Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2′OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine  Much effort has been devoted in the past decade to developing nanostructured molecular probes for RNA live cell imaging Methods for detection of mRNA in living cells allow real time detection of specific mRNA transcripts in vivo may set the gold standard for in vivo real-time imaging modality assisting in tumor diagnosis and staging. It could be used for applications as early detection of mutated cells in the pre-cancerous stage The ability to detect, localize, quantify and monitor such molecular alterations Fluorescently labelled antisense oligonucleotides can bind to natural mRNA in a sequence-specific manner and enable real time monitoring of mRNA transcripts in the cell Several classes of labelled oligonucleotides (ODNs) have been developed for RNA detection in living cells, including tagged linear oligonucleotide, dual linear fluorescence resonance energy transfer (FRET) oligos, dual-labelled hairpin oligos (e.g. molecular beacons); dual FRET molecular beacons and oligos that use fluorescent reporter proteins Various studies indicate the high potency of linear antisense ODNs in visualizing cytoplasmic mRNAs Batish et al. recently described a protocol that enables imaging and localization of endogenous mRNAs in situ using multiple linear antisense probes. The use of multiple probes enabled overcoming the high background produced by the free unbound probes allowing enhancement of the signal to noise ratio Molecular beacons (MB) are antisense molecules possessing complementary sequences on either end of a ODN, enabling the molecule to assume a hairpin configuration in which a fluorophore and quencher are held in close proximity. Hybridization with target nucleic acid sequence opens the hairpin structure and spatially separates the fluorophore from quencher, allowing a fluorescence signal to be generated upon fluorophore excitation only when the MB and target are bound, therefore lowering unspecific background signal. MBs were shown to allow detection of endogenous cytoplasmic mRNA targets in living cells and show advantageous in imaging RNAs owing to the simplicity and specificity of detection Another simple method to enhance the signal in an un-washable living system is by the use of fluorescence resonance energy transfer ODNs (FRET); the detection of FRET upon hybridization of a pair of linear antisense oligos to adjacent sequences on a target mRNA  enables The Hras oncogene appears to be a primary target gene in the formation of Squamous Cell Carcinoma (SCC) in murine skin model and is susceptible to point mutations in several other types of cancers Hras oncogene mRNA in living cells. This system comprises a 14 bp anchor oligo labled with a donor fluorophore, which is complementary to a constant region in the Hras mRNA. The anchor oligo binds both wt (codon CAA) and mutant (CTA) target sequences equally well. Discrimination between wt and mutant targets is achieved by two 8 bp allele-specific probes, labled with an acceptor fluorophore. The probe oligo bind adjacent to the anchor and span the site of the Hras SNP target sequence  and were HPLC purified. Probe sequences were selected from murine Hras entries in the Genebank database [NM_008284]. Alexa Fluor 488 dye (AF488) was attached to the anchor oligo on its 3′-end and either Texas Red (TR) or Alexa Fluor 594 (AF594) dyes were attached to the 5′-end of the allele specific probes. The anchor oligo was made of 2′OMe RNA with PS internucleotide linkages. The allele-specific probes were made of 2′OMe RNA with three consecutive Locked Nucleic Acid (LNA) residues positions at the SNP site, as recommend by You et al. in vivo models for the study of the development of tumors. During the first stage of chemically induced skin carcinogenesis, referred to as ‘initiation’, key genes in epidermal keratinocytes acquire mutations as a result of exposure to a chemical mutagen applied topically, most commonly DMBA . Activating mutations in Hras1 can be detected in the epidermis as early as 3–4 weeks after treatment with DMBA and are observed in the majority of papillomas that develop after the second stage called ‘promotion’ which is induced by topical treatment with TPA (12-O-tetradecanoylphorbol-13-acetate) nd nucleotide of codon 61 in one of the two Hras alleles, as previously described In vivo, these cells will further be subjected to other deleterious mutations and a fraction of them will develop into invasive, metastatic skin tumors during the carcinogenesis process. The cells were a kind gift from Dr. Staurt H. Yuspa  308 cells are BALB/c mouse keratinocytes cell line derived from an early stage papilloma of a well-known SCC skin model. This model of multi-stage chemical carcinogenesis represents one of the best-established 2+. Mouse Embryonic Fibroblasts (MEFs) were purchased from ATCC (SCRC-1008). Culturing was done in full Dulbecco Minimal Essential Medium . Human ovary adenocarcinoma NAR (NCI/Adr-Res) cells were provided by M. Liskowitz  and are also part of the NCI 60 cell lines http://cancer.sanger.ac.uk/cell_lines/cbrowse/nci2.Maintenance was carried in full Minimal Essential Medium growth media  without Ca0.8 µM of the anchor, probe and targets oligos were dissolved in PBS in a black 96 well plate  and fluorescence intensity was measured at 37°C using a Synergy HT plate reader . Three filters were used for detection of donor, acceptor and FRET signals  : , , .Probe specificity was assessed by detecting the FRET signal following hybridization to two 40 bp synthetic RNA oligonucleotide targets corresponding to the mutant and wt alleles as described in 2PO4H2O (Merck), 2 mM K2PO4H2O (Merck) pH 7.4, 137 mM NaCl (Bio lab), 2.7 mM KCl (Merck) and 0.01% Triton (Santa Cruz) in DEPC treated water . Samples were analysed by fluorescence measurements taken every 0.1°C over a gradual temperature increase from 37°C to 75°C on a Roche Lightcycler® 480 . The ROX (577/603) filter was used to detect signal from the Texas Red (583/607) Acceptor Probe and the FAM (492/518) filter was used to detect signal from the Alexa Fluor 488 (493/517) Donor Anchor. Base fluorescence levels of both oligo probes were calibrated to baseline according to 200 nM of each oligo so that increases in fluorescence above this level would account as energy transfer.200 nM of the anchor, probe and target oligonucleotides were annealed in a total volume of 10 µl in phosphate buffered saline  according to manufacturer instructions. 1 µg of the extracted RNA was incubated in a 96 well plate along with 200 nM of anchor and probe T in PBS (−/−) as described.Hras mRNA folding predictions were obtained using the mfold server .Genomic DNA was isolated from cell lines using a DNA extraction solution . 1 µg of DNA was subjected to polymerase chain reaction (PCR) analysis for Hras amplification using PrimeSTAR™ polymerase  according to manufacturer instruction. Primers sequence was as listed below:Forward: ctatagaggtgagctctgcctaccReverse : ctacattgaaacatcagccaagacTo confirm Hras mutation, restriction digestion was carried using XbaI restriction enzyme (Takara). 1 µl of XbaI enzyme was mixed with 2 µl 10x buffer, 0.1% BSA, 5 µl ultra pure water (BI) and 10 µl of purified DNA. After 1 h in 37°C the total volume of the reaction mix was loaded on 1% agarose gel (Hispanagar), stained with ethidium bromide (Bio-lab) and analyzed by gel electrophoresis. Bands were visualized using Gel doc™ XR molecular imager . Hras replicon size was 700 bp and as a result of Hras mutation in codon 61 the total amplicon is expected to yield 2 restriction fragments 300 and 400 bp in size.PCR products were purified using PCR fragments extraction kit  and DNA concentration was measured using Nanodrop spectrophotometer . 70 ng DNA was added to 10 pmole forward primer and 16 µl DDW. Sequencing was carried out using the ABI 3500xl Genetic analyser .Microporation (20) was conducted using Neon™ transfection system . Each sample contained 150 k cells, spun down for 5 min in 0.4 G. Then cells were placed on ice. Supernatant was removed and a reaction mixture containing 10 ul buffer T, 3 µM of each oligo as well as 7 µl PBS were added in the dark. Cells were microporated and immediately placed on ice. The following parameters were used for the microporation: pulse power - 1400 Volt/pulse width - 20 ms/pulse number - 1. After the microporation four identical samples were united into fresh tube containing full growing medium (without antibiotics), spun down and resuspended in 3 ml PBS containing 0.5 ml FCS.Cells were plated on cell culture dishes with glass bottom  that were pre-coated with collagen  for increased adherence. The coat was obtained by adding 10 µg/ml collagen solution to each plate, 1 h incubation in 37°C and two 5 min washes of the plate with PBS. After plating, cells were kept in 4°C until confocal analysis was done.2 throughout the assay. Acceptor photobleaching was performed only in cells that visibly contained both fluorophores. AF488 and TR excitation were performed using 488nm and 561nm lasers, respectively and fluorescence detection was carried out using BP 505–550 and LP 575 detection filters. Photobleaching intensity (Red burn) was 400IT. Red burn intensity was measured accordantly with green fold change in fluorescence.The FRET donor AF488 label and the acceptor TR label were detected in living cells in a single cell resolution by Zeiss LSM 510-META confocal microscope . The cells were kept in a growing chamber in 37°C, 5% COFRET can be detected by exciting the labelled specimen with light of wavelengths corresponding to the excitation spectrum of the donor and detecting light emitted at the wavelengths corresponding to the emission spectrum of the acceptor. There is a need to correct several potential distortions during FRET measurements. These alterations include any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter as well as the dependence of FRET on the concentration of the donor and acceptor. In this assay, we used three filter sets to measure FRET, thus here we used the following formula in order to correct the above distortions. It should be noted that different equations exists for FRET calculation Capital letters represent the filter used  and small letter represents the fluorophors present in the sample .Feff - FRET effective,Ff - The signal received using the FRET filter set from specimen where both fluorophors present.Df - The signal received using the donor filter set from specimen where both fluorophors present.Fd- The signal received using the FRET filter set from specimen where only donor fluorophor is present.Dd- The signal received using the donor filter set from specimen where only donor fluorophor is present.Af- The signal received using the Acceptor filter set from specimen where both fluorophors are present.Fa- The signal received using the FRET filter set from specimen where only acceptor fluorophor is present.Aa - The signal received using the Acceptor filter set from specimen where only acceptor fluorophor is present.Acceptor photobleaching assay measures the increase in green donor emission while red acceptor emission is decreased by photobleaching (sensitized emission). During the course of the photobleaching both the green and the red signals are measured every few seconds before and after the red photobleaching. In this case, since the extent of red decay influences the increase in the green donor intensity, FRET level is corrected accordantly, and is calculated as the ratio between the level of donor green increase and the acceptor red decay as follows:Whereas,ap –F FRET Acceptor photobleaching, D – donor signal, A – acceptor signal, f- final signal (after acceptor photobleach), i– initial signal (before photobleach).t-test was carried on each result before being included in the sample average. One tailed unpaired t-test was used to test the significance between samples mean with p-value of 0.05. Statistical analysis was carried using the Statistical Product and Service Solutions (SPSS) data editor software .Green signal before and after photobleaching was calculated as the mean of two measurements respectively. Samples were analyzed using LSM image browser software  for green and red intensities and FRET output was calculated as indicated. One sample A variety of probe sequences were tested to achieve optimal SNP discrimination. Several spacing variants between the anchor and the specific probe binding sites were also tested to achieve optimal FRET. Previous work have shown that the optimal distance varied with FRET pairs and that FRET pairs with closer emission/excitation wavelengths did better farther apart and those with spectra far apart did better when positional spatially closer. For FAM-ROX (the closest FRET pair to AF488-TexRed used in this study), 5–8 bases spacing was found optimal We synthesized and tested several variants of the chemically modified FRET oligos to optimize specificity. The chemical modifications employed are shown in m). We therefore also tested variants having PS internucleotide linkages only between the terminal residues . 2′OMe residues are susceptible to exonuclease attack but are fairly resistant to mammalian endonucleases The Anchor (Oligo 1) and Allele-specific Probes (Oligos 2–5) were made using PS bonds at all internucleotide linkages to protect the oligonucletoides from nuclease degradation. However, the PS modification makes the oligonucleotides more hydrophobic and increases non-specific interaction with proteins, which can cause toxicity and may influence intracellular distribution (20). Further, the PS modification reduces binding affinity (lowers TThe Anchor (Oligos 1 and 6) had a 3′-AF488 dye which served as the FRET donor. The Allele-specific Probes had either a 5′-TR dye (Oligos 2 and 3) or a 5′-AF594 dye (Oligos 4 and 5) which served as the FRET acceptor. When hybridized to the Hras target mRNA, the 3′-end of the Anchor oligo (FRET donor) will be positioned near the 5′-end of the Allele-specific Probe (FRET acceptor) in an ideal configuration for FRET to occur. The same oligonucleotides free in solution or bound individually to different targets will not be close enough to one another for FRET to occur. When annealed to the same target, excitation of the donor at around 488 nm will lead to fluorescence emission at around 520 nm, transferring energy to the acceptor dye, which will in turn emit a fluorescence signal around 615 nm . The redin vitro, two 40 bp RNA oligonucleotides were synthesized and served as synthetic targets of the probes. Target U oligo was the perfect-matched target of probe T, and represents a short fragment of mutant Hras allele, while target A is a single mismatched target to the T probe which is a part of the wt Hras allele. Similarly, probe A is a perfect match to target A oligo and a single mismatch to the target U oligo.For the purpose of testing the specificity of oligos When comparing mismatch discrimination between the different versions of FRET pairs , Texas Rmfold (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/), they may be inaccurate due to limitations of the biophysical models used, and the limited understanding of protein-RNA interaction It is well known that a functional mRNA molecule in a living cell always has RNA-binding proteins on it, forming a ribonucleoprotein (RNP). Furthermore, an mRNA molecule often has double stranded portions and forms secondary (folded) structures. Although predictions of mRNA secondary structure can be made using software such as Hras folding predictions include several optional secondary structures, ranked in an ascending order of free energy ranging from ΔG = −315.25 up to −306.20 Kcal/mole.In 8 out of 12 structures, including the energetically favoured structure (4D), the probe binding site, including codon 61 (mutation site), is located on the edge of a loop, in positions 384–410. It is unknown whether the probe can bind to the free bases in the loop structure while it is in its folded state or whether the transcript secondary structure opens in the presence of the complimentary high affinity FRET pair. The oligos with modified bases provide higher affinity towards the target sequence than intramolecular complementary unmodified sequences, therefore, probably competition occurred. We next investigated whether the mRNA was accessible for binding in spite of its secondary structure .m). When both oligos are placed with a single mismatch target sequence, the affinity towards the target is not sufficient to obtain both ODN bound to the target, abolishing FRET .Next, we wanted to validate that the signal observed through the emission of the acceptor fluorophore represents a true FRET signal rather than leakage from the emission spectra of the donor or excitation of the acceptor. Moreover, calculation of FRET through measuring the light emitted from the acceptor after excitation of the donor  can be mWhen both anchor and probe are present with no target, the donor green signal is detected while no signal is generated by the red acceptor, thus no FRET has occurred . In the . The Hras point mutation in codon 61 of 308 cells causes appearance of XbaI restriction site . In MEF cells however, FRET response was not detected, due to the single mismatch between T probe and the wt target mRNA.Ras is a frequently mutated oncogene in human cancer ion site  thus theOnce entering the dynamic cellular environment, there are numerous challenges facing the FRET oligos before they can bind their target in a specific and functional manner. The ODNs need to be stable and non-degradable. They are also expected to independently locate and bind their specific target mRNA, which might be folded, wrapped with binding proteins, located in p-bodies or is being translated at a given time point. The FRET pair oligos were transfected into three cell lines representing various allelic combinations.NAR – human, lacking the murine Hras mutation - these cells served as positive control to the target since they were transfected with the synthetic 40 bp target. It should be noted that due to the fast kinetics of the probes (5∼min) it was possible to visualize the binding (FRET) event before the un-modified target ODN was degraded.MEF – murine cells, containing only the wt (A) alleles (match to A/A and mismatch to A/T FRET pair).Finally, the 308 murine cells, containing one mutant Hras allele and one wt allele .Single cells were subjected to acceptor photobleaching (AP) while placed in an incubation chamber during confocal microscopy. Representative images are shown in Average values of FRET signals were calculated as the ratio between the donor signal increase and the acceptor decrease and are summarized in Accordantly, a positive FRET response was observed both in MEF and 308 cells when transfected with A probe  cannot be used In the present study, we have challenged the molecular probes by constructing allele-specific probes aimed not only to identify a certain type of transcript but to discriminate between two alleles differing in only one nucleotide. Therefore, calibrated and selective design of ODN was a crucial step, determining the final outcome of the following studies.m between match and mismatch m using these modified bases against an RNA target, we empirically tested the influence of the different designs  on probes functionality. Each pair underwent multiple rounds of selection hybridization to achieve optimal discrimination under physiological conditions. Then the different designs of the FRET pairs were compared as shown in m between match and mismatch. In this case, temperature is fixed at 37°C, so adjustments can only be made on the oligo sequence/length and choice of chemical modifications employed.Both FRET pair ODNs were modified with 2′OMe residues, phosphorothioate linkages and LNA residues at the SNP site to improve allele discrimination. Use of DNA probes would form a DNA:RNA heteroduplex between the ODNs and the Hras mRNA target, which could lead to RNase H mediated degradation of the mRNA, an undesired outcome m) and improved specificity toward the target in vitro and in living cells under physiological conditions, and most interestingly, can discriminate between two alleles differing in only one nucleotide.For specific detection of the desired transcript, the targeted segment has to be unique, such that it does not have significant sequence homology with other RNA sequences and also it should not have a structural constraint that may prevent hybridization of the probe. It is well known that RNA molecules within living cells are commonly associated with RNA-binding proteins, e.g., ribonucleoproteins (RNPs) and form complex secondary structures. It is preferable to avoid targeting RNA sequences that are double stranded, or occupied by RNA binding proteins; Since the models offered by the different prediction software are often inaccurate, the ability to reliably predict the mRNA folding state and binding proteins is limited As was observed before, ODN probes rapidly enter the cell nucleus after entering the cytoplasm, and specific hybridization patterns can be observed within 1–2 minutes irrespective of the target RNA An optional way to prevent accumulation of antisense ODNs in the nucleus is by attaching macromolecules (e.g. NeutrAvidin) to the ODN so that they are too large to pass through the nuclear pores in vitro FRET measurement to actually visualize the energy transfer between the FRET pair, rather than calculating it out of a fluorescent output which needs to be processed and “repaired” using various formulas different in their level of accuracy. In vitro have shown high specificity of our modified FRET pair towards both exogenous and endogenous targets at 37°C.The qPCR provided a platform for The differential FRET signal seen in living cells indicated that the ODNs survived the cells environment without being trapped in the endosomal pathway. The endosomal escape in our case is attributed to the method of transfection used, i.e. microporation, which was utilized due to previous testimonies in which it was said to contribute to this escape Harvey-ras gene is a highly potent oncogene, which was found to be one of the main prognostic factors along with c-fos, c-myc and p53 of breast carcinoma In conclusion, this work presents a non-invasive method that allows the detection of point mutation in living cells. This proof-of-concept study can be expanded for detection of genetic aberrations in clinical diagnostics as well as a research tool.Figure S1Optimization between the Anchor (donor) and Probe (acceptor) oligonucleotides.(a): alignment of the anchor, probe, and target sequences is shown. (b): FRET signal intensity comparison with 2, 3, 4, or 5 bases separation between the Anchor and Probe oligonucleotides. The Anchor oligonucleotide is the same as Oligo 1 in (PDF)Click here for additional data file.Figure S2Fluorescence resonance energy transfer demonstrated by fluorescence emission spectra. 200 nM Anchor  and T-allele Probe  were incubated and measured at 37°C without target RNA (Blue), with mismatch target Oligo 10 (Green), and with match target Oligo 9 (Red).(PDF)Click here for additional data file.


It is estimated that any progress made towards improving maternal and child health in Mauritania has likely stalled. A lack of reliable and up-to-date data regarding maternal and child health indicators makes it difficult to identify current gaps and adapt international programmes to meet local needs.Using secondary data collected as part of a baseline assessment for a maternal and child health programme being implemented in two health departments, we compared maternal and child health indicators across two different samples of pregnant women and children under-five in M'bagne and Guérou. Descriptive analyses were conducted using a Pearson's Chi-Squared test, assuming a binomial distribution and a confidence level of alpha=0.05.Our results indicated that there were marked regional differences in maternal and child health indicators between these two rural sites, with M'bagne generally performing better across a range of indicators including: immunisation rates, child registration, vitamin A supplementation, deworming, delivery in the presence of a skilled birth attendant, and post-natal care coverage. In Guérou we observed lower rates of fever, diarrhoea, and fast and difficult breathing among children under-five.Though socio-cultural differences may play a part in explaining some of these observed differences, these alone do not account for the observed differences in maternal and child health indicators. Context-specific activities to overcome barriers to care must be designed to address such rural regional differences if we are to see an improvement across maternal and child health indicators and accelerate progress towards MDGs 4 & 5 in Mauritania. Despite early indications that Mauritania had made progress in reducing child and infant mortality rates over the last 30 years, projections estimate that improvements have likely stalled since the mid-1990s . Infant Programme Élargi de Vaccination (Expanded Programme on Immunisation) and one in five children (20%) between the ages of 6-59 months had never received a Vitamin A supplement . OuProgramme Élargi de Vaccination, may have in part contributed to the important progress of vaccination coverage in Mauritania. Significant differences in completed vaccination schedules for children between the ages of 12-23 months indicate greater vaccination coverage in M'bagne. Similarly, our results indicate significantly greater prevalence of children aged 6-59 months having received Vitamin A in M'bagne in the 6 months preceding the survey. Better immunisation rates and greater prevalence of Vitamin A supplements are also consistent with the greater observed prevalence of children aged 12-59 months having received a deworming tablet in M'bagne. Differences between the two sites suggest that there is a need to focus greater resources and increase access to supplementation coverage and vaccination programmes in Guérou. We found no significant differences in rates of malnutrition between Guérou and M'bagne.The implementation of successful national immunization programmes, such as Mauritania's Forfait Obstétrical (Obstetric Risk Insurance) programme reported an increase in the rates of hospital and post-natal care for pregnant women living in the capital . It is therefore important that households in both regions have access to PNC. This is especially true in Guérou where our results show a worryingly low uptake of PNC services after delivery.Though socio-cultural differences exist between Guérou and M'bagne, these alone do not account for the observed regional differences in maternal and child health indicators. Other factors such as geographical inaccessibility, limitations in human resources for health, religious ideologies, and the presence and activities of existing NGOs in the area must also be considered. Insufficient progress in improvements in maternal and child health suggests that there is an important need for the continued support of effective actions and for implementing new programmes aimed at improving child health in rural geographic areas in Mauritania. Context-specific activities to overcome barriers to care must be designed to address such rural regional differences if we are to see an improvement across maternal and child health indicators and accelerate progress towards MDGs 4 & 5 in Mauritania.


Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation . Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment. ZNF703, DDHD2 and FGFR1 genes at 8p12 and CCND1 at 11q13 that are amplified in breast tumours MDM2, CDK4 and HMGA2 at 12q14, which have recently been show to occur on separate amplicons in liposarcomas Leiomyosarcomas belong to a group of sarcomas that are characterised by genetic instability as evidenced by pervasive, seemingly random karyotypic abnormalities. Although the complexity of genomic abnormalities varies significantly among clinically similar cancer cases, certain aberrations have been shown to be recurrent and preserved as the tumours evolve. Accumulating evidence has thus led to the current view that genetic instability is an enabling characteristic that leads to the cancer phenotype and that the resulting recurrent copy number aberrations are an important clue to pathogenetic mechanisms Mapping of genome-wide copy number aberrations can be done by microarray-based comparative genomic hybridisation (array CGH or aCGH). It involves co-hybridising fragments of test and reference genomic DNA that has been differentially labelled with fluorescent dyes to a set of mapped and annotated DNA sequences (probes) on a microarray. By measuring the ratio of fluorescence at each probe, it is possible to detect copy number differences between test (tumour) and reference  DNA at that genomic location. Target probes may be in the form of cDNA sequences, Bacterial Artificial Chromosomes (BACs) or oligonucleotides and depending on the size, type and number of probes on the array, copy number aberrations (CNAs) can be detected at the level of single genes and even specific exons In order to generate reproducible aCGH results, pure high molecular weight DNA  has traditionally been used. Availability of fresh tissue is however limited, particularly in rare tumours like leiomyosarcomas and most tumour tissue available for research is formalin-fixed and paraffin-embedded (FFPE) in order to preserve the tissue structure for histopathology. DNA isolated from such tissues is typically of low quality (low yield and low molecular weight) due to the degradative and fragmenting effects of formalin Another major limitation to the use of FFPE DNA for high-resolution oligonucleotide aCGH is technical difficulty in labelling fragmented DNA. Traditional enzymatic methods for labelling DNA (Nick translation or Random priming) involve a fragmentation step with DNase or restriction digestion respectively, which in the case of FFPE DNA further fragments the DNA. An alternative to enzymatic labelling is the Universal Linkage System (ULS), which directly labels the DNA by a chemical reaction that incorporates platinum-conjugated fluorophores into DNA without the need for fragmentation, making it suitable for low molecular weight DNA such as that from FFPE tissue Recent efforts have been made to optimise the utility of archived FFPE tissues for array CGH after labelling by enzymatic National Research Ethics Committee approval was obtained for the collection and use of fresh and archival tissue samples . Written informed consent was obtained before the collection of fresh tissue samples and all data from archival samples was analysed anonymously. All tissue was collected and stored according to the principles of the Declaration of Helsinki and the use of tissue was in compliance with the Human Tissue Act 2004.Formalin-fixed, paraffin-embedded (FFPE) samples of tumour collected between 1997 and 2011 were obtained for 22 leiomyosarcoma cases  from theStandard H&E-stained slides representative of tumour blocks were marked to identify areas of >70% viable tumour, which were then macro-dissected by scraping off 20 µm sections on glass slides using a scalpel blade. For three of these cases, prior to fixing in buffered formalin and within 30 minutes of surgical excision, tumour and normal tissue (if available) were macroscopically sampled by an experienced sarcoma pathologist and then snap-frozen. Array CGH experiments were done on paired fresh frozen (FF) and FFPE tumour samples. When normal tissue was not available for sampling, pooled normal genomic DNA (Promega®) was used as reference DNA for array CGH experiments.3 of tissue  was heat de-paraffinised at 90°C, followed by overnight treatment with 1M-sodium thiocyanate. This was followed by 48-hour proteinase K treatment and then RNase A treatment. DNA was then purified using the Qiagen® DNeasy kit, substituting the wash buffer AW2 with 80% ethanol, and eluting in nuclease–free water.Genomic DNA was extracted from approximately 25 µg of fresh frozen tissue  using the Qiagen® DNeasy Blood and Tissue Kit, according to manufacturer’s instructions. A modification of the same protocol recommended by the ULS labelling system manufacturer (Agilent®) was used for DNA extraction from FFPE tissues. Briefly, approximately 4 mm260/280 and A260/230 were used to assess DNA purity, and samples with ratios ∼1.80 and >1.90, respectively were regarded as sufficiently pure and suitable for ULS labelling. All DNA samples were visualized on 1.0% agarose gel pre-stained with Ethidium Bromide and fragment sizes were assessed against a 1 Kb DNA ladder (Promega®).Extracted DNA was quantified by spectrophotometry using a Nanodrop ND-1000 (Nanodrop®). Ratios of absorbance, AFor fresh frozen samples, an enzymatic (random priming) labelling system (Agilent®) was used according to manufacturers’ instructions. Briefly, 0.5–1 µg of genomic DNA was digested with Alu1 and Rsa1 restriction enzymes (Promega®) at 37°C for two hours and then random primers were added. Tumour and normal DNA samples were then labelled with Cy5-dUTP and Cy3-dUTP respectively, by incubation with Exo-Klenow (large fragment of E. coli DNA polymerase I) at 37°C for a further two hours. Excess nucleotides were removed using Amicon 30 µm centrifugal filters (Millipore®).FFPE tumour and reference DNA was labelled using an optimised version of the protocol for ULS labelling of FFPE DNA (Agilent®). Prior to labelling, heat fragmentation at 95°C was required when average fragment size was greater than 7.0 Kb. 0.5–1 µg of tumour and reference DNA was then chemically labelled by incubating with ULS-Cy5 and Cy3 respectively (about 1 µl dye for 0.8 µg of DNA) in a thirty-minute reaction. Labelling reactions were prepared in thin-walled 0.2 ml PCR tubes and incubated on a thermal cycler with a heated lid (SensoQuest®). Unreacted dye was then removed using KREApure filters (Agilent®).260 (for DNA), A550 (for Cy5) and A649 (for Cy3) was used for determination of DNA and fluorophore concentrations. The degree of labelling (DoL) is the number of fluorophore molecules per 100 nucleotides, expressed as a percentage and was calculated from the post-labelling DNA yield and fluorophore concentration. According to manufacturer’s recommendations, DoL values between 0.75% and 2.5% were regarded as optimal for Cy5 while values between 1.75% and 3.5% were optimal for Cy3-labelled DNA.Spectrophotometry (Nanodrop ND-2000®) measuring ACy5-Labeled tumour DNA was combined with an equivalent amount of Cy3-labeled reference DNA. In five cases, reference DNA was intentionally sex-mismatched. Repetitive sequences were blocked with human Cot-1 DNA (Invitrogen®) and samples were hybridised onto SurePrint G3 Human CGH Microarrays, 4×180K (Agilent®) according to manufacturer’s instructions. Following hybridisation for 24 hours (FF samples) or 40 hours (FFPE samples), microarray slides were washed according to manufacturer’s instructions and scanned immediately on a DNA Microarray Scanner (Agilent®).2 ratio). It also computes a set of Quality Control (QC) metrics including the average green and red signal intensity at all the probes and using non-hybridising control probes, determines the background signal (noise) and signal-to-noise ratio. Average signal intensity >150 with signal-to-noise ratio >20 were regarded as satisfactory.Scanned images were analysed using Feature Extraction software v10.7.3 (Agilent®), which normalizes the fluorescent intensity of both dyes at each probe and calculates their ratio, expressed on a logarithmic scale .Among other QC metrics, it calculates the Derivative Log Ratio Spread (DLRS), a measure of the variation in the difference between log2 ratio threshold. Log2 ratio thresholds 0.25 and −0.3 were used to identify single copy number gains and losses respectively, and thresholds for gains and losses of two or more copies were set at 1.14 and −1.1 respectively. Significance threshold p-value for aberration calls was set at a minimum of 5.0×10−6, requiring at least three contiguous probes.Feature Extracted Data was then analysed using Nexus Copy Number Software v6.1 (Biodiscovery®). For individual arrays, copy number aberrations were called using the FASST2 Segmentation Algorithm, which uses a Hidden Markov Model (HMM)-based approach that does not aim to estimate the copy number state at each probe but uses many states to cover more possibilities, such as mosaic events. These state values are then used to make calls based on a logFor analysis of common aberrations detected among multiple samples, the GISTIC (Genomic Identification of Significant Targets in Cancer) algorithm was used Two-colour interphase FISH was used to confirm array CGH results in one of the LMS cases (LMS 9). FISH experiments were carried out on short-term cultured tumour cells using the LSI ATM probe  and an α-satellite probe to the centromere of chromosome 11 , as previously described 260/230<1.50 and the DNA was re-purified by sodium acetate-isopropanol precipitation before labelling.The DNA yield in most FFPE LMS tumours was good, exceeding 10 µg in most cases. In two cases however, the yield was low and vacuum centrifugation was used to bring DNA concentration to optimal values. Another FFPE DNA sample had AWhen visualized on agarose gels against a 1 Kb ladder, DNA from the FF samples and commercial pooled genomic DNA showed relatively distinct bands of high molecular weight DNA, while that from FFPE tumours showed a range of fragment sizes that varied from <1.0 Kb on average to as high as 8.0 Kb. The degree of fragmentation appeared to be worse in FFPE samples that were older when compared with the more recent tumours . Tumour Initial attempts at DNA labelling by the ULS method produced a variable degree of labelling. We noted that when labelling reactions were carried out on heat blocks or water baths, the degree of labelling was consistently low and in cases where post-labelling DNA quantitation showed that there was a higher amount of DNA in the reaction than initially estimated, the degree of labelling was either variable or arrays failed due to signal intensities that were below recommended thresholds. Since the ULS system does not amplify the DNA, this suggested that the ratio of DNA to ULS Cy-dye in the reaction was high, resulting in an inefficient labelling reaction. We therefore modified the protocol to use excess ULS Cy-dye relative to amount of DNA (about 0.8 µg of DNA to 1 ml of dye) and carried out reactions in thin-walled tubes on a thermal cycler to ensure uniform optimal temperature for the reactions. All FFPE samples that were labelled in this way had consistently good degree of labelling. Quality assessment of arrays using this DNA showed good red and green signal intensity that was similar to that seen with high quality DNA that was labelled using the enzymatic method. Results of experiments carried out before and after optimisation of the labelling protocol are summarised in Signal intensities of both red and green dyes were much higher than the manufacturer-recommended threshold for all DNA samples regardless of source tissue or labelling method. The Derivative Log Ratio Spread (DLRS) is widely regarded as a robust parameter for measurement of the quality of microarray experiments. It represents the ‘noisiness’ of array data and a low DLRS means that the data has small probe-to-probe variability and better ability to array to identify small aberrations and vice versa. Manufacturer-recommended thresholds for DLRS when using FFPE DNA is <0.4, a value above which array data may be compromised and the array should be failed. DLRS values for all the arrays fell in the recommended pass range and all but five of the 22 FFPE DNA arrays had DLRS <0.3, the threshold recommended for DNA from fresh frozen tissue or cells .2 ratios were 0.58 (p<0.0001) and 0.54 (p<0.0001) for LMS 9 and LMS 10 respectively , paired samples of macroscopically sampled fresh tumour and macro-dissected FFPE tumour were obtained. High molecular weight DNA from the fresh frozen (FF) tissue was labelled by the enzymatic method, while fragmented FFPE DNA from the same tumours was labelled using the one-step ULS method. There was good correlation between paired FF and FFPE samples in two out of the three cases. The Pearson correlation coefficients (r) of overall probe logectively .2 ratios remained similar with amplitude close to the threshold set in the algorithm for CNA detection. An example is shown in Genomic profiles of detected CNAs in both sample types were also were also very similar in both LMS cases with most chromosomes showing near identical loss and gain patterns  and 3. O2 ratio correlation (r = −0.02). Although a few small aberrations were seen in both sample types, most of the CNAs detected in the macro-dissected FFPE tissue were not detected in FF tissue (The third leiomyosarcoma case (LMS 11) compared in this way however, showed poor overall probe logF tissue . Histoloin situ Hybridisation (FISH) in two LMS cases, one on FFPE tissue (not shown) and the other in cultured cells  include whole or near-whole arm deletion in 10p, 10q, 13q, 16q, and deletion of the telomeric end of 11q . Deletioed cells .2 ratio values) of aberrations. It also determines the statistical probability that the common aberrations occur by chance alone and using False Detection Rate correction, determines a q-value. The program also identifies within these regions, ‘peak’ regions that have the highest statistical likelihood of containing affected genes . Using G-score and q-value thresholds of 1.0 and 0.05 respectively, we identified five significant regions of copy number gain covering 331 genes (4 genes in peak region) and seven significant regions of copy number loss covering 452 genes (13 in peak region), shown in Statistical significance of common aberrations was determined using the GISTIC algorithm. A G-score is assigned to each aberrant genomic region based on the frequency and magnitude  that caused underestimation of DNA concentration gave poor labelling results. Using an excess of dye relative to DNA in labelling reactions gave consistent good degree of labelling and successful arrays. This is in keeping with results published in a recent study that showed that estimation of DNA concentration is critical to sample assessment for labelling 2 ratios near the expected values, even in cases where there were few other genomic aberrations. A number of recent studies using FFPE tissues of similar age have reported variable DLRS values Regardless of sample age, all aCGH experiments (FF and FFPE) in this study showed good DLRS values when labelled using the modified protocols. In addition, FFPE samples from female patients that were hybridised against sex-mismatched DNA showed the X-chromosome gain or loss with log2 ratio correlation with Pearson’s coefficients similar to those reported from a similar study 2 ratios was close to the set thresholds for low-level aberration detection, thus explaining why aberrations were differentially called in the two sample types. The calling algorithm thresholds set for analysis of array CGH data in this study were chosen based on previous studies in literature and regarded as valid as they enabled the detection of common aberrations among the LMS cases that concur with previous studies (see below).Two out of three paired FF and FFPE samples from identical tumours that were compared showed good overall probe log2 ratios from both sample types and the results showed that although a few common CNAs were seen, the majority of CNAs detected in the FFPE sample were not detected in the FF one. A minority of the genomic regions in LMS 9 and 10 also showed significant difference in the probe moving average pattern in addition to CNA detected. This prompted a revisit of the histology of all three tumours, which showed that LMS 11 was very heterogenous and contained large areas of haematoma and normal cells, while the former two were composed relatively homogenously of tumour cells.The third case (LMS 11) showed poor statistical correlation of the overall probe logIn any whole genome nucleic acid isolation, the presence of germ-line DNA from normal cells ‘contaminating’ a tumour sample can potentially mask genomic aberrations. Our ability to macro-dissect tumour cells from the FFPE tissues apparently helped to reduce the masking of genomic copy number aberrations. Heterogeneity of tumour cell populations between the two areas of the whole tumour that were sampled independently and represented by the FF and FFPE samples could potentially account for some of the low level differential aberrations detected in these paired samples in the cases that looked more homogenous on histological examination.We carried out common aberration analysis among the 22 FFPE LMS cases in this study. Frequent common aberrations detected were in concordance with those reported in previous studies. Deletions on 10q, 13q and 16q have been reported as frequent among LMS in numerous studies Over a quarter of the cases presented in this study showed CNAs on genomic regions that involve loci for at least three of the five genes mentioned above, and may well represent a subset of leiomyosarcoma, although the small number of cases does not allow any correlation with clinical data to have statistical significance. The potential for expanding such a retrospective study to improve its statistical power cannot however, be overemphasized.At present, the cost per sample of labelling DNA by the ULS method is less than that of the enzymatic method. In addition, DNA labelling and clean-up is complete within one hour, compared with the enzymatic methods that require at least five times that duration. Most importantly however, in a 24-month period that has seen only three operable leiomyosarcoma cases treated in our centre with the possibility of obtaining fresh tissue, we have been able to access the FFPE archives and analyse more than twenty cases for which progression and survival data is available.In summary, we have optimised a reliable method that is cheap, fast and gives us access to long-term archival samples prepared using even non-standard protocols. We have been able to generate results from this archival tissue that are in close concordance with those from multiple previous studies that utilised fresh tumour tissue. We therefore now have the option to select specific LMS and other tumour subtypes including those that are very rare for high-resolution genomic copy number mapping.


However, Foxp3 can also be turned on in conventional T cells as a consequence of antigen exposure in the periphery, under both non-inflammatory and inflammatory conditions. These so-called peripheral Tregs (pTregs) participate in the control of immunity at sites of inflammation, especially at the mucosal surfaces. Although numerous studies have assessed in vitro generated Tregs (termed induced or iTregs), these cells most often do not recapitulate the functional or phenotypic characteristics of in vivo generated pTregs. Thus, there are still many unanswered questions regarding the T cell receptor (TCR) repertoire and function of pTregs as well as conditions under which they are generated in vivo, and the degree to which these characteristics identify specialized features of pTregs versus features that are shared with tTregs. In this review, we summarize the current state of our understanding of pTregs and their relationship to the tTreg subset. We describe the recent discovery of unique cell surface markers and transcription factors (including Neuropilin-1 and Helios) that can be used to distinguish tTreg and pTreg subsets in vivo. Additionally, we discuss how the improved ability to distinguish these subsets provided new insights into the biology of tTregs versus pTregs and suggested differences in their function and TCR repertoire, consistent with a unique role of pTregs in certain inflammatory settings. Finally, these recent advances will be used to speculate on the role of individual Treg subsets in both tolerance and autoimmunity.Thymically derived Foxp3 The importance of Tregs in maintaining peripheral tolerance to self-tissues is evidenced in both mice and humans by the fatal autoimmune disease that results from a loss of function mutation in the Foxp3 gene, the master transcription factor expressed selectively in Tregs –3. Tregsoth mice , 7 and hoth mice . However antigen  as well  antigen  have bee propria . This al propria –14. Howe propria –17. Within vitro, there has been a considerable amount of interest in determining if a similar conversion of conventional T cells into Foxp3+ Tregs takes place in vivo. While Foxp3 is a critical orchestrator of Treg biology, it is not enough by itself to drive their full transcriptional program -specific T cell receptor (TCR) transgenic (Tg) named 1B3 mouse in which pTregs were spontaneously generated in the periphery when these mice were crossed onto the RAG-2 knockout background. Through a series of experiments utilizing pTregs from this strain, we found that Nrp-1 was expressed on tTregs only and that Nrp-1 expression could be used to distinguish tTregs from peripherally generated pTregs in other settings . Consistf pTregs , 31. Thee thymus . Weiss eIn vitro induction of Foxp3 by TGF-β is not sufficient to induce TSDR demethylation, whereas in vivo generated pTregs exhibit variable patterns. Some of the initial studies showed that in vivo generated pTregs have demethylated TSDR  upon antigen encounter in the tissue. In support of this hypothesis, a recent study showed that tissue-resident macrophages play a key role in generation of pTregs in lungs. These macrophages coexpressed TGF-β and retinal dehydrogenases (RALDH1 and RALDH 2) under steady-state conditions and sampling of airborne antigens by these macrophages and presentation to antigen-specific CD4 T cells resulted in the generation of tissue-resident Foxp3+ Tregs , suggesting their thymic origin. However, pTregs have been shown to upregulate Nrp-1 expression, especially in tissues during inflammation. Indeed, pTregs upregulate Nrp-1 during EAE or lung inflammation, and we also observed upregulation of Nrp-1 expression on pTregs during autoimmune response in pancreas (unpublished observations). Thus, Nrp-1-expressing Tregs present in inflamed tissues may not solely be thymically derived but could be generated by conversion. We believe that presence of pTregs could play a critical role in controlling local inflammatory responses in tissues and may have clinical significance for certain human diseases.During inflammation Treg numbers increase in the relevant tissue and could constitute up to 50% of all CD4e tissue . The tere tissue . Tissulae tissue . Unpubli3+ Tregs . In ongoIt has been postulated that tTreg development in the thymus is associated with high affinity TCR/MHC-peptide interactions while pTreg differentiation in the periphery is induced under sub-immunogenic conditions , 42, 43.in vivo inhibits differentiation of antigen-specific pTregs  by expressing transcription factors and chemokine receptors typically associated with this subset. Although consistent expression of Foxp3 is required to reinforce the regulatory program, Treg cells can also undergo stimulus-specific differentiation that is regulated by transcription factors typically associated with the differentiation of conventional CD4+ T cells. This results in effector Tregs with unique migratory and functional properties expressing transcription factors involved in regulation of the corresponding type of effector immune responses. These “effector Tregs” have unique functional properties and are better equipped to control ongoing immune responses , have provided some useful insights  sequencing. To limit the overall diversity of the repertoire for this study, we used MBP.TCR.Tg.Foxp3-GFP mice and isolated Nrp-1hi tTregs, Nrp-1lo pTregs, and Tconv (CD4+Foxp3−) cells, then amplified, cloned, and sequenced a region of the α chain encompassing the CDR3 for Vα2+ TCR. Although it was a limited analysis and may not represent the whole repertoire, it still provided useful information. Out of nearly 290 clones per subset, 175 pTregs, 212 tTregs, and 192 Tconv cells had productive V–J rearrangements. CDR3 amino acid sequence analysis of Vα2 revealed that there was limited overlap between the tTreg and pTreg CDR3 sequences . Another significant difference between human and mouse T cells is that human Tconv cells can express Foxp3 upon transient activation more readily than mouse cells (+ activated T cells do not exhibit any of the canonical markers of human Tregs.We have learned a great deal about the functions of Foxp3c origin . Howeverd organs , 110. Wese cells –113. Whese cells , 114 or se cells –117 assose cells , it make+ Tregs expressing Helios in the peripheral blood. Although the Helios−Foxp3+ Tregs fit the profile of pTregs, whether these cells originate outside thymus remains controversial. Among other evidences, effector cytokine secretion, which has been proposed to be a function of pTregs, has been associated with Helios−Foxp3+ Tregs (− and Helios+ Tregs are of thymic origin by showing that both Helios− and Helios+ Tregs exhibit a demethylated TSDR and express canonical Treg markers such as CD39 and CTLA-4 in human peripheral blood (− Tregs were gated on the CD45RA+ naïve Treg population in that study and may not include the whole Helios−Foxp3+ T cell population. In addition, studies in mice have shown that pTregs are very similar to tTregs in terms of TSDR demethylation and expression of Treg canonical markers, raising the possibility that these parameters may not adequately discriminate the pTreg and tTreg subsets in humans as well. It has also been argued elsewhere that Helios can be expressed in conventional human T cells upon activation (+ Tregs in humans that also expressed IFN-γ. Despite high levels of Foxp3, these IFN-γ+Foxp3+ cells lack Helios expression and show a partially methylated TSDR in the Foxp3 locus, and therefore fit the profile of peripherally generated Tregs. Moreover, since Helios is selectively expressed on IFN-γ− Tregs (Among other markers found on mouse Tregs, Helios expression has correlated very well in humans Tregs. Like mouse, Helios is expressed highly on human Tregs with greater than 70% of Foxp33+ Tregs , 120. Coal blood . Howevertivation . In thisγ− Tregs , ongoingLastly, understanding Treg subsets in humans is also important because Treg dysfunctions have been reported in several human autoimmune diseases , 122–125Ever since the discovery of CD25 and Foxp3 as markers of regulatory T cells, there has been some controversy in the field regarding the existence and development of pTregs. New technologies in gene profiling, cell sorting, and mouse engineering have made it clear that pTregs develop under normal homeostasis as well as under inflammatory conditions. Identification of genes that are differentially expressed between Treg subsets and mouse models of pTreg generation have helped in differentiating characteristics of pTregs from tTregs. Functionally, the role of pTregs in mucosal tolerance is already pretty well established, and it is now becoming increasingly evident that these Tregs have specialized functions in response to non self-antigens during conditions such as asthma and fetal tolerance. However, a number of key questions still remain. What are molecular determinants that contribute to the induction of Foxp3 in the periphery? Is Foxp3 induction in the periphery restricted to a subset of Treg precursors? How does TCR affinity or strength of signal influence Treg generation in the thymus versus periphery? What are the different conditions under which pTregs play an indispensable role? Finally, how can we utilize pTregs to improve Treg therapy in human conditions? The definition of new markers to complement Nrp-1 and Helios and new mouse models and humanized mouse models of pTreg generation will undoubtedly play a part in answering some of these questions in the very near future. A better understanding of the biology of pTregs will in turn provide a clearer view of the respective role of pTregs versus tTregs in a number of human pathologies and will be important in devising optimal therapeutic strategies as Tregs are increasingly being considered as either tools or targets of immunotherapy in many diseases.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Allergic asthma is associated with chronic airway inflammation and progressive airway remodelling. However, the dynamics of the development of these features and their spontaneous and pharmacological reversibility are still poorly understood. We have therefore investigated the dynamics of airway remodelling and repair in an experimental asthma model and studied how pharmacological intervention affects these processes.Using BALB/c mice, the kinetics of chronic asthma progression and resolution were characterised in absence and presence of inhaled corticosteroid (ICS) treatment. Airway inflammation and remodelling was assessed by the analysis of bronchoalveolar and peribronichal inflammatory cell infiltrate, goblet cell hyperplasia, collagen deposition and smooth muscle thickening.Chronic allergen exposure resulted in early (goblet cell hyperplasia) and late remodelling (collagen deposition and smooth muscle thickening). After four weeks of allergen cessation eosinophilic inflammation, goblet cell hyperplasia and collagen deposition were resolved, full resolution of lymphocyte inflammation and smooth muscle thickening was only observed after eight weeks. ICS therapy when started before the full establishment of chronic asthma reduced the development of lung inflammation, decreased goblet cell hyperplasia and collagen deposition, but did not affect smooth muscle thickening. These effects of ICS on airway remodelling were maintained for a further four weeks even when therapy was discontinued.Utilising a chronic model of experimental asthma we have shown that repeated allergen exposure induces reversible airway remodelling and inflammation in mice. Therapeutic intervention with ICS was partially effective in inhibiting the transition from acute to chronic asthma by reducing airway inflammation and remodelling but was ineffective in preventing smooth muscle hypertrophy. Human bronchial asthma is a chronic inflammatory disease of the airways that is characterized by chronic airway inflammation, airway hyperresponsiveness (AHR), and airway remodelling The majority of animal studies are based on models of acute allergic airway inflammation We here have characterised the inflammatory and remodelling events that contribute to the transition from acute to chronic experimental asthma. Furthermore, we have studied the impact of ICS treatment during this transition phase, to specifically identify steroid-sensitive and -resistant pathways. The reversibility of remodelling has been examined following a period of ICS therapy and in the optimal situation of allergen avoidance.Female BALB/c mice aged 6–8 weeks were purchased from Harlan Winkelmann  and were maintained under pathogen-free conditions in isolated ventilated cages with 12 hour light/dark cycles. Water and ovalbumin (OVA)-free diet were supplied ad libitum. All mouse experiments met German and international guidelines and were approved by the Regierungspraesidium Giessen, and all measures were taken to keep animal suffering to a minimum.3  diluted in 200 µl phosphate-buffered saline (PBS). Mice were challenged with OVA (grade V) aerosol (1% wt/vol in PBS) twice a week on 2 consecutive days over a period of up to 18 weeks, control groups received PBS. One group of mice was additionally treated with 50 µl of 200 µg/ml budesonide  intranasally (i.n.) 4 hours before each OVA challenge. Group composition is illustrated in Mice were sensitised to OVA by three intraperitoneal (i.p.) injections of 10 µg OVA grade VI  adsorbed to 1.5 mg Al(OH)Mice were sacrificed and BALF was obtained using 1 ml PBS containing protease inhibitor cocktail  as described previously Lungs were fixed ex situ with 6% (wt/vol) paraformaldehyde via the trachea, removed and stored in 6% paraformaldehyde. Independent uniform random (IUR) sections were then prepared in order to obtain a representative collection of lung tissue samples IL-4, IL-5, IL-13, IFN-γ  and TGF-β  were measured by ELISA in cell-free lavage fluids according to the manufacturers' protocol. The detection limit for each cytokine was 10 pg/ml and 20 pg/ml for TGF-β.H&E-stained tissue sections were viewed and random images collected under 20× objective. Degree of inflammation was expressed as a peribronchial airway inflammation score  compT) was determined as the volume of the respective component, determined by counting all points intercepting the airway epithelium and Sirius Red- and α-SMA-positive components, respectively comp  =  L(P) ×T Σ comp/P(2× Σ biI) L(P) is the line length per test point, compP, the number of points hitting the respective component and blI the number of intersections between the test line and the epithelial basal membrane.PAS-stained sections were viewed and random images collected under 20× objective. The fraction of the analysed basal membrane covered by goblet cells was then evaluated. Inflammation and goblet cells were quantified using a PC-based Olympus light microscope BX51 equipped with a Cell-F System . Paraffin sections stained with sirius red, anti-SMA, were used to quantify changes in airway collagen deposition, smooth muscle cell layer thickening, respectively, using the BX51 microscope equipped with a CAST-Grid System  Graphing and statistical analysis of normally distributed data was performed using Graph Pad Prism 5 . Data are expressed as mean ± SEM or as mean and percentiles. Statistical significance was determined using one-way ANOVA with Tukey's Multiple Comparison Test (for multiple group comparison) or the Student's unpaired t-test for two group comparison. Statistical significance was referred to as follows *p≤0.05, **p≤0.01, ***p≤0.001.The kinetics of airway inflammation was investigated using a chronic mouse model of experimental asthma by challenging mice with aerosolised OVA over an eighteen week period . In contHallmarks of airway remodelling are goblet cell hyperplasia, thickening of the smooth muscle cell layer and extracellular matrix deposition. To quantify changes in airway remodelling immunohistochemical analysis in combination with stereological quantification was performed. Comparison of PAS-stained lung sections from chronically OVA treated mice revealed that ∼45% of all cells lining the bronchial airways throughout the whole observation period were goblet cells. Advanced structural changes, such as collagen deposition and smooth muscle thickening, in the airways were not detected until eight weeks of OVA exposure as determined by collagen and α-SMA staining, respectively. Between 8 and 12 weeks the amount of airway collagen rapidly increased by approximately 3 fold and then remained constant. Thickening of the smooth muscle layer was less pronounced than collagen deposition, but increased approximately two fold between 6 and 12 weeks . TogetheTo mimic the situation of effective allergen avoidance, it was investigated how allergen cessation would affect established airway remodelling and inflammation. Following 12 weeks of OVA challenge mice exhibited robust airway inflammation and fully established remodelling, including thickening of the smooth muscle layers and increased deposition of collagen. Therefore, this time point was chosen as the reference point for the chronic situation and used to investigate the effects of allergen cessation over the following eight week period. After four weeks of resolution, total BALF cell numbers remained constant but the numbers of eosinophils and neutrophils decreased to baseline levels . The num+ T lymphocytes were still present after 4 weeks and only reduced after 8 weeks of allergen cessation , OVA challenge induced a strong bronchoalveolar and tissue inflammation, recruiting eosinophils, neutrophils and lymphocytes to the BALF  resulted in a rapid decrease in inflammatory cells such as eosinophils and neutrophils from the BALF. In contrast, macrophage numbers revealed a different kinetics initially increasing in numbers before returning to baseline levels. This temporary increase in macrophage numbers adds further support to the important role of this cell type in the resolution of inflammation The extensive airway remodelling at twelve weeks of OVA-challenge correlated with high levels of TGF-β in the BALF. TGF-β has important roles in mediating remodelling by inducing the production of extracellular matrix proteins and cell proliferation. It has been shown that TGF-β has a significant role in pulmonary fibrosis ICS are the mainstay of asthma therapy in humans Similar observations were reported in a chronic OVA-induced asthma model when budesonide was given for four weeks following allergen cessation, however in this case no differences in collagen deposition and smooth muscle mass were observed et al. the simultaneous removal of both the allergen and ICS resulted in a marked rebound of goblet cell hyperplasia, which was most apparent after prolonged co-application of budesonide and allergen Our study also expands on works of Kumar and Herbert in which the authors showed that dexamethasone treatment resulted in reduced lung inflammation and collagen deposition The disparity between ICS effects in acute and chronic asthma supports the concept that there is a shift in immune responses throughout disease progression in allergic asthma In conclusion, using a chronic model of experimental asthma we have shown that continuous allergen exposure in mice induces reversible airway remodelling. Treatment of established inflammation and remodelling can be partially accomplished with corticosteroids, however, most prominent beneficial effects are observed by allergen avoidance. This model offers new opportunities to further delineate the cellular and molecular signalling pathways that contribute to the transition from the acute to the chronic phenotype, and to elaborate the pathways of normal repair and structural reorganisation.Figure S1Prolonged allergen challenge results in decreased BALF inflammatory cell recruitment. Differential cell counts from chronic allergic inflammation model following allergen challenge for up to 18 weeks, 6–8 animals per group, *p≤0.05, ***p≤0.001.(TIFF)Click here for additional data file.Figure S2Inhaled corticosteroids attenuates features of acute airway inflammation. (A) Acute airway inflammation was generated in mice via intraperitoneal (i.p.) injection of OVA conjugated to Alum and subsequently challenged with PBS or OVA for three days with or without the prior treatment with inhaled corticosteroids (ICS). Mice were analysed after 48 hrs for; (B) total BALF cells, (C) eosinophils, (D) lymphocytes, (E) neutrophils, (F) macrophages and (G) fraction of basal membrane covered by goblet cells. The results are presented as box and whiskers-plots and show mean and percentiles of 6–8 animals per group, *p≤0.05, **p≤0.01, ***p≤0.001. (BM  =  basement membrane).(TIFF)Click here for additional data file.


Yersinia pestis. Y. pestis mutants lacking the yersiniabactin (Ybt) siderophore-based iron transport system are avirulent when inoculated intradermally but fully virulent when inoculated intravenously in mice. Presumably, Ybt is required to provide sufficient iron at the peripheral injection site, suggesting that Ybt would be an essential virulence factor for flea-borne plague. Here, using a flea-to-mouse transmission model, we show that a Y. pestis strain lacking the Ybt system causes fatal plague at low incidence when transmitted by fleas. Bacteriology and histology analyses revealed that a Ybt-negative strain caused only primary septicemic plague and atypical bubonic plague instead of the typical bubonic form of disease. The results provide new evidence that primary septicemic plague is a distinct clinical entity and suggest that unusual forms of plague may be caused by atypical Y. pestis strains.Plague is a flea-borne zoonosis caused by the bacterium  Yersinia pestis is usually transmitted by infected fleas and produces bubonic plague, characterized by a painful, swollen lymph node, the bubo Y. pestis. However, atypical strains have been isolated from around the world and it remains unclear whether these isolates produce one or another form of plague.Y. pestis strains form red colonies (pigmented or Pgm+) after growth at ≤34°C on media containing Congo red, but white colonies (Pgm–) may be isolated at a frequency of 10−4– mutants result from the deletion of a 102-kb chromosomal region termed the pgm locus hmsHFRS) which is essential for the pigmentation phenotype and for the production of a biofilm in the flea gut that can block normal blood feeding; the blockage of the flea's digestive tract is considered to be an important process for flea-borne transmission pgm locus also contains the Yersinia high-pathogenicity island (HPI), which carries among other genes the irp1-irp2-ybtU-ybtT-ybtE, the ybtP-ybtQ-ybtX-ybtS and the psn loci that encode the yersiniabactin (Ybt) siderophore-based iron acquisition and transport system. The irp genes encode the high molecular weight proteins (HMWP) 1 and 2 which act in concert with YbtU, YbtE, YbtS and probably YbtT to synthesize the Ybt siderophore Y. pestis by the TonB-dependent outer membrane receptor Psn and the inner membrane ABC-transporter YbtP-YbtQ. A critical role of the Ybt system in bubonic plague is indicated by the fact that Ybt–Y. pestis strains are essentially avirulent by the subcutaneous inoculation route that mimics the flea bite, although these strains retain complete or nearly complete virulence when inoculated intravenously Typical pgm locus is subject to complete or partial loss at relatively high frequency by genomic rearrangements; and Pgm– Ybt– and Pgm–Ybt+ strains from natural plague foci have been described Despite the importance of the Hms and Ybt system for flea-borne transmission and for disease in bubonic plague models, respectively, the Y. pestis 195/P strain which was originally isolated from a patient with pneumonic plague irp2 mutant with an in-frame deletion of bases 242 to 5721 were used in this study. The irp2 mutant was generated by allelic exchange using the suicide plasmid pCVD442 and verified by sequencing. Bacteria cultured overnight in Luria broth at 21°C without aeration were quantified by using a Petroff-Hausser bacterial counting chamber, diluted in PBS and inoculated intravenously into the tail vein or intradermally in the upper right thigh to groups of 8–10 week-old female RML Swiss-Webster mice.The fully virulent Y. pestis infectivity after challenge by flea bite Xenopsylla cheopis rat fleas were allowed to feed on heparanized mouse blood containing wild-type Y. pestis or an isogenic irp2 mutant, using an artificial feeding device, and maintained as previously described Y. pestis to block X. cheopis), 49 to 115 fleas were applied to a restrained mouse and allowed to feed for 60 min. Immediately after the challenge, the fleas were examined individually under a dissecting microscope to determine how many infective (blocked) fleas had bitten each mouse Y. pestisA flea-borne transmission model was used to determine All animal experiments were approved by the Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health Biosafety and Animal Care and Use Committees in accordance with National Institutes of Health guidelines .Y. pestis to colonize and block fleas Y. pestis strain and an isogenic Ybt-negative mutant after natural transmission by infected X. cheopis rat fleas. We first tested the virulence of our Ybt– mutant after needle-inoculation of cultured bacteria. The LD50 of the mutant was 10 CFU after intravenous (IV) injection, but >105 CFU after intradermal (ID) inoculation, similar to what has been reported previously for other Ybt–Y. pestis strains – mutant was highly attenuated when inoculated intradermally by needle, fleabites from fleas infected with this mutant produced terminal disease in two of ten mice, at 3 and 6 days post-challenge (irp2 mutant (P = 0.001 by log-rank test).The Ybt system is not required for hallenge . Thus, t10 CFU per ml of blood and spleen respectively). From these results one can infer that six mice had bubonic plague followed by sepsis and 3 mice developed primary septicemic plague (irp2 mutant was significantly different (P = 0.0015 by Fisher's exact test). Neither of the two mice that developed terminal disease after being bitten by fleas infected with the irp2 mutant had typical bubonic plague (10 CFU in the spleen and 3.8 and 7.6 log10 CFU per ml of blood). Hence, one mouse infected with the irp2 mutant had primary septicemic plague, and the other had septicemic plague associated with a mild lymphadenitis. For mice challenged with either wild-type or Δirp2 Y. pestis, disease outcome  did not correlate with the cumulative number of challenges or infective flea bites (P>0.05 by Fisher's exact test).Six of the nine sick mice infected by fleabite with the wild-type strain were diagnosed with typical bubonic plague, characterized by severe lymphadenitis with destruction of the lymph node architecture and the presence of numerous bacteria admixed with cellular debris . The othc plague . In contc plague . Histoloc plague , but thec plague . Lymph nc plague   mutant 5 CFU) is several orders of magnitude higher than the number of CFU transmitted by blocked fleas (median <100 CFU) The present results with a Y. pestis strains are frequently isolated from natural sources, and have been associated with mild cases of human plague pgm locus indicates that Ybt– and Pgm– clones are generated spontaneously in nature with some regularity. These clones would be at a disadvantage because of their decreased transmissibility, but could persist for some time during an epidemic associated with high flea density. The recently described early-phase transmission by fleas might also be more conducive to the circulation of these clones The extent to which atypical forms of plague are attributable to atypical strains rather than the host immune response is unknown, but non-pigmented


Chlamydia trachomatis is the etiological agent of trachoma the world's leading cause of infectious blindness. Here, we investigate whether protracted clearance of a primary infection in nonhuman primates is attributable to antigenic variation or related to the maturation of the anti-chlamydial humoral immune response specific to chlamydial antigens.Genomic sequencing of organisms isolated throughout the protracted primary infection revealed that antigenic variation was not related to the inability of monkeys to efficiently resolve their infection. To explore the maturation of the humoral immune response as a possible reason for delayed clearance, sera were analyzed by radioimmunoprecipitation using intrinsically radio-labeled antigens prepared under non-denaturing conditions. Antibody recognition was restricted to the antigenically variable major outer membrane protein (MOMP) and a few antigenically conserved antigens. Recognition of MOMP occurred early post-infection and correlated with reduction in infectious ocular burdens but not with infection eradication. In contrast, antibody recognition of conserved antigens, identified as PmpD, Hsp60, CPAF and Pgp3, appeared late and correlated with infection eradication. Partial immunity to re-challenge was associated with a discernible antibody recall response against all antigens. Antibody recognition of PmpD and CPAF was destroyed by heat treatment while MOMP and Pgp3 were partially affected, indicating that antibody specific to conformational epitopes on these proteins may be important to protective immunity.C. trachomatis antigen-specific humoral immune response. However, we cannot conclude that antibodies specific for these proteins play the primary role in host protective immunity as they could be surrogate markers of T cell immunity. Collectively, our results argue that an efficacious subunit trachoma vaccine might require a combination of these antigens delivered in their native conformation.Our findings suggest that delayed clearance of chlamydial infection in NHP is not the result of antigenic variation but rather a consequence of the gradual maturation of the  Chlamydia trachomatis is the etiological agent of trachoma the world's leading cause of infectious blindness. In this study, we investigated whether delayed clearance of a primary infection in nonhuman primates was attributable to antigenic variation or related to gradual changes in the humoral immune response specific to chlamydial antigens. We found that antigenic variation was not related to the inability of monkeys to efficiently resolve their infection. However, exploring changes in the immune response as a possible reason for delayed clearance revealed that antibody recognition was restricted to the antigenically variable major surface protein and a few conserved polypeptides. Antibody recognition of the major antigenically variable surface protein correlated with the initial reduction in infectious burdens while recognition of conserved chlamydial antigens occurred late and correlated with infection eradication. These findings suggest that delayed clearance of chlamydial infection is not the result of antigenic variation but a consequence of a gradually evolving humoral immune response specific to different chlamydial antigens. Antibody recognition was at least partially directed against conformational epitopes, indicating that an efficacious subunit trachoma vaccine might require a combination of antigens delivered in their native conformation. Chlamydia trachomatis is the causative agent of blinding trachoma and sexually transmitted diseases. C. trachomatis utilizes a unique biphasic developmental cycle alternating between infectious elementary bodies (EB) and metabolically active reticulate bodies (RB). Multiple serovars exist within C. trachomatis. The ompA gene, coding for the immunodominant major outer membrane protein (MOMP), differentiates these serovars The obligate intracellular bacterial parasite C. trachomatis serovar A, A2497 The nonhuman primate ocular model is the most relevant animal model for studying trachoma. Not only is this ocular model appropriate in its ability to mimic the acute aspects of human trachoma infection but isolation of laboratory animals ensures infection exposure and disease are not related to reinfection. Previously, we used this model to examine infection with a recently isolated virulent Tanzanian clinical strain of C. trachomatis specific humoral immune response might explain the basis of the protracted period of time required to eradicate the primary infection and clinical disease. We found no evidence linking antigenic variation to delayed clearance of primary infection in these animals but observed convincing findings implicating gradual changes in the humoral immune response specific to a few chlamydial antigens as a possible mechanism.In this study, we investigate whether antigenic drift or maturation of the host Macaca fascicularis) maintained at the Rocky Mountain Laboratories were cared for under standard practices implemented by the Rocky Mountain Veterinary Branch/NIAID/NIH. Monkeys were housed separately when being used for experimental studies. All handling procedures were reviewed and affirmed by the Animal Care and Use Committee at Rocky Mountain Laboratories and work was conducted in full compliance with the Guide for Care and Use of Laboratory Animals. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care. Conjunctival chlamydial infections can cause transient local inflammation in the eye and are not associated with pain or significant discomfort; discomfort is usually limited to minor photophobia. During the experimentations we were prepared to treat any animals determined to be under stress or in significant discomfort with appropriate analgesics and antibiotics. We observed no stress or significant discomfort during the entire experiment. At the end of the nonhuman primate re-challenge experiment animals were treated with antibiotics and returned to their colony unharmed.Healthy adult naïve cynomolgus macaques (C. trachomatis strains A2497 (serovar A) and Ba/Ap-2/OT (serovar Ba) were cultured in either HeLa 229 or McCoy cells (ATCC) and elementary bodies (EBs) were purified by density gradient centrifugation 4 IFU per eye directly applied on the conjunctival surface of the upper and lower lids of both eyes with strain A2497. Chlamydial culture and ocular clinical disease scoring was done as previously described All NHP ocular chlamydial infections and re-infections were done using 2×10Journal of Infectious DiseasesThree male cynomolgus monkeys were infected ocularly with the A2497 trachoma strain and the infections were allowed to run their natural self-limiting course (4–6 months). Approximately three months following spontaneous clearance of infection and disease (269 days post primary infections) monkeys were similarly re-challenged and again the infections were allowed to run their natural course. At weekly intervals during the entire experiment, ocular disease was evaluated and scored and samples were collected. The primary infection of these animals has been described before in the ompA PCR and sequencing was performed as previously described http://pbil.univ-lyon1.fr/cap3.php). Using the NCBI website (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi), assembled sequences were compared against the previously reported A2497 ompA sequence At various times post-infection chlamydiae cultured from the conjunctivae of all three monkeys were plaque cloned using McCoy cells 8 organisms as previously described Plaque clones from monkey RML126 were passed in HeLa 229 cells to obtain sufficient organisms to infect six well culture plates. Six 6-well culture plates of HeLa cells were infected with each clone at an MOI of 1. At 48 hpi, chlamydiae were harvested and partially purified on 30% MD-76R (Merry X-Ray Corporation) density gradients C. trachomatis serovar A or serovar Ba at a MOI of 2.5 and fed with RPMI  containing 4 µg/ml of emetine. Twelve hpi cells were washed twice with 1 ml of RPMI minus L-methionine and L-cysteine and then incubated with 300 µCi of 35S L-methionine and L-cysteine in 3 ml of RPMI. At 50 hpi the monolayers were washed 3 times with 5 ml PBS and then lysed in 1 ml of cold RIPA lysis buffer  containing protease inhibitor (Roche Protease Inhibitor Cocktail Tablet). The cell lysate was gently passed through a 25 gauge needle 10 times to shear DNA and centrifuged at 19,000× g at 4°C for 30 min. Intrinsically radiolabelled antigen supernatants were collected, aliquoted, and stored at −20°C, with aliquots used in immunoassays.McCoy cells grown in 6 well tissue culture plates were infected with 2O for 10 min and then placed on filter paper, vacuum dried and exposed to high performance chemiluminescence film (Amersham Hyperfilm ECL).Fifty µl Protein A and 50 µl Protein G magnetic beads (Invitrogen) were used for each RIP reaction according to the manufacturer's protocol. Briefly, beads were washed several times with Ab Binding and Wash Buffer. Intrinsically radiolabeled chlamydial antigen lysates were pre-cleared by incubation with 100 µl of beads for 1 hr at RT with continuous rotation. Beads were then re-suspended in Ab Binding and Wash Buffer. NHP sera were added to the suspension to achieve a final dilution of 1∶80. Tubes were rotated for 1 hr at RT and the supernatants removed. Beads were washed and then incubated with 100 µl of pre-cleared antigen lysate for 2 hours by rotating at RT. Supernatants were removed and the beads were washed four times and then transferred to a clean tube. Beads were suspended in 30 µl of 2× Laemmlie sample buffer (BioRad) and boiled for 10 min at 100°C. Supernatants were removed from the beads, loaded onto 4–15% precast SDS-PAGE gels (BioRad), and electrophoresed at 70 V for 30 min and then 150 V for 70 min. Gels were rinsed in 5 ml of dH35S L-methionine and L-cysteine was added to the culture media. The immunoprecipitation (IP) procedure used was also identical with the exception that the final elution of antigen bound to beads employed the Invitrogen non-denaturing elution protocol using 20 µl elution buffer for 5 min at RT. The elution supernatant was transferred to a new tube and 20 µl of 2× sample buffer was added to each tube. Samples were boiled for 10 min at 100°C and electrophoresed as described above. Proteins were transferred to PVDF (BioRad) membranes for 4 hr at 350 mA. Following transfer, the membranes were blocked overnight, washed twice with diH20, and incubated with mouse monoclonal or rabbit monospecific primary antibody specific to chlamydial proteins at RT for 3 hours. The membrane was washed, incubated with anti-mouse or anti-rabbit secondary antibody at RT for 1 hr, washed repeatedly, and then incubated with 1 ml of chemiluminescent developing solution for 5 mins at RT (Invitrogen). Gels were exposed to high performance chemiluminescence film (Amersham Hyperfilm ECL).McCoy cells were infected with chlamydiae and chlamydial antigen lysates were prepared as described above for intrinsically radiolabeling experiments, except no C. trachomatis strain A2497, a recent trachoma clinical isolate with enhanced virulence characteristics for the nonhuman primate (NHP) eye In our previous study three cynomolgus monkeys were infected ocularly with ompA, the gene encoding the major outer membrane protein (MOMP), an immunodominant neutralizing target and the serotyping antigen of chlamydiae ompA sequence differences were found (data not shown). To look for antigenic variation beyond ompA, 4 clones from the final reactivation period (week 14) and 2 clones from the initial peak shedding period (week 1) in one monkey (RML 126) underwent comparative genomic sequencing. As with ompA, no genetic variation was observed between the genomes of the plaque clones isolated from early and late time points (data not shown).To examine the possible role of antigenic drift in delayed clearance, chlamydia positive ocular swabs from time points following culture negative periods were cultured and chlamydiae were plaque cloned . We initially looked for antigenic variation in To investigate the possible role of an evolving antibody mediated immune response we collected sera from the three monkeys during primary infection and following re-challenge. These sera were analyzed by radioimmunoprecipitation (RIP) with intrinsically radiolabelled labeled chlamydial proteins prepared from non-denaturing detergent lysed infected cells. Despite the complex composition of the lysates, we observed a surprisingly simple antibody recognition profile by RIP that was consistent among the three nonhuman primates . All thrC. trachomatis serovars A2497 and serovar B (Ba/Ap-2), strains differentiated by ompA antigenic variation that are representative of the C and B complex serogroups, respectively. For this experiment we used sera from a single monkey (RML 134) obtained at four representative timepoints: at the days of infection and re-challenge, and at the peak of anti-MOMP reactivity post infection and re-challenge  RIP antigen . AntibodC. trachomatis infection in NHP primates was related to antigenic drift or whether gradual maturation of the host antibody mediated immune response was required to eradicate the infection. We detected no signs of antigenic drift in ompA or other chlamydial genes. This finding is consistent with epidemiological studies showing MOMP does not change rapidly even in trachoma endemic areas In this study we investigated whether a long-duration but self-limiting ocular C. trachomatis serovar B by western blotting C. trachomatis serovar B, a laboratory reference strain of unknown virulence. The strain produced a self-limiting acute conjunctivitis that resolved spontaneously at approximately 8 weeks post-infection without delayed clearance or episodes of reactivation; no culture positivity was seen after week 6. The animals were not re-challenged following clearance of the primary infection so their protective immune status was unknown. Finally, western blotting was used to define anti-chlamydial antibody specificity over the infection period, a method that detects antibodies primarily against denatured protein antigens. In contrast, we employed a recent serovar A trachoma clinical isolate (A2497), a strain shown to have enhanced virulence characteristics for macaques compared to a laboratory adapted C. trachomatis serovar A strain (HAR-13) In a study similar to ours, Caldwell et al. examined the temporal serum antibody response in cynomolgus macaques infected ocularly with C. trachomatis EBs that resulted in a significant 70 fold reduction in infectious ocular burdens early post infection C. trachomatis specific humoral immune response takes so long to fully develop. It is possible that recognition of certain potentially protective epitopes requires long-term antigen stimulation. This would be similar to the natural protective immunity that older individuals develop in hyper endemic areas after years of long-term exposure through repeated reinfection episodes. Nevertheless, we believe a vaccine containing a cocktail of these antigens might generate protective immunity capable of functioning at early and late time points during chlamydial infection. A vaccine with these protective properties would be beneficial in preventing or reducing chlamydial transmission and re-infection, both of which are driving forces in the pathology of blinding trachoma. The difficulty however with this approach is if the native conformation of these proteins is critical to protective immunity, as our findings imply, producing these immunogens as recombinant proteins in heterologous expression systems will be a major yet worthy challenge. An alternative vaccine approach that would negate this requirement is the use of a live-attenuated trachoma vaccine, such as the recently described live-attenuated plasmidless vaccine Our findings imply that an efficacious trachoma vaccine might require multiple chlamydial antigens expressed in their native confirmation. A logical cocktail of recombinant antigens based on our results would consist of MOMP, PmpD, CPAF, and Pgp3, although other low molecular weight antigens associated with infection eradication that were not identified in this study may also be needed. Mechanistically, the kinetics of antibodies specific for these proteins infers that antibody specific to MOMP, a surface exposed and antibody neutralizing target, likely function in reducing early chlamydial burdens, whereas antibodies to CPAF, Pgp3 and PmpD, another surface exposed neutralizing target, are required to eradicate infection. This hypothesis is consistent with previous reports using these antigens as single subunit vaccines in both NHP and murine models. Kari et al. immunized NHP with native-MOMP extracted from