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# Christopher’s Infection Formula Chlamydia

## Duration Of Ct Infection

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The mean duration of infection, , can be expressed as a weighted average of the length of asymptomatic infection A and symptomatic infection S,

with being the proportion of incident infections in which symptoms develop. In the Discussion section we show that results would have been similar if we considered durations of treated and untreated infections instead.

For the duration of asymptomatic CT infections, A, we use an estimate of 1·36 years, based on a previous evidence synthesis of studies on CT duration in asymptomatic women . This was a synthesis of nine studies identified from recent reviews , four that recruited asymptomatic infected women in STD clinic settings, and five studies based on population screening. Evidence was presented that these approximately represented incident and prevalent infections, respectively. The authors fitted mixtures of exponential models to these data. The estimates used here were based on a model that assumed CT infections clear at a constant rate.

Studies of CT duration have the inherent limitation that patients may clear infection and be re-infected during the follow-up period. For this reason we consider same-partner re-infections, which microbiological evidence suggest comprise the great majority of re-infections , to be part of a continuous episode.

## Cell Culture Transfection Knockdown And Infection

HeLa or U2OS cells were routinely cultured in DMEM containing 10% fetal calf serum and antibiotics . Cells were transfected with pEGFP-C2 or pEGFP-IPAM90-268 , using Turbofect according to the manufacturer’s instructions . Cells were transfected with 10nM of siRNA designed against CEP170 sequence or against non-specific sequence, using Hiperfect according to the manufacturer’s instructions . Three siRNAs directed against CEP170 were independently validated by using western blot and immunofluorescence. Cells were infected with C. trachomatis serovar L2 as previously described . When these approaches were used in combination, cells were transfected with plasmids immediately prior to infection , or with siRNA 24h prior to infection. When appropriate, expression plasmids were transfected 48h after transfection of siRNA.

## Incidence Of Chlamydia Trachomatis Infection In Women In England: Two Methods Of Estimation

Published online by Cambridge University Press: 13 June 2013

School of Health and Population Sciences, University of Birmingham, Birmingham, UK
School of Social and Community Medicine, University of Bristol, Bristol, UK
D. DE ANGELIS
Health Protection Agency, Colindale, London, UKMedical Research Council Biostatistics Unit, Cambridge, UK
N. J. WELTON
School of Social and Community Medicine, University of Bristol, Bristol, UK
J. MACLEOD
School of Social and Community Medicine, University of Bristol, Bristol, UK
K. TURNER
School of Social and Community Medicine, University of Bristol, Bristol, UK
P. J. HORNER
Affiliation:School of Social and Community Medicine, University of Bristol, Bristol, UKBristol Sexual Health Centre, University Hospital Bristol NHS Foundation Trust, Bristol, UK
*
* Author for correspondence: Dr M. J. Price, School of Health and Population Sciences, Public Health Building , University of Birmingham,

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## Reverse Transcription And Quantitative Real

Stage 1 with 50°C for 1 min followed by 95°C for 10 min.

Stage 2 with 94°C for 15 s followed by 55°C for 30 s and 72°C for 35 s with data collection for continuous fluorescence reading, repeated 40 times.

Stage 3 with dissociation steps for melting curve analysis of specific and unspecific PCR products.

The end products of a PCR were analyzed on a 2% ethidium bromide agarose gel to verify amplification of a specific product with the corresponding predicted amplicon size. The relative expression of specific products was calculated and statistically evaluated using relative expression software tool from Pfaffl et al. . Data are normally presented as means of triplicates with standard deviations as error bars. Some experiments had only duplicates. Three or more independent experiments were performed with similar results.

## Chlamydia Trachomatis Replicates In Aa M But Not Ca M

CD14+ monocytes were converted to macrophages by incubation in macrophage-colony stimulating factor for 5 days. Non-adherent cells were discarded, and the remaining adherent macrophages were activated with either IL-4 or IFN- to alternatively activated or classically activated macrophages. To verify the activation states, cells were analyzed for cell surface markers, including mannose receptor , high-affinity Fc gamma receptor , and a co-stimulatory molecule CD86. The mean fluorescence intensity of CD206 was 32.9 for resting macrophages, 1.5 for CA m, and 398.1 for AA m . The MFI of CD64 was 57.2 for resting macrophages, 273.4 for CA m, and 35.7 for AA m. The MFI of CD86 was 13.6 for resting macrophages, compared to 17.7 for CA m and 41.7 for AA m. Macrophages activated with IL-4 expressed high levels of CD206, consistent phenotypically with the AA m, while those activated with IFN- displayed high cell surface expression of CD64 . The IL-4 activated macrophages also displayed higher levels of cell surface CD86 relative to the IFN–activated population. The non-activated group displayed low levels of CD206 and CD86 relative to the putative AA m subset. Overall, IL-4 activation of macrophages generated CD206hi CD64lo CD86hi m consistent with alternative activation, while IFN- treatment yielded a population displaying CD206- CD64hi CD86lo phenotype, suggestive of classical activation. Resting m had a CD206lo CD64lo CD86lo phenotype.

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## Management Of Sex Partners

Sex partners should be referred for evaluation, testing, and presumptive treatment if they had sexual contact with the partner during the 60 days preceding the patients onset of symptoms or chlamydia diagnosis. Although the exposure intervals defining identification of sex partners at risk are based on limited data, the most recent sex partner should be evaluated and treated, even if the time of the last sexual contact was > 60 days before symptom onset or diagnosis.

## Ipam A Chlamydia Inclusion Protein Acting On Mts

Using confocal microscopy, we initially confirmed the arrangement of the MT network during the maturation of the inclusion in detail in HeLa cells . At 12h, MTs assemble at the inclusion periphery, and filaments partially cover this early structure. By 24h, MTs have entirely surrounded the inclusion from where some filaments extend and contact the plasma membrane. Later , a dense MT scaffold encircles the inclusion. This scaffold is associated with a nest of MTs that originate at the inclusion and extend towards the plasma membrane. The scaffold and nest MT superstructure was even more evident when human osteosarcoma cells were infected with C. trachomatis . Indeed, these cells have a larger cell volume and consequently a cytoskeleton that is easier to observe in comparison to HeLa cells. In particular, it was easier to discern at later time points that MT actively accumulated around the inclusion periphery in the cell body rather than being compressed against the plasma membrane during inclusion expansion . The initial MT scaffold present at 24hours post infection , formed independently of the previously reported actin filament cage , although inclusion-associated F-actin and MT structures coincided partially at later time points . Thus, host MTs are progressively organized at the inclusion surface, and assemble into an interlinked scaffold and nest superstructure.

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## Models And Data Sources

An attempt was made to identify data sources on incidence and prevalence of CT in the UK. A formal systematic review was not conducted, but papers were identified from recent reviews and synthesis exercises. Only one published report on incidence was identified , and a recent synthesis of UK CT prevalence data was also used . Information on CT duration was based on a recent synthesis described below. The information in represents all the information incorporated in the synthesis. Below we set out the assumptions that were made about the processes that generated the data, and the main features of the synthesis model. We begin by discussing the duration of CT infection which is required for all subsequent analyses.

Table 1. Data on duration of Chlamydia trachomatis infection

CrI, Credible interval.

Table 2. Data derived from tables 2 and 4 from LaMontagne et al. on infection and re-infection rates per 100 women years: numerators r and denominators n

GP, General practitioner FP, family planning STD, sexually transmitted disease clinic.

*n is estimated as the total number of 6-month follow-up periods . This has been calculated from the reported rates and numbers of events.

Table 3. Estimated prevalence of Chlamydia trachomatis in females in the general population reported in table 4 in Adams et al.

CI, Confidence interval.

Table 4. Reported adjusted odds ratios for the effect of setting on Chlamydia prevalence in females in the UK, from table 3 in Adams et al.

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## Detection Of Transferrin Receptors

Viable cells were pulsed for 1 h with Alexa Fluor 594 conjugated to transferrin. In brief, macrophages were plated and grown on coverslips in 24-well plates at a density of 0.5 or 1.0 × 105 cells/well. At the end of the cultivation period, cell culture supernatant was removed and replaced with 100 l PBS with 1:100 dilutions of aqueous 5 mg/ml stock of human transferrin Alexa Fluor 594 . After 1 h of incubation at 37°C in 5% CO2 atmosphere, cells were repeatedly washed with PBS and fixed with 1 ml 4% paraformaldehyde in PBS as described in the IFA procedure. TfR was detected using confocal microscopy. Analysis of uninfected cells and macrophages infected with C. trachomatis showed that infection of macrophages did not interfere TfR detection. The depicted micrographs were normalized by using Adobe Photoshop and selecting input levels from 0 to 165.

Chlamydial infection is the most frequently reported bacterial infectious disease in the United States, and prevalence is highest among persons aged 24 years . Multiple sequelae can result from C. trachomatis infection among women, the most serious of which include PID, ectopic pregnancy, and infertility. Certain women who receive a diagnosis of uncomplicated cervical infection already have subclinical upper genital tract infection.

Asymptomatic infection is common among both men and women. To detect chlamydial infection, health care providers frequently rely on screening tests. Annual screening of all sexually active women aged < 25 years is recommended, as is screening of older women at increased risk for infection . In a community-based cohort of female college students, incident chlamydial infection was also associated with BV and high-risk HPV infection . Although chlamydia incidence might be higher among certain women aged 25 years in certain communities, overall, the largest proportion of infection is among women aged < 25 years .

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## Chlamydia Trachomatis Infection Of Aa M Impacts Its Production Of Il

That C. trachomatis survived and replicated in AA m raised the possibility of pathogen modulation of AA m function. In this regard, we considered that infection would lead to potential changes in cytokine production. In the following experiments, we monitored two major cytokines with opposing effects IL-10 and IL-12p70, which are anti- and pro-inflammatory cytokines, respectively.

When stimulated with E. coli LPS, AA m responded by producing high amounts of anti-inflammatory IL-10 in contrast to CA m .

Figure 8.Chlamydia trachomatis induces IL-10 production in AA m, and IL-12p70 in CA m. M were left untreated or stimulated with Escherichia coli LPS , inoculated with heat-killed or viable Chlamydia trachomatis for one day. IL-10 concentrations of these cell culture supernatants were measured. Means of triplicates with respective standard deviations are depicted. After two-way ANOVA analysis, p-values of post hoc Tukey HSD are depicted. p-values 0.001 are symbolized by . p-values 0.05 are symbolized by . Results representative of one out of four independent experiments are shown. Results for IL-12p70. p-values 0.01 are symbolized by , p-values 0.5 are symbolized by . Results representative of one out of four independent experiments are shown.

## A Second Estimate Of Ct Incidence In England

A second estimate of the annual population incidence can be obtained using data on duration and data on prevalence using the relationship: incidence = prevalence/duration, so that:

Where duration is estimated as previously described and prevalence a,pop is informed directly by the data in Table 1 so $\tilde \lambda\hskip0.5pt _,}^2}$ is estimated for the groups 1819, 2024, 2529, and 3044 years.

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## Infant Pneumonia Caused By C Trachomatis

Chlamydial pneumonia among infants typically occurs at age 13 months and is a subacute pneumonia. Characteristic signs of chlamydial pneumonia among infants include a repetitive staccato cough with tachypnea and hyperinflation and bilateral diffuse infiltrates on a chest radiograph. In addition, peripheral eosinophilia occurs frequently. Because clinical presentations differ, all infants aged 13 months suspected of having pneumonia, especially those whose mothers have a history of, are at risk for , or suspected of having a chlamydial infection should be tested for C. trachomatis and treated if infected.

#### Diagnostic Considerations

Specimens for chlamydial testing should be collected from the nasopharynx. Tissue culture is the definitive standard diagnostic test for chlamydial pneumonia. Nonculture tests can be used. DFA is the only nonculture FDA-cleared test for detecting C. trachomatis from nasopharyngeal specimens however, DFA of nasopharyngeal specimens has a lower sensitivity and specificity than culture. NAATs are not cleared by FDA for detecting chlamydia from nasopharyngeal specimens, and clinical laboratories should verify the procedure according to CLIA regulations . Tracheal aspirates and lung biopsy specimens, if collected, should be tested for C. trachomatis.

#### Treatment

Erythromycin base or ethylsuccinate 50 mg/kg body weight/day orally divided into 4 doses daily for 14 days

Azithromycin suspension 20 mg/kg body weight/day orally, 1 dose daily for 3 days

## The Response Of T3s Promoters To Alterations In Dna Supercoiling Correlates With Their Temporal Expression Pattern

We next examined if there is a relationship between this differential response to DNA supercoiling and the temporal expression of T3S genes. For each promoter, we calculated the relative promoter activity by defining the maximal level of transcription at any superhelical density as 100% and normalizing other transcription levels to this value. To compare the promoters, we graphed the effects of superhelical density on the relative activity of each promoter. The promoters for the three mid-cycle T3S operons were transcribed at low levels from a relaxed template and at much higher levels from more supercoiled templates. Transcription of the cdsC, fliF, and incA promoters was highest at a superhelical density of approximately 0.06 and leveled off or decreased slightly for a of > 0.06 .4A). These superhelical density optima are close to the in vivo superhelicity of 0.063 to 0.077 that we measured for the chlamydial plasmid in the reticulate body stage of the developmental cycle . In addition, this response to DNA supercoiling closely mirrors the supercoiling response pattern for promoters of two other non-T3S chlamydial mid genes, ompA 4D) and pgk . ompA encodes the major outer membrane protein , and pgk is the gene for phosphoglycerol kinase neither is known to be associated with the T3S system in Chlamydia.

Published ahead of print on 16 March 2010.

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## Differential Regulation Of Ido Production In M In Response To Viability And Type Of Bacteria

We next determined the influence of C. trachomatis infection on anti-chlamydial effector molecules such as IDO. RT-PCR for IDO showed an increase of transcript for infected macrophages. The relative IDO expression for uninfected resting macrophages increased 53-fold with infection . The relative IDO expression of uninfected CA m increased 54682-fold upon infection . The relative IDO expression for uninfected AA m increased 10-fold compared to infected cells . To confirm this up-regulation of transcript translates into protein levels, Western blots of IDO were performed. Immunoblots did show detectable IDO only in infected CA m, but not in resting or AA m . The question then arose whether heat-stable components of C. trachomatis or viable C. trachomatis are sufficient to induce IDO protein in macrophages and how it compares to treatment with lipopolysaccharide from Escherichia coli . Interestingly, only viable but not heat-killed C. trachomatis was able to induce IDO. Furthermore, E. coli LPS unlike C. trachomatis led to detectable IDO in not only CA, but also resting and AA m .

## A Chlamydia Effector Recruits Cep170 To Reprogram Host Microtubule Organization

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Present address: Institut Pasteur, Channel Receptor Unit, 25 rue du Dr Roux, Paris 75015, France.

The authors declare no competing or financial interests.

J Cell Sci

Maud Dumoux, Anais Menny, Delphine Delacour, Richard D. Hayward A Chlamydia effector recruits CEP170 to reprogram host microtubule organization. J Cell Sci 15 September 2015 128 : 34203434. doi:

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## Cep170 Influences Mt Organization Through Ipam In Infected Cells

As IPAM is sufficient for CEP170 interaction, we next examined the relative location of endogenous IPAM and CEP170 in cultured HeLa and U2OS cells infected with C. trachomatis. CEP170 was present in patches at the inclusion membrane from 24hpi and multiple patches were evident at 66hpi . Consistent with the in vitro interaction and the effects of IPAM90-268 in cultured cells, colocalization of IPAM and CEP170 was observed at the inclusion, although as might be expected during infection both IPAM and CEP170 were additionally present in isolation .

To probe further how CEP170 functions relate to IPAM, we examined the consequences of GFP-IPAM90-268 overexpression in infected cells in the presence and absence of CEP170. When expressed in infected cells, GFP-IPAM90-268 puncta are enriched at the inclusion, with a minor population present at the cell periphery . Moreover, we observed a more prominent MT scaffold associates with a hyper-developed nest, modifying the cell shape in a distinctive manner in cell expressing endogenous levels of CEP170 . This exaggerated phenotype due to the presence of high levels of GFP-IPAM90-268 in infected cells is indicative of an IPAM-induced overstimulation rather than a dominant negative effect. In CEP170 siRNA cells, GFP-IPAM90-268 overexpression partially reverts the defect in the formation of the MT nest , suggesting that GFP-IPAM90-268 is able to compensate for loss of CEP170 via a secondary or redundant pathway.

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