established in the CNS in advance of viral pathogen penetration. Il1β suppressed REM sleep mimicking aspects of VSV-induced sleep alterations whereas some robustly induced chemokines may be protective of REM. Thus, multiple brain chemokines may mediate sleep across VSV encephalitis via differential somnogenic effects.
Circulation Research, 2016
Forkhead box P3(+) T regulatory cells (Tregs) are key players in maintaining immune homeostasis. ... more Forkhead box P3(+) T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe(-)(/-) mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3(+) Tregs and the accumulation of an intermediate Th1-like interferon (IFN)-γ(+)CCR5(+) Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than from T-effector cells. We show that Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a(-/-) Tregs, we demonstrate that elevated IFNγ(+) Mir146a(-/-) Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe(-/-) mice, in comparison to Mir146a(+/+) Tregs. Finally, via single-cell RNA-sequencing and real-time -polymerase chain reaction, we show that Th1/Tregs possess a unique transcriptional phenotype characterized by coexpression of Treg and Th1 lineage genes and a downregulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. In addition, an ingenuity pathway analysis further implicates IFNγ, IFNα, interleukin-2, interleukin-7, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), T-cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ(+) Th1/Tregs that may permit further arterial inflammation and atherogenesis.
Journal of Infectious Diseases, 2015
We document a unique DNA recombination between polyomavirus JC (JCV) and Epstein-Barr virus (EBV)... more We document a unique DNA recombination between polyomavirus JC (JCV) and Epstein-Barr virus (EBV) at sequences of JCV found infecting the brain. Archetype JCV is present in bone marrow and uroepithelial cells of most adults. During immunosuppression JCV can infect the brain, causing a demyelinating disease, PML. Rearrangements in the archetype control region (NCCR) are necessary for neurovirulence. Two NCCR deletions and a duplication occur at sequences of homology with EBV, present latently in B cells, which may be co-infected with both viruses. Recombination between JCV and EBV occurs in B lymphoblasts at a sequence essential for JCV neurovirulence and in cerebrospinal fluid of immunosuppressed multiple sclerosis patients, those susceptible to PML. Interviral recombination is a model for conferring advantages on JCV in the brain. It can alter a critical NCCR sequence and potentially facilitate use of EBV DNA abilities to transfer among different cell types.
Brain Disorders & Therapy, 2015
Progressive multifocal leukoencephalopathy is a neurodegenerative disease caused by demyelination... more Progressive multifocal leukoencephalopathy is a neurodegenerative disease caused by demyelination in the brain. The demyelination is due to infection of oligodendroglial cells by polyomavirus JC, a circular DNA virus. The virus resides as an archetype form in uroepithelial cells and bone marrow of more than 70% of adults, in whom it seldom causes overt symptoms. The JC viral form infecting the brain differs from the archetype. This viral form contains two deletions and a duplication in the non-coding control region that are thought to be derived from the archetype. These rearrangements are necessary for neurovirulence. This review considers how these rearrangements occur in the context of transit to the brain and adaptation to infect glial cells.
Background / Purpose: JC virus undergoes control region (NCCR) DNA sequence changes as it acquire... more Background / Purpose: JC virus undergoes control region (NCCR) DNA sequence changes as it acquires the ability to infect glial cells in the brain. We describe a sequence of recombination events, the earliest of which occur in a subset of normal individuals and can lead to changes characteristic of neurovirulence. Identification of people with JCV bearing certain specific early changes will help track those predisposed to progressive multifocal leukoencephalopathy. Main conclusion: We have identified two types of JCV NCCR recombination as part of a potential sequence of events that can lead to adaptation to replicate in glial cells. One type of recombination is dependent on fork stalling during JCV DNA replication, while the other type is independent of replication and suggestive of mechanisms specialized in B cell subtypes.
Clinical and Developmental Immunology, 2013
Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML... more Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating infection of oligodendrocytes in the brain. PML, a frequently fatal opportunistic infection in AIDS, has also emerged as a consequence of treatment with several new immunosuppressive therapeutic agents. Although nearly 80% of adults are seropositive, JCV attains an ability to infect glial cells in only a minority of people. Data suggest that JCV undergoes sequence alterations that accompany this ability, and these changes can be derived from an archetype strain by mutation, deletion, and duplication. While the introductory source and primary tissue reservoir of JCV remain unknown, lymphoid cells have been identified as potential intermediaries in progression of JCV to the brain. This review is focused on sequence changes in the noncoding control region (NCCR) of the virus. We propose an adaptive mechanism that involves a sequential series of DNA replication-driven NCCR recombination events involving stalled DNA replication forks at NCCR palindromic secondary structures. We shall describe how the NCCR sequence changes point to a model in which viral DNA replication drives NCCR recombination, allowing JCV adaptation to different cell types in its progression to neurovirulence.
BioProcessing Journal, 2012
BioProcessing Journal, 2011
Clinical Infectious Diseases, 2011
Background. Bacterial meningitis (BM) is a severe infection mainly caused by Streptococcus pneumo... more Background. Bacterial meningitis (BM) is a severe infection mainly caused by Streptococcus pneumoniae and Neisseria meningitidis (NM). However, genetically determined susceptibility to develop severe infections by these microorganisms is variable between individuals. Toll-like receptor 9 (TLR9) recognizes bacterial DNA leading to intracellular inflammatory signaling. Single nucleotide polymorphisms (SNPs) within the TLR9 gene are associated with susceptibility to several diseases, no such association with meningitis has been described.
Journal of Virology, 2008
This study was undertaken to investigate possible immune mechanisms in fatal HSV-1 2 encephalitis... more This study was undertaken to investigate possible immune mechanisms in fatal HSV-1 2 encephalitis (HSE) after HSV-1 corneal inoculation. Susceptible 129S6 (129) but not resistant C57BL/6 3 (B6) mice developed intense focal inflammatory brainstem lesions of primarily F4/80 + macrophages and 4 Gr-1 + neutrophils detectable by MRI as early as day 6 post infection (PI). Depletion of macrophages and 5 neutrophils significantly enhanced survival of infected 129 mice. Immunodeficient B6 (IL-7R -/-Kit w41/w41 ) 6 mice lacking adaptive cells (B6-E mice) transplanted with 129 bone marrow showed significantly 7 accelerated fatal HSE compared to B6-E mice transplanted with B6 marrow or control non-transplanted 8 B6-E mice. In contrast, there was no difference in ocular viral shedding in B6-E mice transplanted with 9 129 bone marrow or B6 bone marrow. Acyclovir treatment of 129 mice beginning day 4 PI (24 h after 10 HSV-1 first reaches the brain stem) reduced nervous system viral titers to undetectable levels but did not 11 alter brainstem inflammation or mortality.
Journal of Virology, 2001
Gender influences the incidence and severity of some bacterial and viral infections and autoimmun... more Gender influences the incidence and severity of some bacterial and viral infections and autoimmune diseases in animal models and humans. To determine a gender-based difference, comparisons were made between male and female mice inoculated with herpes simplex virus type 1 (HSV-1) by the corneal route. Mortality was higher in the male mice of the three strains tested: 129/Sv//Ev wild type, gamma interferon (IFN-␥) knockout (GKO), and IFN-␥ receptor knockout (RGKO). Similarly, in vivo HSV-1 reactivation occurred more commonly in male mice, but the male-female difference in reactivation was restricted to the two knockout strains and was not seen in the 129/Sv//Ev control. Comparison among male mice of the three strains showed a higher mortality of the RGKO mice and a higher reactivation rate of the GKO and RGKO mice than of the 129/Sv//Ev males. In contrast, female RGKO and GKO mice did not differ from female 129/Sv//Ev controls in either mortality or reactivation. HSV-1 periocular and eyelid disease was also more severe in male and dihydrotestosterone (DHT)-treated female mice than in control female mice. These results show a consistent gender difference in HSV-1 infection, with a worse outcome in male mice. In addition, the results comparing GKO and RGKO mice to controls show differences only in male mice, suggesting that some effects of IFN-␥, a key immunoregulatory molecule, are gender specific.
Nature biotechnology, 2004
Small interfering RNAs (siRNA) are potent reagents for directed post-transcriptional gene silenci... more Small interfering RNAs (siRNA) are potent reagents for directed post-transcriptional gene silencing and a major new genetic tool for investigating mammalian cells. When synthetic siRNAs are used for gene silencing, the costs can be substantial because of variations in siRNA efficacies. An alternative to chemically synthesized siRNAs are siRNAs produced by bacteriophage T7 RNA polymerase. We found that siRNAs synthesized from the T7 RNA polymerase system can trigger a potent induction of interferon alpha and beta in a variety of cell lines. Surprisingly, we also found very potent induction of interferon alpha and beta by short single-stranded RNAs (ssRNAs) transcribed with T3, T7 and Sp6 RNA polymerases. Analyses of the potential mediators of this response revealed that the initiating 5' triphosphate is required for interferon induction. We describe here an improved method for T7 siRNA synthesis that alleviates the interferon response while maintaining full efficacy of the siRNAs.
Nature Protocols, 2006
RNA interference is a powerful tool for target-specific knockdown of gene expression. The trigger... more RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21-25 nt with 2-bp 3' overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.
PLoS pathogens, 2015
The establishment of latent infections in sensory neurons is a remarkably effective immune evasio... more The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dos...
Current Eye Research, 2003
Corneal infection with herpes simplex virus (HSV) leads to the recruitment of immune cells to the... more Corneal infection with herpes simplex virus (HSV) leads to the recruitment of immune cells to the eye itself, the trigeminal ganglion and the brainstem. In addition, some resident cells in these target tissues are infected by HSV, activated during the inflammatory response or both. Chemokine signaling is an important component of the regulatory circuit governing the host immune response to virus infection. This review discusses chemokine responses in relation to HSV infection of the cornea emphasizing the role of CXCR3 chemokine signaling by the IFN-gamma inducible ligands MIG, IP10 and I-TAC and includes discussion of their potential role in immunopathology in the nervous system.
Journal of Controlled Release, 2012
Tumor necrosis factor alpha (TNFα) is a classic proinflammatory cytokine implicated in the pathog... more Tumor necrosis factor alpha (TNFα) is a classic proinflammatory cytokine implicated in the pathogenesis of several autoimmune and inflammatory diseases including viral encephalitis. Macrophages being major producers of TNFα are thus attractive targets for in vivo RNA interference (RNAi) mediated down regulation of TNFα. The application of RNAi technology to in vivo models however presents obstacles, including rapid degradation of RNA duplexes in plasma, insufficient delivery to the target cell population and toxicity associated with intravenous administration of synthetic RNAs and carrier compounds.
Journal of Virology, 2003
Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically... more Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G؉C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-␥], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8 ؉ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-␥ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in partic-
Typescript. Thesis (Ph. D.)--University of Wisconsin--Madison, 1998. Includes bibliographical ref... more Typescript. Thesis (Ph. D.)--University of Wisconsin--Madison, 1998. Includes bibliographical references.
Journal of neuroimmunology, Jan 15, 2017
Rapid eye movement (REM) sleep is rapidly and persistently suppressed during vesicular stomatitis... more Rapid eye movement (REM) sleep is rapidly and persistently suppressed during vesicular stomatitis virus (VSV) encephalitis in C57Bl/6J (B6) mice. REM sleep suppression was associated with a complex global brain chemokine/cytokine response with bimodal kinetics although regionally distinct cytokine profiles were readily identified. Cytokine mRNA was translated either immediately or suppressed until the pathogen was cleared from the CNS. Innate signaling pathway (TLRs, RIG-I) activation occurred rapidly and sequentially prior to VSV neuroinvasion suggesting that antiviral states are quickly established in the CNS in advance of viral pathogen penetration. Il1β suppressed REM sleep mimicking aspects of VSV-induced sleep alterations whereas some robustly induced chemokines may be protective of REM. Thus, multiple brain chemokines may mediate sleep across VSV encephalitis via differential somnogenic effects.
Circulation Research, 2016
Forkhead box P3(+) T regulatory cells (Tregs) are key players in maintaining immune homeostasis. ... more Forkhead box P3(+) T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe(-)(/-) mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3(+) Tregs and the accumulation of an intermediate Th1-like interferon (IFN)-γ(+)CCR5(+) Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than from T-effector cells. We show that Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a(-/-) Tregs, we demonstrate that elevated IFNγ(+) Mir146a(-/-) Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe(-/-) mice, in comparison to Mir146a(+/+) Tregs. Finally, via single-cell RNA-sequencing and real-time -polymerase chain reaction, we show that Th1/Tregs possess a unique transcriptional phenotype characterized by coexpression of Treg and Th1 lineage genes and a downregulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. In addition, an ingenuity pathway analysis further implicates IFNγ, IFNα, interleukin-2, interleukin-7, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), T-cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ(+) Th1/Tregs that may permit further arterial inflammation and atherogenesis.
Journal of Infectious Diseases, 2015
We document a unique DNA recombination between polyomavirus JC (JCV) and Epstein-Barr virus (EBV)... more We document a unique DNA recombination between polyomavirus JC (JCV) and Epstein-Barr virus (EBV) at sequences of JCV found infecting the brain. Archetype JCV is present in bone marrow and uroepithelial cells of most adults. During immunosuppression JCV can infect the brain, causing a demyelinating disease, PML. Rearrangements in the archetype control region (NCCR) are necessary for neurovirulence. Two NCCR deletions and a duplication occur at sequences of homology with EBV, present latently in B cells, which may be co-infected with both viruses. Recombination between JCV and EBV occurs in B lymphoblasts at a sequence essential for JCV neurovirulence and in cerebrospinal fluid of immunosuppressed multiple sclerosis patients, those susceptible to PML. Interviral recombination is a model for conferring advantages on JCV in the brain. It can alter a critical NCCR sequence and potentially facilitate use of EBV DNA abilities to transfer among different cell types.
Brain Disorders & Therapy, 2015
Progressive multifocal leukoencephalopathy is a neurodegenerative disease caused by demyelination... more Progressive multifocal leukoencephalopathy is a neurodegenerative disease caused by demyelination in the brain. The demyelination is due to infection of oligodendroglial cells by polyomavirus JC, a circular DNA virus. The virus resides as an archetype form in uroepithelial cells and bone marrow of more than 70% of adults, in whom it seldom causes overt symptoms. The JC viral form infecting the brain differs from the archetype. This viral form contains two deletions and a duplication in the non-coding control region that are thought to be derived from the archetype. These rearrangements are necessary for neurovirulence. This review considers how these rearrangements occur in the context of transit to the brain and adaptation to infect glial cells.
Background / Purpose: JC virus undergoes control region (NCCR) DNA sequence changes as it acquire... more Background / Purpose: JC virus undergoes control region (NCCR) DNA sequence changes as it acquires the ability to infect glial cells in the brain. We describe a sequence of recombination events, the earliest of which occur in a subset of normal individuals and can lead to changes characteristic of neurovirulence. Identification of people with JCV bearing certain specific early changes will help track those predisposed to progressive multifocal leukoencephalopathy. Main conclusion: We have identified two types of JCV NCCR recombination as part of a potential sequence of events that can lead to adaptation to replicate in glial cells. One type of recombination is dependent on fork stalling during JCV DNA replication, while the other type is independent of replication and suggestive of mechanisms specialized in B cell subtypes.
Clinical and Developmental Immunology, 2013
Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML... more Polyomavirus JC (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a demyelinating infection of oligodendrocytes in the brain. PML, a frequently fatal opportunistic infection in AIDS, has also emerged as a consequence of treatment with several new immunosuppressive therapeutic agents. Although nearly 80% of adults are seropositive, JCV attains an ability to infect glial cells in only a minority of people. Data suggest that JCV undergoes sequence alterations that accompany this ability, and these changes can be derived from an archetype strain by mutation, deletion, and duplication. While the introductory source and primary tissue reservoir of JCV remain unknown, lymphoid cells have been identified as potential intermediaries in progression of JCV to the brain. This review is focused on sequence changes in the noncoding control region (NCCR) of the virus. We propose an adaptive mechanism that involves a sequential series of DNA replication-driven NCCR recombination events involving stalled DNA replication forks at NCCR palindromic secondary structures. We shall describe how the NCCR sequence changes point to a model in which viral DNA replication drives NCCR recombination, allowing JCV adaptation to different cell types in its progression to neurovirulence.
BioProcessing Journal, 2012
BioProcessing Journal, 2011
Clinical Infectious Diseases, 2011
Background. Bacterial meningitis (BM) is a severe infection mainly caused by Streptococcus pneumo... more Background. Bacterial meningitis (BM) is a severe infection mainly caused by Streptococcus pneumoniae and Neisseria meningitidis (NM). However, genetically determined susceptibility to develop severe infections by these microorganisms is variable between individuals. Toll-like receptor 9 (TLR9) recognizes bacterial DNA leading to intracellular inflammatory signaling. Single nucleotide polymorphisms (SNPs) within the TLR9 gene are associated with susceptibility to several diseases, no such association with meningitis has been described.
Journal of Virology, 2008
This study was undertaken to investigate possible immune mechanisms in fatal HSV-1 2 encephalitis... more This study was undertaken to investigate possible immune mechanisms in fatal HSV-1 2 encephalitis (HSE) after HSV-1 corneal inoculation. Susceptible 129S6 (129) but not resistant C57BL/6 3 (B6) mice developed intense focal inflammatory brainstem lesions of primarily F4/80 + macrophages and 4 Gr-1 + neutrophils detectable by MRI as early as day 6 post infection (PI). Depletion of macrophages and 5 neutrophils significantly enhanced survival of infected 129 mice. Immunodeficient B6 (IL-7R -/-Kit w41/w41 ) 6 mice lacking adaptive cells (B6-E mice) transplanted with 129 bone marrow showed significantly 7 accelerated fatal HSE compared to B6-E mice transplanted with B6 marrow or control non-transplanted 8 B6-E mice. In contrast, there was no difference in ocular viral shedding in B6-E mice transplanted with 9 129 bone marrow or B6 bone marrow. Acyclovir treatment of 129 mice beginning day 4 PI (24 h after 10 HSV-1 first reaches the brain stem) reduced nervous system viral titers to undetectable levels but did not 11 alter brainstem inflammation or mortality.
Journal of Virology, 2001
Gender influences the incidence and severity of some bacterial and viral infections and autoimmun... more Gender influences the incidence and severity of some bacterial and viral infections and autoimmune diseases in animal models and humans. To determine a gender-based difference, comparisons were made between male and female mice inoculated with herpes simplex virus type 1 (HSV-1) by the corneal route. Mortality was higher in the male mice of the three strains tested: 129/Sv//Ev wild type, gamma interferon (IFN-␥) knockout (GKO), and IFN-␥ receptor knockout (RGKO). Similarly, in vivo HSV-1 reactivation occurred more commonly in male mice, but the male-female difference in reactivation was restricted to the two knockout strains and was not seen in the 129/Sv//Ev control. Comparison among male mice of the three strains showed a higher mortality of the RGKO mice and a higher reactivation rate of the GKO and RGKO mice than of the 129/Sv//Ev males. In contrast, female RGKO and GKO mice did not differ from female 129/Sv//Ev controls in either mortality or reactivation. HSV-1 periocular and eyelid disease was also more severe in male and dihydrotestosterone (DHT)-treated female mice than in control female mice. These results show a consistent gender difference in HSV-1 infection, with a worse outcome in male mice. In addition, the results comparing GKO and RGKO mice to controls show differences only in male mice, suggesting that some effects of IFN-␥, a key immunoregulatory molecule, are gender specific.
Nature biotechnology, 2004
Small interfering RNAs (siRNA) are potent reagents for directed post-transcriptional gene silenci... more Small interfering RNAs (siRNA) are potent reagents for directed post-transcriptional gene silencing and a major new genetic tool for investigating mammalian cells. When synthetic siRNAs are used for gene silencing, the costs can be substantial because of variations in siRNA efficacies. An alternative to chemically synthesized siRNAs are siRNAs produced by bacteriophage T7 RNA polymerase. We found that siRNAs synthesized from the T7 RNA polymerase system can trigger a potent induction of interferon alpha and beta in a variety of cell lines. Surprisingly, we also found very potent induction of interferon alpha and beta by short single-stranded RNAs (ssRNAs) transcribed with T3, T7 and Sp6 RNA polymerases. Analyses of the potential mediators of this response revealed that the initiating 5' triphosphate is required for interferon induction. We describe here an improved method for T7 siRNA synthesis that alleviates the interferon response while maintaining full efficacy of the siRNAs.
Nature Protocols, 2006
RNA interference is a powerful tool for target-specific knockdown of gene expression. The trigger... more RNA interference is a powerful tool for target-specific knockdown of gene expression. The triggers for this process are duplex small interfering RNAs (siRNAs) of 21-25 nt with 2-bp 3' overhangs produced in cells by the RNase III family member Dicer. We have observed that short RNAs that are long enough to serve as Dicer substrates (D-siRNA) can often evoke more potent RNA interference than the corresponding 21-nt siRNAs; this is probably a consequence of the physical handoff of the Dicer-produced siRNAs to the RNA-induced silencing complex. Here we describe the design parameters for D-siRNAs and a protocol for in vitro and in vivo intraperitoneal delivery of D-siRNAs and siRNAs to macrophages. siRNA delivery and transfection and analysis of macrophages in vivo can be accomplished within 36 h.
PLoS pathogens, 2015
The establishment of latent infections in sensory neurons is a remarkably effective immune evasio... more The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dos...
Current Eye Research, 2003
Corneal infection with herpes simplex virus (HSV) leads to the recruitment of immune cells to the... more Corneal infection with herpes simplex virus (HSV) leads to the recruitment of immune cells to the eye itself, the trigeminal ganglion and the brainstem. In addition, some resident cells in these target tissues are infected by HSV, activated during the inflammatory response or both. Chemokine signaling is an important component of the regulatory circuit governing the host immune response to virus infection. This review discusses chemokine responses in relation to HSV infection of the cornea emphasizing the role of CXCR3 chemokine signaling by the IFN-gamma inducible ligands MIG, IP10 and I-TAC and includes discussion of their potential role in immunopathology in the nervous system.
Journal of Controlled Release, 2012
Tumor necrosis factor alpha (TNFα) is a classic proinflammatory cytokine implicated in the pathog... more Tumor necrosis factor alpha (TNFα) is a classic proinflammatory cytokine implicated in the pathogenesis of several autoimmune and inflammatory diseases including viral encephalitis. Macrophages being major producers of TNFα are thus attractive targets for in vivo RNA interference (RNAi) mediated down regulation of TNFα. The application of RNAi technology to in vivo models however presents obstacles, including rapid degradation of RNA duplexes in plasma, insufficient delivery to the target cell population and toxicity associated with intravenous administration of synthetic RNAs and carrier compounds.
Journal of Virology, 2003
Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically... more Recently, prokaryotic DNAs containing unmethylated CpG motifs have been shown to be intrinsically immunostimulatory both in vitro and in vivo, tending to promote Th1-like responses. In contrast, CpG dinucleotides in mammalian DNAs are extensively methylated on cytosines and hence immunologically inert. Since the herpes simplex virus (HSV) genome is unmethylated and G؉C rich, we predicted that CpG motifs would be highly prevalent in the HSV genome; hence, we examined the immunostimulatory potential of purified HSV DNA in vitro and in vivo. Mouse splenocyte cultures treated with HSV DNA or HSV-derived oligodeoxyribonucleotides (ODNs) showed strong proliferative responses and production of inflammatory cytokines (gamma interferon [IFN-␥], tumor necrosis factor [TNF], and interleukin-6 [IL-6]) in vitro, whereas splenocytes treated with mammalian CV-1 DNA or non-CpG ODN did not. After immunization with ovalbumin (OVA), only splenocytes from mice immunized with HSV DNA or HSV-ODN as the adjuvants proliferated strongly and produced typical Th1 responses, including CD8 ؉ cytotoxic T-lymphocyte responses, upon restimulation with OVA. Furthermore, HSV-ODN synergized with IFN-␥ to induce nitric oxide (NO), IL-6, and TNF production from macrophages. These results demonstrate that HSV DNA and HSV-ODN are immunostimulatory, driving potent Th1 responses both in vitro and in vivo. Considering that HSV DNA has been found to persist in nonneuronal cells, these results fuel speculation that HSV DNA might play a role in pathogenesis, in partic-