Identification of a new group of Chlamydia psittaci strains called TWAR.

A new group of Chlamydia psittaci strains has been identified. They are called TWAR after the laboratory designation of the first two isolates. Twelve strains were isolated from pharyngeal swabs of different persons with acute respiratory disease in Seattle, Wash., during 1983 to 1986. One strain was obtained from the eye of a child during the trachoma vaccine study in Taiwan in 1965. Nine strains were characterized in this study. TWAR organisms formed intracytoplasmic inclusions in HeLa cells which were morphologically typical of C. psittaci and iodine stain negative (contained no glycogen). Immunological analysis with various chlamydia-specific monoclonal antibodies revealed that TWAR strains belong to the genus Chlamydia, are distinct from C. trachomatis, and are serologically unique among C. psittaci. All TWAR strains so far isolated appear identical serologically. TWAR organisms grew poorly in egg and cell cultures and demonstrated low virulence to mice by intracerebral, intranasal, and intravenous inoculation. Available data suggest that the TWAR strain is a primary human pathogen.     

Characterization of the new Chlamydia agent, TWAR, as a unique organism by restriction endonuclease analysis and DNA-DNA hybridization.

Several molecular techniques were used for comparison of the novel Chlamydia agent, TWAR, with Chlamydia trachomatis and Chlamydia psittaci. Unlike all serotypes of C. trachomatis and most strains of C. psittaci, the eight TWAR isolates examined did not contain extrachromosomal DNA. TWAR was readily distinguished from C. trachomatis or C. psittaci by restriction endonuclease analysis, whereas identical or nearly identical restriction patterns were observed among the TWAR isolates. Southern blot analysis with a gene encoding a portion of the C. trachomatis serovar L2 major outer membrane protein as the probe showed that TWAR, like C. psittaci, contained sequences homologous to this gene. However, while the hybridization patterns were identical for all TWAR isolates, they differed from those of any of the other Chlamydia species tested. A PstI gene bank containing TWAR DNA was constructed in pUC19. Random fragments were purified and used for probing Chlamydia chromosomal digests. All of the five probes tested were TWAR specific, with the TWAR isolates showing identical patterns of homology. Qualitative studies of the DNA homology revealed that TWAR did not have significant homology to any of the Chlamydia strains assayed. Collectively, these results demonstrate that the TWAR isolates represent a single strain or closely allied genotypes and are clearly distinct from any of the other chlamydiae tested.     

A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections

During a 2 1/2-year period, we studied 386 University of Washington students with acute respiratory disease, to determine whether a Chlamydia psittaci strain, here designated TWAR, is an important respiratory pathogen. Serologic evidence of recent TWAR infection was found in 13 students, and the organism was isolated from 8 of these. TWAR infection occurred in 12 percent of the students who had pneumonia (9 of 76), 5 percent of those with bronchitis (3 of 63), and 1 percent of those with pharyngitis (1 of 150). The TWAR infections occurred throughout the study period. Pharyngitis, often accompanied by laryngitis, was a common first symptom. Clinically, the infections resembled those with Myco-plasma pneumoniae; therefore, the patients were given courses of erythromycin used for the treatment of M. pneumoniae infections. This therapy proved to be inadequate. The limited data available suggest that the TWAR strain is a "human" C. psittaci that is spread from human to human, without a bird or animal host.     

Chlamydia pneumoniae (TWAR).

Chlamydia pneumoniae (TWAR) is a recently recognized third species of the genus Chlamydia that causes acute respiratory disease. It is distinct from the other two chlamydial species that infect humans, C. trachomatis and C. psittaci, in elementary body morphology and shares less than 10% of the DNA homology with those species. The organism has a global distribution, with infection most common among children between the ages of 5 and 14 years. In children, TWAR infection is usually mild or asymptomatic, but it may be more severe in adults. Pneumonia and bronchitis are the most common clinical manifestations of infection, and TWAR is responsible for approximately 10% of cases of pneumonia and 5% of cases of bronchitis in the United States. The microimmunofluorescence serologic assay is specific for TWAR and can distinguish between recent and past infections. The organism can be isolated in cell culture; however, PCR techniques have recently facilitated its detection in tissues and clinical specimens.     

急性冠脉综合征病人肺炎衣原体抗体滴度的临床价值

目的：探讨肺炎衣原体TWAR IgG,IgM抗体滴度对急性冠脉综合征（ACS）的临床预测价值。方法：应用间接微量免疫荧光法，测定了102例ACS组病人和60例对照组受试者血清TWAR IgG,IgM,抗体滴度，并随访6个月。结果：ACS组TWAR IgG既往感染予性率和平均几何滴度均显著高于对照组，P＜0．01，高抗体水平发生ACS风险相对增高，ACS病人随访中，非感染者心血管事件发生率较感染者明显降低，P＜0．05，结论：肺炎衣原体感染与ACS有关，TWAR IgG抗体滴度对判断ACS病人预后有较好的临床价值。     

Factors affecting viability and growth in HeLa 229 cells of Chlamydia sp. strain TWAR.

Two prototype isolates (TW-183 and AR-39) of Chlamydia sp. strain TWAR were used to study factors affecting growth of this organism in HeLa 229 cells. The results showed that an incubation temperature of 35 degrees C was better than one of 37 degrees C for growth. The burst size after 3 days of incubation at 35 degrees C was found to be small (13 to 52), which partially explains the difficulty of serial passage in cell culture. Application of a higher centrifugal force (1,700 X g versus 900 X g) at the time of inoculation enhanced growth 2.2 to 3.6 times. Infectivity was enhanced by treatment of cells with DEAE-dextran (2.4 times) or poly-L-lysine (1.6 times), but not with Polybrene or polyethylene glycol. The viability of the TWAR organism in chlamydia transport medium SPG was also studied. It was shown that the organism was rapidly inactivated at room temperature (22 degrees C); only 1% remained viable after storage for 24 h. The viability was preserved at 4 degrees C, and 70% remained viable after storage for 24 h. Freezing at -75 degrees C inactivated 23% of the organisms when the organisms were frozen within 4 h after harvesting and stored at 4 degrees C before freezing.     

Prevalence of Chlamydia pneumoniae antibodies in patients with acute respiratory infections in Israel.

AIMS--To evaluate the prevalence of antibodies to Chlamydia pneumoniae (TWAR) in relation to other aetiological agents of acute respiratory infections in Israeli patients. METHOD--Serum samples from 604 patients (183 children and 421 adults) were collected over three years. Antibodies to C pneumoniae, C trachomatis, and Legionella sp were evaluated using the microimmunofluorescence (MIF) assay. Antibodies to Mycoplasma pneumoniae were detected using the Serodia Myco II test. RESULTS--Antibodies to TWAR were detected in 319 (51.3%) sera. Twenty one patients had MIF results indicative of recent infection. TWAR prevalence and antibody titres in children (aged 1-10 years) were low, gradually increased in teenagers (11-18 years), and were highest in adults and elderly patients. In contrast to the consistently noted TWAR antibody prevalence and serological evidence of recent infection during the study period, a significant decrease in those variables was recorded for C trachomatis. Six patients had serological evidence of recent infection with both C pneumoniae and C trachomatis. The presence of antibodies to Mycoplasma pneumoniae and Legionella sp was tested in 473 of the patients; 29 had antibodies to M pneumoniae and 23 to Legionella sp. Six patients (including five children) had serological evidence of recent infection with M pneumoniae and four with Legionella sp. CONCLUSION--C pneumoniae should be considered in patients with acute respiratory diseases. MIF is the preferred method for monitoring the presence of antibodies to this organism.     

空斑法筛选具有中和活性的抗衣原体粘连蛋白McAb

本研究制备了抗１８ｋＤａ粘连蛋白的单克隆抗体，并证实在不同血清型之间，甚至不同种衣原体之间１８ｋＤａ粘连蛋白具有一定的共同抗原表位。我们建立的空斑形成试验和单克隆抗体空斑减少中和试验，实验结果明显、客观，易于判定，稳定性好。用此筛选出县有中和活性的２Ｂ９ＭｃＡｂ。该ＭｃＡｂ不同于目前已报道的ＭｃＡｂ，其中和活性不依赖于补体，而可能是抑制衣原体的粘附。     

Laboratory diagnosis of human chlamydial infections.

Chlamydia trachomatis is a human pathogen that causes ocular disease (trachoma and inclusion conjunctivitis), genital disease (cervicitis, urethritis, salpingitis, and lymphogranuloma venereum), and respiratory disease (infant pneumonitis). Respiratory chlamydioses also occur with infection by avian strains of C. psittaci or infection by the newly described TWAR agent. Diagnosis of most acute C. trachomatis infections relies on detection of the infecting agent by cell culture, fluorescent antibody, immunoassay, cytopathologic, or nucleic acid hybridization methods. Individual non-culture tests for C. trachomatis are less sensitive and specific than the best chlamydial cell culture system but offer the advantages of reduced technology and simple transport of clinical specimens. Currently available nonculture tests for C. trachomatis perform adequately as screening tests in populations in which the prevalence of infection is greater than 10%. A negative culture or nonculture test for C. trachomatis does not, however, exclude infection. The predictive value of a positive nonculture test may be unsatisfactory when populations of low infection prevalence are tested. Tests that detect antibody responses to chlamydial infection have limited utility in diagnosis of acute chlamydial infection because of the high prevalence of persistent antibody in healthy adults and the cross-reactivity due to infection by the highly prevalent C. trachomatis and TWAR agents. Assays for changes in antibody titer to the chlamydial genus antigen are used for the diagnosis of respiratory chlamydioses. A single serum sample that is negative for chlamydial antibody excludes the diagnosis of lymphogranuloma venereum.     

Role of sulfated glycans in adherence of the microsporidian Encephalitozoon intestinalis to host cells in vitro

Microsporidia are obligate intracellular opportunistic protists that infect a wide variety of animals, including humans, via environmentally resistant spores. Infection requires that spores be in close proximity to host cells so that the hollow polar tube can pierce the cell membrane and inject the spore contents into the cell cytoplasm. Like other eukaryotic microbes, microsporidia may use specific mechanisms for adherence in order to achieve target cell proximity and increase the likelihood of successful infection. Our data show that Encephalitozoon intestinalis exploits sulfated glycans such as the cell surface glycosaminoglycans (GAGs) in selection of and attachment to host cells. When exogenous sulfated glycans are used as inhibitors in spore adherence assays, E. intestinalis spore adherence is reduced by as much as 88%. However, there is no inhibition when nonsulfated glycans are used, suggesting that E. intestinalis spores utilize sulfated host cell glycans in adherence. These studies were confirmed by exposure of host cells to xylopyranoside, which limits host cell surface GAGs, and sodium chlorate, which decreases surface sulfation. Spore adherence studies with CHO mutant cell lines that are deficient in either surface GAGs or surface heparan sulfate also confirmed the necessity of sulfated glycans. Furthermore, when spore adherence is inhibited, host cell infection is reduced, indicating a direct association between spore adherence and infectivity. These data show that E. intestinalis specifically adheres to target cells by way of sulfated host cell surface GAGs and that this mechanism serves to enhance infectivity.     

In vitro studies of rickettsia-host cell interactions: ultrastructural changes induced by Rickettsia rickettsii infection of chicken embryo fibroblasts.

Secondary chicken embryo fibroblasts infected with the Sheila Smith strain of Rickettsia rickettsii and grown in monolayer culture undergo rapid morphological alterations. Transmission electron microscopic examination of cells at intervals after infection showed several progressive host cell lesions, including widespread dilatation of the rough endoplasmic reticulum and outer nuclear envelope and the accumulation of electron-dense material within the cisternae of intracellular membranes. Dilatation of the rough endoplasmic reticulum is a common, early reversible manifestation of other forms of cell injury. However, the severity of the damage to the host cell resulting from the progressive distention of intracellular membranes and the subsequent formation of small segments of membrane-bound host cytoplasm within the cisternae of these membranes is unknown. Early in the infection cycle, the rickettsiae were found free in the host cell cytoplasm, within invaginations of the nuclear envelope, occasionally free in the space between the outer and inner nuclear membranes, and in the host nucleoplasm, but not within cisternae formed by swollen endoplasmic reticulum. As a consequence of intracisternal swelling and fusion of intracellular membranes later in the infection cycle, the majority of the rickettsiae were found surrounded by host cytoplasm bound by host-derived internal membranes and appeared to persist in this state until cell lysis. The overall cytopathological changes in cells infected with R. richettsii appear dramatic and, from other studies in our laboratory, are significantly different from those observed in cells infected with Rickettsia prowazekii.     

Experimental infection of mouse peritoneal mesothelium with scrub typhus rickettsiae: an ultrastructural study.

The infection cycle of Rickettsia tsutsugamushi in mouse peritoneal mesothelial cells, observed late in the course of an established infection, intimately involved the host cell plasma membrane. Organisms multiplied in the cytoplasm, moved to the cell periphery, and acquired a host-membrane coat as they budded from the cell surface. Rickettsiae enveloped by this membrane entered other mesothelial cells, apparently by a phagocytic mechanism. Organisms escaped from the phagocytic vacuole as the vacuole membrane and host membrane coat disintegrated. Free rickettsiae replicated by binary fission in the cell cytoplasm. Rickettsial infection of mesothelial cells induced conspicuous cellular hypertrophy with increased numbers of unaltered cytoplasmic organelles.     

Current knowledge onChlamydia pneumoniae,strain TWAR,an important cause of pneumonia and other acute respiratory diseases

This article reviews current knowledge ofChlamydia pneumoniae strain TWAR, a newly recognizedChlamydia organism that causes acute respiratory infection, especially atypical pneumonia. Information is included on the microbiology, classification and laboratory diagnosis of the organism. Details of a series of studies of both endemic and epidemic respiratory infections are reviewed to present information on both the clinical and epidemiological characteristics of infection with strain TWAR. Laboratory studies of antibiotic sensitivity and recommendations for treatment are presented.     

Analysis of the hamster polyomavirus infection in vitro: host-restricted productive cycle

In a search for a host fully permissive for the hamster polyomavirus (HaPV) productive cycle in cell culture, the replication of the viral genome has been assayed in a panel of murine and hamster cell types. These experiments led to the conclusion that hamster cells represent the most permissive host for HaPV DNA replication although some murine cells also permit replication of the viral DNA. A single burst of infectious particles is demonstrable in some replication-competent cells, but the outcome of the infection appears to be clearly host dependent. In one hamster cell line (GD36), the virus can be propagated by successive productive cycles. In other hamster cells, despite a successful initial virus burst following transfection of viral DNA, a block in the production of virus particles seems to prevent the spread of infection. This type of restriction level may play a role in the in vivo host range of HaPV.     

Localization of Chlamydia trachomatis hypothetical protein CT311 in host cell cytoplasm

The chlamydia-specific hypothetical protein CT311 was detected both inside and outside of the chlamydial inclusions in Chlamydia trachomatis-infected cells. The extra-inclusion CT311 molecules were distributed in the host cell cytoplasm with a pattern similar to that of CPAF, a known Chlamydia-secreted protease. The detection of CT311 was specific since the anti-CT311 antibody labeling was only removed by absorption with CT311 but not CPAF fusion proteins. In addition, both anti-CT311 and anti-CPAF antibodies only detected their corresponding endogenous proteins without cross-reacting with each other or any other antigens in the whole cell lysates of C. trachomatis-infected cells. Although both CT311 and CPAF proteins were first detected 12 h after infection, localization of CT311 into host cell cytosol was delayed until 24 h while CPAF secretion into host cell cytosol was already obvious by 18 h after infection. The host cell cytosolic localization of CT311 was further confirmed in human primary cells. CT311 was predicted to contain an N-terminal secretion signal sequence and the CT311 signal sequence directed secretion of PhoA into bacterial periplasmic region in a heterologous assay system, suggesting that a sec-dependent pathway may play a role in the secretion of CT311 into host cell cytosol. This hypothesis is further supported by the observation that secretion of CT311 in Chlamydia-infected cells was blocked by a C16 compound known to inhibit signal peptidase I. These findings have provided important molecular information for further understanding the C. trachomatis pathogenic mechanisms.     

Host cell death due to enteropathogenic Escherichia coli has features of apoptosis

Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen.     

Is the follicular dendritic cell a primarily stationary cell?

The cell nature of follicular dendritic cells (FDC), a member of dendritic cell group, was examined to see whether or not they are recirculating cells on splenic implantation study. Slices of BALB/c mouse spleen were implanted into C57BL/6 mice neonatally thymectomized and reconstructed by F1 (BALB/c x C57BL/6) thymus grafting. On H-2 class I immunohistology 6 months later, host spleens consisted of only the cells including FDC of host (C57BL/6) type. On the other hand, FDC of regenerated splenic grafts were of splenic donor (BALB/c) origin and haematogenic cells including germinal centre lymphocytes were of the host (C57BL/6) origin. The fact that the FDC in the regenerated splenic grafts reside irrespective of the replacement by host recirculating cells indicates that FDC belong primarily to stationary cell populations but not recirculating cell populations.     

Ultrastructural cytochemical evidence for the activation of lysosomes in the cytocidal effect of Chlamydia psittaci.

The cytopathic effect of the polyarthritis strain of Chlamydia psittaci was studied in cultured bovine fetal spleen cells and found to be mediated by the release of lysosomal enzymes into the host cytoplasm during the late stages of chlamydial development. Ultrastructural cytochemical analysis and cell fractionation studies of infected cells revealed a close relationship between the stage of chlamydial development, fine structural features of the host, and localization of lysosomal enzyme activities. After adsorption, chlamydiae entered the host cells by endocytosis. The endocytic vacuoles containing individual chlamydiae and later the inclusion vacuoles containing the different chlamydial developmental forms were always free from lysosomal enzyme activity. Even after extensive multiplication of chlamydiae, lysosomal enzymes remained localized within lysosomes or their precursors in the host cell. Coincident with the process of chlamydial maturation, lysosomal enzymes were released into the host cytoplasm and were always associated with disintegration of host cell constituents and lysis. The chlamydiae appeared to be protected from this lysosomal enzyme activity by the inclusion membrane. After release from the inclusion, elementary bodies maintained their fine structural features, whereas all other chlamydial developmental forms lost their ultrasturctural integrity.     

Transplantation of adipose derived stromal cells into the developing mouse eye

Adipose derived stromal cells (ADSCs) were transplanted into a developing mouse eye to investigate the influence of a developing host micro environment on integration and differentiation. Green fluorescent protein-expressing ADSCs were transplanted by intraocular injections. The age of the mouse was in the range of 1 to 10 days postnatal (PN). Survival dates ranged from 7 to 28 post transplantation (DPT), at which time immunohistochemistry was performed. The transplanted ADSCs displayed some morphological differentiations in the host eye. Some cells expressed microtubule associated protein 2 (marker for mature neuron), or glial fibrillary acid protein (marker for glial cell). In addition, some cells integrated into the ganglion cell layer. The integration and differentiation of the transplanted ADSCs in the 5 and 10 PN 7 DPT were better than in the host eye the other age ranges. This study was aimed at demonstrating how the age of host micro environment would influence the differentiation and integration of the transplanted ADSCs. However, it was found that the integration and differentiation into the developing retina were very limited when compared with other stem cells, such as murine brain progenitor cell.