Shinsaku Sakurada

Moderate hypothermia delays proinflammatory cytokine production of human peripheral blood mononuclear cells.

Objective To clarify the influence of moderate hypothermia on the production of proinflammatory cytokines. Design Controlled in vitro study. Setting Research laboratory. Subjects Peripheral blood mononuclear cells from healthy adult human subjects. Interventions Stimulation with 1 &mgr;g/mL lipopolysaccharide at 33°C and 37°C. Measurements Concentrations of released tumor necrosis factor-&agr;, interleukin-1&bgr;, and interleukin-6 were measured chronologically by enzyme immunoassay. The number of mRNA copies of these cytokines was determined by competitive reverse transcriptase-polymerase chain reaction analysis, and nuclear factor-&kgr;B activations were assessed by electrophoretic mobility shift assay. Main Results Significant reduction of the released-tumor necrosis factor-&agr; concentration was observed 1 and 2 hrs after the stimulation with lipopolysaccharide at 33°C compared with 37°C. The peak release of interleukin-1&bgr; at 33°C was delayed 12 hrs later than that at 37°C. A delayed peak in the release of interleukin-6 also was observed at 33°C. The peaks of cytokines were confirmed at the mRNA expression level by competitive reverse transcriptase-polymerase chain reaction analysis at both temperatures. The peak of the tumor necrosis factor-&agr; mRNA expression level was observed at 1 hr after the stimulation at 37°C and 2 hrs after the stimulation at 33°C. In the interleukin-1&bgr; mRNA expression, at 37°C the first peak appeared 1 hr and the second 6 hrs after the stimulation. In contrast, at 33°C, the first peak appeared 2 hrs and the second 12 hrs after the stimulation. Whereas interleukin-6 mRNA expression at 37°C peaked 6 hrs after the stimulation, no definite peak was observed at 33°C and the expression level was approximately half of that at 37°C. The maximum intensity of nuclear factor-&kgr;B activation at 33°C was delayed by 1.5 hrs compared with that at 37°C. Conclusions Moderate hypothermia delays the induction of proinflammatory cytokines in human peripheral blood mononuclear cells.

Regulation of NF-kappa B and disease control: identification of a novel serine kinase and thioredoxin as effectors for signal transduction pathway for NF-kappa B activation.

We have identified novel signal transduction cascades in activating NF-kappa B, as well as its pathogenetic roles in various disease processes. By applying the basic knowledge obtained through these studies, we hope to find new therapeutic measures against currently incurable diseases such as hematogenic cancer cell metastasis, rheumatoid arthritis, and AIDS. We also propose a novel strategy in screening effective inhibitors against transcription factors. Elucidation of the cis-regulatory element for expression of pathogenetic genes and identification of the responsible transcription factor will not only facilitate the study of pathogenesis but will also promote the development of effective therapy. Recognition of control mechanisms of the NF-kappa B activation pathway has explained the therapeutic efficacy of various compounds with different pharmacologic actions. A similar strategy may be applicable for other inducible transcription factors. From the medical point of view, one of the purposes of these approaches is to find small molecular weight compounds that can be administered orally and that are effective in controlling gene expression of pathogenetic genes.

Effective Human Herpesvirus 8 Infection of Human Umbilical Vein Endothelial Cells by Cell-Mediated Transmission

ABSTRACT Cell-free transmission of human herpesvirus 8 (HHV-8) to human cells in vitro has been reported to be difficult, if not impossible. The present experiments were conducted with the idea that cell-cell contact may produce much more effective transmission, so-called cell-mediated transmission. Primary human umbilical vein endothelial cells (HUVECs) were cocultured with an HHV-8-infected lymphoma cell line, BCBL-1 cells. When a ratio of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated BCBL-1 cells to HUVECs of 10:1 was used, more than 20% of HUVECs were found to express the HHV-8 latency-associated nuclear antigen (LANA) 48 h after the start of coculturing; this value increased to more than 30% after 72 h. HHV-8-encoded ORF26, K8, K8.1, K10, K11, ORF59, and ORF65 proteins were not detected in these HHV-8-infected HUVECs until 72 h. The HHV-8 antigens were not observed in HUVECs cocultured with TPA-treated BCBL-1 cells separated by a membrane. Thirty days after removal of the BCBL-1 cells from the cell-mediated transmission experiment, the HUVECs still expressed LANA and the HHV-8 genome was detected by PCR in these cells. Moreover, the ORF59 protein, a DNA replication-associated protein of HHV-8, was expressed in such HUVECs in the presence of TPA stimulation. These results indicated a far more effective transmission mechanism, cell-cell contact, suggesting the possibility that such a mechanism works in vivo.

Cysteine protease activity and histamine release from the human mast cell line HMC-1 stimulated by recombinant streptococcal pyrogenic exotoxin B/streptococcal cysteine protease.

ABSTRACT We constructed the expression vector pSK-SCP containing the streptococcal exotoxin B gene (spe b) which expressed protease activity. We showed that the recombinant streptococcal pyogenic exotoxin B/streptococcal cysteine protease (rSPE B/SCP) was secreted into the culture supernatant of the transformant and retained its SCP activity, which was equivalent to or greater than that of the naturally occurring molecule. The secreted rSPE B/SCP induced histamine release and degranulation of the human mast cell line HMC-1. This study may contribute to the understanding of the pathogenic role of SPE B/SCP in streptococcal infection and streptococcal toxic shock syndrome.

Involvement of Vascular Endothelial Growth Factor in Kaposi's Sarcoma Associated with Acquired Immunodeficiency Syndrome

To examine the role of vascular endothelial growth factor (VEGF) in the development of edema associated with Kaposis sarcoma (KS) in acquired immunodeficiency syndrome (AIDS), we exploited animal model systems to detect the activity that induces vascular hyper‐permeability (VHP) using cultured AIDS‐KS spindle cells. Cultured AIDS‐KS spindle cells and conditioned medium (AIDS‐KS‐CM) that had been semi‐purified through a heparin affinity column were tested for the ability to induce VHP in animals. The AIDS‐KS spindle cells and AIDS‐KS‐CM induced VHP that was histamine‐independent. The VHP‐inducing activity was detected in the 0.5 M NaCl fraction from the heparin affinity column and was blocked by anti‐VEGF neutralizing antibody. In addition, the production of VEGF was demonstrated in fresh AIDS‐KS tissue as well as in cultured AIDS‐KS cells, while control cells were negative for VEGF production. From these observations, we concluded that AIDS‐KS cells produce a factor(s) that promotes VHP, and this factor could be VEGF.

Alteration of the cellular response to interleukin-1ß by SV40 large T antigen in rheumatoid synovial fibroblasts

SummaryThe large T antigen of SV40 (LT) has been widely used to immortalize primary cells for various studies. In this study, synovial fibroblasts of a patient from rheumatoid arthritis (RA) were transformed with LT gene to analyze the effect of SV40-mediated transformation on the production of cytokines, such as IL-6, IL-8, and GM-CSF, that are under the control of interleukin-1β (IL-1β), a physiological inducer of nuclear factor κB (NF-κB). It was noted that the basal levels of GM-CSF and IL-8 were upregulated, whereas that of IL-6 was downregulated. Moreover, the extents of induction of these cytokines in response to IL-1β were markedly downregulated in synovial fibroblasts transformed by LT as compared from parental cells. Although IL-1β could translocate NF-κB to the nucleus in all cells, some of the transformed cells exhibited nuclear translocation of NF-κB even before the stimulation with IL-1β, suggesting that transformation of LT resulted in the constitutive activation of NF-κB, either directly or indirectly. In order to examine whether LT downregulate the κB-dependent gene expression, we performed the transient luciferase gene expression assay. We found that co-transfection of LT did not downregulate the κB-dependent gene expression that was stimulated with L-1β. These observations suggest that the apparent inhibitory effect of LT on the IL-1-induced expression of cytokines may not be through its direct action on the NF-κB transactivation.

Inhibition of Cytokines and ICAM-1 Induction in Rheumatoid Fibroblasts by anti-NF-κB Reagents

The role of the transcription factor, nuclear factor NF-κB, in the induction of cytokines and intercellular adhesion molecule-1 (ICAM-1) on stimulation with interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) was studied in primary rheumatoid synovial fibroblasts (RSF) obtained from patients with rheumatoid arthritis (RA). The production of GM-CSF, IL-6, and IL-8 and the expression of ICAM1 were augmented after nuclear translocation of NF-κB induced by treatment with IL-1 or TNF-α. We examined the effects of N-acetyl-l-cysteine (NAC) and acetylsalicylic acid (aspirin) on the induction of proinflammatory cytokines and ICAM-1. Pretreatment of RSF with NAC inhibited nuclear translocation of NF-κB completely, and the induction of these cytokines and ICAM-1 was markedly suppressed. On the other hand, the effect of aspirin was only partial. These observations indicate the pivotal role of NF-κB in RA pathogenesis, thus highlighting the possibility of a novel therapeutic strategy.

Redox Regulation of the Nuclear Factor Kappa B (NF-κB) Signaling Pathway and Disease Control

Nuclear factor kappa B (NF-κB) is an inducible cellular transcription factor that activates various cellular and viral genes. NF-κB usually exists as a molecular complex with an inhibitory molecule, IκB, in the cytosol. On stimulation of the cells, such as by proinflammatory cytokines IL-1 and tumor necrosis factor (TNF), IkB is dissociated and NF-κB is translocated to the nucleus and activates the expression of the target genes. We found that a redox control mechanism is involved in the DNA-binding activity of NF-κB and that a cellular-reducing catalyst thioredoxin (Trx), together with kinases, is primarily involved as an effector molecule in this signaling pathway. Trx was recently demonstrated to associate with the redox-sensitive cysteine within the DNA-binding loop of NF-κB. Effects of antioxidants in blocking NF-κB activation can be explained by the involvement of radical oxygen intermediates (ROI) in this pathway. These findings support the idea that redox regulation involving ROI and Trx plays a crucial role in the signal transduction pathway leading to NF-κB activation, thus contributing substantially to understanding of the pathogenetic processes of various diseases including AIDS, hematogenic cancer cell metastasis, and rheumatoid arthritis (RA).