Herpes simplex virus US3 protein kinase regulates host responses and determines neurovirulence.

The US3 of HSV encodes a serine/threonine protein kinase that is highly conserved among members of the alphaherpesviruses. It is an accessory gene that is not required for viral replication in cultured cells but appears essential for viral survival in humans. Although accumulating in vitro evidence suggested that the viral protein kinase is multifunctional, little information is available about its functions in vivo. Several reports point out that, upon invasion into the peripheral nervous system, HSV blocks virus-induced neuronal apoptosis, while presumably subverting host immune responses, largely through actions of the US3 protein kinase. In addition, the US3 protein kinase confers the viral neurovirulence. In the present article, functions of the HSV US3 protein kinase are briefly reviewed, with special attention given to its role in regulating host responses and neurovirulence.

Herpes simplex virus type 1 persists in the aged brain through hypothetical expression of accessory genes.

Herpes simplex virus type 1 persists in the brain of most aged individuals and may contribute to the pathogenesis of Alzheimer's disease. The virus likely utilizes accessory genes for neural spread within the nervous system and herpes simplex virus type 1 may regulate various host responses through an array of accessory genes. This mini-review focuses on these viral accessory genes that may shed light on the potential mechanisms of this enigmatic phenomenon in the elderly brain.

The olfactory bulb: A link between environmental agents and narcolepsy, from the standpoint of autoimmune etiology.

Narcolepsy type 1 is a lifelong sleep disorder characterized by the loss of hypocretin-producing neurons in the brain. Environmental agents, including influenza, neurotoxic metals, and combustion smoke, have been implicated in the pathogenesis, especially in carriers of the human leukocyte antigen class II DQB1*06:02 allele. Sensitive experimental approaches have recently revealed hypocretin-autoreactive CD4+ and CD8+ T cells in the blood of narcoleptic patients. However, such potentially harmful cells are also detectable, to a lesser degree, in control DQB1*06:02 carriers, suggesting that the integrity of the blood-brain barrier (BBB) provides a neuroprotective effect. Here, we present the hypothesis that external toxic agents induce neuroinflammation in the olfactory bulb and concomitant overproduction of proinflammatory cytokines (e.g., tumor necrosis factor-α and interferon-γ); this, in turn, compromises the BBB, allowing autoimmune cells to access and kill hypocretinergic neurons. Such sequential pathological alterations could occur insidiously, passing unnoticed and consequently being underestimated. The elevated number of autoreactive T cells in narcoleptics relative to controls might reflect externally induced immunomodulation rather than a direct disease trigger.

The olfactory bulb: A link between environmental agents and narcolepsy.

Narcolepsy with cataplexy is a lifelong sleep disorder associated with orexin/hypocretin deficiency in the central nervous system. In addition to a genetic predisposition, a variety of environmental factors, such as influenza viruses, have been implicated in the pathogenesis of the disease. In this article, a hypothesis is proposed that environmental agents access the olfactory bulb and trigger neuroinflammation, which in turn induces neurodegeneration of orexinergic neurons in the lateral hypothalamus and other neuronal subpopulations regulating the sleep-wake cycle, which triggers the development of narcolepsy.

Oncolytic virotherapy for malignant melanoma with herpes simplex virus type 1 mutant HF10.

BACKGROUND: Many viruses have been engineered and evaluated for their potential as therapeutic agents in the treatment of malignant neoplasm, including malignant melanoma. OBJECTIVE: In this study, we investigated the efficacy of HF10, an attenuated, replication-competent HSV, in immunocompetent animal models with malignant melanoma. METHODS: For in vitro study, viral cytotoxicity assays and replication assays were performed both in human and mouse melanoma cells. For the study in vivo, intraperitoneally disseminated or subcutaneous melanoma models were prepared in DBA/2 mice using clone M3 cells, then HF10 was inoculated intraperitoneally or intratumorally. Therapeutic efficacy of HF10 was assessed by survival, tumor growth, and histopathological analysis. RESULTS: HF10 infection produced cytolytic effects in melanoma cells at various multiplicities of infection (MOI). In the intraperitoneal melanoma model, all mice survived when given intraperitoneal injections of HF10 compared with 100% fatality in the control mice. In the subcutaneous tumor model, intratumoral inoculation of HF10 significantly reduced tumor growth. Histology and immunohistochemistry showed tumor lysis and inflammatory cell infiltration after intratumoral HF10 inoculation. Viral antigen was retained at the inoculation site until 7 days post-infection. HF10-treated intraperitoneal tumor mice were also protected against tumor rechallenge. HF10 also affected the non-inoculated contralateral tumor when injected into the ipsilateral tumor of mice, suggesting that HF10 can induce systemic antitumor immune responses in mice. CONCLUSION: Oncolytic viral therapy using HF10 was effective in melanoma mouse models, and intratumoral injection of HF10 induced systemic antitumor responses. These results suggest that HF10 is a promising agent for the treatment of advanced melanoma.

Changes in Basic Movement Ability and Activities of Daily Living After Hip Fractures: Correlation Between Basic Movement Scale and Motor-Functional Independence Measure Scores.

OBJECTIVE: The aim of this study was to examine the correlation between basic movement ability and activities of daily living (ADL) in elderly patients after hip fracture surgery and predict ADL outcomes from changes in basic movement ability. DESIGN: Fifty-four patients receiving rehabilitation after hip fracture surgery were collected prospectively. Ambulatory ability was evaluated using a Basic Movement Scale (BMS), and ADL was evaluated using the motor subscale of the Functional Independence Measure (motor-FIM). From the results of evaluating BMS and motor-FIM weekly, the important postoperative period to regain ADL was investigated. RESULTS: There was a close correlation between BMS and motor-FIM scores at each evaluation point (r = 0.971, P < 0.001) and a significant correlation between weekly BMS and motor-FIM gains (r = 0.741, P < 0.001). Cluster analysis of BMS scores from postoperative week (POW) 2 to 12 showed three patterns of change, with BMS scores at POW 2 reflecting the outcome. CONCLUSIONS: The very strong correlation between BMS and motor-FIM scores suggests that BMS is a favorable indicator of changes in ADL. Because basic movement ability at POW 2 also reflected the prognosis, constructive interventions should be implemented early to help patients ambulate and regain other basic movements by no later than POW 2.

Lipopolysaccharide enhances interferon-gamma-induced nitric oxide (NO) production in murine vascular endothelial cells via augmentation of interferon regulatory factor-1 activation.

Lipopolysaccharide (LPS) enhances the production of nitric oxide (NO) in interferon (IFN)-gamma-stimulated vascular endothelial cells. We studied the mechanism by which LPS enhances IFN-gamma-induced NO production by using the murine vascular endothelial cell line, END-D. LPS enhanced IFN-gamma-induced NO production via augmented expression of inducible type NO synthase (iNOS) mRNA. LPS significantly augmented the activation of interferon regulatory factor (IRF)-1 in IFN-gamma-stimulated END-D cells, although it did not affect the activation of either MyD88-dependent nuclear factor (NF)-kappaB or MyD88-independent IRF-3. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), prevented the nuclear translocation of IRF-1 in LPS and IFN-gamma-stimulated END-D cells, and inhibited the iNOS expression and NO production in those cells. Therefore, it is proposed that LPS enhanced NO production in IFN-gamma-stimulated END-D cells via augmenting p38 MAPKmediated IRF-1 activation.

5-Fluorouracil prevents lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells by inhibiting Akt-dependent nuclear factor-kappaB activation.

The effect of 5-fluorouracil (5-FU) on the production of nitric oxide (NO) in macrophages was examined by using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. 5-FU at non-toxic concentrations significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition by 5-FU was mediated by attenuated expression of an inducible NO synthase protein and mRNA. 5-FU inhibited the activation of nuclear factor (NF)-kappaB and the subsequent nuclear translocation. Furthermore, 5-FU inhibited the phosphorylation of Akt, an upstream molecule of NF-kappaB signaling. 5-FU did not affect a series of mitogen-activated protein kinases. Therefore, 5-FU was suggested to inhibit the LPS-induced NO production in activated macrophages through preventing Akt-dependent NF-kappaB activation.

MnTBAP, a synthetic metalloporphyrin, inhibits production of tumor necrosis factor-alpha in lipopolysaccharide-stimulated RAW 264.7 macrophages cells via inhibiting oxidative stress-mediating p38 and SAPK/JNK signaling.

Antioxidants are able to inhibit inflammatory gene expression in response to lipopolysaccharide via down-regulating generation of intracellular reactive oxygen species (ROS) as second messengers. The effect of manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a synthetic metalloporphyrin with antioxidant activity, on tumor necrosis factor (TNF)-alpha production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells was examined. MnTBAP prevented the generation of intracellular ROS in lipopolysaccharide-stimulated RAW 264.7 cells and further inhibited lipopolysaccharide-induced TNF-alpha production. MnTBAP exclusively prevented the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK/JNK) whereas it did not affect the phosphorylation and activation of nuclear factor-kappaB and extracellular signal regulated kinase 1/2. MnTBAP was suggested to inhibit lipopolysaccharide-induced TNF-alpha production by the prevention of intracellular ROS generation and subsequent inactivation of p38 MAPK and SAPK/JNK.

Differential expression of autophagy in Hodgkin lymphoma cells treated with various anti-cancer drugs.

AIM: autophagy is a pivotal physiological process for survival during starvation, differentiation and normal growth control. It is defined as the process of sequestrating cytoplasmic proteins or even entire organelles into the lytic compartment (lysosome/vacuole). This study investigates the expression of autophagy in Hodgkin lymphoma cells treated with various anti-cancer drugs. METHODS: Hodgkin's lymphoma cells (HD-My-Z cells) were cultured with various anti-cancer drugs, such as bleomycin, adriamycin, gemcitabine and paclitaxel. Autophagy was detected by fluorescent pattern of light chain 3(LC3) proteins and the apoptotic cell death was determined by annexin V binding. RESULTS: autophagy was detected in HD-My-Z cells treated with gemcitabine, but not with bleomycin, adriamycin and paclitaxel. Adriamycin exhibited the strongest cytotoxic action, and the cytotoxic action of bleomycin and gemcitabine was less marked compared with adriamycin. Paclitaxel did not cause significant cell death in the cells. CONCLUSION: autophagy was differentially expressed in Hodgkin lymphoma cells treated with anti-cancer drugs and the expression did not correspond to the apoptotic cell death.

Olfactory vector hypothesis for encephalitis lethargica.

Viruses have long been implicated in the pathogenesis of classical encephalitis lethargica, which was first described by Constantin von Economo in 1917. In this article, I propose the hypothesis that an airborne virus travels along the olfactory conduit to infect the olfactory bulb; this local infection or induced neuroinflammation, in turn, retrogradely targets certain neuronal populations with sleep-wake regulatory functions in the hypothalamus and midbrain, leading to the development of wakeful inactivity, a hallmark clinical feature of the disease. Furthermore, the olfactory vector hypothesis may also explain the pathomechanism of the debilitating complication of the disease, i.e., postencephalitic parkinsonism, in terms of a recently discovered nigro-olfactory projection.

Viremic attack explains the dual-hit theory of Parkinson's disease.

The dual-hit theory of Parkinson's disease proposes that an airborne pathogen attacks both the olfactory and enteric nervous systems to initiate the Lewy pathology, gradually leading to devastating neurodegenerative processes within the brain. Based on published literatures, this article proposes a hypothesis that viruses with viremic potential can simultaneously attack both of these nervous systems via viremia due to the lack of a blood-nerve barrier in these tissues, thereby explaining the dual-hit theory. Understanding the precise mechanisms underlying the neuropathology will facilitate development of better prophylactic and early intervention strategies against Parkinson's disease.

Defective responsiveness of CD5+ B1 cells to lipopolysaccharide in cytokine production.

Previously, we found that mouse TH2.52 cells possess the characteristic of CD5(+) B1 cells and proliferate in response to lipopolysaccharide (LPS). The effect of LPS on cytokine production by TH2.52 B1 cells was studied. TH2.52 cells constitutively produced a small amount of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and TNF-alpha and IL-6 production was markedly enhanced by LPS stimulation. Although interferon (IFN)-gamma caused the production of various cytokines, such as IL-2, IL-4, IL-6 and TNF-alpha in TH2.52 cells, LPS did not cause the production of such cytokines. LPS did not induce IFN-beta production in TH2.52 cells and TH2.52 cells lacked the expression of several molecules participating in the MyD88-independent pathway in LPS signaling. Defective responsiveness of TH2.52 B1 cells to LPS in cytokine production might be responsible for the failure of IFN-beta production due to the lack of molecules participating in the MyD88-independent pathway.

Accessory genes define the relationship between the herpes simplex virus and its host.

Accessory genes of herpes simplex virus are implicated in the interplay between the virus and host responses to infection, ensuring the survival of the virus in the host and maintaining its transmission cycle in human populations. They will provide vital clues about novel vaccination strategies and gene and oncolytic therapies.

Herpes simplex virus US3 protein kinase regulates virus-induced apoptosis in olfactory and vomeronasal chemosensory neurons in vivo.

A role for the US3 protein kinase of herpes simplex virus (HSV) in regulating virus-induced neuronal apoptosis was investigated in an experimental mouse system, in which wild-type HSV invades the central nervous system (CNS) via the olfactory and vomeronasal systems upon intranasal infection. Wild-type HSV-2 strain 186 infected a fraction of olfactory and vomeronasal chemosensory neurons without inducing apoptosis and was transmitted to the CNS, precipitating lethal encephalitis. In sharp contrast, an US3-disrupted mutant, L1BR1, induced neuronal apoptosis in these peripheral conduits upon infection, blocking viral transmission to the CNS and causing no signs of disease. An US3-repaired mutant, L1B(-)11, behaved similarly to the wild-type virus. Only 5 p.f.u. of L1BR1 was sufficient to compromise mice when the mutant virus was introduced directly into the olfactory bulb, a viral entry site of the CNS. These results suggest that the US3 protein kinase of HSV regulates virus-induced neuronal apoptosis in peripheral conduits and determines the neuroinvasive phenotype of HSV. Furthermore, virus-induced neuronal apoptosis of peripheral nervous system cells may be a protective host response that blocks viral transmission to the CNS.

Lipopolysaccharide prevents doxorubicin-induced apoptosis in RAW 264.7 macrophage cells by inhibiting p53 activation.

The effect of lipopolysaccharide on doxorubicin-induced cell death was studied by using mouse RAW 264.7 macrophage cells. Pretreatment with lipopolysaccharide at 10 ng/mL prevented doxorubicin-induced cell death and the inhibition was roughly dependent on the concentration of lipopolysaccharide. Posttreatment with lipopolysaccharide for 1 hour also prevented doxorubicin-induced cell death. Lipopolysaccharide inhibited DNA fragmentation and caspase-3 activation in doxorubicin-treated RAW 264.7 cells, suggesting the prevention of doxorubicin-induced apoptosis. Lipopolysaccharide did not significantly inhibit doxorubicin-induced DNA damage detected by single-cell gel electrophoresis (comet) assay. Lipopolysaccharide definitely inhibited the stabilization and nuclear translocation of p53 in doxorubicin-treated RAW 264.7 cells. Lipopolysaccharide, as well as being an inhibitor of p53, abolished doxorubicin-induced apoptosis. Therefore, p53 was suggested to play a pivotal role in the prevention of doxorubicin-induced apoptosis in RAW 264.7 cells by lipopolysaccharide.

Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide.

BACKGROUND: Recently it has been reported that, toll-like receptors (TLRs) are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer. Although some cancer cells like melanoma cells are known to respond to lipopolysaccharide (LPS) via TLR4, not all cancer cells are positive for TLR4. There is little information on the expression and function of TLR4 in neuroblastoma cells. In this study, we investigated the expression of TLR4 in human neuroblastoma NB-1 cell line. METHODS: Expression and localization of TLR4 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis, respectively. Activation of nuclear factor (NF)-kappaB by LPS was detected by degradation of IkappaB-alpha and NF-kappaB luciferase assay. Activation and expression of mitogen-activated protein (MAP) kinase and interferon regulatory factor (IRF)-3 was detected by immunoblot analysis. RESULTS: Human NB-1 neuroblastoma cells expressed intracellular form of TLR4, but not the cell surface form. Further, NB-1 cells express CD14, MD2 and MyD88, which are required for LPS response. However, LPS did not significantly induce NF-kappaB activation in NB-1 cells although it slightly degraded IkappaB-alpha. NB-1 cells expressed no IRF-3, which plays a pivotal role on the MyD88-independent pathway of LPS signaling. Collectively, NB-1 cells are capable to avoid their response to LPS. CONCLUSION: Although human NB-1 neuroblastoma cells possessed all the molecules required for LPS response, they did not respond to LPS. It might be responsible for intracellular expression of TLR4 or lack of IRF-3.