Hisashi Ohkuni

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.

Efficacy of gentian violet in the eradication of methicillin-resistant Staphylococcus aureus from skin lesions

The efficacy of gentian violet (Gv) in eradicating methicillin-resistant Staphylococcus aureus (MRSA) in decubitus ulcers was investigated. Decubitus ulcers (a total of 18 cases) were scrubbed with Gv aqueous solution 0.1% and ointment containing Gv 0.1% was applied daily. MRSA was not detected in these lesions for 3-34 days (average, 10.5 +/- 2.5 days) after the application of Gv ointment. Before this trial, all patients were treated with povidone-iodine and antibiotics; however, those treatments were not effective in eradicating MRSA from skin lesions. Skin irritation and other systemic side effects caused by Gv were not observed. Our data suggest that Gv is a useful agent for treatment of the decubitus ulcers infected with MRSA.

Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov., new members of the Streptococcus mitis group, isolated from human clinical specimens

Taxonomic studies were performed on eight strains of alpha-haemolytic streptococci that showed very low DNA-DNA hybridization similarity values with all established members of the mitis group of the genus Streptococcus. These strains were isolated from the tooth surface and pharynx of humans. 16S rRNA gene sequence analysis showed that these strains belonged to the mitis group, but that they fell into two new branches. DNA-DNA hybridization demonstrated two new similarity groups. From the results of the present study, the names Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov. are proposed for these new groups. The type strains are O-66T (= GTC 848T = JCM 10158T) and O-122T (= GTC 849T = JCM 10157T), respectively.

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.

Immunological studies of post-streptococcal sequelae. Evidence for presence of streptococcal antigens in circulating immune complexes.

Since elevated levels of circulating complexes have been noted to occur in the sera of patients with post-streptococcal sequelae, the possibility that these complexes contained streptococcal antigens within the complex was investigated. Sera from these patients were precipitated with polyethylene glycol to extract a fraction rich in these complexes, which was then injected into rabbits. The rabbit sera were then reacted with both cellular and extracellular fractions obtained from streptococcal strains associated with either acute post-streptococcal nephritis (APSGN) or acute rheumatic fever (ARF) by using immunoelectrophoresis and ELISA techniques. The data demonstrate that both ARF and APSGN complexes contain streptococcal antigens. However, APSGN complexes react uniquely to certain extracellular antigens present in those strains associated with nephritis, while ARF complexes react specifically to certain streptococcal extracellular antigens excreted by strains associated with rheumatic fever. Neither of the two groups of complexes appear to contain streptococcal antigens related to any cellular antigens derived from the group A streptococcus. Additionally, a rabbit serum immunized with streptococcal extracellular products reacted directly with complexes isolated from nephritis patients. Removal of the gamma globulin by absorption with an anti-human Fc serum resulted in the concomitant loss of reactivity with the anti-streptococcal serum, strongly suggesting an intimate association of the streptococcal antigen with these complexes. The presence of streptococcal antigens within the circulating immune complex of patients with APSGN coupled with their specific presence in those strains associated with post-streptococcal glomerulonephritis argues strongly for a causal role of these antigens in the disease process.

Characterization of recombinant Streptococcus mitis‐derived human platelet aggregation factor

Ohkuni H, Nagamune H, Ozaki N, Tabata A, Todome Y, Watanabe Y, Takahashi H, Ohkura K, Kourai H, Ohtsuka H, Fischetti VA, Zabriskie JB. Characterization of recombinant Streptococcus mitis‐derived human platelet aggregation factor. APMIS 2012; 120: 56–71.

Possible Pathogenic Effect of Streptococcus mitis Superantigen on Oral Epithelial Cells

Viridans and non-hemolytic streptococci in oral cavity have been suggested to be involved in the occurrence of some oral disorders and systemic diseases such as Behcet’s disease and Kawasaki disease. However, little is known about the pathogenic roles of extracellular products of these streptococci. Recently, we isolated a strain of Streptococcus mitis, strain 108, from the tooth surface of a male infant with aseptic meningitis and prepared two fractions, F-1 and F-2, from the concentrated culture supernatant. The fraction F-1 induced inflammatory cytokine production in human peripheral blood cell and gingival fibroblast cultures (1). The fraction F-2 had superantigenicity: it induced proliferation of human T cells bearing Vb2 and Vb5.1 T cell receptors in the presence of irradiated autologous peripheral blood mononuclear cells (2). In this study, we examined the cytotoxic effects of human peripheral blood T cells activated with the F-2 on oral epithelial cells and discussed the possibility that the superantigen were associated with the pathogenesis of oral mucosal disorders.

The presence of tumor necrosis factor-α and its antibody in the sera of cachexic patients with gastrointestinal cancer

Although cancer cachexia has been shown to involve several cytokines, the tumor necrosis factor-α (TNF) has rarely been detected in such patients. In this study, sera from 21 patients with cancer cachexia were examined for the presence of TNF and the anti-TNF antibody using an enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. All of the patients had recurrent cancer and manifested the characteristics of progressive body weight loss. TNF was found in the sera of four patients (20%) at levels ranging from 10.4 to 53.1 pg/ml, while a positive reaction for the anti-TNF antibody was detected in the sera from six patients (30%), two of whom showed both TNF and its antibody. Thus, either TNF or the anti-TNF antibody was present in the sera from 8 of 21 patients (40%). The results of this study indicate that TNF may be present in the circulation of at least 40% of cachexic patients, and suggest that it may be one of the main mediators of cancer-associated cachexia.

Correlation between epidermal growth factor receptor concentration and the growth of human gastric cancer xenografts in nude mice

SummarySeven human gastric cancer xenografts with different concentrations of EGF receptor were established in nude mice. The expression of EGF receptor in the tumors was demonstrated by Western blotting with anti-EGF receptor antibody, binding assay with125I-EGF and immunohistochemistry with anti-EGF receptor antibody. Western blotting revealed EGF receptor doublet bands at molecular masses of 150 KDa and 170 KDa in all of the samples. The concentration of125I-EGF binding activity in the tumors ranged from 36.0 to 11,000 fmol/mg protein, with a mean of 345 fmol/mg protein. EGF receptor was also demonstrated immunohistochemically on the apical border of the glands and the cell membrane of the tumor cells. There seemed to be a close correlation between the concentration of125I-EGF binding activity and the doubling time of these tumors in nude mice (γ= -0.68). However, no definite correlation was observed between EGF ligand binding and histological features of intestinal type or diffuse type. The expression of EGF receptor appears to facilitate the growth of human gastric cancer xenografts in nude mice.