Surgery for trachomatous trichiasis (TT) is a key component of the SAFE Strategy for trachoma control. Unfortunately, recurrent TT following surgery is common, probably due to various surgical and disease factors. To develop strategies to reduce recurrence rates it is necessary to understand its pathological basis. In this study we investigated the relationship between recurrent trichiasis and the expression of various cytokines and fibrogenic genes during a two-year follow-up period.S100A7, IL1B, CXCL5, TNFA, NOS2A, CTGF, MMP7, MMP9 and MMP12. Thirteen hundred individuals were enrolled and underwent surgery. By two years 122 had developed recurrent TT with at least 3 lashes touching the eye. Recurrent TT was consistently associated across multiple time points with about a 2-fold increase in S100A7 expression (p = 0.008). Clinically visible conjunctival inflammation was associated with increased S100A7, IL1B, CXCL5, MMP9 and MMP12 expression.Individuals undergoing surgery for TT were examined at baseline (pre-operative), 6, 12, 18 and 24 months. Conjunctival swab samples were collected from the tarsal conjunctiva for RNA isolation on each occasion. Individuals who developed recurrent TT with at least 3 lashes touching the eye on one or more occasion were designated “cases” and an equal number of “controls” were randomly selected from those without recurrent TT, frequency matched for age and baseline TT severity. The expression of the following genes was measured by quantitative RT-PCR: S100A7 expression was associated with trachomatous conjunctival scarring and may be linked to the pathophysiology of recurrent TT. S100A7 expression could be a potential biomarker for this disease process. As part of the epithelial innate immune response S100A7 has multiple actions, potentially contributing to a chronic pro-inflammatory response, which may lead to ongoing tissue damage and increased scarring.Increased  S100A7) before surgery and on multiple occasions during a two-year follow-up period. S100A7 is able to promote inflammation and may contribute to the development of the scarring process in trachoma.Trachoma causes blindness through corneal damage from in-turned eyelashes (trachomatous trichiasis [TT]). Trichiasis is treated surgically to correct the anatomical abnormality. Unfortunately, TT frequently returns following surgery, which again puts the person at risk of sight loss. Recurrent trichiasis is multifactorial. We investigated the possible role of various immuno-fibrogenic factors. To do this we operated on 1300 people with TT and followed them up every six months for a two-year period. On each occasion a conjunctival swab was collected for human gene expression analysis. We measured various factors that are thought to be important in inflammation and scarring diseases. The gene expression profile of people who developed recurrent TT was compared to a sample of those that did not have a recurrence. Recurrent TT was associated with increased expression of psoriasin ( Chlamydia trachomatis infection that are characterised by inflammation. However, the pathophysiology of this scarring process is poorly understood; it is unclear which immuno-fibrogenic mechanisms are most important and what factors drive disease progression, particularly in the late stages when C.trachomatis can rarely be detected Trachoma remains the leading infectious cause of blindness worldwide Surgery is performed to correct TT. Unfortunately, TT frequently recurs, particularly when performed under operational conditions Early recurrence is probably attributable to a combination of factors including pre-operative disease severity, how well the surgery was performed, and possibly early post-operative wound healing responses C. trachomatis infection in people with scarring trachoma, and it has not been found to be a risk factor for recurrent TT C. trachomatis prevalence, recurrence was reduced, although this was not linked to C. trachomatisC. trachomatis, post-operative azithromycin did not affect the recurrence rate Late recurrence is likely due to immune-mediated conjunctival scarring disease, however, the pathological basis of progressive cicatrisation in trachoma remains to be elucidated. It may be driven by recurrent infection, yet, it is relatively unusual to detect S100A7 or Psoriasin, which was increased 15-fold in trichiasis cases before surgery, compared to controls MMP1/TIMP1 ratio A better understanding of the immuno-fibrogenic basis of recurrent TT may help in developing interventions that reduce recurrence that is attributable to the scarring disease process. We have previously examined the conjunctival transcriptome in scarring trachoma in a case-control study Recently we reported the results of a randomised controlled trial of absorbable (vicryl) versus silk sutures for TT surgery; there was no difference in outcome between the two treatment arms This study adhered to the tenets of the Declaration of Helsinki and was approved by three science and ethics committees: the National Health Research Ethics Review Committee, Ministry of Science and Technology, Ethiopia, the London School of Hygiene and Tropical Medicine Ethics Committee (UK) and Emory University Institutional Review Board . Potential participants were provided with both written and oral information in Amharic about the trial. For those agreeing to participate, written informed consent in Amharic was required prior to enrolment. If the participant was unable to read and write, the information sheet and consent form were read to them and their consent recorded by witnessed thumbprint, which was approved by the Ethics Committees.This nested case-control study was conducted within a surgical trial of absorbable versus silk sutures in the treatment of upper eyelid TT in Amhara Region, Ethiopia, which has been reported separately Following the baseline pre-operative clinical assessment (described below), participants were randomised to one of two surgical intervention groups, which differed only in the type of sutures that were used. The posterior lamellar tarsal rotation procedure was used in all cases to correct upper eyelid TT, using the randomly allocated suture material: (1) PLTR with 4/0 silk sutures  or (2) PLTR with 5/0 polyglactan-910  Participants were re-examined at 6, 12, 18 and 24 months after surgery. To be eligible for analysis in this study a complete follow-up series was required; cases and controls were only selected from those individuals who were examined on all occasions. Individuals who developed any recurrent TT (defined as one or more lashes touching the eye) at any point during the course of the two-year follow-up period were designated recurrent TT “cases” and those without any trichiasis recurrence or evidence of epilation at any time point were designated non-recurrent “controls”. We further limited recurrent TT cases to those that had 3 or more lashes touching the eye at some point during follow-up, as this provided just over 100 recurrent TT cases for analysis. The non-recurrent controls were selected at random from operated individuals who had not developed recurrent TT at any time point during follow-up; these controls were frequency matched with the recurrent cases for age and baseline trichiasis disease severity (determined by the number of lashes touching the eye before surgery).At all assessments participants were examined for clinical signs of trachoma, graded using 2.5× binocular loupes according to the detailed WHO trachoma grading system later (Life Technologies). Samples were kept on ice packs until frozen later the same day at −20°C.Conjunctival swab samples were collected by the examiner from the upper tarsal conjunctival surface for RNA isolation at all time-points, except 3-months. The ocular surface was anaesthetised with preservative-free proxymetacaine 0.5% eye drops . A dacron polyester-tipped swab (Hardwood Products Company) was passed horizontally across the conjunctival surface 4 times with a quarter turn between each pass. Swabs were then placed directly into a tube containing 0.3 ml of RNAIL1B], tumour necrosis factor-α [TNFA], psoriasin-1 [S100A7], chemokine (CXC) ligand-5 [CXCL5], nitric oxide synthase-2A [NOS2A]), (ii) various matrix metalloproteinases  and (iii) connective tissue growth factor (CTGF).The abundance of nine selected transcripts was estimated by quantitative RT-PCR. The choice of targets was informed by our previously published conjunctival transcriptome analysis conducted on pre-operative samples from TT patients in the same population and matched normal controls HPRT-1 as the reference gene) were designed by Sigma Life Science (www.Sigma.com/designmyprobe) using Beacon Designer 7.60 . The thermal cycle protocol used the following conditions: 95°C for 15 minutes, followed by 45 cycles of (1) denaturation at 94°C for 30 seconds, (2) annealing and extension at 60°C for 30 seconds. Fluorescence data was acquired at the end of each cycle. The relative efficiency of the component reactions was assessed using standards containing all targets in a sequence of tenfold serial dilutions. Reactions were performed in duplicate, in a total volume of 25 µl, which contained 2 µl of sample or standard. Probe and primer sequences are available on request.Total RNA was extracted from the swab samples using the RNeasy Micro Kit (Qiagen). Reverse transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's instructions. Multiplex real-time quantitative PCR was performed on a Rotor-Gene 6000  using the QuantiTect Multiplex NoROX Kit (Qiagen), according to the manufacturer's instructions. Multiplex assays of up to four separate targets , age (in years), case-control status, conjunctiva inflamed (P2/P3) at baseline and the number of lashes touching the eye at baseline. A stepwise selection process was performed to fit each model, retaining terms if the p-value for omission was <0.2 and their p-value in the model was <0.2, with p-values assessed by the likelihood ratio test. Similar models were fitted for each follow-up time point. There was no evidence of a differential effect over time, assessed by likelihood ratio testing with and without an interaction with time . Therefore, we combined the data from the four follow-up time points, using random-effects linear models. An un-conditional logistic regression model was developed for the association between recurrent trichiasis and baseline conjunctival inflammation and the expression levels of all nine genes. Again a stepwise selection process was performed to fit each model, retaining terms if the p-value for omission was <0.2 and their p-value in the model was <0.2, with p-values assessed by the likelihood ratio test. The correlation in the expression of the various targets at each time point was investigated using biplots and partial correlation coefficients. To adjust for multiple comparisons, we calculated critical significance thresholds for each table using the conservative Bonferroni correction. Although we make several comparisons, these are un-likely to be truly independent of each other, as one would expect some of these genes to interact in biological networks.Data were managed in Access (Microsoft) and analysed in STATA 11 (StataCorp). Cases and controls were comparable in terms of age, sex and pre-operative trichiasis severity, therefore, the relative level of expression of each target was compared between them using unadjusted unpaired A total of 1300 individuals with major TT were recruited into the trial and received trichiasis surgery There were 740 people (seen and sampled five times) who had no evidence of recurrent TT at any point during the follow-up period. Overall, the non-recurrent group tended to be younger  and had less severe pre-operative trichiasis  compared to the recurrent TT cases. To ensure that the non-recurrent “control” group was of a similar age and preoperative trichiasis severity, 122 individuals were randomly selected from the potential 740, constrained by frequency matching from within the same combination of age group  and pre-operative trichiasis severity group (0 (epilating), 1–5, 6–9, 10–19, 20+ lashes) as the 122 recurrent TT cases. The baseline distribution of age and trichiasis severity groups were the same for cases and controls, except in one instance, where there was one too few controls to frequency match . There wA total of 1220 samples had been collected across the five time points from these 244 participants. The nine targets were measured in each of these. There were a small number of samples from each time point for which it was not possible to perform quantitative PCR due to an inadequate RNA sample . For the purpose of this analysis these samples are treated as missing data.S100A7 (at the 0.05 level), on 4 out of 5 occasions. No other targets had a consistent association with recurrent TT  and matrix metalloproteinases  across multiple time points  IL1B and CXCL5, (3) IL1B and TNFA, (4) NOS2A and MMP7, and (5) MMP9 and MMP12.Partial correlation coefficients were calculated for each target with each other for each time point. The total number of significant partial correlations at the 0.05 level is represented in Recurrent trichiasis is a challenging problem for prevention of blindness programmes as it renders the surgical intervention less effective, increasing the risk of sight loss. Multiple factors likely contribute to recurrence, which can broadly be subdivided into disease related and surgery related factors. Refinements of the operative procedure and improvements in surgical quality are anticipated to lead to a reduction in early recurrent trichiasis IL1B, TNFA, S100A7, CXCL5, NOS2A) and tissue remodelling factors , which were associated with conjunctival scarring and trichiasis before surgery. Therefore, these were chosen as candidates to be investigated in this longitudinal study of recurrent trichiasis following surgery.The choice of targets for this longitudinal gene expression study was guided by findings from the baseline case-control study of the conjunctival transcriptome, which compared individuals with established TT to people without disease from the same population S100A7 (Psoriasin). There was an approximately 2-fold increase in its expression on four out of the five time-points; this association remained significant after adjustment was made for other potential explanatory factors. Clinically visible conjunctival inflammation (P2/P3) was also associated with increased expression of S100A7. Multivariable models indicated that the associations between recurrent trichiasis, S100A7 expression and inflammation were independent. Unsurprisingly, the expression of several pairs of factors showed a degree of association on multiple occasions.In the present study the only gene found to have consistently increased conjunctival expression (both before and after surgery) in individuals who developed recurrent trichiasis was S100A7 to be the most strongly up-regulated gene in the conjunctiva in TT cases relative to normal controls, in the same Ethiopian population and in Tanzanians with conjunctival scarring in the absence of TT S100A7 expression to be significantly increased in children with signs of clinically active trachoma We previously found S100A7: carcinoma of the breast, lung, prostate, stomach and cutaneous squamous cell carcinoma. Aggressive tumour behaviour (invasion and metastasis) has been particularly associated with its expression Psoriasin is a member of the S100 family, a diverse group of calcium binding, low molecular weight proteins, which are expressed in multiple tissues, particularly epithelial surfaces The functions of S100A7 have only recently begun to be explored in detail. It is an anti-microbial peptide, protecting epithelial surfaces from bacterial infection Little is known about the regulation of S100A7. Some bacterial components, such as flagellin, trigger increased production of S100A7, probably through TLR5 IL1B, CXCL5, MMP9 and MMP12 were increased in the presence of clinical inflammation on several occasions. We have previously reported associations between IL1B and MMP9 and conjunctival inflammation following surgery TNFA was not consistently associated with inflammation (only one occasion), which is a pattern we have found in two earlier studies Chronic conjunctival inflammation is an important component of the pathophysiology of trachoma and it is a relatively frequent finding in people with cicatricial disease C. trachomatis because in this population (which has an active azithromycin mass distribution programme) at baseline we have previously reported that infection was very rare in adults with and without TT (0.1%) and therefore considered it unlikely to be informative This study has a number of potential limitations. The outcome of interest was recurrent trichiasis. This can be a variable sign especially if the patient is practicing epilation. However, careful note was made of any evidence of epilation on examination. Secondly, measuring the expression of a gene does not necessarily equate to functional activity of its protein product. Positive findings will need further validation with alternative approaches, such as immunohistochemistry to detect the protein. We did not perform PCR testing for S100A7 was consistently associated with recurrent trichiasis indicates that it may have a role in this disease process. In the light of new information becoming available about the contribution of S100A7 to other diseases, our findings suggest that this molecule warrants further investigation in trachoma.Anti-scarring therapies are increasingly used in ophthalmic surgery to limit cicatricial complication


Breast cancer is the second leading cause of cancer-related deaths in western countries. Colony-Stimulating Factor-1 (CSF-1) and its receptor (CSF-1R) regulate macrophage and osteoclast production, trophoblast implantation and mammary gland development. The expression of CSF-1R and/or CSF-1 strongly correlates with poor prognosis in several human epithelial tumors, including breast carcinomas. We demonstrate that CSF-1 and CSF-1R are expressed, although at different levels, in 16/17 breast cancer cell lines tested with no differences among molecular subtypes. The role of CSF-1/CSF-1R in the proliferation of breast cancer cells was then studied in MDAMB468 and SKBR3 cells belonging to different subtypes. CSF-1 administration induced ERK1/2 phosphorylation and enhanced cell proliferation in both cell lines. Furthermore, the inhibition of CSF-1/CSF-1R signaling, by CSF-1R siRNA or imatinib treatment, impaired CSF-1 induced ERK1/2 activation and cell proliferation. We also demonstrate that c-Jun, cyclin D1 and c-Myc, known for their involvement in cell proliferation, are downstream CSF-1R in breast cancer cells. The presence of a proliferative CSF-1/CSF-1R autocrine loop involving ERK1/2 was also found. The wide expression of the CSF-1/CSF-1R pair across breast cancer cell subtypes supports CSF-1/CSF-1R targeting in breast cancer therapy. However, the expression of CSF-1R and/or CSF-1 has been documented in several human cancers, including carcinomas of breast, female reproductive tract, prostate and kidney www.clinicaltrials.org). Elucidation of the involvement of CSF-1R in breast cancer cell proliferation would strengthen the rationale of CSF-1R targeting in CSF-1R expressing cancers.Targeting receptor tyrosine kinases with kinase inhibitors  has recently opened a new era in the treatment of hematologic malignancies and solid tumors such as gastrointestinal stromal tumors In this work, we characterized the role of CSF-1R in the proliferation of breast cancer cells and found that CSF-1R is widely expressed in breast cancer cell lines at both mRNA and protein levels. Interfering with the CSF-1/CSF-1R signaling pathway, either by CSF-1R inhibition or by inhibition of autocrine CSF-1, impaired MDAMB468 and SKBR3 cell proliferation. In addition, exposure to ectopic CSF-1 stimulated MDAMB468 and SKBR3 growth. We found ERK1/2, c-Jun, cyclin D1 and c-Myc, known for their involvement in cell proliferation, to be downstream CSF-1R in breast cancer cells. The wide expression of CSF-1/CSF-1R pair across breast cancer cell subtypes supports CSF-1/CSF-1R targeting in breast cancer therapy.www.lgcstandards-atcc.org) periodically tested in our laboratory by western blotting for the presence of EGFR protein) were cultured in DMEM, while human chronic myeloid leukemia K562 cells (www.lgcstandards-atcc.org); periodically tested in our laboratory by western blotting or Q-PCR for the expression of BCR/Abl) in RPMI, supplemented with 4 mM glutamine and 10% fetal bovine serum (FBS). Human primary macrophages were obtained after informed consent as previously described 2.NIH/3T3 murine fibroblasts expressing ectopic human CSF-1R  Data were obtained mapping CSF-1 and CSF-1R gene symbols to GSE2603 (probes 2078082_at and 203104_at) Culture plates were placed on ice, cell monolayers rapidly washed 3 times with ice-cold PBS containing 100 mM orthovanadate and cells lysed by scraping in Laemmli buffer  and incubating at 95°C for 10 minutes in the presence of 100 mM 2-mercaptoethanol. Lysates were then clarified by centrifugation .30–60 µg of total proteins was separated by SDS-PAGE in 9–15% polyacrylamide gel and transferred onto PVDF membranes (Millipore) by electroblotting. Membranes were incubated  in Odyssey Blocking Buffer diluted 1∶1 with PBS, and then in the same buffer containing 0.1% Tween-20 and the primary antibody . After extensive washing with PBS/0.1% Tween-20, membranes were incubated in Odyssey Blocking Buffer diluted 1∶1 with PBS containing IRDye®800CW- or IRDye®680-conjugated secondary antibody . Bands were visualized by infrared imaging  and images recorded as TIFF files for quantification with Adobe Photoshop software. Rabbit α-phospho-T202/Y204-ERK1/2 ; rabbit α-ERK1 ; rabbit polyclonal α-CSF-1R C-20 ; goat α-CSF-1 antibody N-16 ; α-phospho-723-CSF-1R ; mouse α-vinculin ; rabbit α-phospho-S63/73-c-Jun ; mouse monoclonal α-cyclin D1 ; mouse monoclonal α-myc .Cells were detached by incubation in PBS containing 0.2% EDTA (pH 7.2), washed with PBS, pelleted and incubated in 20 µl of an anti-CSF-1R antibody  CSF-1 was measured by ELISA (RayBio ELISA KIT Human MCSF), according to the manufacturer's instructions. The manufacturer claims that the minimum detectable dose of CSF-1 is typically less than 5 pg/ml (sensitivity of the methods). Supernatants were collected at cell confluency from cultures in complete medium. Each sample (100 µl) was assessed in duplicate.Silencing was performed as previously described 2B4O7 was added. Cells were washed with PBS and permeabilized by a 5 minute incubation in PBS containing 0.2% Triton X-100. After three washes in PBS cells were incubated with 10% horse serum in PBS/1%BSA for 45 minutes and then washed in PBS and incubated overnight at 4°C in a 1∶250 dilution of a mouse monoclonal α-BrdU antibody  in PBS/1%BSA, washed with PBS and incubated with a 1∶800 dilution of an α-mouse Cy3-labelled secondary antibodies . Cells were washed in PBS and incubated with 5 µg/ml Hoechst 33258 (Sigma) nuclear dye in PBS for 10 minutes. Following two washes in PBS, coverslips were mounted with propyl-thiogallate on glass slides and cells observed with a Leica DC200 microscope. Pictures were taken from 6 different field/sample (>600 cells were scored for each treatment) and the percentage of cells undergoing DNA synthesis was calculated by the ratio of the number of BrdU-positive cells to the total number of cells determined by Hoechst 33258 staining. Incubation with secondary antibody alone did not produce any significant fluorescence.Cells were seeded onto glass coverslips in complete medium for 24 hours and then incubated in the absence of FBS for 24 hours before being incubated in DMEM with or without 25 ng/ml CSF-1 or a 1∶50 dilution of a goat α-CSF-1 blocking or pre-immune serum Cells were seeded in 12 multi-well plates and incubated for 24 hours in complete medium before serum starvation for further 24 hours. Cells were then treated in serum-free medium with imatinib  for 45 minutes and then with CSF-1 for further 48 hours. Cell were then washed twice with PBS and incubated for 10 minutes at RT with a Crystal Violet solution . After extensive water washing, plates were allowed to dry and dye extracted by incubating with 1% SDS. Densitometric measurement was then performed at 550 nm.5′-CCTCGCTTCCAAGAATTGCA-3′, rev: 5′-CCCAATCTTGGCCACATGA-3′ ; CSF-1R, N-term, for: 5′-GGAGGCTGCCCAGATCGT-3′ rev: 5′-GCGAGCTTGGTGTTGTTGTG-3′ ; cyclin D1 (CCND1), for: 5′-GAGAGGAAGCGTGTGAGGCGGTAG-3′, rev: 5′-GGATGCTGGAGGTGCGAGGA-3′; cyclin G1 (CCNG1), for: 5′-CCAGCTGAATGCCCTGTTG-3′, rev: 5′-AGTCTCAAACCACAGACCTTTGG-3′; cyclin H (CCNH), for: 5′-GAGGAGCAGCTGGCAAGACT-3′, rev: 5′-ACGGCTTTGCATCTGAATTTG-3′; cyclin I (CCNI), for: 5′-CATCTCAACATTTGGCAGTCCTT-3′, rev: 5′-GAAGTTGGTTGCAGGCCATAC-3′; rRNA 18S, for: 5′-CGGCTACCACATCCAAGGAA-3′, rev: 5′-GCTGGAATTACCGCGGCT-3′ (amplicon size 180 bp); GAPDH, for: 5′-AACAGCC TCAAGATCATCAGCAA-3′, rev: 5′-CAGTCTGGGTGGCAGTGAT-3′. CSF-1R mRNA expression was assessed by Q-PCR  with the ABI Prism 7500 Sequence Detection System (Applied Biosystem) using Power SYBR® Green PCR master mix (Applied Biosystem). A melting curve analysis was performed to discriminate between specific and non-specific PCR products. Alternatively, amplified product size was verified by running in 3–4% agarose gels. The housekeeping 18S rRNA and/or GAPDH genes were used as internal references for normalization. The relative expression of CSF-1R, with respect to SKBR3 cells chosen as calibrator, was calculated by using a comparative threshold cycle method and the formula 2(−ΔΔCt)After total RNA extraction by TRIzol (Invitrogen) as specified by the manufacturer, 1 µg of total RNA/sample was submitted to reverse transcription with SuperScriptVILO-Reverse Transcriptase (Invitrogen) for 10 minutes at 25°C, 1 hour at 42°C and 5 minutes at 85°C utilizing 50 pmol random hexameric primers. The primers used were as follows: CSF-1R, C-term, for: http://array.lonza.com/plate/00188263/. The housekeeping rRNA 18S gene was used as internal reference for normalization. Fold change was calculated as described above. Data reported (+SEM) were obtained from four independent experiments.SKBR3 cells were transfected with the indicated siRNA and cultured in complete medium for 72 hours. Cells were then lysed and total RNA extracted. Gene expression analysis was performed with Q-PCR Arrays . A list of these 96 genes is available online at CSF1R is expressed, although at different levels, in all cell lines tested.The expression of CSF-1R and/or CSF-1 in human breast carcinomas has been documented in both cell lines and tumors samples The presence of cell surface CSF-1R protein was then verified by flow cytometry , S1A. AlThe cell lines were also tested for CSF-1 expression by measurement of secreted CSF-1 (by ELISA and western blotting) and of CSF-1 in cell lysates (by western blotting). CSF-1 was detected in all the cell lines tested . It is oin silico analysis on publicly available gene expression profiling datasets  among breast cancer subtypes in either cell lines or in tumors samples. Moreover, in silico analysis of comparative genomic hybridization datasets relative to 49 breast cancer cell lines The data reported in datasets  [41], [4To determine whether CSF-1R transduces proliferative signals in breast cancer cells we chose two cell lines from different subtypes . When baTo further characterize CSF-1R involvement in breast cancer cell proliferation we treated breast cancer cells  with CSFTo test whether an autocrine CSF-1/CSF-1R loop exists in breast cancer cells  we used The fact that CSF-1 promotes proliferation in breast cancer cells prompted us to determine whether their growth is sensitive to tyrosine kinase inhibitors used in the clinic. Imatinib (IM) is known to inhibit CSF-1R activity Although the expression of CSF-1/CSF-1R has been previously documented in breast cancer and shown to correlate with poor prognosis, few studies have been performed to understand the role of CSF-1R-dependent signaling in the proliferation of breast cancer cells or other solid tumors CSF-1R sustains breast cancer cells proliferation, as highlighted in two cell lines of different molecular subtypes. Indeed, interfering with CSF-1/CSF-1R signaling, either by targeting the receptor or by blocking the ligand binding, impacts on breast cancer cell proliferation. This proliferation was induced either by ectopic CSF-1, that mimic CSF-1 produced by fibroblasts and monocytes/macrophages associated with the tumor, or autocrine CSF-1. We found that CSF-1R activation sustained the expression of cyclin D1 and c-Myc and activated c-Jun, which are established CSF-1 downstream targets in other cell types We also found that CSF-1R-induced proliferation involves ERK1/2 activation in SKBR3 and MDAMB468 cells. Our data are in line with previous studies in which breast cancer cell lines expressing ectopic CSF-1R showed increased expression of cyclin D1 as a consequence of ERK1/2 activation upon CSF-1 administration CSF1 and CSF1R expression is a general feature of breast cancer cells. These findings support the possibility to target CSF-1R signaling in a large proportion of breast cancers, independently of their molecular subtype. This is particularly relevant for tumors that are classify as of the basal-like subtype. Triple-negative  breast cancers make up the majority of this subgroup Our data show that 16 cell lines among 17 tested express CSF-1 and CSF-1R, although at different levels. Moreover, gene expression profiling datasets show that in silico analysis indicate that CSF-1R is not overexpressed (when compared to human monocytes) nor amplified The data obtained from our experiments and IM is a tyrosine kinase inhibitor in use for the clinical management of chronic myeloid leukemia and has been shown to inhibit several different kinases, including CSF-1R. We found that IM impairs the proliferation of breast cancer cell lines in the presence of CSF-1. As IM targets kinases other than CSF-1R our experiments do not prove that the effects shown are only due to CSF-1R inhibition. However, they support the concept that targeting CSF-1R with tyrosine kinase inhibitors can effectively block CSF-1-dependent growth stimulation. This is particularly relevant when considering strategies to interfere with autocrine CSF-1-dependent proliferation.In conclusion, the evidence we provided for the expression and functional role of the CSF-1/CSF-1R pair in breast cancer, together with the established role of CSF-1/CSF-1R in breast cancer motility and invasiveness Figure S1Settlement of CSF-1 or CSF-1R targeting using NIH/3T3 cells expressing ectopic CSF-1R. (A) NIH/3T3-Fms cells were transfected with the indicated siRNA. Total protein lysates obtained at the indicated times were subjected to immunoblotting with the indicated antibodies. Densitometric values of bands  are reported as ratios between the siCSF1R and the siNT value, set as 1. 72 hours post-transfection cells were analyzed by flow cytometry. Percentages of CSF-1R-positive cells are reported. (B) NIH/3T3-Fms cells were transfected with the indicated siRNA and incubated for 24 hours. Cells were then serum-starved for further 24 hours and treated with (CSF-1) or without (−) 25 ng/ml CSF-1 for 24 hours, and tritiated thymidine uptake measured. Data represent mean (± SEM) of one of 3 representative experiments; ** and ***, Student's t test: p<0.01, p<0.001, respectively. (C) NIH/3T3-Fms cells were incubated with or without 25 ng/ml CSF-1 for 10 minutes. Before cell treatment, CSF-1 had been incubated for 1 hour at 37°C in the absence (−) or the presence of a 1∶50 dilution of a CSF-1-blocking anti-serum (I) or pre-immune serum (PI). Total protein lysates were subjected to immunoblotting with the indicated antibodies. Densitometric values of bands  are reported as ratios between the siCSF-1R and the correspondent siNT value, set as 1. (D) NIH/3T3-Fms cells were cultured for 24 hours without serum and then for 45 minutes with the indicated doses of imatinib (IM) before treatment with or without 25 ng/ml CSF-1 for 10 minutes. Cells were then lysed and protein subjected to immunoblotting with the indicated antibodies.(TIF)Click here for additional data file.Figure S2CSF-1 and CSF-1R gene copy number in breast cancer cell lines and tumor samples. Data have been collected from CGH experiments performed by others with breast cancer cell lines (left) (TIF)Click here for additional data file.Figure S3CSF-1R signaling induces ERK1/2 phosphorylation in breast cancer cell lines. Serum-deprived cells (24 hours) were incubated with or without CSF-1 (25 ng/ml) for 10 minutes and lysed in RIPA buffer. Total protein lysates were subjected to immunoblotting with the indicated antibodies. N3F: NIH/3T3-Fms cells. Densitometric values of bands  are reported as ratios between the CSF-1-treated and the untreated value, set as 1. Threshold for activation was arbitrary set at ≥1.2.(TIF)Click here for additional data file.


Background. Blood-brain barrier (BBB) dysfunction may have a significant role in the pathogenesis of Alzheimer's disease (AD). Modifiable factors associated with BBB function may have therapeutic implication. This study tested the hypothesis that dyslipidemia is associated with BBB impairment in mild-to-moderate AD. Methods. Thirty-six subjects with AD were followed for 1 year. Fasting CSF and plasma were collected with clinical assessments at baseline and 12 months. BBB impairment was defined as CSF albumin index ≥9. Independent t-tests and linear regression assessed the relationship between plasma lipoproteins and BBB integrity. Results. Dyslipidemia was prevalent in 47% of the population, and in 75% of those with BBB impairment. Subjects with BBB impairment had significantly higher mean plasma triglyceride and lower HDL cholesterol . Plasma triglycerides explained 22% of the variance in BBB integrity and remained significant after controlling for age, gender, ApoE-4 genotype, blood pressure, and statin use. Conclusion. Dyslipidemia is more prevalent in AD subjects with BBB impairment. Plasma triglyceride and HDL cholesterol may have a role in maintaining BBB integrity in mild-to-moderate Alzheimer's disease. The CSF albumin index is an established measure of blood-brain barrier (BBB) integrity in living patients . This inIf BBB function plays a role in the pathogenesis of AD, then modifiable factors associated with it may have therapeutic potential. A clinical trial of B vitamin supplementation has suggested that the BBB may be a modifiable entity in subjects with hyperhomocysteinemia and mild cognitive impairment . Lipids The study participants were recruited from the clinic population of the NIA—Layton Aging and Alzheimer's Disease Center of Oregon Health and Science University and have been described previously . BrieflyThirty-six subjects with mild-to-moderate AD were evaluated by medical history, neurological and general examination, the Mini-Mental State Examination (MMSE) , the CliLumbar punctures were performed in the morning under standardized conditions at L3 to L4 or L4 to L5 interspaces, immediately aliquoted, and snap frozen at −70°C until assayed. These samples had normal cell count and glucose levels, and the aliquots analyzed were matched sequentially by draw to control for CSF content drift by sequence. A CSF-to-serum ratio of albumin (CSF Albumin Index) ≥ 9.0 was considered BBB impairment. Reproducibility of the CSF albumin index in AD over 1-year has been established (intraclass correlation coefficient =  .96) . Laboratt-tests compared the mean differences in CSF albumin Index by subjects classified as atherogenic dyslipidemia .P for difference = 0.251). Total and LDL cholesterol were not different between the two groups . However, mean plasma triglycerides were higher and HDL cholesterol lower in subjects with BBB impairment  . Subjects with atherogenic dyslipidemia (n = 15) had mean CSF Albumin Index 2.69 units higher than those without (n = 21) (P = .029). Subjects with metabolic dyslipidemia (n = 17) had mean CSF Albumin Index 2.43 units higher than those without (n = 19) (P = 0.48).The overall prevalence of “atherogenic” and “metabolic” dyslipidemia was 42% and 47%, respectively. The prevalence of atherogenic dyslipidemia was more frequent in AD subjects with BBB impairment than in subjects without  (P = 0.040). Total cholesterol, LDL cholesterol, and HDL cholesterol were not associated with CSF Albumin Index in the regression model (data not shown).Linear regression analysis indicated that each 10 mg/dL increase in plasma triglyceride content was associated with a 0.13 unit increase in CSF Albumin Index (= 0.004) . Plasma These findings demonstrate a higher prevalence of dyslipidemia in Alzheimer's subjects with BBB impairment. Plasma triglycerides and HDL cholesterol were the lipids most associated with BBB integrity. Plasma triglycerides explain the most variation in BBB integrity compared to HDL, LDL, and total cholesterol.A relationship between triglycerides and dementia has been reported in one large study . This grAlthough sample size in this study is a limitation, we believe that the study has particular strengths worth noting. Sixty four percent of this study population has autopsy confirmed AD, and the entire sample had probable AD confirmed by consensus diagnosis. Another strength is the stability of the CSF Albumin Index as a measure of BBB integrity in living subjects  and the The possibility that dyslipidemia is causally related to BBB impairment may be clinically significant since dyslipidemia is treatable. If this evidence is confirmed in other populations, then the next step may involve a lipid-modifying strategy to modify the natural history of AD. While it is true that statin therapy has been unsuccessful in altering the course of AD, these current findings place emphasis on modifying triglyceride and HDL cholesterol, ideally in subjects selected on the basis of BBB impairment at baseline. Perhaps a dietary pattern or supplementation with omega-3 PUFAs and niacin would offer one strategy, since they favorably modify triglyceride and HDL cholesterol metabolism, respectively . The eme


The epidemiology of pneumococcal meningitis in the African ‘meningitis belt’ is poorly studied. In order to ensure an effective vaccination strategy and post-vaccination surveillance, we examined the serotype distribution patterns of pneumococcal meningitis in Niger over the period 2003–2011.Cerebrospinal fluid (CSF) samples were collected from different health facilities throughout Niger in the frame of the national microbiological surveillance of meningitis. Determination of the serotype of CSF positive for pneumococci was performed using a sequential multiplex PCR method (SM-PCR) adapted with a national algorithm in which 32 different serotypes were covered and grouped into eight consecutive PCR.The SM-PCR assay could predict the Sp serotype for 779 CSF (88.7%), 98 CSF (11.3%) were not-typeable in our national-adapted algorithm. In total, 26 different serotypes were identified. Serotype 1 (n = 393) was the most prevalent and accounted for 45.3% of infections, followed by serogroups/serotypes 12F/(12A)/(44)/(46) (7.3%), 6/(6A/6B/6C/6D) (5.4%), 14 (5.2%), 5 (4.6%), 23F (4.2%), 45 (3.6%), 2 (3.1%), 18/(18A/18B/18C/18F) (2.9%) and 17 others serotypes with a prevalence of less than 2%. The proportion of serotype 1 in infants(<2 years old) represented only 4.3% of the cases affected by this serotype. In contrast, serotypes 5, 6, 14, 19A and 23F were only detected in very young children.The proportion of serotype 1 in the pneumococcal meningitis cases and the theoretical vaccine coverage across all age groups advocates for the introduction of a conjugate vaccine (PCV10 or 13) into the Expanded Programme on Immunization (EPI) in Niger. Post-vaccine introduction surveillance supported by molecular approaches will be essential to provide a comprehensive picture of the impact of the vaccine on the burden reduction of pneumococcal meningitis and on pneumococcal serotype distribution. Neisseria meningitidis (Nm) particularly serogroup A, responsible for the majority of the disease burden and accounting for several thousand cases every year. Streptococcus pneumoniae (Sp) was not supposed to occur in epidemics, however, pneumococcal meningitis (PM) epidemics have been well described in Senegal (serotype 5) in 1983 Historically, meningitis epidemics in sub-Saharan Africa have been due to The introduction of the MenAfriVac™ vaccine (conjugate vaccine against serogroup A meningococci) in Burkina Faso, Mali and Niger has already contributed to a substantial decline in the number of meningitis cases due to meningococci irrespective of the ability of currently circulating serogroups W In the countries of the meningitis belt, case fatality rates (CFR) associated with pneumococcal meningitis generally exceeded those of meningococcal meningitis and can be as high as 50% Currently, over 90 pneumococcal serotypes are recognized and approximately a quarter of these serotypes are responsible for the majority of cases of invasive pneumococcal disease In this study, the serotype distribution pneumococci responsible for meningitis from 2003 to 2011 was investigated to predict the potential coverage of the 10 or 13-valent pneumococcal conjugate vaccine (PCV10 or PCV13) scheduled for introduction in the Nigerien vaccination programme in 2014 and to assess the potential for serotype replacement post vaccine introduction.Meningitis surveillance in Niger is undertaken by the Direction of Surveillance and Response to Epidemics (DSRE), Ministry of Public Health (MoPH). Quantitative data on the morbidity and the mortality by age group for all clinically suspected cases of meningitis meeting a standard case definition are notified on a weekly basis. This surveillance is coupled to the cerebrospinal fluid (CSF) microbiological surveillance across Niger performed by the Centre de Recherche Médicale et Sanitaire (CERMES) based in Niamey. Aliquots of CSF samples were collected daily during the epidemic season from all national hospitals within Niamey. Additionally, between November and June each year from 2003–2010, a monthly collection of clinically well-defined samples occurred from hospitals/health facilities within a radius of 300 km from Niamey (Dosso and Tillabéry Regions) by CERMES. Frozen or refrigerated CSF samples from the rest of the country were sent on a voluntary basis to CERMES or DSRE by mandated transport companies.N. meningitidis (crgA gene), S. pneumoniae (lytA gene) and H. influenzae (bexA gene) in the conditions described by Chanteau et al. Laboratory confirmation of bacterial meningitis was performed by CERMES using culture and/or PCR techniques on CSF or CSF-inoculated trans-isolates (TI) as previously described lytA gene were retained for typing.Positive CSF detected by the multiplex PCR targeting the et al.Determination of the serotype of pneumococci in CSF was performed using the SM-PCR method initially described by Pai All PCR primer pairs were validated by multiplex PCR reactions against DNA from known serotyped strains obtained from the Biological Resource Center of the Pasteur Institute  or isolated in Niger and serotyped by the NRCP.lytA positive CSF were first screened with a cpsA PCR amplification and cpsA positive CSF were subsequently analysed in the sequential PCRs all including four primer pairs for different serotypes. Remaining lytA positive/cpsA negative CSF were screened in simplex PCR for serotypes 25F and 38.All Statistical analysis with the open-source R2.14.0. (2011) software  were used to describe the epidemiology of pneumococcal meningitis using both data from epidemiological surveillance ensured by MoPH and from microbiological surveillance performed by CERMES.All specimens (CSF) were collected by clinicians with patient or family oral consent after being informed why their CSF was sampled. This was done as part of the routine clinical management of patients according to the national guidelines in Niger. By no means, additional CSF sampling for research purpose was done in the frame of this study. Remaining aliquots of CSF after routine CSF examination by health facilities for case management were sent on a voluntary basis to CERMES to meet enhanced microbiological surveillance of meningitis guidelines. CERMES is a Technical and Scientific National Institution from the Ministry of Public Health (Decree n° 2005-060/PRN/MSP/LCE) with a Board of Directors in which several representatives of the Ministry, as well as a representative of the National Ethics Committee, are sitting. CERMES was designated and committed by a decree from the Ministry of Public Health as National Reference Laboratory (NRL) for meningitis in Niger (249/MSP/DGSP/DPHL/MT). Furthermore, an official written consent of the Ministry of Public Health was obtained for collection and microbiological analysis of CSF (2011/000699/MSP/DGSP). Confidentiality on patients' identity was also guaranteed. As such, written consent was not sought and approval from the national ethics committee was not required.st January 2003 to 31st December 2011, the MoPH of the Republic of Niger reported a total of 42,013 suspected cases of meningitis and 19,223 CSF were collected and analysed (45.8%) at CERMES. Molecular analyses of all CSF samples obtained from 2003 to 2011 revealed 1010/19,223 CSF positive results by lytA PCR: 5.3% of all CSF, 12.6% of those having a known aetiology . Among those 1010 lytA positive CSF, 86 were either missing or the CSF volume was depleted. All other CSF (n = 924) were screened again with a simplex PCR for lytA and cpsA genes. A total of 80 CSF were negative for cpsA .The 57 lytA negative, cpsA negative CSF were not included in the SM-PCR but the 23 lytA positive, cpsA negative CSF were analysed separately in simplex PCR for serotypes 25F and 38. The years recording the highest recovery rate of Sp were 2004, 2005 and 2007 with 26.1, 37.7 and 36.4% of all CSF positive for a known aetiology , respectively.From 1Our adapted SM-PCR covering 32 different serotypes provided the Sp serotype for 779 CSF (88.7%), with 98 CSF (11.3%) remaining not-typeable (SM-PCR NT). In total, 26 different serotypes were identified . Serotypst PCR reaction was able to detect the serotype in 53.6% of all positive CSF tested. Reactions 1 and 2 covered 69.1%, reactions 1–3 79.2% and reactions 1–4 84.2%. Serotype 1 represented 59.3% of all positive CSF in 2003. Serotypes 14, and 18 were not detected in 2003, serotype 19A was not detected in 2003, 2008, 2010 and 2011 and serotype 2 was not detected in 2011. On the 23 lytA positive, cpsA negative CSF, 13 were positive for the serotype 38 but all negative for serotype 25F. These additional analyses rose the % of serotype 38 from 0.1 to 1.63%. The 10 remaining negative CSF were included in the SM-PCR NT CSF.The 1Serotype 1 was detected primarily amongst older children, teenagers and young adults (5 to 20 years old) . The proS. pneumoniae from meningitis cases is essential for accurate epidemiological surveillance in the context of vaccine introduction.Accurate serotype determination of et al.The rollout of pneumococcal vaccines in the developing world through the considerable support of the GAVI alliance is now underway across three continents and Niger is one of the eligible countries for support in 2014. This paper presents the most comprehensive data to date detailing the national baseline serotype distribution for Niger as a basis to assess the potential coverage and future impact of the pneumococcal-conjugated vaccine selected for introduction. However there were, as with any long term surveillance programme, some limitations to our data. The national meningitis surveillance system, relies on biological samples (mainly CSF) received by the National Reference Laboratory which are either refrigerated or frozen. Yet as a consequence of poor infrastructure at peripheral laboratories  most of the samples were not viable on arrival. CSF recently withdrawn and kept at room temperature or seeded in Trans-isolates allowed the culture of aetiological agents but samples of this type were rare from sentinel sites. As such molecular typing is the only workable option to determine the serotype of Sp in CSF in our context. Although a suitable, adaptable and increasing used method, the simple and schematic sequence-based system of sequential multiplex PCR (SM-PCR) has some disadvantages. The main disadvantage is its inability to discriminate closely related serotypes, however some improvements have been made to circumvent this lytA positive CSF from PM and in total, 26 different serotypes were identified between 2003–2011. Supplementary PCR amplification on 23 lytA positive, cpsA negative CSF revealed 13 cases of serotype 38 bringing its representativity to 1.63%. This phenomenon has been observed previously with serotypes 38 and 25F generally negative for cpsAet al.Our Niger adapted SM-PCR was able to determine the Sp serotype in 88.7% of nd most predominant circulating serotype) is not included in any of the conjugate vaccines.If we consider the coverage of PCV10 across all age groups, 68.2% of circulating serotypes in Niger would be covered and this includes all serotypes frequently associated with infants in our setting with the exception of 19A. This serotype is included in PCV13 and so coverage would increase to 68.7% . HoweverConjugate vaccines have been generally recommended for routine use in neonates and children under than 2 years of age whereas the pneumococcal polysaccharide vaccine (PPV) is generally recommended for all adults 65 years and older and for 5 to 64 year olds who have a chronic disease or illness, an impaired immune system, or who live in areas or among social groups where there is an increased risk for pneumonia or meningitis. Our results showed that serotype 1  is more commonly identified in children aged >5 year, in teenagers and young adults (5 to 20 years old). The proportion of serotype 1 in those under 2 years old, represented just 4.3% of all serotype 1 cases. Although they are often found in disease, serotype 1 pneumococci are seldom carried or are simply hard to detect in carriage studies due to technical reasons. If carried in very young children, the introduction of the conjugated vaccine in the Expanded Programme on Immunization (EPI) could induced a herd immunity effect and consequently reduce the burden encountered in older children and young adults. On the other side, the proportion of other serotypes such as 5, 6, 14, 19A and 23F in under 2 years old children represented 61.1%, 76.7%, 80.5%, 80.0% and 62.9% of cases respectively. These data would therefore support the introduction of a conjugate vaccine (PCV10 or PCV-13) into the EPI. In the future it would be beneficial to see the development or expansion of formulations which are of relevance to Africa to provide greater coverage in an area suffering from the highest burden.Streptococcus pneumoniae involved in meningitis cases is essential in the context of vaccine introduction and epidemiological surveillance. Our study represents the first comprehensive report outlining the national baseline serotype distribution of PM in Niger prior to the introduction of a pneumococcal conjugate vaccine in 2014.Accurate serotype determination of Across the study period (2003–2011), serotype 1 was the most commonly identified serotype and accounted from 35.1% to 59.3% of all CSF investigated dependent on year. This report also underlines the importance of having sustained and high quality microbiological surveillance of PM complemented by accurate analytical methodologies for determining pneumococcal serotypes in Niger.


A 65-year-old Japanese male with therapy-related myelodysplastic syndrome was admitted for unrelated cord blood transplantation. A cord blood unit from a male donor was obtained from the Japan Cord Blood Bank Network. The patient then received a conditioning regimen consisting of fludarabine, intravenous busulfan, and total body irradiation. Successful engraftment was obtained. The bone marrow examination on day 28 revealed trilineage engraftment, and chimerism analysis by variable number of tandem repeat polymerase chain reaction confirmed complete donor chimerism. At that time, conventional cytogenetics of the bone marrow aspirate showed 20 out of 20 metaphases with the 47, XXY karyotype characteristic of Klinefelter syndrome. Klinefelter syndrome is the most common genetic cause of human male infertility with a reported prevalence of 0.1–0.2% in the general population. In Japan Cord Blood Bank Network, there is no informed consent from parents about the possibility that post-unrelated cord blood transplantation patient evaluation may reveal donor-origin inherited diseases including cytogenetic abnormality. It is desirable to have opportunities in Japan discussing whether parents will be notified of the possibility that post-unrelated cord blood transplantation evaluation may reveal donor-derived illness incidentally. The use of unrelated cord blood (CB) as a source of hematopoietic stem cells has been increasing in Japan. Indeed, more than a thousand unrelated cord blood transplantations (UCBTs) are performed annually in Japan . His first relapse occurred in 2004 and treatment consisted of three courses of rituximab and the CHASE regimen , intravenous busulfan (3.2 mg/kg on days –6 to –3), and total body irradiation (4 Gy on day –2) . Engraftment was obtained. A neutrophil count of > 0.5 × 103/L and a platelet count of > 20 × 103/L were achieved on days 14 and 42, respectively. He developed acute GVHD grade II of the gut and was successfully treated with hydrocortisone. The bone marrow examination on day 28 revealed trilineage engraftment, and chimerism analysis by variable number of tandem repeat using the AmpFLSTR® SGM Plus® PCR Amplification Kit (Applied Biosystems) confirmed the establishment of complete donor chimerism. At that time, conventional cytogenetics of the bone marrow aspirate showed 20 out of 20 metaphases with 47, XXY  score was Int-2  (Klinefelter et al. We described donor-derived cytogenetic abnormalities of 47, XXY in the recipient of UCBT. It is desirable to have opportunities in Japan discussing whether parents will be notified of the possibility that post-UCBT evaluation may reveal donor-derived illness incidentally.Written informed consent was obtained from the patient for the publication of this report and any accompanying images.


The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values. The advent of recombinant DNA technologies allowed the production of a large number of proteins with potential applications as therapeutic drugs. However, several shortcomings might limit their usefulness as biopharmaceuticals. First of all, proteins have to be stable under storage conditions in order to avoid the formation of protein aggregates that could be associated to higher immunogenicity. Furthermore, proteins can be degraded in vivo by cellular proteases and/or rapidly excreted by kidneys, leading to a short circulating half-life that reduces their therapeutic efficacy Contrary to the first generation of random pegylation, not always able to guarantee protein homogeneity, stability and activity, the second generation is mostly a site-directed pegylation, which enables the production of conjugated proteins with better defined characteristics. The most employed pegylation strategies for site-specific conjugation involve the addition of PEG to the N-terminal residue of the protein chain, or to a free cysteine, naturally present or genetically introduced. Moreover, in the case of site-directed enzymatic pegylation, it is possible to increase the choice of the pegylation target, making it possible to conjugate the polymer to the suitable protein site Interestingly, site-specific pegylation obtained following different approaches could in principle lead to isomeric products with different characteristics, such as in the case of the human granulocyte colony stimulating factor (G-CSF), whose aggregation propensity has been found to be differently affected when pegylation was performed following different strategies In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific pegylated forms of recombinant human methionyl-G-CSF, also known as Filgrastim, a protein of 18,799 Dalton belonging to the family of hematopoietic cytokines. Filgrastim is widely used as therapeutic agent in chemotherapy and bone marrow transplantation, since it regulates the production and the differentiation of hematopoietic progenitor cells We performed our studies on the site-specific mono-pegylations of Met-G-CSF at the N-terminal methionine (Met-G-CSF-Met1-PEG) and at glutamine 135 (Met-G-CSF-Gln135-PEG), in order to compare products with a significant difference in the pegylation site.Our investigations show that both the site-specific mono-pegylations of Met-G-CSF preserve the protein secondary structure and strongly increase its thermal stability, as indicated by circular dichroism (CD), Fourier transform infrared (FTIR) and intrinsic fluorescence spectroscopies. Moreover, we found that both kinds of pegylation reduce the Filgrastim aggregation propensity and prevent its precipitation, as revealed by dynamic light scattering (DLS), CD and FTIR spectroscopies. In particular, by means of DLS we found that the two pegylation strategies drastically reduced the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation.The products Met-G-CSF and Met-G-CSF-Met1-PEG, respectively corresponding to Filgrastim and to the commercial N-terminally mono-pegylated Filgrastim, were prepared according to the methods described in the literature The localization of the pegylation sites on the protein three-dimensional structure 50 = 343.4±6.8 pg/ml), while Met-G-CSF-Met1-PEG of 13.8% (EC50 = 435.8±10.4 pg/ml), in comparison to non-pegylated Met-G-CSF, taken as 100% of activity (EC50 = 60.3±3.9 pg/ml).The biological activity of the products, measured by a proliferation assay, indicated that Met-G-CSF-Gln135-PEG had an in vitro residual proliferative activity of 17.6%  at 295 nm, during the thermal treatment. The emission intensity at 330 nm of the three Met-G-CSF variants, between 20°C and 95°C, is compared in To better evaluate the protein mid-point of transition, we reported the ratio between the fluorescence emission intensity at 330 nm and at 350 nm oC, suggests that large aggregates were forming upon bringing Met-G-CSF at temperatures of the order of 50oC. It is likely that precipitation of the largest aggregates took place at 55oC, a process that causes the observed slow decrease of the average intensity at this temperature. Noteworthy, the two pegylated variants Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG displayed almost an identical profile of their scattering intensity, that is characterized instead by a slow monotonous increase in intensity that reached a plateau value significantly lower than that of the non-pegylated variant  heating the samples at 55°C, close to the transition mid-point of the Met-G-CSF non-pegylated form. We monitored the protein aggregation kinetics at 55°C for about 7 hours, following the scattering intensity of the three samples as a function of time. As reported in  variant .h = 3.4±0.4 nm at equilibrium at T = 37oC and to rise to Rh = 39.4±4 nm after 7 hours incubation at 55°C. It is noteworthy that the observed monotonous increase in the scattering intensity as a function of the incubation time (h = 39.4±4 nm) versus time, rather than to an increase in their size.The two pegylated proteins displayed a very similar behavior in terms of hydrodynamic radius that was found to be Rion time  correspooC  is a therapeutic recombinant protein approved under the brand name of Neupogen® for the treatment of febrile neutropenia often associated to cancer chemotherapy Bio-Ker has produced an isomeric form of Met-G-CSF-Met1-PEG by a site-specific enzymatic pegylation of Filgrastim, catalysed by a bacterial transglutaminase, to obtain the glutamine 135 pegylated Met-G-CSF  G-CSF site-specific monoconjugates. EU Patent EP2049566 B1) . InteresIn this work, we characterized by several complementary biophysical approaches the non-pegylated and the two pegylated variants of Met-G-CSF, with the aim to disclose the effects of these modifications on the structural properties, stability and aggregation of the proteins.First of all, we assessed, by FTIR and CD, that the two pegylation strategies didn’t affect the protein secondary structure. This point is, indeed, crucial since the protein function is known to be strictly related to the preservation of its native structure.Concerning the protein stability, the thermal unfolding experiments performed by CD and fluorescence spectroscopies showed that both kinds of pegylation increase the stability of Met-G-CSF in the same way for the two pegylated products. This result is of great relevance since - as reported in the literature - pegylation could lead to products with different stability depending on the pegylation strategies In our work, we found that the thermal unfolding of the Met-G-CSF variants was associated to protein aggregation, as revealed by DLS, CD and FTIR spectroscopies. In particular, the non-pegylated protein underwent aggregation immediately after the temperature increase to 55°C, forming insoluble aggregates. Instead, the two pegylated proteins were characterized by a lower aggregation kinetics, that also led to soluble aggregates. Moreover, as studied by DLS, the aggregation at 55°C of the two pegylated products was found not to be associated to the formation of assemblies of increasing size, but to that of homogeneous size particles, with hydrodynamic radius of about 40 nm. Indeed, opposite to what found for the unmodified protein, both kinds of pegylation significantly reduced the polydispersity of the protein even at 55°C, up to the end of our observations .These results, overall, indicate that the coupling of polyethylene glycol to Met-G-CSF allowed to obtain a considerable reduction in protein aggregation compared to the non-pegylated protein, both in terms of amount and size of aggregates, and in particular without the formation of insoluble aggregates. These properties enable to prepare a more concentrated pharmaceutical product with an improved shelf-life.The different behavior of the two pegylated variants compared to the unmodified protein could be explained considering the steric hindrance of the PEG moiety, that reduces the aggregation rate, while the solvation by water molecules of the polar PEG groups increases the solubility of the pegylated protein assemblies.The possibility to control protein aggregation represents an important challenge in the development of protein for therapeutic use. Indeed, aggregation, that can occur during different stages of recombinant protein productions - from the expression in the host cells to the storage conditions - strongly affects protein bioavailability, immunogenicity and pharmacokinetics The results reported in this work show that both the site-specific pegylation strategies employed in this study led to two isomeric mono-pegylated Met-G-CSF derivatives with comparable structural properties and improved stability, associated to a lower aggregation propensity compared to the non-pegylated protein. Interestingly, these results are in agreement with preclinical studies , that demonstrated for the two pegylated products superimposable pharmacodynamic and toxicological characteristics.In this view, the biophysical approach described here might enable to evaluate protein stability and aggregation propensity of modified proteins, in the early stages of the development, offering a useful tool to identify samples with potential high therapeutic values.E. coli strain exploiting a fusion protein technology, as previously reported 8PNP20-Glu-Ser-Ser-Gly-Leu-Phe-Lys-Arg-Met-GCSF was expressed by high biomass fermentation as insoluble inclusion bodies that, after cell breakage, were recovered by centrifugation. After isolation of inclusion bodies, the fusion protein was dissolved in 7M guanidine buffer, refolded by dilution, dialysed and treated with the patented recombinant endoprotease ss-Kex1-C611  Novel soluble endoproteases for the in vitro processing of recombinant proteins. EU Patent EP1334183 B1), which cleaved exactly at the carboxyterminal side of dipeptide Lys-Arg. The released Met-G-CSF was purified to homogeneity by three orthogonal column chromatographies and concentrated by ultrafiltration to about 3 mg/ml of Met-G-CSF.Methionyl-G-CSF  was produced by fermentation of a recombinant The N-terminal pegylated Met-G-CSF , corresponding to the commercial PegFilgrastim Neulasta®, was prepared by reductive alkylation of Met-G-CSF, as previously reported  N-terminally chemically modified protein compositions and methods. U.S. Patent 5,824,784). Briefly, monomethoxy-PEG-propionaldehyde 20 kDa  was added under stirring to a cooled solution of Met-G-CSF in 100 mM pH 5 phosphate buffer containing 20 mM sodium cianoborohydride, at a molar ratio protein:PEG reagent of 1∶5. The one-pot reaction includes the PEG binding preferentially to the α-amino group as a Schiff base, which is reduced to a secondary amine by sodium cianoborohydride.After overnight incubation under stirring, excess reagents and by-products (traces of di- and tri-pegylated derivatives) were separated by cation-exchange chromatography on a CM Sepharose column eluted with a NaCl linear gradient (from 0 to 500 mM in 15 column volumes). The fraction pool containing the purified Met-G-CSF-Met1-PEG was concentrated by ultrafiltration to about 10 mg/ml (expressed as protein moiety).Gln135 pegylated Met-G-CSF  was prepared by a specific enzymatic transamidation between 20 kDa monomethoxy-PEG-alkylamine  and the side chain of Met-G-CSF glutamine 135, catalysed by a microbial transglutaminase, as previously described  G-CSF site-specific monoconjugates. EU Patent EP2049566 B1). Briefly, to a 2 mg/ml Met-G-CSF solution in 20 mM pH 8.1 potassium dihydrogen phosphate buffer, also containing 20 mg/ml of 20 kDa monometoxy-PEG-alkylamine, was added a solution of Activa WM microbial transglutaminase  to a final enzymatic activity of 0.25 U/ml. The solution was kept under stirring for 5 hours in refrigerator, diluted in 20 mM, pH 5.0 sodium acetate buffer and purified by cation exchange chromatography on a Macrocap SP column eluted with 98 mM sodium acetate buffer pH 5.0. The fraction pool containing Gln135 mono-pegylated Met-G-CSF was dialysed against 10 mM acetic acid pH 4.5 buffer and concentrated by ultrafiltration to about 13 mg/ml (expressed as protein moiety).2 atmosphere. After 48 hours, 20 µl of tetrazolium salt WST-1 were added to the cells and incubation continued further for 4 hours under the same conditions. The absorbance of formazan derived from WST-1 cleavage by cellular mitochondrial dehydrogenases was measured using an ELISA microplate reader , equipped with a 420–480 filter, against a buffer background. The biological activity of each sample was calculated from proliferation curve and expressed as EC50 value .Biological activity of non-pegylated and pegylated Met-G-CSF derivatives was measured in vitro according to a described method −1 spectral resolution, 25 kHz scan speed, 512 scan coadditions, and triangular apodization. The second derivatives of the measured spectra For FTIR measurements in attenuated total reflection (ATR), 5 µl of Met-G-CSF, Met-G-CSF-Met1-PEG or Met-G-CSF-Gln135-PEG, diluted at 2.7 mg/ml in 10 mM sodium acetate buffer pH 4.5, were deposited on the diamond plate of the single reflection ATR device . Spectra were recorded after solvent evaporation to allow the formation of a hydrated protein film CD spectra in the far-UV region (185–260 nm) were recorded on a Jasco J-815 spectropolarimeter . Non-pegylated and pegylated Met-G-CSF variants were measured at 0.1 mg/ml, in 10 mM sodium acetate buffer pH 4.5, in a 0.1 cm path length quartz cell at a scanning speed of 10 nm/min and by the averaging of 4 scans.For thermal unfolding experiments, the samples were heated from 20 to 95°C at a rate of 1°C /min and the ellipticity and the high tension voltage (HT[V]) at 222 nm were monitored every 0.5°C. CD spectra in the full far UV range were then collected at 20°C, 95°C, and at 20°C after heating up to 95°C.Intrinsic fluorescence measurements were performed with a Cary Eclipse Varian fluorimeter , with an excitation wavelength of 295 nm (slit 5 nm). Non-pegylated and pegylated Met-G-CSF variants were measured at 0.1 mg/ml, in 10 mM sodium acetate buffer pH 4.5 and in a 1 cm path length quartz cell.For thermal unfolding experiments, the samples were heated from 20 to 95°C at a rate of 1°C/min and the fluorescence emissions were monitored at 330, 340, and 350 nm (slit 5 nm).oC. The temperature was monitored by a thermocouple placed just below the scattering cell.Dynamic light scattering measurements have been performed for non-pegylated and pegylated Met-G-CSF variants on a home-made spectrometer The normalized intensity autocorrelation functions (ACFs) were computed by an ISS FCS board  and they were fitted to a multi-exponential decay law according to the following relation k is the translational diffusion coefficient of the k-th diffusing species, q is the wave vector, q = (4πn)/(λ)sin(θ/2), n is the refraction index of the solution, λ = 514 nm is the laser light wavelength and θ = 90o is the scattering angle. The parameter fcoh depends on the ratio between the detector and the coherence area, which was left as a free fitting parameter, typically in the range 0.35–0.15. The values of Dk provide us the average size of the diffusing objects, according to the Einstein relation:where Dwhere KB is the Boltzmann constant, T and η are the solution temperature and viscosity and Rh is the hydrodynamic radius.An alternative way to analyze the DLS data is to use the cumulant analysis. In order to gain information on the solution polydispersity where σ is the polydispersity of the sample. It must be noted that the absolute value of the polydispersity index σ depends critically on the number of points used for the analysis of the autocorrelation function decay and the number of cumulants used to describe the decay. Here we have fitted Eq.3 to the autocorrelation function decay in the range 2–30 µs of lag times.


Cardiac troponin is a specific biomarker for cardiomyocyte necrosis in acute coronary syndromes. Troponin release from the coronary circulation remains to be determined because of the lower sensitivity of the conventional assay. We sought to determine basal and angina-induced troponin release using a highly sensitive troponin assay.th, 75th percentiles)] to CS  in 96 (91.4%) patients and the difference was 1.1  pg/mL, which reflected basal transcardiac troponin release (TTR). TTR was positively correlated with PV levels . Male sex, left ventricular hypertrophy determined by echocardiography, T-wave inversion, and CAD correlated with elevated TTR defined as above: median, 1.1 pg/mL. A significant increase in TTR was noted in 17 patients with coronary spasms  but not in 16 patients without spasms  after the acetylcholine provocation.The cardiac troponin T levels in serum sampled from the peripheral vein (PV), the aortic root (AO), and the coronary sinus (CS) were measured in 105 consecutive stable patients with coronary risk factor(s) and suspected coronary artery disease (CAD) and in 33 patients without CAD who underwent an acetylcholine provocation test. At baseline, there was a significant increase in the troponin levels from AO [9.0  pg/mL for median . This sensitive assay detected myocardial ischemia-induced increases in TTR caused by coronary spasms. Cardiac troponin is well established as a specific biomarker for cardiomyocyte necrosis in individuals with acute coronary syndromes (ACS) but not in individuals with angina pectoris The present study was approved by the Ethics Review Committee of Kumamoto University . Signed informed consent was obtained from each patient before participation. This study is registered at the UMIN protocol registration system (UMIN000005099). This study was conducted in accordance with the ethical principles originating in the Declaration of Helsinki.https://center.umin.ac.jp ID: UMIN000005099.The cardiac troponin T levels in serum sampled from the peripheral vein, the aortic root, and the coronary sinus were measured using a highly sensitive assay in 105 consecutive stable patients who had at least 1 coronary risk factor, were suspected of having coronary artery disease (CAD), and underwent coronary angiography from April 2008 to September 2009. In the same period, samples collected before and after the ACh provocation test were also assessed in 33 patients without CAD.A 6-F catheter was placed in the coronary sinus to sample blood during coronary angiography. CAD was defined to be ≥75% stenosis (according to the classification set by the American Heart Association) on conventional coronary angiography analyzed quantitatively by software . The ACh test was indicated for patients with chest discomfort at rest in the absence of CAD. Patients with heart failure were excluded from the ACh test. Incremental doses  of ACh were injected into the left coronary artery, and angiography was performed 1 min after each injection. Then, 50 µg of ACh was injected into the right coronary artery. At baseline and either ACh-induced coronary spasm or 1 min after injection of the maximum dose of ACh, paired samples were collected simultaneously from the aortic root and coronary sinus. This method is described in the current guidelines of vasospastic angina Blood was processed immediately and frozen at 80°C until it was assayed by a commercial highly sensitive troponin T assay method . Because of the enhanced sensitivity, this assay is reported to have a coefficient of variation of 8% at 10 pg/mL 2 for men and >122 g/m2 for women in echocardiography L, and V3–V6Blood was drawn after an overnight fast. Diabetes mellitus (DM) was diagnosed based on criteria set by the World Health Organization or the use of hypoglycemic agents or insulin. Hypertension was defined as a systolic blood pressure ≥140 mmHg, a diastolic blood pressure ≥90 mmHg, or the use of an antihypertensive treatment. The estimated glomerular filtration rate was calculated using a modified formula from the Modification of Diet in Renal Disease study equation, which was proposed by the Japanese Society of Nephrology th–75th percentiles, as appropriate. Differences in troponin T levels between the aortic root and the coronary sinus were analyzed by the Wilcoxon rank sum test. Pearson correlations between troponin T in peripheral veins and transcardiac troponin release among patients with significant transcardiac release (N = 91) were analyzed after log-transformation of each variable. Differences in transcardiac troponin release between two groups with respect to coronary risk factors and patient diseases were analyzed by the Mann–Whitney U test. Univariate and multivariate logistic regression analyses for higher basal transcardiac troponin release defined as above: median, 1.1 pg/mL, were undertaken among certain risk factors. Bootstrapping was used for esvaluation of the internal validity of the variable selection in the multivariate models. We also calculated the Hosmer–Lemeshow goodness-of-fit statistic. P<0.05 denoted statistical significance, and all tests were two-tailed. Variables were log-transformed if they had a skewed distribution. All analyses were undertaken using SPSS 17.0J for Windows  and SAS software, version 9.3 .Data are expressed as mean ± standard deviation or median and 25th, 75th percentile) = 10.3  pg/mL] compared to the aortic root  in 96 (91.4%) patients. The difference in the troponin T levels between the coronary sinus and the aortic-root level was 1.1  pg/mL; the difference reflected basal transcardiac troponin release (2), hypertension, DM, lower estimated glomerular filtration rate , current smoking status, and left ventricular hypertrophy (as determined by echocardiography and electrocardiography) ( release . Transca release  after loography) . We asseography) . The HosTranscardiac troponin release was also assessed before and after provocation in patients without CAD who underwent the ACh test (N = 33). Basal transcardiac troponin release in the coronary circulation was observed in most of the patients with suspected CAD and coronary risk factor(s). Male sex, left ventricular hypertrophy determined by echocardiography, T-wave inversion, and CAD correlated with elevated troponin release under stable physical conditions. This sensitive assay detected significant angina-like transient myocardial ischemia-induced significant increase in transcardiac troponin release in patients with coronary spasms in the absence of CAD.We confirmed the existence of basal transcardiac troponin release, which had never been detected by the conventional troponin assay. The basal troponin release was shown to be correlated with peripheral serum levels of troponin. Furthermore, we originally identified factors that are significantly associated with elevated transcardiac troponin release using univariate and multivariate logistic regression analysis, whereas the earlier studies were based on only peripheral troponin revels Cardiac troponins exist in a structural form with tropomyosin and actin and in a free cytosolic pool We used the ACh-test to provoke myocardial ischemia because we intended to exclude exercise-induced myocardial stretch, increased production of oxidative radicals, altered acid–base balance, or double product-dependent myocardial damage We used a highly sensitive assay from Roche Diagnostics. This assay has been commonly used in recently reported large observational studies The study population was relatively small because of the invasive nature of coronary sinus sampling. Although transcardiac troponin production was comparable between patients with and without hypertension or DM, the small number of patients might have caused a lack of statistically significant difference in this analysis. We assessed the “basal” transcardiac troponin release during angiography, but this method could not exclude silent ischemia because of the lack of functional studies such as the measurement of lactate in coronary sinus. The weak correlation between transcardiac troponin release and peripheral levels does not allow to draw definitive conclusions. The diagnostic value of transcardiac troponin release was insufficient for the purpose of CAD detection in clinical practice. The ACh test may cause total occlusion of the epicardial coronary artery with ST segment elevation, which is rarely observed in most CAD patients but which may occur in certain situations such as variant angina and acute coronary syndromes with non-obstructive coronary artery disease. The lack of follow-up data for the peripheral-vein levels of troponin just after the ACh-test or on the next day was a major limitation for all clinical applications. The appropriateness of the point of blood sampling from coronary sinus (1 minute after ACh-induced spasm) is uncertain. Serial sampling is necessary to determine the optimal time point. We did not have data on cardiovascular outcome, and the prognostic impact of transcardiac troponin release was not determined in the present study.Basal troponin release in the coronary circulation was observed in most patients with suspected CAD and coronary risk factor(s). Male sex, left ventricular hypertrophy determined by echocardiography, T-wave inversion, and CAD correlated with elevated troponin release in stable physical conditions. This sensitive assay could be used to detect significant increases in transcardiac troponin release provoked by coronary spasm-induced transient myocardial ischemia in patients without CAD.


The purpose of this study is to report a case of Kyrieleis plaques  associated with cytomegalovirus (CMV) retinitis.A 47-year-old female with recently diagnosed human immunodeficiency virus and a CD4 count of 55 cells/µl presented with decreased vision and floaters in her left eye. Ophthalmic examination revealed an advancing border of white granular CMV retinitis extending into the macula. Intraocular aqueous specimen contained 420,000 copies/ml of CMV DNA by polymerase chain reaction. The patient was treated with intravitreal foscarnet and oral valganciclovir.Kyrieleis plaques involving the retinal arteries were noted on presentation and increased during the first 6 weeks of treatment as the retinitis faded. The plaques on fluorescein angiography did not leak fluorescein dye and slowly faded over 5 months.Kyrieleis plaques can be seen in the setting of CMV retinitis. These plaques can be differentiated from vascular sheathing and frosted branch angiitis by its occurrence only in the retinal arteries and the absence of leakage of fluorescein dye. Toxoplasma gondii chorioretinitis being the most common. They have also been reported with Rickettsia conorii, Mycobacterium tuberculosis, Treponema pallidum, and varicella-zoster virus (VZV) infections [Kyrieleis plaques, also referred to as segmental retinal periarteritis, were first described in an eye with tuberculosis uveitis by Kyrieleis in 1933 . These pfections .The etiology of Kyrieleis plaques has not been well established. Orzalesi and Ricciardi suggested these lesions are an immune response to an infectious agent and result from the deposition of immune cells and inflammatory debris in the arterial walls . Others The purpose of our report is to describe the presence of Kyrieleis plaques, distinct from vascular sheathing and frosted branch angiitis, associated with cytomegalovirus (CMV) retinitis. To our knowledge, this association appears to be rarely acknowledged and not previously reported.A 47-year-old Hispanic female with untreated human immunodeficiency virus (HIV) presented with decreased vision and floaters in her left eye for 1-week duration. She was diagnosed with both HIV and type II diabetes mellitus 4 months prior. On ophthalmic examination, best-corrected visual acuity was 20/25 in the right eye and 20/60 in the left eye. The anterior chambers were deep and quiet, and the intraocular pressures were 18 mm Hg in both eyes. A few inflammatory cells were present in the vitreous of the left eye. Funduscopic examination revealed moderate non-proliferative diabetic retinopathy in both eyes and an advancing border of white granular retinitis surrounding the inferior and temporal macula of the left eye. Kyrieleis plaques were present on the retinal arteries without any significant sheathing or involvement of the retinal veins Fig. . FluoresT. gondii, VZV, herpes simplex virus (HSV), T. pallidum, and tuberculosis. CD4 T cell count was 55 cells/μL, and HIV viral load was 193,065 copies/ml. Polymerase chain reaction of the aqueous was positive for CMV DNA  and negative for HSV and T. gondii DNA.Laboratory evaluation revealed an elevated serum CMV IgG antibody with a negative systemic work up for Mycobacterium avium complex prophylaxis, and atovaquone for Pneumocystis jiroveci pneumonia prophylaxis. Over the first 6 weeks as the retinitis resolved, the Kyrieleis plaques increased in number and become confluent along parts of the retinal arteries (Fig. The CMV retinitis was treated with intravitreal injections of foscarnet sodium and oral valganciclovir. The patient was also started on HAART, antiglycemic therapy, azithromycin for ies Fig. . The Kyries Fig. . On follCMV retinitis typically presents with focal areas of retinal necrosis with primary lesions usually located adjacent to blood vessels, secondary to hematogenous spread of the virus . On fundVascular involvement in CMV retinitis can result in vascular sheathing. Exudates around retinal vessels, more commonly veins, can result in focal areas of fluffy white cuffing or sheathing, with or without skip areas . When thKyrieleis plaques have been primarily reported in association with toxoplasmosis chorioretinitis. Although the cause of Kyrieleis plaques is unclear, the increase in these plaques following treatment and immune recovery in our case supports the theory of an immune response to an infectious agent and deposition of inflammatory debris as the etiology of these plaques. Kyrieleis plaques, although not specifically noted in many reports of infectious chorioretinitis, may be under reported. While many authors have concluded that Kyrieleis plaques are rare, Griffin and Bodian in 1959 felt that they may be more common than the literature suggests . We have