CD14-mediated induction of interleukin-8 and monocyte chemoattractant protein-1 by a heat-resistant constituent of Porphyromonas gingivalis in endothelial cells.

Viable and inactivated Porphyromonas gingivalis dose-dependently induced interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) secretion in human umbilical vein endothelial cells (HUVECs). The inactivated P. gingivalis, in comparison with viable bacteria, tended to enhance the production of both chemokines more strongly. The production of MCP-1 protein began increasing immediately after stimulation by P. gingivalis, and there was a nearly linear increase from 0 to 8 h of incubation, whereas IL-8 production showed a linear increase between 4 and 12 h of incubation. The IL-8 and MCP-1 mRNA expressions in HUVECs as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) or Quantikine mRNA colorimetric quantification kits were found to be enhanced by P. gingivalis. Furthermore, the time courses of IL-8 and MCP-1 mRNA expressions were in accordance with those of protein production. Addition of polymyxin B or boiling did not weaken the stimulatory effect of P. gingivalis, which inhibited the effect of Escherichia coli lipopolysaccharide (E. coli LPS) and tumour necrosis factor-alpha (TNF-alpha), respectively. In contrast, the induction of IL-8 and MCP-1 by P. gingivalis was significantly reduced by anti-CD14 antibody. Our results suggest that some heat-stable component of P. gingivalis, including LPS, may be responsible for the induction of IL-8 and MCP-1 in HUVECs by a CD14-dependent mechanism. These effects might be involved in the accumulation and activation of neutrophils and monocytes at an early stage of the periodontal pathogenesis.

A heat-stable component of Bartonella henselae upregulates intercellular adhesion molecule-1 expression on vascular endothelial cells.

Bartonella henselae upregulated the expression of intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). The induction level of ICAM-1 depended on the inoculation bacterial dose. ICAM-1 expression began increasing 4 h after infection and reached a sustained peak beginning at 12 h after B. henselae infection; this time course was similar to that of lipopolysaccharide (LPS) of Escherichia coli. The stimulatory effect was abolished when live B. henselae were separated from HUVECs by a filter membrane. The nonpiliated strain, which is unable to invade endothelial cells, induced ICAM-1 expression to the same extent as the piliated strain. Inactivation of B. henselae by ultraviolet (UV) irradiation, heat (56 degrees C, 30 min), or sonication did not alter its stimulatory activity. Polymyxin B, which strongly inhibited the effect of LPS, did not exert any influence on the stimulatory activity of B. henselae. Furthermore, the effect of sonicated B. henselae was not inhibited even by boiling, which was also the case with LPS. Our data suggest that some heat-stable component of B. henselae binds to the endothelial cell surface, inducing ICAM-1 expression. Though the participation of LPS could not be completely ruled out, we suppose that some unidentified heat-stable proteins, lipids, or polysaccharides may be the stimulatory factor(s). The ability of B. henselae to enhance the expression of adhesion molecules on endothelial cells may be an important mechanism in the pathogenesis of B. henselae infection.

Intracellular infection by the human granulocytic ehrlichiosis agent inhibits human neutrophil apoptosis.

In patients with human granulocytic ehrlichiosis (HGE), the HGE agent has been seen only in the peripheral blood granulocytes, which have a life span too short for ehrlichial proliferation. To determine if the HGE agent delays the apoptosis of human peripheral blood neutrophils for its advantage, peripheral blood granulocytes consisting mostly of neutrophils were incubated with freshly freed host cell-free HGE agent in vitro. The HGE agent induced a significant delay in morphological apoptosis and the cytoplasmic appearance of histone-associated DNA fragments in the granulocytes. This antiapoptotic effect was dose dependent. Although much weaker than the HGE agent freshly freed from the host cells, noninfectious purified HGE agent stored frozen and thawed also had antiapoptotic effect, which was lost with proteinase K treatment but not with periodate treatment. Treatment of neutrophils with a transglutaminase inhibitor, monodansylcadaverine, blocked the antiapoptotic effect of the HGE agent. Addition of oxytetracycline, however, did not prevent or reverse the antiapoptotic effect of the HGE agent. These results suggest that binding of a protein component(s) of the HGE agent to neutrophils and subsequent cross-linking and/or internalization of the receptor and ehrlichiae are required for antiapoptotic signaling, but ehrlichial protein synthesis and/or proliferation is not required. MG-132, a proteasome inhibitor, and cycloheximide accelerated the apoptosis of neutrophils and overrode the antiapoptotic effect of the HGE agent. Studies with specific inhibitors suggest that protein kinase A, NF-kappaB, and interleukin 1beta are not involved in the antiapoptotic mechanism of the HGE agent.

Live Bartonella henselae enhances endothelial cell proliferation without direct contact.

The proliferation of human umbilical vein endothelial cells (HUVECs) cocultivated with live B. henselae was enhanced in a bacterial dose-dependent manner, and the stimulatory effect was specific to vascular endothelial cells. The inactivation of B. henselae by UV or heat treatment abolished its stimulatory activity, suggesting that live bacteria is necessary for the growth stimulation effect. To investigate the role of direct contact, live B. henselae were separated from HUVECs by a filter membrane (Millicell-CM insert). Even under this condition, an enhanced proliferation of HUVECs was observed. However, no morphological changes in the HUVECs were apparent compared to the B. henselae -infected cells. Furthermore, we isolated a nonpiliated strain of B. henselae that is unable to attach to and enter into endothelial cells. The nonpiliated strain possessed the ability to stimulate the proliferation of cocultivated HUVECs the same as the piliated strain. Moreover, the culture supernatants of B. henselae were also able to induce HUVEC proliferation. Our results indicate that the stimulation of HUVEC proliferation by B. henselae is mediated by soluble factor(s) secreted from the bacteria.

Induced production of nitric oxide and sensitivity of alveolar macrophages derived from mice with different sensitivity to Coxiella burnetii.

We compared in vitro sensitivities to Coxiella burnetii of alveolar macrophages, derived from mice sensitive and resistant to C. burnetii, respectively, and examined the role of nitric oxide (NO) in the C. burnetii infection. Alveolar macrophages of sensitive A/J mice showed a larger population of C. burnetii antigen-positive cells than those of resistant C57BL/6 mice. C. burnetii induced NO production in alveolar macrophages, but N-methyl-L-arginine and sodium nitroprusside (SNP), NO inhibitor and donor, respectively, did not inhibit the infection. Thus the NO induction seems to be independent of the cell defense mechanism against the C. burnetii infection.

A subcloned human esophageal squamous cell carcinoma cell line with low thrombomodulin expression showed increased invasiveness compared with a high thrombomodulin-expressing clone--thrombomodulin as a possible candidate for an adhesion molecule of squamous cell carcinoma.

Thrombomodulin (TM) is an endothelial cell surface glycoprotein which converts thrombin from a procoagulant protease to an anticoagulant. We have previously reported that TM is a useful marker for immunohistochemical diagnosis of angiogenic tumors and also have reported that TM is expressed on squamous cell carcinoma (SCC) of the human esophagus. In addition, the expression of TM is significantly decreased in metastatic foci in lymph nodes compared with that in primary lesions. In order to reveal the biological significance of TM in SCC, we subcloned and established two different cell lines, i.e. TM-high-expressing (TE3HTM) cells and TM-low-expressing (TE3LTM) cells, from a human SCC cell line, TE3, using fluorescence-activated cell sorter (FACS) and examined the biological characteristics of these variant cell lines. These tumor cells revealed very similar morphological figures in ordinary cultured conditions and showed almost equal growth rates under various cultured conditions. By the invasion assay of these tumor cells using matrigel, we found that TE3LTM cells showed significantly increased invasive ability compared with that of TE3HTM cells. Characteristic intercellular localization of TM and a different manner of invasiveness between TE3LTM cells and TE3HTM cells suggest that TM may act as a cell-to-cell interaction molecule.

High concentration of recombinant murine interferon-beta enhances the growth of Orientia (formerly Rickettsia) tsutsugamushi Gilliam in mouse L929 cells.

We studied the effect of recombinant murine interferons (rMuIFNs) on the growth of Orientia (formerly Rickettsia) tsutsugamushi Gilliam in mouse L929 cells. Rickettsial growth was measured by flow cytometry. rMUIFN-gamma inhibited the growth of O. tsutsugamushi at the concentrations of 100 i.u./ml and 1,000 i.u./ml in accord with previous reports. Relatively low concentrations (10 i.u./ml and 100 i.u./ml) of rMUIFN-beta also inhibited the growth of O. tsutsugamushi. On the other hand, high concentrations (1,000 i.u./ml and 10,000 i.u./ml) of rMuIFN-beta enhanced the growth of the rickettsia. This enhancement of rickettsial growth was blocked by anti-murine IFN-beta monoclonal antibody (MoAb). rMuIFN-beta also enhanced the growth of Rickettsia sibirica 246 in L929 cells to some extent.

[Clinical and serological study of tsutsugamushi disease in northern Osumi, Kagoshima Prefecture].

We report 34 cases of tsutsugamushi disease seen from 1989 to 1993 at Yagi Clinic, northern Osumi, Kagoshima Prefecture. Nineteen patients (55.9%) showed the highest antibody titers against the Kawasaki strain Orientia tsutsugamushi (Ot) and 13 (38.2%) against the Kuroki strain Ot. It is suggested that two antigenic types (Kawasaki and Kuroki) of Ot were distributed in Kagoshima Prefecture, and the Kawasaki type Ot more or less dominates Kuroki type Ot. There was no difference in clinical features between the two groups of patients.

Retrospective survey of chronic Q fever in Japan by using PCR to detect Coxiella burnetii DNA in paraffin-embedded clinical samples.

We used PCR to detect Coxiella burnetii DNA in paraffin-embedded tissues obtained from patients with chronic endocarditis in which the etiological agent had been unknown. On the basis of the published nucleotide sequence of the C. burnetii htpB gene, primers were chosen to produce an amplified fragment of 285 bp. A total of 60 samples from 56 patients were tested for the presence of C. burnetii DNA. Five samples from four patients were found to be positive. All of the amplified DNA fragments possessed a TthHB8I restriction site, as predicted from the published sequence of C. burnetii. In one of the four positive patients, rickettsia-like particles were found in sections of tissue stained by Gimenez's method. This is the first report of chronic Q fever in Japan.

Dietary Curcuma xanthorrhiza Roxb. increases mitogenic responses of splenic lymphocytes in rats, and alters populations of the lymphocytes in mice.

Rhizomes of Curcuma xanthorrhiza Roxb. (C. xanthorrhiza), a medicinal plant in Indonesia, has been shown to exert diverse physiological functions. Hitherto, a little attention has been paid to its effect on immune functions. This study was carried out to determine the effect of this medicinal plant on mitogenic response of splenic lymphocytes in rats and population of splenic lymphocytes and macrophages and peripheral blood macrophages in mice. Mitogenic responses of splenocytes to phytohemagglutinin, concanavalin A, and pokeweed mitogens were examined in rat fed C. xanthorrhiza for 3 weeks. The medicinal plant increased the blastogenesis to these mitogens. Flow cytometric analysis was carried out for mice fed the medicinal plant for 3 to 5 weeks. C. xanthorrhiza increased the proportion of the splenic T cells throughout the experimental period, but exerted a variable effect on B cells and T cell subsets, that is, elevations of B cells at 3 weeks and of Th cells at 4 weeks without any elevation of Ts cells. The effect of this medicinal plant on a proportion of macrophages from the spleen and peripheral blood was not consistent. Thus, the present study suggests that C. xanthorrhiza contains some principle(s) activating T and B cell-mediated immune functions.

[In vivo and in vitro growth of the TK-1 strain of Coxiella burnetii].

All A/J, BALB/c and C57BL/6 murine strains were resistant against the intraperitoneal infection with TK-1 strain, but an enhancement of susceptibility of mice were revealed by the administration of cyclophosphamide (CPA) in BALB/c and A/J strains. CPA-treated BALB/c mice allowed an increase of TK-1 strain up to 1.7 x 10(6) coxiellar particles/mg spleen. But athymic nude mice of BALB/c strain showed only a slight increase of coxiellar particles in spleen. The resident peritoneal macrophages from BALB/c and A/J, but C57BL/6, showed proliferation of the TK-1 strain in the large infected cell population, and a part of the infected macrophages allowed TK-1 strain to survive. On the other hand, the elicited peritoneal macrophages from resistant C57BL/6 showed the largest infected cell population, number of intracellular coxiellar particles, and following decrease of TK-1 strain in later stage of infection. These in vitro infectivity of TK-1 strain seemed to relate to the in vivo infectivity in mice, and indicated existence of macrophage subpopulation, in which destruction or proliferation of TK-1 strain occurred.

[Application of trichloroacetic acid-treated antigen for serodiagnosis of Q fever by indirect immunofluorescence technique].

It is well known that the etiologic agent, Coxiella burnetii, exhibits an antigenic phase variation (phase I to phase II), and the diagnostic significance of the relative antibody titers against phase I and phase II antigens is pointed out. Therefore both phase I and phase II antigens are necessary for the serological examination of Q fever. But it is not so easy to prepare and maintain the phase II antigen by the conventional method. In the present study we tried to prepare the phase II antigen for immunofluorescence test by chemical treatment of the phase I antigen. As a result, treatment of the TK-1 strain of C. burnetii (phase I) with 10% trichloroacetic acid for 4 hr at 4 degrees C modified the antigenicity. The modified antigen reacted strongly with anti-phase II antibody.

Serological evidence that the Q fever agent (Coxiella burnetii) has spread widely among dairy cattle of Japan.

Serological examination of bovine and human sera for antibodies against Coxiella burnetti was carried out by the immunofluorescence technique. Twenty to 30% of the cows examined were antibody-positive. Sera from two veterinarians also had antibody against C. burnetii. These results suggest an increase in the number of infected cows with C. burnetii in Japan since 1954, and also imply the possibility of the prevalence of acute Q fever in the human population, which had been underestimated and undiagnosed for the last three decades.

Isolation of a Coxiella burnetii strain that has low virulence for mice from a patient with acute Q fever.

Coxiella burnetii was isolated from a patient with Q fever. It could not be determined whether this was an imported case or an indigenous one. Identification of the isolate was made by electron microscopic morphology and the indirect fluorescent antibody test with convalescent-phase serum from a Q fever patient having a known titer of antibody to C. burnetii. The isolated strain, named TK-1, caused no symptoms in ddY and BALB/c mice except when the mice were treated with cyclophosphamide.

Endoscopic evaluation of the porta hepatis in patients with biliary atresia.

Endoscopic inspection of the porta hepatis was undertaken through external enterostomy in nine postoperative patients with biliary atresia. The endoscopic appearance of bile flow in the porta hepatis was classified into three types. 1) Ductal type (D-type), which revealed good bile flow from distinct bile duct orifices (4 cases); 2) Oozing type (O-type), which showed adequate bile flow, but no definite bile duct (3 cases); and 3) Covered type (C-type), in which the porta hepatis was covered with bile "clots" and fibrous tissue (2 cases). The clinical course of the D-type was excellent with immediate disappearance of jaundice and relatively good liver function. In the case of the O-type, jaundice disappeared in 2 but persisted in one. Two patients with C-type died of hepatic failure or sepsis in the early postoperative period. In the D-type intrahepatic bile ducts were well visualized by cholangiography; however, those of the O-type were not so clear. Endoscopic inspection of the porta hepatis is very useful for evaluation of the postoperative state of bilioenteric fistulae in patients with biliary atresia.