Susceptibility of Mice to Vaginal Infection with Chlamydia trachomatis Mouse Pneumonitis Is Dependent on the Age of the Animal

Mice from three strains, BALB/c (H-2(d)), C3H (H-2(k)), and C57BL/6 (H-2(b)), ranging from 5 to 14 weeks of age, were inoculated intravaginally with different doses of the Chlamydia trachomatis mouse pneumonitis serovar. Vaginal swabs taken at weekly intervals showed that the percentage of animals with positive cultures and the number of inclusion-forming units recovered per mouse were higher in the younger animals. Furthermore, vaginal shedding lasted longer in the young mice than in the older mice. In addition, following mating higher rates of infertility and a decrease in the number of embryos were observed in the infected young mice. In conclusion, susceptibility to a chlamydial vaginal infection is dependent on the age of the mice, with the older animals being more resistant.     

Chlamydia trachomatis Persistence in the Female Mouse Genital Tract: Inducible Nitric Oxide Synthase and Infection Outcome

It was previously reported that female mice resolve a primary Chlamydia trachomatis urogenital infection independent of inducible nitric oxide synthase (iNOS). We now report that although iNOS-deficient (NOS2(-/-)) mice resolve culture-apparent infection in a fashion similar to that of normal control (NOS2(+/+)) mice, they sustain significantly increased rates of disease, as assessed by hydrosalpinx formation. PCR amplification of ompA followed by Southern blot detection of amplicands revealed the presence of chlamydial DNA in the lower genital tracts of both NOS2(-/-) and NOS2(+/+) mice at > or =120 days postinfection and in upper genital tract tissues at >120 days postinfection. However, only NOS2(-/-) mice shed low numbers of viable chlamydiae from the lower genital tract after immunosuppressive treatment at 120 days postinfection. When cultured primary murine lung fibroblasts were activated in the presence of gamma interferon (IFN-gamma), inhibition of chlamydial growth occurred in both NOS2(+/+) and NOS2(-/-) cells, but the inhibition was reversible after removal of the cytokine in the NOS2(-/-) primary cell culture only. The iNOS-independent inhibition was microbistatic but was independent of 2,3-indoleamine dioxygenase activity. We conclude that chlamydial DNA and antigens persist in mice subsequent to culture-apparent resolution. In addition, IFN-gamma induces in vivo inhibition of chlamydial growth through microbistatic mechanisms in the absence of iNOS activity, but in the presence of iNOS activity, IFN-gamma is microbicidal and effects eradication.     

Comparable Genital Tract Infection,Pathology, and Immunity in Rhesus Macaques Inoculated with Wild-Type or Plasmid-Deficient Chlamydia trachomatis Serovar D

Rhesus macaques were studied to directly address the potential for plasmid-deficient Chlamydia trachomatis to serve as a live attenuated vaccine in the genital tract. Five repeated cervical inoculations of rhesus macaques with wild-type serovar D strain D/UW-3/Cx or a plasmid-deficient derivative of this strain, CTD153, resulted in infections with similar kinetics and induced comparable levels of protective immunity. After all animals received five challenges with D/UW-3/Cx, levels of inflammation observed grossly and histologically were similar between the groups. Animals in both groups developed evidence of oviduct dilatation; however, reduced oviduct dilatation was observed for “controllers,” i.e., animals without detectable chlamydial DNA in the fimbriae at weeks 5 and 12. Grouping animals into “ascenders” and “controllers” revealed that elevated early T cell responses were associated with protection, whereas higher antibody responses were associated with ascension. Protected animals shared common major histocompatibility complex (MHC) alleles. Overall, genetic differences of individual animals, rather than the presence or absence of the chlamydial plasmid in the primary infecting strain, appeared to play a role in determining the outcome of infection.     

Route of Infection That Induces a High Intensity of Gamma Interferon-Secreting T Cells in the Genital Tract Produces Optimal Protection against Chlamydia trachomatis Infection in Mice

The induction of local T helper type 1 (Th1)-mediated cellular immunity is crucial for resistance of mice to genital infection by the obligate intracellular bacterium Chlamydia trachomatis. We tested the hypothesis that the route of immunization that elicits relatively high numbers of chlamydia-specific, gamma interferon (IFN-γ)-secreting T lymphocytes (ISTLs) in the genital tract would induce optimal protective immunity against reinfection. Female BALB/c mice were infected intravaginally (i.v.), intranasally (i.n.), orally (p.o.), or subcutaneously (s.c.) with C. trachomatis. At days 7, 14, 21, and 28 postinfection, T cells isolated from the genital tract tissues were restimulated with chlamydial antigen in vitro, and the amounts of IFN-γ induced were measured by a sandwiched enzyme-linked immunosorbent assay method. At day 7 postinfection, i.n.- and i.v.-immunized mice had high levels of chlamydia-specific ISTLs in their genital tracts (203.58 ± 68.1 and 225.5 ± 12.1 pg/ml, respectively). However, there were no detectable ISTLs in the genital tracts of p.o.- or s.c.-infected mice. When preinfected mice were challenged i.v. 70 days later, animals preexposed by the i.n. route were highly resistant to reinfection, with greatly reduced chlamydial burden, and suffered an attenuated infection that resolved by day 6 postchallenge. Animals preexposed by the i.v. route were modestly protected, whereas p.o. and s.c. groups were indistinguishable in this regard from control mice. The resistance of i.n.-immunized mice (and to some extent the i.v.-exposed mice) to reinfection was associated with early appearance (within 24 h) of high levels of genital ISTLs compared with mice preinfected by other routes. Furthermore, although i.n. and i.v.-immunized mice had comparable levels of chlamydia-specific immunoglobulin A (IgA) antibodies in their vaginal washes, the levels of IgG2a were four- sixfold higher in i.n.-immunized mice than in any of the other groups. The results suggested that immunization routes that foster rapid induction of vigorous genital mucosal cell-mediated immune (CMI) effectors (e.g., IFN-γ), the CMI-associated humoral effector, IgG2a, and to some extent secretory IgA produce protective immunity against chlamydial genital infection. Therefore, i.n. immunization is a potential delivery route of choice in the development of a vaccine against Chlamydia.     

Immunization with the Chlamydia trachomatis Mouse Pneumonitis Major Outer Membrane Protein Can Elicit a Protective Immune Response against a Genital Challenge

Infertility, ectopic pregnancy, and chronic abdominal pain are frequent complications of genital infections with Chlamydia trachomatis. In an attempt to produce a vaccine to protect against this pathogen we purified and refolded the C. trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP). This preparation, mixed with Freund's adjuvant using vortexing or sonication, was used to immunize BALB/c mice that were subsequently challenged in the upper genital tract. Vaginal cultures were taken on a weekly basis, and mice were mated 6 weeks after the challenge. Gels of the vortexed MOMP showed a predominant band with a molecular size of 62 kDa and weaker bands at 42 and 132 kDa, while the sonicated MOMP had a single band with a molecular size of 42 kDa. Following immunization with these two preparations, strong humoral and cell-mediated immune responses were detected in the mice inoculated with the vortexed MOMP. On the other hand, mice immunized with the sonicated MOMP had a strong humoral immune response but a relatively weak cell-mediated immune response, as determined by a T-cell lymphoproliferative assay and level of cytokine production by splenocytes. Vaginal cultures showed that the mice immunized with the vortexed MOMP were significantly protected, as determined by a decrease in the number of animals with positive cultures, the length of time the mice shed viable organisms, and the number of inclusion-forming units recovered per mouse. Animals immunized with the sonicated MOMP, on the other hand, showed a weaker level of protection based on the same three parameters. After mating, the number of fertile animals and number of embryos per mouse were significantly higher for the mice immunized with vortexed MOMP, but not for the mice immunized with sonicated MOMP, compared to those of the control groups. In conclusion, immunization with a purified and refolded preparation of the C. trachomatis MoPn MOMP confers a significant level of protection in mice against a genital challenge.     

Plasmid CDS5 Influences Infectivity and Virulence in a Mouse Model of Chlamydia trachomatis Urogenital Infection

The native plasmid of both Chlamydia muridarum and Chlamydia trachomatis has been shown to control virulence and infectivity in mice and in lower primates. We recently described the development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis that for the first time provides a platform for the molecular dissection of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In the present study, we transformed a plasmid-free lymphogranuloma venereum isolate of C. trachomatis, serovar L2, with either the original shuttle vector (pGFP::SW2) or a derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene (pCDS5KO). Female mice were inoculated with these strains either intravaginally or transcervically. We found that transformation of the plasmid-free isolate with the intact pGFP::SW2 vector significantly enhanced infectivity and induction of host inflammatory responses compared to the plasmid-free parental isolate. Transformation with pCDS5KO resulted in infection courses and inflammatory responses not significantly different from those observed in mice infected with the plasmid-free isolate. These results indicate a critical role of plasmid CDS5 in in vivo fitness and in induction of inflammatory responses. To our knowledge, these are the first in vivo observations ascribing infectivity and virulence to a specific plasmid gene.     

Lipid Metabolism of Monkey Kidney Cells (LLC-MK-2) Infected with Chlamydia trachomatis Strain lymphogranuloma venereum

Lipid metabolism of monkey kidney (LLC-MK-2) cells and cells infected with a Chlamydia trachomatis strain lymphogranuloma venereum (LGV) was studied. The protein-to-lipid ratio of normal MK-2 cells was found to increase linearly over a 60-h period of incubation. The protein-to-lipid ratio of the infected cells was similar to that in normal cells until 36 h after infection, when a plateau in the ratio was observed. Lipid synthesis of the infected cells was found to be inhibited after 48 h of infection. Turnover of host lipids did not appear to be markedly altered by infection with LGV over a 48-h period of incubation. An anteiso branched chain of 15:0 fatty acid was found in infected cells but not in normal cells. The appearance of this fatty acid, correlated with a rise in the infectivity of LGV, suggests that synthesis of specific lipids was associated with the infection.     

Chlamydia trachomatis Infection in the Female Reproductive Tract of the Rat: Influence of Progesterone on Infectivity and Immune Response

As the most common cause of sexually transmitted disease in women, chlamydial infections can lead to pelvic inflammatory disease, infertility, and ectopic pregnancy. To better understand the role played by sex hormones in modulating the immune response of the genital tract to microbial infections, we have developed a rat model to study Chlamydia trachomatis infection. Inbred female Lewis rats were primed with progesterone and inoculated by intrauterine instillation of C. trachomatis (mouse pneumonitis strain MoPn) into each uterine horn. When infected animals were examined for the presence of chlamydial antigens 14 days postinfection, both the uterus and vagina were found to be positive compared to those of saline-treated animals, which did not show specific staining. The involvement of local and systemic immune systems following chlamydial infection was determined by analyzing major histocompatibility complex (MHC) class II expression in the reproductive tract and lymphocyte proliferation in response to mitogenic and chlamydia-specific stimulation of cells from the spleen and lymph nodes (LN) draining the reproductive tract. Enhanced proliferation was observed in LN following mitogenic but not antigenic (MOMP [major outer membrane protein]) stimulation. In contrast, spleen cell proliferation was lower in chlamydia-infected rats than in saline-treated controls. MHC class II expression, an indicator of inflammatory responses, was upregulated in the uterus, on glandular epithelial cells, and adjacent to glands in response to chlamydial infection. In other experiments, when rats were infected at estrus and diestrus without prior progesterone priming, chlamydial inclusions were not detected in either the uterus or vagina. However, enhanced lymphocyte proliferation was observed in response to mitogenic and MOMP stimulation in the reproductive tract-draining LN from estrous and diestrous animals. These findings indicate that under appropriate endocrine conditions, the rat uterus is susceptible to C. trachomatis infection and that immune responses to this pathogen can be detected locally and systemically. Further, they suggest that clearance of the infection from the reproductive tract involves immune cells from the LN draining the reproductive tract.

Chlamydia trachomatis is an obligate intracellular gram-negative bacteria that is known to infect the genital tract and ocular epithelium. Disease caused by this organism can range from acute self-limiting infection to chronic inflammatory conditions, which can result in pelvic inflammatory disease, infertility, and ectopic pregnancy (12). Chlamydial infection of the urogenital tract is currently the leading cause of sexually transmitted disease in adolescents and women in the United States and Europe (11). Despite the recognition of chlamydial infection as a major public health concern, immune responses to human chlamydial infection are not well understood. Both T-cell-mediated and humoral responses are known to be involved in resolution of chlamydial infection. However, protective immunity developed as a result of chlamydial infection is transient, and reinfections are common (12).Animal models to study chlamydial infections have been developed in the mouse, guinea pig, marmoset, and nonhuman primate (13, 15). In mice, for example, inoculations of chlamydiae via intravaginal and intrauterine routes are known to cause cervicitis or salpingitis, respectively (17, 18). In this species, intravaginal infection with human serovars of chlamydiae was dependent on pretreatment with progesterone (18). In the guinea pig, however, pretreatment with estradiol markedly enhanced the course of infection with guinea pig inclusion conjunctivitis (16). In all cases, endocrine balance at the time of exposure to chlamydiae appeared to play a crucial role for host susceptibility.Previous work from our laboratory has demonstrated that sex hormones regulate the mucosal immune response in the female reproductive tract. Using the rat as a model system, we and others have shown that stage of the estrous cycle and treatment with sex hormones, specifically estradiol and progesterone, influence both the afferent and efferent arms of the immune system (for a review see reference 19). For example, antigen presentation, immunoglobulin A (IgA) transport, immune cell traffic into the reproductive tract, and cytokine production in the uterus and vagina have been shown to be under hormonal control. The aims of the present study were (i) to determine if infection could be successfully established in rats following intrauterine exposure to chlamydiae with or without progesterone treatment and (ii) to determine if immune responses occur locally and at sites distal to the reproductive tract in response to chlamydial infection.     

In Vivo and Ex Vivo Imaging Reveals a Long-Lasting Chlamydial Infection in the Mouse Gastrointestinal Tract following Genital Tract Inoculation

Intravaginal infection with Chlamydia muridarum in mice can ascend to the upper genital tract, resulting in hydrosalpinx, a pathological hallmark for tubal infertility in women infected with C. trachomatis. Here, we utilized in vivo imaging of C. muridarum infection in mice following an intravaginal inoculation and confirmed the rapid ascent of the chlamydial organisms from the lower to upper genital tracts. Unexpectedly, the C. muridarum-derived signal was still detectable in the abdominal area 100 days after inoculation. Ex vivo imaging of the mouse organs revealed that the long-lasting presence of the chlamydial signal was restricted to the gastrointestinal (GI) tract, which was validated by directly measuring the chlamydial live organisms and genomes in the same organs. The C. muridarum organisms spreading from the genital to the GI tracts were detected in different mouse strains and appeared to be independent of oral or rectal routes. Mice prevented from orally taking up excretions also developed the long-lasting GI tract infection. Inoculation of C. muridarum directly into the upper genital tract, which resulted in a delayed vaginal shedding of live organisms, accelerated the chlamydial spreading to the GI tract. Thus, we have demonstrated that the genital tract chlamydial organisms may use a systemic route to spread to and establish a long-lasting infection in the GI tract. The significance of the chlamydial spreading from the genital to GI tracts is discussed.     

儿童呼吸道感染者肺炎支原体及肺炎衣原体特异性抗体检测临床意义

目的 探究儿童呼吸道感染者肺炎支原体及肺炎衣原体特异性抗体检测临床意义。方法 选择我院2018年1月~8月收治的85例呼吸道感染患儿设为研究组,另选择同期在我院进行健康体检的85例儿童设为对照组,对照组采用ELISA法检测肺炎支原体及衣原体,研究组在此基础上行咽拭子培养法检测,比较两组肺炎支原体、衣原体抗体检出率,ELISA法与咽拭子培养法对肺炎支原体、衣原体抗体的阳性检出率,分析研究组呼吸道感染性疾病中肺炎支原体、衣原体抗体阳性分布情况。结果 研究组患儿肺炎支原体抗体MP-IgM抗体、MP-IgA抗体、MP-IgG抗体及肺炎衣原体特异性抗体CP-IgM抗体、CP-IgG抗体的检出率均高于对照组儿童,差异有统计学意义（P＜0.05）;ELISA法对肺炎支原体特异性抗体阳性的检出率为54.12%高于咽拭子培养法的36.47%,组间对比具有统计学意义（P＜0.05）;ELISA法对肺炎衣原体特异性抗体阳性的检出率为58.82%高于咽拭子培养法的37.65%,差异有统计学意义（P＜0.05）;肺炎支原体、肺炎衣原体抗体阳性在各种儿童呼吸道感染性疾病中均有表现,其中,肺炎和支气管肺炎的阳性检出率较咽炎、扁桃体炎均较高。结论 肺炎支原体、肺炎衣原体是引发肺炎和支气管肺炎感染的主要病原菌。     

Cytopathologic detection of Chlamydia trachomatis in vaginopancervical (Fast) smears

The value of the Papanicolaou-stained vaginopancervical (Fast) smear in the detection of chlamydial infection has been disputed. We examined 116 satisfactory Fast smears from 203 women enrolled in the Johns Hopkins Fertility Control Clinic and compared tissue-culture results with cytopathologic detection using various published morphologic criteria. All Chlamydia culture-positive cases were reviewed, and certain cytologic features considered helpful in the detection of chlamydial infection in cervical smears obtained from this selected high-risk population were identified. The changes that had the highest correlation with tissue culture included fine vacuolation of metaplastic endocervical cells, giving their cytoplasm a rarefied "moth-eaten" appearance. Using these criteria, cytopathologic changes of chlamydial infection were observed in 24 of 28 cases of tissue-culture-positive cases and in 8 of 88 tissue-culture-negative cases. The sensitivity and specificity of the Fast-smear cytodiagnosis of Chlamydia infection utilizing these morphologic changes and compared with tissue culture were 86% and 91%, respectively. Other cytologic features, including inflammatory background and intracytoplasmic structures consistent with initial and intermediate chlamydial bodies within the metaplastic cells, were found to be useful although less specific and less sensitive. The implications of these diagnostic features, the conditions to be considered in their differential diagnosis, and the pitfalls of chlamydial cytodiagnosis and the chlamydia culture studies have been critically reviewed. Study design and the high unsatisfactory cervical smear rate are discussed.     

CT043, a Protective Antigen That Induces a CD4+ Th1 Response during Chlamydia trachomatis Infection in Mice and Humans

Despite several decades of intensive studies, no vaccines against Chlamydia trachomatis, an intracellular pathogen causing serious ocular and urogenital diseases, are available yet. Infection-induced immunity in both animal models and humans strongly supports the notion that for a vaccine to be effective a strong CD4⁺ Th1 immune response should be induced. In the course of our vaccine screening program based on the selection of chlamydial proteins eliciting cell-mediated immunity, we have found that CT043, a protein annotated as hypothetical, induces CD4⁺ Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. DNA priming/protein boost immunization with CT043 results in a 2.6-log inclusion-forming unit reduction in the murine lung infection model. Sequence analysis of CT043 from C. trachomatis human isolates belonging to the most representative genital serovars revealed a high degree of conservation, suggesting that this antigen could provide cross-serotype protection. Therefore, CT043 is a promising vaccine candidate against C. trachomatis infection.Chlamydia trachomatis is an obligate intracellular human pathogen which exists in two highly specialized morphological forms: the infectious elementary body (EB) and the replicative reticulate body (RB). The pathogen has been classified into 19 different serotypes (serovars), on the basis of which variant of the major outer membrane protein (MOMP) is expressed on its surface. Worldwide, C. trachomatis is responsible for more than 92 million sexually transmitted infections and 85 million ocular infections per year (45). These infections cause several types of diseases (35, 44), including trachoma-induced blindness (serovars A to C), pelvic inflammatory disease, ectopic pregnancy and infertility (serovars D to K), and lymphogranuloma venereum (serovars L1 to L3). Furthermore, recent studies indicate that chlamydia infections facilitate the transmission of both human immunodeficiency virus (28) and human papillomavirus (16).Although effective antibiotic treatments are available, they are often unsuccessful in halting the spread of the infection or inadequate to prevent the chlamydia-mediated long-term sequelae for a number of reasons. First, urogenital tract infections are frequently asymptomatic and therefore not properly treated in due time. Second, although C. trachomatis is generally sensitive to a panel of antibiotics, multiple-antibiotic-resistant strains of chlamydia have been reported (34). Third, there are indications from in vitro studies that antibiotic treatment could lead to the formation of aberrant chlamydia forms that remain dormant within inclusions and may eventually turn into EBs under favorable environmental conditions (9, 47). Finally, epidemiological data in industrialized countries indicate that the rate of chlamydia reinfection is rising, and this has been attributed to the interference of early antibiotic treatment with the acquisition of immunity against chlamydia (2).For these reasons, the development of an effective vaccine against C. trachomatis infection is urgently needed. Early trials with heat-killed preparations of whole EBs have been shown to elicit short-term protection, but a few cases of immunopathological reactions upon reexposure to chlamydia have also been reported (2). Based on these early findings, further efforts have been focused on the development of subunit vaccines (2, 13, 14). In particular, several studies have described the use of MOMP, the immunodominant chlamydial antigen accounting for 60% of the total mass of the chlamydia outer membrane (6). Immunization with MOMP purified from C. trachomatis elicited an immune response that fully protected mice against an intra-ovarian bursa chlamydia challenge (27). However, only correctly folded MOMP appears to provide protection, and no adequate, scalable processes for the production of soluble and properly folded recombinant MOMP have been developed yet. This has so far hampered the use of MOMP for vaccine development. Moreover, due to the sequence variability of MOMP, a broadly protective chlamydial vaccine will probably require the use of other immunogenic antigens in addition to, or in place of, MOMP.The search for other chlamydial antigens has been driven by the elucidation of the mechanisms of immune responses to chlamydia infection and the demonstration of the importance of CD4⁺ T cells in natural immunity. Animal models have shown that protective immunity to C. trachomatis depends on the elicitation of a Th1-polarized cell-mediated immune response, in particular, gamma interferon (IFN-γ)-secreting CD4⁺ lymphocytes. In mice, the depletion of CD4⁺ T cells results in the loss of protective immunity and adoptive transfer of chlamydia-specific CD4⁺ T cells confers protection against C. trachomatis challenge (36). Studies in primates have also emphasized the role of Th1 cells in immunity to chlamydia (42, 43).Here we report experimental evidence that CT043, a protein annotated as hypothetical, is a novel protective chlamydial antigen. This antigen appears to be associated with the bacterial cell surface and is expressed within the chlamydia inclusion in infected HeLa cells throughout the infection cycle. We show that CT043 is the target of CD4⁺ Th1 cells both in chlamydia-infected mice and in human patients with diagnosed C. trachomatis genital infection. Moreover, by using a DNA priming/protein boost immunization protocol, we show that the antigen significantly reduces the bacterial load in a mouse model of intranasal (i.n.) infection. Finally, sequence analysis of the CT043 gene in 22 C. trachomatis strains belonging to the most representative genital serovars revealed a high degree of conservation (99.4% amino acid identity), suggesting that the antigen could provide cross-serotype protection.Altogether, our study demonstrates that this antigen is a promising candidate to be included in a subunit-based vaccine against C. trachomatis.     

The Combination of the Gastrointestinal Integrin (α4β7) and Selectin Ligand Enhances T-Cell Migration to the Reproductive Tract During Infection with Chlamydia trachomatis

Problem  Chlamydia trachomatis causes sexually transmitted infection and reproductive dysfunction worldwide. Identifying a population of endocervical T-cells to target in vaccine development is likely to enhance efficacy of a vaccine and reduce reproductive tract dysfunction.
Method of study  Endocervical samples were obtained from young women and flow cytometric analysis was used to identify lymphocytes that appeared in the genital tract in response to sexually transmitted bacterial infections caused by C. trachomatis.
Results  Increased numbers of α4β7+CLA+ memory T-cells, a unique T-cell phenotype, were found in the endocervix of human female subjects infected with C. trachomatis .
Conclusion A unique population of memory T lymphocytes expressing both α4β7 and CLA gain access to reproductive tract tissues during a sexually transmitted infection with C. trachomatis and should be considered in development of vaccines against sexually transmitted infections.     

Persistent Infection of Mouse Fibroblasts (L Cells) with Chlamydia psittaci: Evidence for a Cryptic Chlamydial Form

When monolayers of mouse fibroblasts (L cells) were infected with enough Chlamydia psittaci (strain 6BC) to destroy most of the host cells, 1 in every 10(5) to 10(6) originally infected cells gave rise to a colony of L cells persistently infected with strain 6BC. In these populations, the density of L cells and 6BC fluctuated periodically and reciprocally as periods of host cell increase were followed by periods of parasite multiplication. Successive cycles of L-cell and 6BC reproduction were sustained indefinitely by periodic transfer to fresh medium. Isolation of L cells and 6BC from persistent infections provided no evidence that there had been any selection of variants better suited for coexistence. Persistently infected populations consisting mainly of inclusion-free L cells yielded only persistently infected clones, grew more slowly, and cloned less efficiently. They were also almost completely resistant to superinfection with high multiplicities of either 6BC or the lymphogranuloma venereum strain 440L of Chlamydia trachomatis. These properties of persistently infected L cells may be accounted for by assuming that all of the individuals in these populations are cryptically infected with 6BC and that cryptic infection slows the growth of the host cell and makes it immune to infection with exogenous chlamydiae. According to this hypothesis, the fluctuations in host and parasite density occur because some factor periodically sets off the conversion of cryptic chlamydial forms into reticulate bodies that multiply and differentiate into infectious elementary bodies in a conventional chlamydial developmental cycle.     

Chlamydia pneumoniae Infection of Human Endothelial Cells Induces Proliferation of Smooth Muscle Cells via an Endothelial Cell-Derived Soluble Factor(s)

An association of Chlamydia pneumoniae with atherosclerosis and coronary heart disease has been determined epidemiologically and by the detection of C. pneumoniae organisms in atherosclerotic lesions in both humans and animal models of atherosclerosis. Previously, it has been shown that C. pneumoniae is capable of replicating in cell types found within atheromatous lesions, viz., endothelial cells, smooth muscle cells (SMC), and macrophages, yet the role of C. pneumoniae in the pathogenesis of atherosclerosis has not been determined. Since intimal thickening is a hallmark of atherosclerosis, we investigated whether C. pneumoniae infection of human umbilical vein endothelial cells (HUVEC) could induce the expression of a soluble factor(s) with mitogenic potential for SMC by using [3H]thymidine incorporation and direct cell counting. Conditioned medium harvested from HUVEC infected with C. pneumoniae stimulated SMC replication in a time- and dose-dependent fashion. Infection studies using various multiplicities of infection (MOIs) ranging from 0.001 to 1 demonstrated a dose-dependent production of the soluble factor(s). At an MOI of 1, SMC stimulation indices were 8.4 (P < 0.01) and 12.2 (P < 0.01) for conditioned media harvested at 24 and 48 h, respectively. To determine whether viable C. pneumoniae was required for production of the soluble factor(s), HUVEC were infected with heat-inactivated C. pneumoniae or with viable organisms in the presence of chloramphenicol. Both treatments produced stimulation indices similar to those for live C. pneumoniae in the absence of chloramphenicol (P > 0.05), indicating that the factor(s) was produced by HUVEC and not by C. pneumoniae and that signal transduction events following chlamydia endocytosis may be important in the production of a soluble factor(s). The ability of C. pneumoniae to elicit an endothelial cell-derived soluble factor(s) that stimulates SMC proliferation may be important in the pathogenesis of atherosclerosis.