Purification on Renografin Density Gradients of Chlamydia trachomatis Grown in the Yolk Sac of Eggs

Howard, Lawrence C.; Orenstein, Neil S.; King, Norval W.

doi:10.1128/aem.27.1.102-106.1974

Cited by 69 publications

(33 citation statements)

References 8 publications

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“…One of the major factors that have permitted the use of radiolabeled compounds to begin studying the growth characteristics of anaplasma has been the adaption of the Renografin gradient techniques developed for the study of rickettsia and chlamydia (2,5,8,25) for use with anaplasma. This has permitted isolation of anaplasma in sufficient purity to allow the detection of specific incorporation of radiolabeled compounds.…”

Section: Discussionmentioning

confidence: 99%

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Synthesis of DNA and protein by Anaplasma marginale in bovine erythrocytes during short-term culture

Davis¹,

Talmadge²,

Parish³

et al. 1978

Infect Immun

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Bovine erythrocytes infected with Anaplasma marginale were cultured for 1 to 5 days in a CO2 incubation chamber, pulse-labeled with [3H]thymidine and [14C]methionine, lysed, and fractionated by differential centrifugation and continuous density gradient centrifugation in Renografin. Anaplasma and associated fragments of stroma formed two distinct bands in the dense region of the gradient. Electron microscopic examination of pelleted material from the bands from cells cultured for 1 day revealed the presence of organisms that were morphologically intact or in various states of degeneration. Examination of fractions from the gradient for incorporation of label revealed that analplasma present in erythrocytes can incorporate both [3H]thymidine and [14C]methionine. Subsequent experiments demonstrated that organisms cultured for 3 and 5 days incorporated the radiolabeled compounds also, but to a lesser extent. The experiments demonstrate that it is possible to culture analplasma in vitro for short periods of time and monitor their growth characteristics.

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Section: Discussionmentioning

confidence: 99%

“…The supernatant, containing erythrocyte stroma and anaplasma, was centrifuged at 10,-000 x g for 60 min. The pellets were resuspended and placed on a continous density gradient of Renografin (2,5,8,25) (20 to 55% in Hanks balanced salt solution) and centrifuged at 25,000 rpm on a Beckman SW41 rotor for 70 min.…”

mentioning

confidence: 99%

Synthesis of DNA and protein by Anaplasma marginale in bovine erythrocytes during short-term culture

Davis¹,

Talmadge²,

Parish³

et al. 1978

Infect Immun

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show abstract

“…Growth and isolation of bacteria. Chlamydia trachomatis serovar L2 (434/Bu; ATCC) was propagated in McCoy cells as described previously (9,28). Aliquots of purified elementary bodies were stored at -80°C in SPG buffer (250 mM sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid) and thawed immediately prior to use.…”

Section: Methodsmentioning

confidence: 99%

Gamma Interferon Is Required forChlamydiaClearance but Is Dispensable for T Cell Homing to the Genital Tract

Helble

González

Andrian

et al. 2020

mBio

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While there is no effective vaccine against Chlamydia trachomatis infection, previous work has demonstrated the importance of C. trachomatis-specific CD4+ T cells (NR1 T cells) in pathogen clearance. Specifically, NR1 T cells have been shown to be protective in mice, and this protection depends on the host’s ability to sense the cytokine gamma interferon (IFN-γ). However, it is unclear what role NR1 production or sensing of IFN-γ plays in T cell homing to the genital tract or T cell-mediated protection against C. trachomatis. Using two-photon microscopy and flow cytometry, we found that naive wild-type (WT), IFN-γ−/−, and IFN-γR−/− NR1 T cells specifically home to sections in the genital tract that contain C. trachomatis. We also determined that protection against infection requires production of IFN-γ from either NR1 T cells or endogenous cells, further highlighting the importance of IFN-γ in clearing C. trachomatis infection. IMPORTANCE Chlamydia trachomatis is an important mucosal pathogen that is the leading cause of sexually transmitted bacterial infections in the United States. Despite this, there is no vaccine currently available. In order to develop such a vaccine, it is necessary to understand the components of the immune response that can lead to protection against this pathogen. It is well known that antigen-specific CD4+ T cells are critical for Chlamydia clearance, but the contexts in which they are protective or not protective are unknown. Here, we aimed to characterize the importance of gamma interferon production and sensing by T cells and the effects on the immune response to C. trachomatis. Our work here helps to define the contexts in which antigen-specific T cells can be protective, which is critical to our ability to design an effective and protective vaccine against C. trachomatis.

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“…Chlamydia trachomatis serovar L2 (LGV 434) were grown in HeLa 229 cells, and EBs were purified by Renografin density gradient centrifugation as previously described (43). All cell lines were obtained from the American Type Culture Collection (ATCC).…”

Section: Organisms and Cell Culturementioning

confidence: 99%

Chlamydia trachomatis Infection Causes Mitotic Spindle Pole Defects Independently from its Effects on Centrosome Amplification

Knowlton

Brown

Richards

et al. 2011

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Chlamydiae are gram negative, obligate intracellular bacteria, and Chlamydia trachomatis is the etiologic agent of the most commonly reported sexually transmitted disease in the United States. Chlamydiae undergo a biphasic life cycle that takes place inside a parasitophorous vacuole termed an inclusion. Chlamydial infections have been epidemiologically linked to cervical cancer in patients previously infected by human papillomavirus (HPV). The inclusion associates very closely with host cell centrosomes, and this association is dependent upon the host motor protein dynein. We have previously reported that this interaction induces supernumerary centrosomes in infected cells, leading to multipolar mitotic spindles and inhibiting accurate chromosome segregation. Our findings demonstrate that chlamydial infection causes mitotic spindle defects independently of its effect on centrosome amplification. We show that chlamydial infection increases centrosome spread and inhibits the spindle assembly checkpoint delay to disrupt centrosome clustering. These data suggest chlamydial infection exacerbates the consequences of centrosome amplification by inhibiting the cells’ ability to suppress the effects of these defects on mitotic spindle organization. We hypothesize that these combined effects on mitotic spindle architecture identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation.

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Purification on Renografin Density Gradients of Chlamydia trachomatis Grown in the Yolk Sac of Eggs

Cited by 69 publications

References 8 publications

Synthesis of DNA and protein by Anaplasma marginale in bovine erythrocytes during short-term culture

Synthesis of DNA and protein by Anaplasma marginale in bovine erythrocytes during short-term culture

Gamma Interferon Is Required forChlamydiaClearance but Is Dispensable for T Cell Homing to the Genital Tract

Chlamydia trachomatis Infection Causes Mitotic Spindle Pole Defects Independently from its Effects on Centrosome Amplification

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