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Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The genome of Orientia tsutsugamushi has revealed multiple ORFs encoding tetratricopeptide-repeat (TPR) proteins. The TPR protein family has been shown to be involved in a diverse spectrum of cellular functions such as cell cycle control, transcription, protein transport, and protein folding, especially in eukaryotic cells. However, little is known about the function of the TPR proteins in O. tsutsugamushi. To investigate the potential role of TPR proteins in host-pathogen interaction, two oriential TPR proteins were expressed in E. coli and applied for GSTpull down assay. DDX3, a DEAD-box containing RNA helicase, was identified as a specific eukaryotic target of the TPR proteins. Since the RNA helicase is involved in multiple RNA-modifying processes such as initiation of translation reaction, we performed in vitro translation assay in the presence of GST-TPR fusion proteins by using rabbit reticulocyte lysate system. The TPR proteins inhibited in vitro translation of a reporter luciferase in a dose dependent manner whereas the GST control proteins did not. These results suggested TPR proteins of O. tsutsugamushi might be involved in the modulation of eukaryotic translation through the interaction with DDX3 RNA helicase after secretion into host cytoplasm.

Absorption of posaconazole oral suspension is influenced by several factors including diet, medications, and mucosal integrity. However, there are few prospective data about which is the most important modifiable factor in routine clinical practice. We prospectively analyzed clinical risk factors associated with low posaconazole trough concentrations in 114 patients receiving anticancer chemotherapy due to acute myeloid leukemia or myelodysplastic syndrome who received posaconazole oral suspension. In multivariate analyses, risk factors for drug level

Background Scrub typhus is a mite-borne febrile disease caused by O. tsutsugamushi infection. Recently, emergence of scrub typhus has attracted considerable attention in several endemic countries in Asia and the western Pacific. In addition, the antigenic diversity of the intracellular pathogen has been a serious obstacle for developing effective diagnostics and vaccine. Methodology/Principal findings To understand the evolutionary pathway of genotypic diversification of O. tsutsugamushi and the environmental factors associated with the epidemiological features of scrub typhus, we analyzed sequence data, including spatiotemporal information, of the tsa56 gene encoding a major outer membrane protein responsible for antigenic variation. A total of 324 tsa56 sequences covering more than 85% of its open reading frame were analyzed and classified into 17 genotypes based on phylogenetic relationship. Extensive sequence analysis of tsa56 genes using diverse informatics tools revealed multiple intragenic recombination events, as well as a substantially higher mutation rate than other house-keeping genes. This suggests that genetic diversification occurred via frequent point mutations and subsequent genetic recombination. Interestingly, more diverse bacterial genotypes and dominant vector species prevail in Taiwan compared to other endemic regions. Furthermore, the co-presence of identical and sub-identical clones of tsa56 gene in geographically distant areas implies potential spread of O. tsutsugamushi genotypes. Conclusions/Significance Fluctuation and diversification of vector species harboring O. tsutsugamushi in local endemic areas may facilitate genetic recombination among diverse genotypes. Therefore, careful monitoring of dominant vector species, as well as the prevalence of O. tsutsugamushi genotypes may be advisable to enable proper anticipation of epidemiological changes of scrub typhus., Author summary Scrub typhus, caused by Orientia tsutsugamushi infection, is a mite-borne febrile illness endemic in the Asia-Pacific region. Recent emergence and continuous local outbreaks in many of the endemic countries make it a serious public health issue. In addition, the antigenic diversity of the tsa56 gene, encoding a major outer membrane protein, hampers the development of effective diagnostics and vaccine. Here, we extensively analyzed tsa56 sequences and their spatiotemporal information to elucidate the evolutionary pathway of genotypic diversification, as well as the environmental basis associated with the epidemiological changes of scrub typhus. Based on various informatics analyses, we found that genetic diversification of tsa56 might have been attained via frequent point mutations and subsequent genetic recombination among diverse genotypes. Prevalence of numerous bacterial genotypes and dominant vector species in Taiwan also suggest that the subtropical area located at the center of endemicity, may serve as a local mixing ground for genotype diversification. In addition, detection of identical and sub-identical clones of tsa56 genes in geographically distant countries indicates a potential spreading of bacterial genotypes. Continuous monitoring of dominant vector species and the associated O. tsutsugamushi genotypes might be required for developing better diagnostics and an effective vaccine for scrub typhus.

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular pathogen. After entry into host cells, the bacterium rapidly escapes from the endosomal pathway and replicates in the cytosol of eukaryotic host cells. Here we show that O. tsutsugamushi infection efficiently promotes cellular autophagy, a cell-autonomous defense mechanism of innate immunity. However, most of the internalized bacteria barely colocalized with the induced autophagosomes, even when stimulated with rapamycin, a chemical inducer of autophagy. Treatment of infected cells with tetracycline suppressed bacterial evasion from autophagy and facilitated O. tsutsugamushi targeting to autophagosomes, suggesting that the intracellular pathogen may be equipped with a bacterial factor or factors that block autophagic recognition. Finally, we also found that chemical modulators of cellular autophagy or genetic knockout of the atg3 gene does not significantly affect the intracellular growth of O. tsutsugamushi in vitro. These results suggest that O. tsutsugamushi has evolved to block autophagic microbicidal defense by evading autophagic recognition even though it activates the autophagy pathway during the early phase of infection.

Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the main causes of acute febrile illness in the Asian-Pacific region. Although early diagnosis and immediate antibiotic treatment are critical for reducing disease severity and mortality, current diagnostic methods using serological and molecular approaches have some limitations in sensitivity and applicability in clinical laboratories. In this study, we identified and characterized O. tsutsugamushi surface cell antigen ( sca ) family genes encoding autotransporter proteins in order to test them as novel diagnostic targets. We evaluated antibody responses against the Sca proteins in scrub typhus patient sera and examined the genetic diversity of these genes in different strains after PCR amplification. Specific antibody responses against ScaA and ScaC were observed in patients with high indirect immunofluorescence assay titers (≥1:640), whereas specific responses against ScaB and ScaE were relatively low. Genetic analysis using genomic DNAs revealed the sca genes to be quite variable among the different strains. In contrast to scaA , scaC , and scaD , which were detected in all of the tested strains, scaB and scaE were amplified differentially from the different strains, suggesting a differential presence of the genes in the genomes. Among the members of the gene family, the sequence of scaC is the most highly conserved between the different strains, and the size of scaD is the most variable due to the presence of different numbers of internal repeat sequences. These results suggest that the sca genes of O. tsutsugamushi may be valuable targets for use in combination with classical assay methods for scrub typhus diagnosis.

(2010) and have showed possibility of Korean 3D cinema production. From the experience, many problems were deduced in terms of 3D cinema techniques and work-flow. this study is analysis of `s production. The analysis is focused on the problems from the 3D cinema production and pipeline of . By making an analysis the experience, trial and error of production, it will be giving practical information and analysis result to staffs who are willing to create an next Korean 3D cinema. As a result of analysis, has failed to achieve technical accomplishment and to build effective production pipeline. Never the less, it is positively evaluated that it has tried to establish the foundation of Korean 3D cinema production.

Recently prestressed concrete bridges are generally used instead of reinforced concrete. PSC is more durable than RC because it can reduce crack problems, reinforcement corrosion, leakage and carbonation etc. And also PSC is more effective because there is no crack in tension area, and the entire concrete section is considered in section analysis. And it can reduce section size because vertical component by prestressing force can reduce the shear force. However, using high strength concrete can increase the self weight of bridge because of it`s higher density. So the hollowed PPC girder with light weight aggregate can be a alternative. In this study the hollowed PPC girder with light weight aggregate is designed and the performance of hollowed PPC girder is evaluated by experimental tests as well as numerical analysis. As a result, The hollowed PPC girder of light aggregate behaved fully elastically under service load of 110kN, and the plastic behavior was showed after elastic behavior through experimental test, and it can be also estimated by numerical analysis.

Calcium signaling has been implicated in various steps in bacterial pathogenesis. Here, we investigated the role of Ca(2+) signaling in intracellular invasion of non-phagocytic host cells infected with Orientia tsutsugamushi, the causative agent of scrub typhus. The bacteria induced a transient Ca(2+) increase in HeLa cells immediately after infection and the source of the mobilized Ca(2+) appears to be intracellular stores, not the extracellular milieu, as determined using extracellular (EGTA) or intracellular (BAPTA-AM) Ca(2+) chelators. O. tsutsugamushi rapidly induced activation of PLC-γ1, as indicated by tyrosine phosphorylation. PLC-γ1 activity increased within 1 min of infection and returned to the basal level by 5 min. Pretreatment of host cells with inhibitors of PLC-γ1 (U73122) or IP3R channel activity (2-APB) significantly blocked bacterial entry without affecting bacterial attachment. In addition, these chemical inhibitors were effective in suppressing not only the activation of ERK MAP kinase but also the expression of the chemokine MCP-1. Taken together, Ca(2+) signaling induced by O. tsutsugamushi may play a crucial role in bacterial pathogenesis including inflammatory reactions as well as intracellular invasion into non-phagocytic host cells.

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular pathogen. Previously, we reported that the 56-kDa type-specific antigen (TSA56), a major outer membrane protein of O. tsutsugamushi , binds to fibronectin and facilitates bacterial entry into the host cell, potentially via an interaction with integrins. Here, we demonstrated that O. tsutsugamushi colocalizes with integrin α5β1 and activates integrin signaling effectors, including focal adhesion kinase, Src kinase, and RhoA GTPase, and also recruits signaling adaptors, such as talin and paxillin, to the site of infection. Inhibition of protein tyrosine kinases or RhoA reduced intracellular invasion. We also observed substantial actin reorganization and membrane protrusions at the sites of infection of nonphagocytic host cells. Finally, we identified a region in the extracellular domain of TSA56 that binds to fibronectin. A peptide containing this region was able to significantly reduce bacterial invasion. Taken together, these results clearly indicate that O. tsutsugamushi exploits integrin-mediated signaling and the actin cytoskeleton for invasion of eukaryotic host cells.

This study revealed the presence of various rickettsial agents in mites from wild rodents collected in Southern Jeolla Province, Korea, by nested polymerase chain reaction (PCR) and sequence analysis of a partial citrate synthase and rickettsia outer membrane protein B genes. Rickettsial agents closely related to the Rickettsia species TwKM02, R. australis, and the Rickettsia species Cf15 were successfully identified in this study, for the first time in Korea, and R. japonica, R. akari, R. conorii, R. felis, and R. typhi were also detected, as previously described. The data presented in this paper extend knowledge on the geographic distribution of SFG rickettsiae in eastern Asia and it may necessary to consider the role of mites in rickettsial transmission.

Background Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi. Methodology/Principal Findings We examined the potential role of ScaA protein, an autotransporter protein of O. tsutsugamushi, in bacterial pathogenesis and evaluated the protective attributes of ScaA immunization in lethal O. tsutsugamushi infection in mice. Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells. In addition, immunization with ScaA not only provides protective immunity against lethal challenges with the homologous strain, but also confers significant protection against heterologous strains when combined with TSA56, a major outer membrane protein of O. tsutsugamushi. Conclusions/Significance Immunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far., Author Summary Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the common febrile illness in the Asia-Pacific region, accounting for up to 20% of febrile hospital admissions in rural areas of southern Asia. It has been estimated that one billion people are at risk and one million cases occur annually in the Asian-Pacific region. Despite continuous efforts to develop a vaccine against scrub typhus during the last several decades, all approaches have failed to induce effective immunity. The main issue for the development of a scrub typhus vaccine is the selection of proper antigens that cover a broad range of antigenic strains and induce long-lasting immunity. Here, we examined the potential use of ScaA protein as a vaccine antigen. Our findings demonstrate that ScaA protein functions as a bacterial adhesion factor and an antibody against ScaA significantly inhibits bacterial infection into host cells. In addition, ScaA vaccination provides protective immunity against lethal challenges of the homologous strain, and also confers better protection against heterologous strains when combined with TSA56, the major outer membrane protein that was previously used as a potential vaccine antigen. These results indicate that ScaA proteins could be used as a novel vaccine target for scrub typhus.

Lipid rafts are membrane microdomains that are proposed to function as platforms for both receptor signaling and trafficking. Our previous studies have demonstrated that Tip of herpesvirus saimiri (HVS), which is a T-lymphotropic tumor virus, is constitutively targeted to lipid rafts and interacts with cellular Lck tyrosine kinase and p80 WD repeat-containing endosomal protein. Through the interactions with Lck and p80, HVS Tip modulates diverse T-cell functions, which leads to the downregulation of T-cell receptor (TCR) and CD4 coreceptor surface expression, the inhibition of TCR signal transduction, and the activation of STAT3 transcription factor. In this study, we investigated the functional significance of Tip association with lipid rafts. We found that Tip expression remarkably increased lipid raft fractions in human T cells by enhancing the recruitment of lipid raft-resident proteins. Genetic analysis showed that the carboxyl-terminal transmembrane, but not p80 and Lck interaction, of Tip was required for the lipid raft localization and that lipid raft localization of Tip was necessary for the efficient downregulation of TCR and CD4 surface expression. Correlated with this, treatment with Filipin III, a lipid raft-disrupting agent, effectively reversed the downregulation of CD3 and CD4 surface expression induced by Tip. On the other hand, Tip mutants that were no longer present in lipid rafts were still capable of inhibiting TCR signaling and activating STAT3 transcription factor activity as efficiently as wild-type (wt) Tip. These results indicate that the association of Tip with lipid rafts is essential for the downregulation of TCR and CD4 surface expression but not for the inhibition of TCR signal transduction and the activation of STAT3 transcription factor. These results also suggest that the signaling and targeting activities of HVS Tip rely on functionally and genetically separable mechanisms, which may independently modulate T-cell function for viral persistence or pathogenesis.

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1 alpha (MIP-1 alpha ), MIP-2, monocyte chemoattractant protein 1 (MCP-1), and cytokines gamma-interferon (IFN-gamma ), interleukin-12 (IL-12), IL-10, and tumor necrosis factor alpha (TNF-alpha ) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible-infection model (BALB/c-Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.

Multiplex-nested PCR and sequencing analysis indicated rickettsialike agents in serum specimens from febrile patients., The presence of the nucleic acid of the spotted fever group (SPG) and typhus group (TG) rickettsiae was investigated in 200 serum specimens seropositive for SFG rickettsiae by multiplex-nested polymerase chain reaction with primers derived from the rickettsial outer membrane protein B gene. The DNA of SFG, TG, or both rickettsiae was amplified in the 24 serum specimens, and sequence analysis showed Rickettsia conorii, R. japonica, and R. felis in the specimens. R. conorii and R. typhi were found in 7 serum specimens, which indicated the possibility of dual infection in these patients. These findings suggest that several kinds of rickettsial diseases, including boutonneuse fever, rickettsialpox, R. felis infection, and Japanese spotted fever, as well as scrub typhus and murine typhus, are occurring in Korea.

To investigate the prevalence of spotted fever group rickettsioses in Korea, a serosurvey of Japanese spotted fever rickettsiosis in patients with acute febrile illness was conducted with an indirect immunofluorescence assay. Overall, 19.88% of the patients were found to have polyvalent antibody against Rickettsia japonica . This study is the first documentation of spotted fever group rickettsiosis in Korea.

Cell surface heparan sulfate proteoglycans (HSPGs) play a critical role in the cellular invasion of intracellular bacteria and are presumed to have a role in the infection of host cells by Orientia tsutsugamushi. Previously, we showed that O. tsutsugamushi infection decreased markedly after treating host cells with heparinase, which suggests that HSPGs play an important role in oriential infection. We tested oriential infection in REF-Syn4 cells over-expressing syndecan-4, and in REF-Syn4AS cells in which the expression of syndecan-4 was down regulated by transfecting with anti-sense syndecan-4 cDNA. Oriential infection was found to be dependent on the expression level of syndecan-4 on the cell surface. Furthermore, the infectivity of O. tsutsugamushi was specifically reduced by treating O. tsutsugamushi with the purified recombinant core protein of syndecan-4 (Syn4E). These results suggest that the core protein of syndecan-4 and the heparin/heparan sulfate chain of syndecan play an important role in oriential infection by O. tsutsugamushi.

Orientia tsutsugamushi , an obligate intracellular bacterium, is the causative agent of scrub typhus which is histopathologically characterized by inflammatory manifestations, indicating that rickettsiae induce mechanisms that amplify the inflammatory response. To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production after infection with O. tsutsugamushi in mice. The mRNAs that were upregulated included lymphotactin, RANTES ( r egulated upon a ctivation, n ormal T -cell e xpressed and s ecreted), macrophage inflammatory proteins 1α/β (MIP-1α/β), MIP-2, monocyte chemoattractant protein 1, lymphotoxin β, tumor necrosis factor α, interleukin-6, γ-interferon, transforming growth factor β1, and migration inhibition factor. Peak expression of these chemokines and cytokines was observed between 4 and 8 days after infection. Gene induction was followed by the secretion of chemokine and cytokine proteins. Chemokine profile in infected mice was well correlated with kinetics of inflammatory cell infiltration. Thus, O. tsutsugamushi appears to be a strong inducer of chemokines and cytokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation observed in scrub typhus.

The purpose of this study was to suggest the guideline for scientific and quantitative skill acquisition methods of badminton smashing so that the novice could learn the motions of badminton smashing efficiently. The subject participated consisted of total 6 middle school students of the stilled(3) and the unskilled(3). The cinematographical experimental & Analysis equipments used in the study were composed of two Model D-5100 Video Cameras, a control point, a synchronization timer and Kwon3d motion analysis system. The conclusions obtained were as follows, 1. It was necessary for the unskilled to learn the smashing impact phase in situation of max. velocity of COG in order to minimize the timing loss phase. 2. Particularly. The power from segments of Power Zone must participate in utilization in sequence of order of trunk-upper arm- forearm-hand-racket segment in both the skilled and the unskilled.. 3. Therefore it was necessary for the unskilled to do smashing practice for cumulated sum of absolute & relative velocity transfer into shuttle cock after adjusting anterior-posterior tilting angle and max timing, in sequence of segment recruited.

Human macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-κB and AP-1.Orientia tsutsugamushicauses vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated thecis-acting andtrans-acting elements involved inO. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-κB activation was observed in HUVEC infected withO. tsutsugamushi, inhibition of NF-κB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position −69 to −63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position −69 to −63−96 to −90 or deletion of NF-κB-binding site at position −69 to −63−88 to −79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-κB binding sites at positions −2640 to −2632, −2612 to −2603 in the enhancer region, or the AP-1 biding site at position −2276 to −2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position −69 to −63 in proximal promoter and TRE at position −2276 to −2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected withO. tsutsugamushi. Although NF-κB activation is not essential per se, the κB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the involvement of the NF-κB may be due to the function of chromatin structures and other transcription cofactors in the regulation of MCP-1 gene expression in response toO. tsutsugamushiinfectioin.

Background: Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium. Previously, a large number of genes that encode proteins containing eukaryotic protein-protein interaction motifs such as ankyrin-repeat (Ank) domains were identified in the O. tsutsugamushi genome. However, little is known about the Ank protein function in O. tsutsugamushi. Methodology/Principal Findings: To characterize the function of Ank proteins, we investigated a group of Ank proteins containing an F-box-like domain in the C-terminus in addition to the Ank domains. All nine selected ank genes were expressed at the transcriptional level in host cells infected with O. tsutsugamushi, and specific antibody responses against three Ank proteins were detected in the serum from human patients, indicating an active expression of the bacterial Ank proteins post infection. When ectopically expressed in HeLa cells, the Ank proteins of O. tsutsugamushi were consistently found in the nucleus and/or cytoplasm. In GST pull-down assays, multiple Ank proteins specifically interacted with Cullin1 and Skp1, core components of the SCF1 ubiquitin ligase complex, as well as the eukaryotic elongation factor 1 alpha (EF1 alpha). Moreover, one Ank protein co-localized with the identified host targets and induced downregulation of EF1 alpha potentially via enhanced ubiquitination. The downregulation of EF1 alpha was observed consistently in diverse host cell types infected with O. tsutsugamushi. Conclusion/Significance: These results suggest that conserved targeting and subsequent degradation of EF1 alpha by multiple O. tsutsugamushi Ank proteins could be a novel bacterial strategy for replication and/or pathogenesis during mammalian host infection.

Scrub typhus, caused byOrientia tsutsugamushi, is characterized by local as well as systemic inflammatory manifestations. The main pathologic change is focal or disseminated multiorgan vasculitis, which is caused by the destruction of endothelial cells and perivascular infiltration of leukocytes. We investigated the regulation of chemokine induction in transformed human dermal microvascular endothelial cells (HMEC-1) in response toO. tsutsugamushiinfection. The monocyte chemoattractant protein-1 (MCP-1) and interleukin 8 (IL-8) mRNAs were induced, and their levels showed a transitory peak at 3 and 6 h, respectively. The RANTES transcript was detected at 6 h after infection, with increased levels evident by 48 h. The induction of the MCP-1 and IL-8 genes was not blocked by cycloheximide, suggesting that de novo protein synthesis of host cell proteins is not required for their transcriptional activation. Heat- or UV-inactivatedO. tsutsugamushiinduced a similar extent of MCP-1 and IL-8 responses. The induction of MCP-1 and IL-8 transcripts in the endothelial cells byO. tsutsugamushiwas not blocked by the inhibitors of NF-κB. Furthermore, the activation of NF-κB was not detected in HMEC-1 stimulated withO. tsutsugamushi. These results demonstrate that heat-stable molecules ofO. tsutsugamushiinduce the MCP-1 and IL-8 genes and the induction of the chemokine genes may be mediated by an NF-κB independent mechanism. We also showed that another major transcription factor, activator protein-1 (AP-1), was up-regulated in HMEC-1 afterO. tsutsugamushiinfection. This suggests the possible involvement of AP-1 in the chemokine gene expression.

Orientia tsutsugamushi, an obligate intracellular bacterium, was isolated for the first time in 1930. Infections by virulent strains are characterized by fever, rash, eschar, pneumonia, myocarditis, and disseminated intravascular coagulation. Here we review the general aspects of O. tsutsugamushi and immune responses in terms of inflammation, protective immune mechanisms, and immunogenic antigens.

Scrub typhus, caused byOrientia tsutsugamushiinfection, is characterized by local as well as systemic inflammatory manifestations. Inflammation is initiated byO. tsutsugamushi-infected macrophages and endothelial cells in the dermis. We investigated the regulation of chemokine induction in macrophage cell line J774A.1 in response toO. tsutsugamushiinfection. The mRNAs for macrophage inflammatory proteins 1α/β (MIP-1α/β), MIP-2, and macrophage chemoattractant protein 1 were induced within 30 min, and their levels showed a transitory peak for 3 to 12 h. However, the lymphotactin, eotaxin, gamma interferon-inducible protein 10, and T-cell activation gene 3 mRNAs were not detected by RNase protection assays. Heat-killedO. tsutsugamushiinduced a similar extent of chemokine responses. Induction of the chemokine genes was not blocked by the eukaryotic protein synthesis inhibitor cycloheximide, suggesting that de novo synthesis of host cell protein is not required for these transcriptional responses. The induction of chemokine mRNAs byO. tsutsugamushiwas blocked by the inhibitors of NF-κB activation. Furthermore,O. tsutsugamushiinduced the nuclear translocation and activation of NF-κB. These results demonstrate that heat-stable molecules ofO. tsutsugamushiinduce a subset of chemokine genes and that induction involves activation of the transcription factor NF-κB.

PROBLEM: The pathogenesis of uterine leiomyomata is still unclear. Recently it has been suggested that mac25 plays a tumor-suppressive role in various tumors. The aims of this study were to evaluate a possible involvement of mac25 in the growth of leiomyoma and in the mechanism of a gonadotropin-releasing hormone agonist (GnRHa) inducing shrinkage of leiomyoma. METHODS OF STUDY: Mac25 mRNA transcript was measured by Northern blot in total RNA extracted from the paired specimens of leiomyoma and adjacent myometrium from untreated patients (n = 25) and from leiomyoma specimens from GnRHa-pretreated patients (n = 10). RESULTS: Mac25 mRNA expression was significantly lower in large leiomyoma (more than 150 cm 3 in volume) than in adjacent myometrium and small lciomyoma (less than 120 cm 3 in volume) from untreated patients. There was no difference in this expression between the proliferative and secretory phases of the menstrual cycle. Leiomyoma from GnRHa-pretreated patients had mac25 gene expression levels similar to myometrium and small leiomyoma from untreated patients. CONCLUSIONS: Mac25 may be involved in the growth of uterine leiomyoma and the action of GnRHa may, in part, be mediated by mac25.

We analyzed homotypic and heterotypic antibody responses to a type-specific antigen (Tsa), a 56-kDa protein of Orientia tsutsugamushi , by using sera from mice immunized with strains Gilliam, Karp, Kato, and Boryong. We generated a series of deletion constructs of the tsa gene and expressed them as MalE fusion proteins. Variable domain I (VD I) showed strong responses to homotypic antibodies. Antigenic domain II (AD II) from Boryong and Karp showed cross-reactivities to each other. VD III showed no responses to any of the antibodies. Sera from Kato-immunized mice showed only homotypic responses to AD III. On the other hand, sera of the mice immunized with Gilliam, Karp, or Boryong showed homotypic as well as heterotypic responses to this region. VD IV showed the strongest heterotypic antibody responses among the fragments tested. These data suggest that VD I is important in homotypic antibody responses and that AD II, AD III, and VD IV are important in heterotypic antibody responses of the mice to Tsa.

In order to investigate whether the iron acquisition mechanisms of Staphylococcus aureus are induced by iron restriction in vitro, we examined S. aureus ATCC 6538 for production of siderophore and expression of transferrin-binding protein (SA-tbp) in normal or deferrated brain heart infusion broth (BHI). Siderophore production was earlier and greater in the deferrated BHI. The SA-tbp, detected by ligand blot assay, was expressed only in the deferrated BHI. When human transferrin was added to the deferrated BHI, siderophore production was later and lower than when transferrin was not present. In conclusion, both iron acquisition mechanisms of S. aureus were found to be iron-repressible and via both of them, human transferrin-bound iron was utilized for growth under iron-restricted condition.

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.

Anti-oriential antibody inhibits Orientia tsutsugamushi attachment to, and penetration of, host cells. However, O. tsutsugamushi antigens that induce the production of a neutralizing antibody have not been identified. The authors immunized mice and rabbits with the recombinant 56 kDa protein of O. tsutsugamushi fused to the maltose binding protein of Escherichia coli (MBP-Bor56) and analysed their effect on O. tsutsugamushi attachment to or penetration of L929 cells. O. tsutsugamushi attachment and penetration were measured by using an indirect immunofluorescent antibody assay (IFA). O. tsutsugamushi growth in L929 cells was determined by [3H]thymidine uptake assay. By IFA, we observed a 96% reduction of attachment or penetration of O. tsutsugamushi treated with rabbit anti-MBP-Bor56 sera. [3H]thymidine uptake showed that mouse anti-MBP-Bor56 sera caused a 91% reduction in O. tsutsugamushi growth, when compared to mouse anti-MBP sera. These results suggest that the 56 kDa protein of O. tsutsugamushi plays an important role in O. tsutsugamushi attachment to or penetration of cells.

Orientia tsutsugamushi, the etiological agent of scrub typhus, is an antigenically diverse organism and many serologically distinct strains have been identified. The 56 kDa protein of O. tsutsugamushi, a major protein in the outer membrane, has been thought to be responsible for this antigenic variability. A strain of O. tsutsugamushi isolated in Korea cross-reacted with both Gilliam strain-specific and Karp strain-specific monoclonal antibodies. When its 56 kDa protein gene was cloned and analyzed, its sequence showed variation especially between 1,200 and 1,250 bp, showing that this isolate is a new O. tsutsugamushi strain.

Forty-five hybridoma cell lines producing monoclonal antibodies against Hantaan virus, the etiologic agent of hemorrhagic fever with renal syndrome, were generated by fusion of P3-X63-Ag8.V653 myeloma cells with spleen cells of mice immunized with inactivated Hantaan virus vaccine. Among these, 38 antibodies were identified as binding to the 48-kDa nucleocapsid protein by immunoblot assay or radioimmunoprecipitation. Twenty-six of them were of the immunoglobulin G1 (IgG1), nine were of the IgG2a, and three were of the IgA isotype. According to cross-reactivities with other serotypes of the genus Hantavirus, the antibodies were classified into three groups: 6 antibodies specific to the Hantaan serotype (group I), 20 antibodies cross-reacting with Hantaan and Seoul serotypes (SR-11, Tchoupitoulas, and R22) (group II), and 12 antibodies cross-reacting with Hantaan, Seoul, and Prospect Hill serotypes (group III). None of the antibodies cross-reacted with the Puumala serotype. With a panel of antibodies of different cross-reactivities, serotypes of Hantavirus could be differentiated. Thirty-eight monoclonal antibodies against Hantaan virus nucleocapsid protein which have different cross-reactivities between serotypes were developed. These results confirmed the presence of multiple serotype-specific epitopes on the nucleocapsid protein of Hantaan virus, which can be utilized in differentiation of serotypes.

Background Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the main causes of febrile illness in the Asia-Pacific region. Although cell-mediated immunity plays an important role in protection, little is known about the phenotypic changes and dynamics of leukocytes in scrub typhus patients. Methodology/Principal Findings To reveal the underlying mechanisms of immunological pathogenesis, we extensively analyzed peripheral blood leukocytes, especially T cells, during acute and convalescent phases of infection in human patients and compared with healthy volunteers. We observed neutrophilia and CD4+ T lymphopenia in the acute phase of infection, followed by proliferation of CD8+ T cells during the convalescent phase. Massive T cell apoptosis was detected in the acute phase and preferential increase of CD8+ T cells with activated phenotypes was observed in both acute and convalescent phases, which might be associated or correlated with elevated serum IL-7 and IL-15. Interestingly, peripheral Treg cells were significantly down-regulated throughout the disease course. Conclusions/Significance The remarkable decrease of CD4+ T cells, including Treg cells, during the acute phase of infection may contribute to the loss of immunological memory that are often observed in vaccine studies and recurrent human infection., Author Summary Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi infection. It has been estimated that one billion people are at risk and one million new cases arise each year in Asian-pacific region. Despite of aggressive attempts to develop a prophylactic vaccine against scrub typhus during the last several decades, all approaches have failed to generate long lasting immunity. In addition, little is known about the immunological pathogenesis of scrub typhus. To understand the pathogenic mechanisms of this infectious disease, we extensively analyzed peripheral leukocytes, especially T cells, in Korean scrub typhus patients and compared with healthy volunteers. We observed neutrophilia and CD4+/T lymphopenia in the acute phase of infection, followed by proliferation of CD8+ T cells during the convalescent phase. Massive T cell apoptosis was detected in the acute phase and a preferential increase of CD8+ T cells with activated phenotypes was observed in both acute and convalescent phases. The remarkable decrease of CD4+ T cells, including Treg cells, during the acute phase of infection may contribute to the loss of immunological memory and generate helpless but unregulated cytotoxic T cell responses observed in vaccine studies and recurrent human infection.

Herpesvirus saimiri (HVS), a T-lymphotropic monkey herpesvirus, induces fulminant T-cell lymphoma in non-natural primate hosts. In addition, it can immortalize human T-cells in vitro. HVS tyrosine kinase-interacting protein (Tip) is an essential viral gene required for T-cell transformation both in vitro and in vivo. In this study, we found that Tip interacts with the STAT6 transcription factor and induces phosphorylation of STAT6 in T-cells. The interaction with STAT6 requires the Tyr127 residue and Lck-binding domain of Tip, which are indispensable for interleukin (IL)-2-independent T-cell transformation by HVS. It was also demonstrated that Tip induces nuclear translocation of STAT6, as well as activation of STAT6-dependent transcription in Jurkat T-cells. Interestingly, the phosphorylated STAT6 mainly colocalized with vesicles containing Tip within T-cells, but was barely detectable in the nucleus. However, nuclear translocation of phospho-STAT6 and transcriptional activation of STAT6 by IL-4 stimulation were not affected significantly in T-cells expressing Tip. Collectively, these findings suggest that the constitutive activation of STAT6 by Tip in T-cells may contribute to IL-2-independent T-cell transformation by HVS.

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular pathogen whose mechanism of cellular adhesion and invasion is poorly characterized. Bioinformatic analyses of two O. tsutsugamushi genomes revealed the presence of a group of genes that encode autotransporter proteins. In this study, we identified 10 autotransporter gene products and categorized them into five groups of orthologs (ScaA to ScaE) based on their sequence similarities. Sequence homology was highest between members of ScaC group, suggesting the functional conservation of bacterium-host interactions. ScaC was actively expressed on the surface of O. tsutsugamushi and induced antibody responses in scrub typhus patients. Experiments using microbeads conjugated to recombinant ScaC or a surrogate Escherichia coli expression system showed that ScaC was sufficient to mediate attachment to, but not invasion of, nonphagocytic mammalian cells. In addition, preincubation of host cells with recombinant ScaC significantly inhibited their interaction with O. tsutsugamushi . Finally, fibronectin was identified as a potential receptor for ScaC by using yeast two-hybrid screening, and this was confirmed using a glutathione S -transferase (GST) pulldown assay. Taken together, these results demonstrate that ScaC is involved in the interaction of O. tsutsugamushi with mammalian host cells and suggest that ScaC may play a critical role in bacterial pathogenesis.

The genes encoding the 56-kDa polypeptides were amplified by polymerase chain reaction from the genomic DNAs of three serotypes of Rickettsia tsutsugamushi, Gilliam, Karp, and Boryong. The amplified products were cloned into expression vector pIH821, and the recombinant antigens were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant 56-kDa polypeptides were purified by affinity chromatography for the sensitization of sheep erythrocytes. The recombinant 56-kDa polypeptides were evaluated with 89 serum specimens from health blood donors, 94 serum specimens from scrub typhus patients, and 31 serum specimens from patients with other febrile diseases by a passive hemagglutination assay (PHA). Among the scrub typhus patients diagnosed by indirect immunofluorescent-antibody testing, the antibodies to R. tsutsugamushi were detected in 93 patients (99%). One serum specimen from a healthy person showed a false-positive reaction by this method. The recombinant PHA showed no cross-reactions with sera obtained from other febrile patients with diseases such as murine typhus, hemorrhagic fever with renal syndrome, and leptospirosis. In conclusion, this recombinant PHA could be substituted for the conventional indirect immunofluorescent-antibody test and the immunoperoxidase test.