Evaluation of the benefits of neutral bicarbonate ionized water baths in an open-label, randomized, crossover trial.

We previously demonstrated that neutral bicarbonate ionized water (NBIW) bathing enhances blood flow by bicarbonate ions and described the underlying mechanism. However, additional clinical investigation was warranted to investigate the efficacy of NBIW bathing. Hence, we performed a randomized, open-label, crossover trial to examine the effects of NBIW bathing on mental stress, sleep, and immune function. Participants who regularly felt stressed were randomly assigned to NBIW or regular bathing for 4 weeks. Mental stress was assessed with the Brief Job Stress Questionnaire (BJSQ) and the Profile of Mood States Second Edition; sleep quality, with the Pittsburgh Sleep Quality Index Japanese version (PSQI-J) and actigraphy; and immune function, with laboratory tests. PSQI-J scores and actigraphy sleep latency and bed out latency improved significantly more with NBIW bathing than with regular bathing (p < 0.05). Furthermore, NBIW bathing reduced both stress-induced fluctuations in CD4+ and CD8+ T cell counts and fluctuations in the naive to memory T cell ratio and neutrophil phagocytosis, indicating improved immune function. These findings suggest that daily NBIW bathing could improve mental stress, sleep quality, and immune function and bring about positive health effects in those who experience stress in their daily lives.

The effects of bathing in neutral bicarbonate ion water.

Percutaneously absorbed carbon dioxide enhances blood flow. The mechanism by which it does so is unclear, but we hypothesized that it involves bicarbonate ions. BALB/c mice were bathed in neutral bicarbonate ionized water (NBIW) and showed increased blood bicarbonate levels and blood flow via phosphorylation of peripheral vascular endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO). Phosphorylation of eNOS and NO production were also increased in human umbilical vein endothelial cells cultured in medium containing NBIW, and NBIW showed reactive oxygen species scavenging activity. In a double-blind, randomized study in men and women aged 30 to 59 years with subjective cold intolerance, bathing in NBIW elevated body temperature faster than bathing in a control solution and improved chills and sleep quality. Taken together, our results show that percutaneously absorbed carbon dioxide changes to bicarbonate ions, which act directly on endothelial cells to increase NO production by phosphorylation of eNOS and thus improve blood flow.

Characterization of JC Polyomavirus Derived from COS-IMRb Cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system affecting immunocompromised patients. The study of PML-type JCPyV in vitro has been limited owing to the inefficient propagation of the virus in cultured cells. In this study, we carried out long-term culture of COS-7 cells (designated as COS-IMRb cells) transfected with PML-type M1-IMRb, an adapted viral DNA with a rearranged non-coding control region (NCCR). The JCPyV derived from COS-IMRb cells were characterized by analyzing the viral replication, amount of virus by hemagglutination (HA), production of viral protein 1 (VP1), and structure of the NCCR. HA assays indicated the presence of high amounts of PML-type JCPyV in COS-IMRb cells. Immunostaining showed only a small population of JCPyV carrying COS-IMRb cells to be VP1-positive. Sequencing analysis of the NCCR of JCPyV after long-term culture revealed that the NCCR of M1-IMRb was conserved in COS-IMRb cells without any point mutation. The JCPyV genomic DNA derived from a clone of COS-IMRb-3 cells was detected, via Southern blotting, as a single band of approximately 5.1 kbp without deletion. These findings suggest the potential of using COS-IMRb-3 cells as a useful tool for screening anti-JCPyV drugs.

Establishment of COS-JC cells persistently producing archetype JC polyomavirus.

JC polyomavirus (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunocompromised patients. Archetype JCPyV circulates in the human population. There have been several reports of archetype JCPyV replication in cultured cells, in which propagation was not enough to produce high titers of archetype JCPyV. In this study, we carried out cultivation of the transfected cells with archetype JCPyV DNA MY for more than 2 months to establish COS-7 cells (designated COS-JC cells) persistently producing archetype JCPyV. Moreover, JCPyV derived from COS-JC cells was characterized by analyzing the viral propagation, size of the viral genome, amount of viral DNA, production of viral protein, and structure of the non-coding control region (NCCR). Southern blotting using a digoxigenin-labeled JCPyV probe showed two different sizes of the JCPyV genome in COS-JC cells. For molecular cloning, four of five clones showed a decrease in the size of complete JCPyV genome. Especially, clone No. 10 was generated the large deletion within the Large T antigen. On the other hand, the archetype structure of the NCCR was maintained in COS-JC cells, although a few point mutations occurred. Quantitative PCR analysis of viral DNA in COS-JC cells indicated that a high copy number of archetype JCPyV DNA was replicated in COS-JC cells. These findings suggest that COS-JC cells could efficiently propagate archetype JCPyV MY and offer a useful tool to study persistent infection of archetype JCPyV in a kidney-derived system.

CPT11 prevents virus replication in JCI cells persistently infected with JC polyomavirus.

JC polyomavirus (JCPyV) is the causative agent of the demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML), which occurs in immunocompromised patients. Moreover, patients treated with natalizumab for multiple sclerosis or Crohn disease can develop PML, which is then termed natalizumab-related PML. Because few drugs are currently available for treating PML, many antiviral agents are being investigated. It has been demonstrated that the topoisomerase I inhibitors topotecan and ß-lapachone have inhibitory effects on JCPyV replication in IMR-32 cells. However, both of these drugs have marginal inhibitory effects on virus propagation in JC1 cells according to RT-PCR analysis. In the present study, the inhibitory effect of another topoisomerase I inhibitor, 7-ethy-10-[4-(1-piperidino)-1-piperidino] carbonyloxy camptothecin (CPT11), was assessed by investigating viral replication, propagation, and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using real-time PCR combined with Dpn I treatment in IMR-32 cells transfected with JCPyV DNA. It was found that JCPyV replicates less in IMR-32 cells treated with CPT11 than in untreated cells. Moreover, CPT11 treatment of JCI cells persistently infected with JCPyV led to a dose-dependent reduction in JCPyV DNA and VP1 production. Additionally, the inhibitory effect of CPT11 was found to be stronger than those of topotecan and ß-lapachone. These findings suggest that CPT11 may be a potential anti-JCPyV agent that could be used to treat PML.

Suppressive effect of topoisomerase inhibitors on JC polyomavirus propagation in human neuroblastoma cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system, in immunocompromised patients. Because no drugs have been approved for treating PML, many antiviral agents are currently being investigated for this purpose. The inhibitory effects of the topoisomerase I inhibitors topotecan and ß-lapachone were assessed by investigating viral replication, propagation and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using the human neuroblastoma cell line IMR-32 transfected with the JCPyV plasmid and RT- PCR combined with Dpn I treatment. Dpn I digests the input plasmid DNA containing methylated adenosine, but not newly replicated JCPyV DNA, in IMR-32 cells. It was found that JCPyV replicates less in IMR-32 cells treated with topotecan or ß-lapachone than in untreated cells. Moreover, drug treatment of JCI cells, which are IMR-32 cells persistently infected with JCPyV, led to a reduction in the amount of JCPyV DNA and population of VP1-positive cells. These results demonstrate that topotecan and ß-lapachone affects JCPyV propagation in human neuroblastoma cell lines, suggesting that topotecan and ß-lapachone could potentially be used to treat PML.

Replication of IMR-32-adapted JC virus clones in human embryonic kidney cells.

It has been difficult to study JCV replication because of its restricted host range. In this study, JCV replication was examined using different clones in 293 cells. RT-PCR assay revealed that large T antigen expression in cells transfected with IMR-32-adapted JCVs was significantly greater than in those transfected with Mad-1 or CY. DNA replication assay and viral load verified that the IMR-32-adapted JCVs were replication-competent in 293 cells, but not Mad-1 or CY JCVs. These results suggest that a 293 culture system with IMR-32-adapted JCVs may be a useful tool for assessing replication of JCV in vitro.

TNF-α stimulates efficient JC virus replication in neuroblastoma cells.

JC polyomavirus (JCV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS) in immunocompromised patients, and particularly in the severe immunosuppression associated with acquired immunodeficiency syndrome (AIDS). HIV-1 can lead to the production of tumor necrosis factor-alpha (TNF-α) in the CNS. Our aim was to examine the effects of TNF-α on JCV gene expression and replication using a human neuroblastoma cell line, IMR-32, transfected with JCV DNA, M1-IMRb. Quantitative RT-PCR analysis of JCV large T antigen and VP1 mRNA, the viral DNA replication assay, and the DNase protection assay were carried out. TNF-α treatment of IMR-32 cells transfected with JCV DNA induced large T antigen mRNA and JCV DNA replication, while other effects on VP1 mRNA expression and virus production were marginal. In addition, ELISA analysis of the nuclear p65 subunit of nuclear factor κB (NF-κB), which is a hallmark of NF-κB pathway activation, of IMR-32 cells upon TNF-α treatment showed that TNF-α treatment activated the NF-κB pathway in IMR-32 cells. Taken together, our results suggest that TNF-α stimulation could induce JCV replication associated with the induction of JCV large T antigen mRNA through the NF-κB pathway in IMR-32 cells transfected with JCV DNA. Our findings may contribute to further understanding of the pathogenesis of AIDS-related PML.

Suppressive effect of PARP-1 inhibitor on JC virus replication in vitro.

The incidence of progressive multifocal leukoencephalopathy (PML) has increased due to the AIDS pandemic, hematological malignancies, and immunosuppressive therapies. Recently, the number of cases of monoclonal antibody-associated PML has increased in patients treated with immunomodulatory drugs such as natalizumab. However, no common consensus regarding PML therapy has been reached in clinical studies. In order to examine the suppression of JC virus (JCV) replication by 3-aminobenzamide (3-AB), a representative PARP-1 inhibitor, a DNA replication assay was carried out using the neuroblastoma cell line IMR-32 and IMR-adapted JCV. The suppression of JCV propagation by 3-AB was also examined using JCI cells, which are a carrier culture producing continuously high JCV titers. The results indicated that PARP-1 inhibitors, such as 3-aminobenzamide (3-AB), suppress JCV replication and propagation significantly in vitro, as judged by DNA replication assay, hemagglutination, and real-time PCR analysis. It has been also shown that 3-AB reduced PARP-1 activity in IMR-32 cells. According to the results of the MTT assay, the enzyme activity of 3-AB-treated cells was slightly lower than that of DMSO-treated cells. However, the significant suppression of JCV propagation is not related to the slight decrease in cell growth. To our knowledge, this is the first report that PARP-1 inhibitor suppresses the replication of JCV significantly in neuroblastoma cell lines via the reduction of PARP-1 activity. Thus, PARP-1 inhibitors also may be a novel therapeutic drug for PML.

Exogenous human immunodeficiency virus-1 protein, Tat, enhances replication of JC virus efficiently in neuroblastoma cell lines.

The high incidence of progressive multifocal leukoencephalopathy (PML) among individuals with acquired immunodeficiency syndrome (AIDS) is similar to the incidence of other immunocompromised diseases. The pathogenic JC virus (JCV) with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease in the brains of immunocompromised patients. In a previous study, Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), markedly enhanced the expression of a reporter gene under control of the JCV late promoter. In order to examine the enhancement of JCV replication by Tat protein, the neuroblastoma cell line IMR-32 was used because it enables IMR-32-adapted JCV. The extent of JCV replication in IMR-32 cells treated with Tat protein was significantly higher than that in untreated IMR-32 cells. The enhancement of JCV propagation by Tat protein was also examined using IMR-32-derived JCV producing (JCI) cells which continuously produce JCV. Treatment of JCI cells with Tat protein led to a significant increase in the titers of progeny viruses. It has also been shown that Tat protein leads to a decrease in the expression of purine-rich element binding protein α (Purα) as an important mediator of JCV replication in IMR-32 cells. Thus, it is probable that Tat protein enhances JCV replication in IMR-32 cells via the down-regulation of Purα expression and cell proliferation. To our knowledge, this is the first report that exogenous Tat protein enhances the replication of JCV efficiently in neuroblastoma cell lines.

Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1.

The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.

Archetype JC virus efficiently propagates in kidney-derived cells stably expressing HIV-1 Tat.

Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter. In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.

Inhibitory effect of serotonin antagonists on JC virus propagation in a carrier culture of human neuroblastoma cells.

Human polyomavirus, JCV, causes fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). It has been shown that 5HT(2A)R acts as a cellular receptor for JCV on human glial cells. In the current study, we examined the inhibitory effects of 5HT(2A)R antagonists, ketanserin and ritanserin, both on JCV infection and on propagation by using human neuroblastoma cells IMR-32 and JCI, which continuously produce JCV. Transcriptional analysis revealed that 5HT(2A)R was constitutively expressed in JCI cells. Treatments with 5HT(2A)R antagonists led to a significant reduction in the titers of progeny viruses and the population of infected JCI cells. In addition, the amount of JCV genomic DNA was decreased in JCI cells in the presence of 5HT(2A)R antagonists. These results indicate that 5HT(2A)R antagonists have an inhibitory effect on JCV infection and reproduction, and JCI cells are applicable to an experimental model for pharmacological evaluation of antiviral agents against JCV.

Alkalization of blood pH is responsible for survival of cancer patients by mild hyperthermia.

The effect of mild hyperthermia on venous blood pH was examined in 6 cancer patients. Mild hyperthermia was induced by continuation of a rectal temperature of 39.5 degrees C for 30 min. All 6 patients were diagnosed as suffering from advanced cancer with or without surgery and chemotherapy pretreatments. In Cases 1 to 5, but not Case 6, venous blood pH was alkalized up to pH 7.7 by this mild hyperthermia and the effect was reproduced depending on the application of hyperthermia. At this time, alkalized pH was accompanied by increased PO2 and decreased PCO2 in the blood. These patients showed good physical conditions and improved clinical data. On the other hand, hyperthermia could not be continued in Case 6 due to his worsened physical condition. The present data suggest that mild hyperthermia is a useful method to improve circulation failure, physical condition and clinical data.

Comparison of protective efficacies of plasmid DNAs encoding Japanese encephalitis virus proteins that induce neutralizing antibody or cytotoxic T lymphocytes in mice.

Mice immunized with a plasmid DNA encoding the premembrane (prM) and envelope (E) proteins of Japanese encephalitis (JE) virus (designated pcJEME) produce neutralizing antibodies and are protected from JE. To determine the role of the immune response to other viral proteins in protection, we constructed plasmid DNAs encoding other JE virus proteins and made a direct comparison among these plasmids using a mouse model. Cytotoxic T lymphocytes (CTLs) were induced by plasmids encoding capsid (C) or nonstructural proteins, NS1, NS2A, NS2B, NS3 or NS5. However, these plasmids provided only a partial protection against intraperitoneal challenge with a lethal dose of JE virus, whereas mice immunized with pcJEME were fully protected. In mice inoculated with CTL-inducing plasmids, high virus titers were detected in plasma immediately (1h) following challenge and in brain on day 4 post-challenge, but no virus infectivity was detected in plasma and brain of pcJEME-immunized mice during the 5 days following challenge. These results indicate that protection provided by the prM/E-encoding DNA consists of neutralizing antibody that prevents virus dissemination from the peripheral site to the brain, and that this antibody-mediated mechanism of protection is more efficient than the immunity induced by plasmids that generate CTL responses capable of killing JE virus-infected cells.

Enhancing effect of vaxfectin on the ability of a Japanese encephalitis DNA vaccine to induce neutralizing antibody in mice.

Vaxfectin, a recently developed adjuvant, was evaluated for its enhancing effect on immunogenicity of a Japanese encephalitis (JE) DNA vaccine plasmid encoding the JE virus premembrane (prM) and envelope (E) genes (designated pcJEME), using BALB/c and ICR mice. Formulation of pcJEME with Vaxfectin provided > or =8-fold higher neutralizing antibody titers than those induced by pcJEME alone and reduced the amount of pcJEME to one-tenth to induce comparable levels of neutralizing antibody. Use of Vaxfectin did not alter a Th1 type IgG isotype immune response (IgG1 < IgG2a) induced by pcJEME in mice. These results indicate that Vaxfectin has an ability to enhance immunogenicity of pcJEME and is considered as a useful adjuvant for DNA vaccines in murine experimental models.