Recurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation.in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered “superfertile”, defined by a mean TTP of 3 months or less.Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL. Miscarriage is the most common complication of pregnancy. It is estimated that 30% of embryos are lost prior to implantation (pre-implantation loss) and a further 30% before 6 weeks gestation  Early pregnancy loss is widely viewed as a dichotomous disorder, attributed either to maternal factors or chromosomal errors in the conceptus. On the maternal side, numerous anatomical, endocrine, immunological, thrombophilic and genetic perturbations have been invoked to explain RPL, yet none are specific or prevalent et alFor most of the menstrual cycle, the endometrium is not primed for implantation. It only transiently acquires a receptive phenotype, starting approximately 6 days after the postovulatory progesterone surge and is estimated to last between 2 to 4 days et al. in this journal.Although the luminal endometrial epithelium is the primary barrier in the implantation process, the progesterone responses in this cellular compartment that underpin the receptive phenotype are mediated by signals derived from the underlying stromal cells Based on these observations, we hypothesized that impaired decidualization of the endometrium prior to conception predisposes for subsequent pregnancy failure, either by prolonging the implantation window, thereby disabling natural embryo selection, or by disrupting the maternal responses to embryonic signals. If correct, impaired decidualization and lack of natural embryo selection should not only lead to RPL but also be associated with paradoxical superfecundity, defined by persistent very short time-to-pregnancy (TTP) intervals.et alWe speculated that impaired decidualization of the stromal compartment may facilitate delayed implantation of compromised embryos by prolonging the window of endometrial receptivity, as suggested by the population study of Wilcox in vivo observations, we established primary cultures from 9 RPL patients and 12 controls , one of the earliest and most abundant glycoproteins secreted by embryonic trophoblast, on cultures . ESCs wecultures . In contcultures . The datP = 0.01 when adjusted for maternal age). In the absence of a suitable control group, we compared the observed incidence of achieving 3 or more pregnancies within 1, 3, or 6 months with the predicted likelihoods, based on an average MFR of 20% RPL patients often report short periods between pregnancies, which is the predictable clinical correlate of impaired embryo recognition and selection. To substantiate this observation, we analysed the time-to-pregnancy (TTP), expressed in months, in 2076 pregnancies reported by 560 women with a history of 3 or more consecutive miscarriages. Woman-based analysis of the mean and mode TTP showed that many RPL patients are highly fecund . MultivaPRL, a highly sensitive and specific decidual marker gene in the endometrium in vivo as well in primary cultures subjected to a decidualizing stimulus. However, we also obtained evidence that uterine receptivity is enhanced and prolonged in RPL patients, which in turn may facilitate delayed implantation of compromised embryos. Differentiating ESCs abundantly express PROK1, a cytokine that promotes embryo-uterine interaction via induction of leukaemia inhibitory factor (LIF) in endometrial epithelial cells Our study provides compelling evidence that impaired decidual programming of the endometrium is an important mechanism underlying RPL. This is based primarily on the observation that expression of We also found that RPL is further associated with paradoxical endometrial responses to embryonic hCG signalling, characterized by induction rather than inhibition of PRL and PROK1 expression in decidualizing ESCs. The ability of hCG to inhibit decidual PRL expression is in agreement with a previous study PRL and PROK1, the data demonstrate that endometrial programming in preparation of pregnancy is both quantitatively and qualitatively different in RPL, characterized by impaired decidualization of resident stromal cells, prolonged endometrial receptivity and a dysregulated maternal response to embryonic signals. This pathological pathway explains several established clinical features of RPL. For example, in view of the excess of chromosomally abnormal preimplantation human embryos Although our expression analysis was confined to Biologic fertility is measured using TTP PRL and PROK1 in timed endometrial biopsies from RPL patients is recapitulated upon differentiation of purified ESCs maintained in prolonged cultures. Such cellular ‘memory’ suggests that the endometrial decidual response is subject to epigenetic programming in humans A striking observation is that the aberrant expression of In summary, decidualization of uterine tissues is indispensable for placenta formation in pregnancy. We now provide evidence that failure to express an adequate decidual phenotype disables embryo recognition and selection upon implantation, which may lead to shorter TTP intervals but also predisposes to persistent pregnancy failure. Established risk factors for RPL, such as antiphospholipid antibodies and endocrine perturbations, may directly impact on uterine function, although our data suggest that the primary defect lies in the ability of ESCs to mount a decidual response. Together, these findings establish a novel pathological pathway that unifies maternal and embryonic causes of RPL.The Research Ethics Committee of each participating centre approved the study: Hammersmith and Queen Charlotte's & Chelsea Research Ethics Committee (1997/5065) and the Liverpool (Adult) Research Ethics Committee (REC reference number: 05/Q1505/147). Written informed consent was obtained from all participating subjects. All patients were investigated according to the standard clinic protocols Women were asked to use commercially available urine LH kits and to contact the research team at the time of their LH surge. All samples were obtained 6–10 days after the LH surge with a pipelle sampling device. Each biopsy was divided and one portion snap-frozen in liquid nitrogen for RNA analysis. The other portion was fixed in formalin for histological dating using standard criteria. The demographic details of the RPL and control groups are summarize in −6 M) for 2, 4 or 8 days. At each time-point, supernatants were collected and frozen and cells harvested for mRNA analysis. Additional cultures were established from control and RPL patients  and L19-antisense (5′-GCA GCC GGC GCA AA-3′); PRL-sense (5′-AAG CTG TAG AGA TTG AGG AGC AAA C-3′) and PRL-antisense (5′-TCA GGA TGA ACC TGG CTG ACT A-3′); PROK1-sense(5′-GTG CCA CCC CGG CAG-3′) and PROK1-antisense (5′-AGC AAG GAC AGG TGT GGT GC-3′). Secreted PROK1 and PRL levels were determined using ELISAs .Total RNA was extracted from frozen tissue or cell cultures using Trizol Reagent , treated with DNAseI , reversed transcribed, and the resulting cDNA subjected to real-time quantitative PCR analysis using an ABI Prism 7700 Sequence Detection System  with the following gene-specific primer pairs: L19-sense , were adjusted for maternal age at time of pregnancy. Sensitivity analyses excluding women with other pregnancy complications  showed similar effects. The unit of analysis was a pregnancy.t-test and Mann-Whitney U test were used to determine statistical significance between two groups. For multiple comparisons, ANOVA test with Bonferroni correction was used. P<0.05 was considered significant. The association between the relevant pregnancy outcome and TTP was explored using logistic regression analysis, with effects on risk being estimated by odds ratios with 95% confidence intervals. Since women could have more than one pregnancy outcome in the analysis, a robust method based on the “sandwich estimate” Student's Figure S1RPL is associated with impaired PROK1 and PRL secretion by decidualizing ESCs. Secreted PROK1 (A) and PRL (B) levels accumulated over 48 hours in the supernatants of confluent primary ESC cultures decidualization with 8-Br-cAMP and MPA for 8 days. Primary cultures were established from 10 RPL patients and 10 control (CON) subjects. Horizontal bars indicate the median expression in each group.(0.61 MB EPS)Click here for additional data file.Table S1Analysis of timed endometrial biopsies - patient characteristics. The data presented are mean ± standard deviation. * indicates P<0.001.(0.03 MB DOC)Click here for additional data file.Table S2Analysis of fetal versus biochemical RPL - patient characteristics. The data presented are mean ± standard deviation. * indicates P<0.05. A ‘fetal’ loss was defined as a pregnancy failure between 6–13 weeks gestation with prior ultrasound evidence of fetal development. A ‘biochemical’ loss was defined as a miscarriage at 4–6 weeks gestation with ultrasound evidence of either an intrauterine pregnancy sac with no fetus or retained products of conception.(0.03 MB DOC)Click here for additional data file.Table S3Time-course analysis - patient and culture characteristics. The data presented are mean ± standard deviation. LMP  =  last menstrual period. * indicates P<0.001.(0.03 MB DOC)Click here for additional data file.Table S4hCG analysis - patient characteristics. The data presented are mean ± standard deviation. LMP  =  last menstrual period. * indicates P<0.001.(0.03 MB DOC)Click here for additional data file.Table S5Analysis of time-to-pregnancy (TTP) in women with RPL. SD  =  standard deviation; NS  =  not significant.(0.03 MB DOC)Click here for additional data file.


Silene vulgaris exhibits high mitochondrial diversity and occasional paternal inheritance of mitochondrial markers. Here we address whether variation in DNA molecular markers is correlated with variation in transcription of mitochondrial genes in S. vulgaris collected from natural populations.Although rapid changes in copy number and gene order are common within plant mitochondrial genomes, associated patterns of gene transcription are underinvestigated. Previous studies have shown that the gynodioecious plant species cox1 and atp1, in offspring of ten plants from a natural population of S. vulgaris in Central Europe. We also investigated transcription profiles of the atp1 and cox1 genes. Most DNA haplotypes and transcription profiles were maternally inherited; for these, transcription profiles were associated with specific mitochondrial DNA haplotypes. One individual exhibited a pattern consistent with paternal inheritance of mitochondrial DNA; this individual exhibited a transcription profile suggestive of paternal but inconsistent with maternal inheritance. We found no associations between gender and transcript profiles.We analyzed RFLP variation in two mitochondrial genes, S. vulgaris.Specific transcription profiles of mitochondrial genes were associated with specific mitochondrial DNA haplotypes in a natural population of a gynodioecious species Our findings suggest the potential for a causal association between rearrangements in the plant mt genome and transcription product variation. Despite numerous investigations of the structure and dynamics of the plant mitochondrial (mt) genome over the last decades, little is known about whether patterns of DNA sequence variation are associated with variation in transcription products in plants from natural populations. Unlike animals that possess a small, compact, gene-dense, circular mt genome, land plants have mt genome which is organized as a collection of circular and linear molecules of various sizes . Plant mArabidopsis thaliana cytochromatp1 gene in heteroplasmic individuals, which might have been overlooked by PCR-RFLP screening. The a tp1 haplotype KovA differs from haplotypes KovB and KovC by the presence of one SmaI site. When a plant is homoplasmic for the atp1 haplotype KovA, SmaI cuts all atp1 copies, revealing two bands on an agarose gel, and PCR amplification of genomic DNA pre-digested with SmaI does not generate any fragments. If the atp1 haplotypes KovB or KovC are found in very low copy number, however, SmaI restriction sites are not found in all copies of atp1 and PCR amplification of genomic DNA pre-digested by SmaI will be positive. For the knock-back analyses, we digested about 200 ng (1 μl) of genomic DNA with SmaI at 30°C in a final volume of 20 μl for 6 hours according to the manufacturer's directions (Fermentas). In a control reaction, water replaced the restriction enzyme. Two μl of digestion reaction mixture was used in PCR reactions with atp1 specific primers. The atp1 PCR fragment was produced if (1) genomic DNA contained rare copies of the atp1 gene different from the KovA haplotype, or if (2) genomic DNA of homoplasmic individual of the KovA haplotype was partially digested. As the case (2) represents an artifact and could be incorrectly interpreted as evidence for heteroplasmy; we confirmed the absence of SmaI site in atp1 PCR fragment by an additional SmaI digestion. Only those individuals, which provided an atp1 PCR fragment not cleavable by SmaI, were considered heteroplasmic.The "knock-back" approach developed by  was adopcox1 and atp1 genes in two separate assays. This kind of mt markers is referred to as Southern-RFLP markers to distinguish them from PCR-RFLP markers located in the coding regions. Five hundred ng of genomic DNA was digested with either HindIII or EcoRI, electrophoretically separated overnight on a 0.9% agarose gel, transferred to a membrane and hybridized with non-radioactively labeled cox1 and atp1 probes as described by [cox1 and atp1 Southern-RFLP patterns were estimated using the same membrane. Relative intensity of individual bands in the same run was determined using Phosphoimager FLA7000.RFLP variation was assessed for the HindIII and EcoRI restriction sites flanking the ribed by . The comcox1 or atp1 homologs, either full length or truncated, that are present either in the nucleus or in the mitochondrion, although the location in chloroplast cannot be also excluded. We also did not identify any atp1 or cox1 haplotypes that contained EcoRI or HindIII restriction sites, therefore one Southern band corresponds to one gene copy (full length or truncated).Faint bands were present in many Southern-RFLP patterns, in varying positions. Because the post-hybridization washes were of very high stringency, these bands cannot represent non-specific targets. These bands identify Total RNA was extracted from the flower buds (1 - 4 mm in size) of the greenhouse grown plants using an RNeasy Plant Mini kit (Qiagen). One μg of total RNA was loaded on agarose gel . RNA loading buffer was composed of 50% formamide (deionised), 6% formaldehyde, 1 × MOPS buffer, 10% glycerol and 0.05% bromophenolblue (w/v). After overnight electrophoresis, RNA was transferred to a positively charged membrane Hybond N+ (Amersham) by capillary blotting. The same probes as used for Southern hybridization were applied in EasyHyb buffer (Roche). Membranes were hybridized at 52°C overnight, washed at very high stringency , and detected using CDPStar (Roche) as a substrate. Exposure times of < 30 min were sufficient to detect strong bands on Hyperfilm (Amersham). If necessary, membranes were stripped with deionised formamide at 80°C according to the manufacturer's protocol.S. vulgaris plants. KM ran and optimized Northern hybridizations and contributed to raw data interpretations. MSO helped to interpret the results and made large contributions to manuscript writing. HS collected the plant material, designed the experiments and drafted the manuscript. All authors read and approved the final manuscript.HOA performed Southern hybridizations and PCR-RFLP screens of


The transduction efficiency of NIH-OVCAR3, MDAH 2774 and SKOV3 cells with rAAV-GFP particles was low (< 1%) and did not improve by increasing the number of particles/cell. Repeated administration and continued exposure of NIH-OVCAR3 and MDAH 2774 improved transduction to over 3%. In contrast, these cell lines were more efficiently transduced by rAAV-GFP in the presence of adenovirus (~15%) and by rAd-GFP (> 50%). These results indicate that in contrast to rAd vectors, rAAV particles are not suitable for therapeutic gene transfer in ovarian cancer cells unless efficient help can be provided to mediate ss to ds DNA conversion.© 2001 Cancer Research Campaign  http://www.bjcancer.comRecombinant adeno-associated virus (rAAV) vectors have emerged as vehicles for gene therapy. In addition, anti-neoplastic properties have been attributed to wild-type AAV. To take advantage of both features and to overcome technical problems associated with rAAV preparation, we developed a production method in which rAAV particles are amplified in an infectious cycle in the presence of wtAAV. This results in a 10


The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV). Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection.We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6), capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60%) and AAV6.Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina. As a therapeutic device, adeno-associated viral (AAV) mediated ocular gene therapy holds tremendous promise for treating and potentially curing a variety of inherited retinal degenerative diseases, such as glaucoma, age-related macular degeneration (AMD), retinitis pigmentosa, and other blinding diseases. The therapeutic potential of AAV-mediated gene delivery to the retina has best been illustrated by the positive results from three phase I clinical trials for Leber's Congenital Amaurosis (LCA), a rare autosomal recessive blinding disease caused by loss of function mutations in the visual cycle enzyme retinol isomerase (RPE65) required to synthesize 11-cis retinal http://www.sph.uth.tmc.edu/retnet). This large genetic heterogeneity, leading to a common phenotype of photoreceptor cell death, suggests that a mutation-independent neuroprotective strategy may be the most practical solution to a clinical treatment. Therefore, there is a strong need for a simple, safe, and broad-based ocular gene delivery strategy that provides sustained production of a neuroprotective or anti-angiogenic therapeutic for these conditions.Importantly, genetic heterogeneity is a key feature of retinal degeneration conditions, and to date, over 130 genes with mutations causing one or more forms of inherited orphan retinal degenerative diseases have been cloned, and over 50 more have been identified based on candidate gene studies or linkage mapping  that assemble into a 60-mer capsid shell. This viral capsid mediates the ability of AAV vectors to overcome many of the biological barriers of viral transduction–including cell surface receptor binding, endocytosis, intracellular trafficking, and unpackaging in the nucleus Several viral vector systems have been evaluated for ocular gene delivery; however, AAV offers distinct advantages in safety, stability, and efficiency. AAV belongs to the Subretinal administration of many of these AAV variants has led to efficient gene expression in a number of retinal neurons and epithelia, including photoreceptors and RPE Despite these benefits, intravitreal injections pose significant challenges. First, dilution of vector into the vitreal fluid leads to lower vector concentrations compared to subretinal injections, a particular concern for larger mammals. However, intravitreal vector administration into macaque eyes showed that AAV is still able to overcome this challenge and lead to retinal transduction + and water homeostasis, free radical scavenging and oxidative protection, neurotrophic secretion, and neurotransmitter and retinoid uptake and recycling Müller cells serve numerous, significant physiological functions in the retina including but not limited to: metabolic support of neurons, KRecently, the efficiency of this approach has been demonstrated as Müller cell secretion of neurotrophin-4 (NT-4) was shown to protect photoreceptors from oxidative stress in a mouse model of neovascularization through intravitreal injection in vitro and in vivo relative to their parent serotypes as well as variants with enhanced permissivity to astrocytes in vivo through selection in primary human astrocytes in vivo. Efficient and specific intravitreal transduction of Müller cells by this vector will permit the development of more innocuous and effective treatments for retinal degenerative conditions through panretinal secretion of neuroprotective or anti-angiogenic factors, as well as new approaches to investigate fundamental questions in the biological role of this cell type in the retina.Recently, we applied directed evolution to select variants from combinatorial libraries that demonstrate a diverse range of cellular tropisms in vitro and rat astrocytes in vivo using highly diverse AAV libraries (>107) in vitro) with several distinct libraries: (1) an AAV2 random mutagenesis library generated via error prone PCR cap genes of 7 natural human and non-human AAV serotypes We recently evolved several novel AAV capsids that efficiently transduced both primary human astrocytes in vitro astrocyte infectivity  library, that showed a dramatic increase in specificity and efficiency for Müller cells relative to controls at three weeks post-injection . To analyze the contributions of each of these mutations to ShH10's novel phenotype, single point mutants and all potential double mutants were generated from the AAV6 cap gene via site-directed mutagenesis. Each resulting variant was used to package rAAV-CMV-GFP and was purified via iodixanol gradient ultracentrifugation. To characterize the in vitro infectivity of these mutants, and in particular their glycan dependence in light of the substantial role proteoglycans and glycoproteins play in AAV transduction ShH10 is highly Müller cell selective, while AAV6 yields no detectable retinal expression upon intravitreal administration, yet intriguingly ShH10 differs from AAV6 at only four residues: I319V, N451D, D532N, and H642N  reveals that ShH10 does not share mutations with the other evolved variants except for mutation H642N with variant ShH13, which has been shown in our previous work to have an enhanced tropism for Müller cells upon subretinal injection The Importantly, in beginning to mechanistically investigate viral infection in the retina, our previous studies have shown that AAV binding at the inner limiting membrane (ILM) is implicated in mediating transduction via intravitreal injection of AAV serotype 2, and to a lesser extent 8 and 9 Fig. S2) may then suggest a potential dueling role in achieving optimum transduction, as too high of an affinity may also encumber efficient viral penetration in the retina.In first binding to the ILM, the presence of high levels of heparan sulfate in the proteoglycan matrix of the ILM may mediate viral accumulation at this site and act as a ‘sink’ Once it traverses the ILM, ShH10 must bind Müller cells. Recent studies have implicated the epidermal growth factor receptor (EGFR), which is expressed on the surface of Müller cells, as a coreceptor for AAV6 transduction Preliminary results suggest that the N451D mutation may partly be responsible for such potentially improved receptor interactions, as this substitution is sufficient to confer a limited Müller cell intravitreal infectivity Fig. 7. ShH10 exhibited enhanced transduction of Müller cells underlying areas of retinal vasculature, suggesting that its infectivity of retinal astrocytes may also offer a possible mechanistic explanation for its tropism. Whether this is just a result of commonalities between these glial subtypes that allow for ShH10 permissivity in both or whether infection of one type may facilitate adjacent infection of the other through localized binding remains to be analyzed. Regardless, this property would be useful in infecting the activated Müller cells in neovascularized tissue to deliver neurotrophic factors to sites of vascular abnormalities in vivo through Müller-specific AAV-mediated RNAi-knockdown of factors implicated in 11-cis-retinol generation Overall, efficient and selective intravitreal transduction of Müller cells by AAV may provide safer and more effective treatments for retinal degenerative conditions through secretion of neuroprotective factors, while also enabling basic investigations to gain a greater understanding of Müller cell physiology. For instance, the uncertain role of Müller cells in the visual cycle of cone photoreceptors can be examined in vivo selections could provide a more powerful approach to directly target specific cell types in the retina. For example, the targeting of engineered light gated channels to specific subpopulations of retinal cells, such as ON bipolar cells or ON/OFF retinal ganglion cells For future studies, the use of highly diverse AAV libraries coupled with Vectors were produced by the plasmid co-transfection method 12 vg/ml of AAV dsCAG-GFP, was made with direct observation of the needle in the center of the vitreous cavity.Adult wild type Spaque Dawley rats were used for all studies. All animal procedures were conducted according to the ARVO Statement for the Use of Animals and the guidelines of the Office of Laboratory Animal Care at the University of California, Berkeley. Before vector administration, rats were anesthetized with ketamine (72 mg/kg) and xylazine (64 mg/kg) by intraperitoneal injection. An ultrafine 30 1/2-gauge disposable needle was passed through the sclera, at the equator and next to the limbus, into the vitreous cavity. Injection of 5 µl, containing 1−5×10Fundus imaging was performed one to eight weeks after injection with a fundus camera  equipped with a wide angle 130° retinopathy of prematurity (ROP) lens to monitor eGFP expression in live, anesthetized rats. Pupils were dilated for fundus imaging with phenylephrine (2.5%) and atropine sulfate (1%).One to eight weeks after vector injection, rats were humanely euthanized, the eyes were enucleated, a hole was introduced in the cornea, and tissue was fixed with 10% neutral buffered formalin for 2–3 hours. The cornea and lens were removed. The eyecups were washed in PBS followed by 30% sucrose in the same buffer overnight. Eyes were then embedded in optimal cutting temperature embedding compound  and oriented for 10 µm thick transverse retinal sections.Tissue sections were rehydrated in PBS for 5 min, followed by incubation in a blocking solution of 1% BSA, 0.5% Triton X-100, and 2% normal donkey serum in PBS for 2–3 hours. Slides were then incubated with commercial monoclonal antibodies raised against glutamine synethetase in rabbit (Sigma G2781) at a 1∶3000 dilution, calbindin (Abcam ab11426-50) in rabbit at a 1∶1000 dilution, vimentin in mouse (Dako M0725) at a 1∶1000 dilution, or laminin in rabbit  at 1∶100 in blocking solution, overnight at 4°C.The sections were then incubated with Cy3-conjugated secondary anti-rabbit or anti-mouse antibody (Molecular Probes) at a 1∶1000 dilution in blocking solution for 2 hours at room temperature. The results were examined by fluorescence microscopy using an Axiophot microscope  equipped with a Xcite PC200 light source and QCapturePro camera or a confocal microscope . Transduction profiles were analyzed by counting individual cells from whole retinas in 15 µm cryosections (n = 6) using fluorescence microscopy. Efficiencies were calculated by dividing the total number of these transduced Müller cells by the total number of Müller cells in the retinal slice (mm) in which these cells were present (n = 6).4 cells  in 12-well plates. Cells were transduced with rAAV GFP vectors at a gMOI of 103–105 (n = 3), and the percentage of GFP-expressing cells was determined by flow cytometry 48 hours post-infection.Transduction studies using rAAV CMV-GFP were performed with 5×10Figure S1Sequence analysis of novel AAV variants. Sequence comparison between ShH10 and ShH13 along with parent AAV serotypes 2 and 6.(0.03 MB PDF)Click here for additional data file.Figure S2Heparin binding affinity of ShH10, AAV2, and AAV6. Elution profile from a heparin column for ShH10, AAV2, and AAV6. Y-axis values represent the fraction of virus eluted, and the X-axis represents the concentration of NaCl in the eluant (mM).(8.32 MB TIF)Click here for additional data file.


Nine patients receiving the CRC Phase II study schedule of temozolomide were assessed with [18F]fluorodeoxyglucose ([18F]FDG) dynamic positron emission tomography (PET) scans prior to and 14 days after treatment with temozolomide given as 750–1000 mg m−2over 5 days every 28 days. Tumour MRGlu was calculated and compared with objective response at 8 weeks. Pretreatment MRGlu was higher in responders than non-responders. The responding patient group had a greater than 25% reduction in MRGlu in regions of high focal tumour uptake (HFU). Whole tumour changes in MRGlu did not correlate with response. Percentage change in HFU standardized uptake value (SUV) did discriminate the responding from the non-responding patients, but not as well as with MRGlu. Large differences also occurred in the normal brain SUV following treatment. Thus, MRGlu appeared to be a more sensitive discriminator of response than the simplified static SUV analysis. Changes in MRGlu may reflect the degree of cell kill following chemotherapy and so may provide an objective, quantitative subclinical measure of response to therapy. © 2000 Cancer Research CampaignQuantitation of metabolic changes in tumours may provide an objective measure of clinical and subclinical response to anticancer therapy. This pilot study assesses the value of quantitation of metabolic rate of glucose (MRGlu) measured in mmol min


Key to understanding the epidemiology and pathogenesis of prion diseases, including chronic wasting disease (CWD) of cervids, is determining the mode of transmission from one individual to another. We have previously reported that saliva and blood from CWD-infected deer contain sufficient infectious prions to transmit disease upon passage into naïve deer. Here we again use bioassays in deer to show that blood and saliva of pre-symptomatic deer contain infectious prions capable of infecting naïve deer and that naïve deer exposed only to environmental fomites from the suites of CWD-infected deer acquired CWD infection after a period of 15 months post initial exposure. These results help to further explain the basis for the facile transmission of CWD, highlight the complexities associated with CWD transmission among cervids in their natural environment, emphasize the potential utility of blood-based testing to detect pre-clinical CWD infection, and could augur similar transmission dynamics in other prion infections. Chronic wasting disease (CWD) is a fatal transmissible spongiform encephalopathy (TSE), or prion disease, marked by a high transmission efficiency among cervids. Since its identification in cervid populations of northern Colorado and southeastern Wyoming We undertook the present studies to confirm our earlier findings on prion excretion through bioassays in white-tailed deer, but also to determine: (a) whether CWD prions are present in body fluids and excretions during the pre-clinical phase of infection, and (b) whether repeated environmental fomite exposure alone, vs. direct animal-to-animal contact, was sufficient to transmit CWD in deer.CWD tonsil biopsy positive at 12 months pi, but not at earlier sampling intervals (CWD in the medulla oblongata at the level of the obex (medulla at obex) and in lymphoid tissue  , 2. Two ntervals . At 19 md tissue .CWD was detected in tonsil of 1 of the 3 inoculated deer at 12 months pi, but not at earlier time points. By 19 months pi, study termination, a second animal was CWD+, by detection of PrPCWD in brain and lymphoid tissue. The remaining deer was of necessity terminated at 16 months pi due to unmanageable aggressive behavior. This animal was CWD negative as determined by extensive western blot and IHC analysis  oral doses of urine and feces from CWD+ source deer ranging from pre-clinical to advanced clinical disease , 2. All y course , 2.CWD-positive at 15 months pi. At study termination, 19 months pi, both animals were CWD+ , or 12 months (n = 2) pi  pi , 2.Project-dedicated caretakers intimately familiar with each animal observed the deer daily. Subtle clinical signs consistent with CWD were detected 12–20 months pi in 3 of 4 positive control animals (cohort 5), 2 of 3 animals receiving blood transfusion (cohort 1) and 1 of 3 deer orally inoculated with saliva (cohort 2). Clinical disease onset manifested primarily as perceived body muscle-mass reduction and gradual weight loss, which reached ≥20% of maximum body weight over 2 to 8 months. Other clinical signs included: rough hair coat due to piloerection and a body stance characterized by a low head position and wide leg stance. Changes in behavior included hyperphagia and polydipsia despite weight loss, and stereotypic movements including head tossing, repetitive and exaggerated lifting of the legs, diminished alertness, and occasionally aggressive behavior in the advanced stage of disease. Animals were euthanized when they displayed advanced clinical signs of CWD or at 19 months pi.CWD deposition in infected deer CWD correlated with each other and with previous positive tonsil-biopsy results in 10 of 13 animals  deer and 2 of 3 cohort 1 (blood) tonsil-biopsy-positive deer were euthanized due to advanced clinical signs of CWD at 13, 24, 26 and 29 months pi, respectively. Earlier studies established that medulla at obex, tonsil and retropharyngeal lymph node had highest frequency of PrP animals . In 3 annecropsy . Westernnecropsy  in all cpsy  in alimentary tract tissue, and have suggested saliva as a possible vehicle for prion transmission et al reported the presence of the pathological prion protein in both major and minor salivary glands of naturally and experimentally infected sheep The presence of infectious CWD prions in saliva suggests the potential for disease transmission via grooming interactions, shared water sources and communal feeding grounds—especially in high density cervid populations, such as those that exist for some cervid species during the breeding season, at baiting sites, in captivity, and in low predation situations. Other investigators have detected the presence of the aberrant misfolded prion protein  et al reported PMCA amplification of PrPSC from the blood of scrapie-infected sheep CWD test negative animals that may harbor infectious prions not manifested in the observation periods used in our CWD studies.Direct detection of blood-borne PrPRES has been found in peripheral organs of some sporadic CJD patients RES is not limited to vCJD. In an ongoing study of sixty-six individuals who received blood products from asymptomatic blood donors who later developed vCJD RES was detected in lymphoid tissue but not brain, thus providing presumptive evidence for a case of subclinical infection Hunter and colleagues in vitro conversion studies have indicated an inefficient conversion of human PrP into a protease-resistant form Additional cases of subclinical human prion disease may exist. While et al in the natural environment CWD detection, was seen in as little as 15 months post initial exposure. These results are also consistent with the findings of Georgsson Previous studies have confirmed direct animal-to-animal contact—horizontal transmission—as an efficient mode for prion disease transmission CWD in the three deer inoculated with urine and feces. Our earlier report prnp gene G/S polymorphism at codon 96 remained negative 19 mo. pi. In the present study all three deer inoculated with urine and feces expressed the G/G polymorphism at prnp codon 96, which is associated with susceptibility to CWD infection CWD in the obex or lymphoid tissues of deer with either G/G or G/S polymorphisms at 19 mo pi. Although both of our bioassay studies in deer have failed to transmit CWD infection by oral exposure to urine and feces from CWD-infected deer, these results must still be interpreted with caution in light of ongoing PMCA and cervid transgenic mouse intracerebral bioassay studies which suggest that very low concentrations of prions may be present in urine and feces of CWD+ cervids CWD, or a larger dose of inoculum by the oral route is necessary for efficient passage of prions across the alimentary mucosa.Previous studies have postulated that environmental contamination by excreta from infected cervids seems the most plausible explanation for the dissemination of CWD In summary, the results reported here reconfirm that blood and saliva are sources of infectious CWD prions, consistent with previous findings All animals were handled in strict accordance with good animal practice as defined by relevant national and/or local animal welfare bodies, and all animal work was approved by Colorado State University Animal and Care Use Committee .White-tailed deer fawns were provided by the Warnell School of Forestry and Natural Resources, University of Georgia, Athens (UGA)—a region where CWD has not been detected. The deer fawns were hand-raised and human and indoor-adapted before overnight transport directly to the Colorado State University (CSU) CWD research indoor isolation facility without contact with the native Colorado environment. The 4-month-old fawns were adapted to the facility housing conditions and diet for 2 months before study start.All white-tailed deer were genotyped to determine GG/GS (codon 96) status by the laboratory of Dr. Katherine O'Rourke, USDA-ARS, Pullman, WA.Protocols to preclude extraneous exposure and cross-contamination between cohorts of animals as previously described Groups of n = 2 to 4 six-month-old fawns  were houThe following inocula were used: Blood , saliva  were obtained from serial sample collections of pre-clinical CWD-inoculated, tonsil biopsy positive deer, while urine and feces  were obtained from serial sample collections of pre-clinical and clinical CWD-inoculated, tonsil biopsy positive deer housed in the CSU CWD isolation facilityCWD).All animals were monitored for evidence of CWD infection by serial tonsil biopsies taken at 3, 6, 12 months pi, and at study termination. Tonsil tissue was divided and equal portions either stored at −70°C or fixed in 10% formalin for 24 hours before processing for IHC. At the same sampling intervals, blood, saliva, feces, and urine were collected from each animal and stored at −70°C. At necropsy, palatine tonsils, brainstem (medulla at the obex) and retropharyngeal lymph nodes, as well as other tissues, were collected for examination by IHC and western blotting (WB) to identify the presence of the protease-resistant prion protein associated with CWD . Twenty percent (20%) w/v homogenates were prepared in NP-40 buffer  by Fastprep™ disruption at setting 6.5 for 45 seconds. Twenty-five µl of each homogenate was mixed with 5 µl proteinase K (PK) (Invitrogen) to a final concentration of 25 µg/ml and incubated for 30 minutes at 37°C with shaking. Proteinase K activity was stopped with 4 µl 200 mM Pefablock SC and an equivalent volume of each sample was mixed with 10 µl sample buffer , 5 µl NP-40 buffer , heated to 95°C for 5 minutes and separated by 12% Bis-Tris precast polyacrylamide gel electrophoresis (PAGE) (Invitrogen) at 150 volts for 2.5 hours in 1× MOPS (Invitrogen). PAGE separated proteins were transferred to polyvinylidene fluoride (PVDF) membrane for 1 hour at 100 volts in transfer buffer . After the PVDF membranes were blocked overnight at room temperature in Pierce Blocker™ they were probed with the PrP specific antibody BAR224 (kindly supplied by Dr. J. Grassi) followed by horse radish peroxidase (HRP)-goat anti-mouse IgG diluted in Pierce Blocker™. Membranes were washed for 1 hour after blocking and between antibodies with wash buffer . To visualize PrP bands the PVDF membranes were developed with the Amersham™ ECL detection system and a digital GelDoc™ (Fuji Intelligent dark box) using LAS-3000 Lite ImageReader software.Immunohistochemistry (IHC) was performed by employing protocols as described by Spraker et alCWD specific primary antibody BAR224 and a biotinylated secondary goat anti mouse antibody (Ventana). After autostaining, the slides were quickly rinsed in a warm water detergent solution, passed through a series of dehydration baths and cover-slipped.


Post-operative outcomes may be improved by the use of flow related end-points for intra-venous fluid and/or low dose inotropic therapy. The mechanisms underlying this benefit remain uncertain. The objective of this study was to assess the effects of stroke volume guided intra-venous fluid and low dose dopexamine on tissue microvascular flow and oxygenation and inflammatory markers in patients undergoing major gastrointestinal surgery.Randomised, controlled, single blind study of patients admitted to a university hospital critical care unit following major gastrointestinal surgery. For eight hours after surgery, intra-venous fluid therapy was guided by measurements of central venous pressure (CVP group), or stroke volume (SV group). In a third group stroke volume guided fluid therapy was combined with dopexamine (0.5 mcg/kg/min) (SV & DPX group).n = 45 per group). In the SV & DPX group, increased global oxygen delivery was associated with improved sublingual (P < 0.05) and cutaneous microvascular flow (P < 0.005) . Microvascular flow remained constant in the SV group but deteriorated in the CVP group (P < 0.05). Cutaneous tissue oxygen partial pressure (PtO2) (Clark electrode) improved only in the SV & DPX group (P < 0.001). There were no differences in serum inflammatory markers. There were no differences in overall complication rates between the groups although acute kidney injury was more frequent in the CVP group (CVP group ten patients (22%); pooled SV and SV & DPX groups seven patients (8%); P = 0.03) .135 patients were recruited (Stroke volume guided fluid and low dose inotropic therapy was associated with improved global oxygen delivery, microvascular flow and tissue oxygenation but no differences in the inflammatory response to surgery. These observations may explain improved clinical outcomes associated with this treatment in previous trials.ISRCTN 94850719 Complications are common following major non-cardiac surgery and represent an important cause of avoidable morbidity and mortality -3. Estim2), reduced venous oxygen saturation (mixed venous haemoglobin saturation with oxygen (SvO2) central venous haemoglobin saturation with oxygen (ScvO2)) and poor outcomes following major surgery is well recognised [The association between low cardiac output, inadequate global oxygen delivery . Written informed consent was obtained from all patients prior to surgery. Participants were randomly allocated to one of three treatment groups by computer-generated random sequence in blocks of nine. Groups were stratified according to surgical procedure . Study group allocations were placed in serially numbered opaque envelopes.2 (> 94%), temperature (36 to 37°C), serum base excess (-2 to +2 mmol/l) and partial pressure of arterial carbon dioxide . Cardiac output monitoring was not used during surgery. Following surgery, all patients were admitted to critical care. For the eight-hour intervention period, either a doctor (SJ) or nurse  administered one of three allocated haemodynamic protocols as described below. These protocols are similar to those used in a previous trial [General anaesthesia was standardised and included intravenous fentanyl, propofol and atracurium for induction of anaesthesia and maintenance with inhaled isoflurane in oxygen-enriched air and epidural analgesia. Clinical staff administered intravenous fluids, blood products and, if required, vasoactive drugs in order to maintain routine physiological, haematological and biochemical parameters within normal ranges as follows: pulse rate (60 to 100 bpm), mean arterial pressure (60 to 100 mmHg), central venous pressure (CVP) (6 to 12 mmHg), urine output (> 25 ml/hr), haemoglobin (> 8 g/dl), SpOus trial .Intravenous lactated Ringer's solution was administered at 1 ml/kg/hr for maintenance requirements. Patients received additional 250 ml fluid challenges with intravenous colloid solution  to achieve an optimal value of CVP. Colloid solution was administered in one or more rapid boluses to achieve a sustained rise in CVP of at least 2 mmHg for 20 minutes or more. If CVP decreased, fluid challenges were repeated to establish whether the patient was fluid responsive.Intravenous lactated Ringer's solution was administered at 1 ml/kg/hr. Patients received additional 250 ml fluid challenges with intravenous colloid solution to achieve an optimal value of stroke volume. Colloid solution was administered in one or more rapid boluses to identify whether the patient was fluid responsive. A stroke volume response to fluid was defined as a sustained rise in stroke volume of at least 10% for 20 minutes or more. When a patient was identified as stroke volume responsive to fluid, further 250 ml boluses of fluid were administered until a plateau value was achieved. If stroke volume decreased, fluid challenges were repeated to establish whether the patient was fluid responsive.2 index (DO2I) but was decreased or discontinued in patients with evidence of myocardial ischaemia or tachycardia .Intravenous lactated Ringer's solution was administered at 1 ml/kg/hr. Patients received additional fluid challenges with colloid solution to achieve an optimal value of stroke volume in an identical fashion to patients in the SV group. In addition, a continuous intravenous infusion of dopexamine was administered at 0.5 μg/kg/min . This infusion rate was not adjusted to achieve a specific value for cardiac output or DOOnly the member of the research team who delivered the intervention was aware of the study group allocation. Cardiac output data were made available to clinical staff only on specific request. The reasons for this and any subsequent changes in treatment were documented by research staff. Dummy infusions were used in patients not allocated to receive dopexamine. All other management decisions were taken by clinical staff.Sublingual microvascular flow was evaluated before surgery and at 0, 2, 4, 6 and 8 hours immediately after surgery using sidestream darkfield (SDF) imaging with a ×5 objective lens  . Image a2) was measured before surgery and at hour 0, 2, 4, 6 and 8 hours after surgery at two sites on the abdominal wall using a Clark electrode . PtO2 probes warm the skin to 44°C minimising artefact due to local vasoconstriction.Cutaneous red blood cell flux was measured before surgery and at 0, 4 and 8 hours after surgery at two sites on the forearm by laser Doppler flowmetry . Baseline red cell flux on the forearm was measured and following this, the post-occlusive hyperaemic response was examined by inflating a cuff around the upper arm to 20 mmHg above systolic pressure for three minutes and measuring the changes in red cell flux on releasing the pressure in the cuff. The difference between baseline flux and peak hyperaemia was then evaluated at each time point. Cutaneous tissue oxygen partial pressure .Arterial and central venous blood samples were taken at hour 0, 2, 4, 6 and 8 after surgery from indwelling catheters for analysis of arterial haemoglobin saturation with oxygen, ScvOSerum samples were obtained from all patients following induction of anaesthesia but prior to surgery. Further serum samples were obtained immediately following surgery, at the end of the intervention period and 24 hours after the end of surgery. These samples were centrifuged at 3,000 g for 10 minutes and stored at -80°C. Subsequent analysis of IL1 beta, IL6, IL8, and TNFα was performed using a multi-array electro-chemiluminescence technique . Levels of soluble inter-cellular adhesion molecule 1 (ICAM-1) were quantified using a similar technique.Clinical outcomes data for each patient were collected by a member of the research team who was unaware of study group allocation and then verified by the senior investigator who was also unaware of the study group allocation. Estimated glomerular filtration rate (eGFR) was calculated preoperatively and on day seven after surgery from serum creatinine, age, race and gender using the Modification of Diet in Renal Disease equation . Patient2 between each of the intervention groups and the control group. Trends in physiological variables over time within groups were tested using one-way repeated measures analysis of variance (ANOVA) or Friedmann test. Differences in physiological variables between groups were tested using two-way repeated measures ANOVA, the t test and one-way ANOVA with post hoc t-test with Bonferroni correction. Categorical variables were tested with the Chi squared or Fisher's exact tests. Statistical analysis was performed using GraphPad Prism version 4.0 . Analysis was performed on an intention-to-treat basis including all randomised patients. Significance was set at P < 0.05. Data are presented as mean (standard deviation) where normally distributed or median (interquartile range) where not normally distributed.Assuming a 5% type I error rate and an 80% type II error rate, it was calculated that 45 patients would be required in each group to detect a 10 mmHg difference in PtOP = 0.11). One patient randomised to the SV & DPX group developed myocardial ischaemia during surgery and, in accordance with the study protocol, did not receive dopexamine. In five patients the dose of dopexamine was reduced because of an increase in heart rate and in one patient, dopexamine was subsequently discontinued. On only one occasion, a clinician asked to view a patient's cardiac output data because of concern that poor cardiac function might have been complicated by pulmonary oedema. This information did not prompt any changes in treatment. No patients received additional inotropic therapy during the intervention period.Between December 2007 and February 2009, 135 patients were recruited . Consequently, a post hoc analysis of the predefined renal outcome was performed. Fewer patients developed acute kidney injury in the pooled SV and SV & DPX groups within seven days of surgery  associated with stroke volume-guided therapy. This finding is consistent with a recent meta-analysis suggesting that haemodynamic optimisation protects renal function in surgical patients [This randomised controlled trial used physiological end-points and was not designed to identify differences in clinical outcomes although a patients . There wIn common with all trials of complex interventions, it was not possible to fully blind clinical staff to study group allocation. We did, however, conceal study group allocation from all investigators apart from the member of the research team delivering the intervention. This included concealment of cardiac output data and the use of dummy infusions. All complications, including acute kidney injury, were assessed according to prospectively defined criteria and verified by the principal investigator who was unaware of study group allocation. Lastly our stratified randomisation procedure ensured that the three groups were comparable.The importance of using cardiac output-derived data to guide a carefully prescribed and consistently applied clinical intervention is illustrated by the findings of a previous multi-centre randomised trial in which perioperative pulmonary artery catheterisation, in the absence of improved haemodynamics, failed to influence outcome . In the 2 and ScvO2 to values previously associated with improved clinical outcomes [2, with no change in heart rate or ScvO2, microvascular flow was better maintained than in the CVP-guided therapy group. The incremental effects of low dose dopexamine on both microvascular flow and tissue oxygenation are likely to relate to the β2-adrenoceptor-mediated inotropic and vasodilator actions of this agent. It is therefore possible that changes in microvascular flow relate to direct effects on the microcirculation as well as global cardiac output.The relation between derangements in cardiac output-related variables and complications following major surgery is well described -11. The outcomes -11. We sInterestingly, in a recent randomised trial, low-dose nitroglycerin had no effect on sublingual microvascular flow in resuscitated patients with severe sepsis . These c2) remains constant, cutaneous PtO2 will decrease thus linking peripheral perfusion and tissue oxygenation [The simultaneous use of three different modalities to assess different aspects of tissue microvascular function was an important strength of this investigation. SDF imaging is a non-invasive technique that provides a real-time video image of the intact microcirculation. However, this technique is limited by semi-quantitative analysis and the fact that it can only be used to image the microcirculation under mucosal surfaces. Laser Doppler flowmetry is a technique based on the Doppler shift of reflected laser light from moving red blood cells. This method cannot distinguish the size or type of microvessel, direction of flow or heterogeneity of flow, all of which may be important in critically ill or high-risk surgical patients. These limitations can be addressed through the measurement of post-occlusion reactive hyperaemia, which provides a reproducible assessment of endothelium-dependant microvascular response . The cutgenation . The conA treatment algorithm incorporating stroke volume-guided fluid therapy plus low-dose dopexamine infusion was associated with significant improvements in microvascular flow and tissue oxygenation but no change in the inflammatory response to surgery. These physiological changes may explain the beneficial effects of cardiac output-guided haemodynamic therapy demonstrated in previous clinical trials. Our findings strongly support the need for large multi-centre trials to evaluate the clinical effectiveness of cardiac output-guided haemodynamic therapy. Several such trials are now under way in patients with severe sepsis, those undergoing major surgery and in potential organ donors.• Peri-operative haemodynamic therapies guided by cardiac output monitoring have been associated with improved clinical outcomes in small clinical trials.• The mechanism of therapeutic benefit is believed to relate to improved tissue perfusion and oxygen delivery but this theory has not previously been tested.• In this study, stroke volume-guided fluid therapy and low-dose dopexamine infusion was associated with improvements in tissue microvascular flow and oxygenation but clinical outcomes were similar between groups.• These findings may explain the improved clinical outcomes reported in previous studies. Large randomised trials are now required to confirm the clinical benefits of this treatment approach.2: oxygen delivery; DO2I: oxygen delivery index; DPX: dopexamine; eGFR: estimated glomerular filtration rate; ICAM-1: inter-cellular adhesion molecule 1; IL: interleukin; MFI: microvascular flow index; PaCO2: partial pressure of arterial carbon dioxide; PaO2: partial pressure of arterial oxygen; PtO2: tissue oxygen partial pressure; SaO2: arterial haemoglobin saturation with oxygen; ScvO2: central venous haemoglobin saturation with oxygen; SpO2: arterial haemoglobin saturation; SV: stroke volume; SvO2: mixed venous haemoglobin saturation with oxygen; SDF: sidestream darkfield imaging; TNF: tumour necrosis factor.ANOVA: analysis of variance; CVP: central venous pressure; DORP has received a research grant and equipment loans from LiDCO Ltd and honoraria from BBraun, Edwards Lifesciences, Covidien and Pulsion Medical Systems. SJ, CH and RP are named inventors on a lapsed patent application relating to the peri-operative use of dopexamine.RP formulated the hypothesis and developed the protocol with CH and DW. The investigation was performed by SJ, AVS and SLA, at The Royal London Hospital, UK. SJ, RP, DW and CH assisted in the data analysis. The manuscript was drafted by SJ and RP. All authors read and approved the final version.


In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the “wet” form of AMD. In contrast, very little is known about the mechanisms of the predominant, “dry” form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD.−/− mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%–300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36−/− mice express reduced levels of COX2 and VEGF in vivo, and COX2−/− mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency.We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies. Florian Sennelaub and colleagues show that CD36 deficiency leads to choroidal involution, a key feature of "dry" age-related macular degeneration, via COX-2 down-regulation in the retinal pigment epithelium. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in industrialized countries. The macula is the central region of the retina, the tissue at the back of the eye that detects light and converts it into electrical messages that are sent to the brain. In the commonest form of AMD—“dry” AMD—the light-sensitive cells in the retina (the photoreceptors) gradually die. This degeneration might occur because of damage to the retinal pigment epithelium (RPE). This layer of dark cells lies between the photoreceptors and the choroid, the layer of the eye that contains blood vessels and brings oxygen to the retina. The RPE keeps the retina healthy by transferring the right amount of oxygen and nutrients from the choroid to the retina and by removing worn-out photoreceptor outer segments  in a process called phagocytosis (engulfment and digestion). In addition to photoreceptor degeneration and RPE shrinkage, a layer of the choroid rich in small blood vessels (the choriocapillaris) also shrinks in dry AMD. For affected individuals, all these changes  mean that the sharp central vision that is needed for reading and driving is destroyed, leaving only dim, burred images or a black hole at the center of the vision.Little is known about the molecular mechanisms that underlie dry AMD and, consequently, there is no cure for it. In this study, the researchers have tested whether a molecule called CD36, which is expressed on the surface of RPE cells, is involved in dry AMD. CD36 is a scavenger receptor—which means it binds many potentially harmful molecules including oxidized fats (which are present in the photoreceptor outer segments) and is involved in their phagocytosis. Phagocytosis itself induces the expression of several proteins in the RPE cells, including COX2, a “proangiogenic” protein that stimulates the growth of blood vessels. Putting this information together, the researchers hypothesized that a defect in CD36 might cause the characteristic retinal atrophy (by preventing the phagocytosis of worn-out photoreceptor outer segments) and choroidal involution (by preventing the induction of COX2 expression and consequently the maintenance of the blood vessels in the choroid) of dry AMD.The researchers first show that retinal degeneration occurs in rats and mice that express no CD36. This degeneration  was seen in old but not young animals. Choroidal involution was also seen in these CD36-deficient animals. This change was present in young mice and rats but increased with age so that by one year old, the choriocapillaris looked moth-eaten. Next, the researchers show that although RPE cells taken from normal animals and grown in dishes were able to make COX2 in response to exposure to purified photoreceptor outer segments, RPE cells from CD36-deficient animals did not. The expression of vascular endothelial growth factor  showed a similar pattern. Finally, the researchers report that COX2 deficiency in mice caused similar age-dependent choroidal involution and similar effects on VEGF expression in RPE cells as CD36 deficiency.These findings show that CD36 deficiency leads to progressive, age-related degeneration of photoreceptors and choroidal involution in rats and mice. They also show that CD36 deficiency causes this choroidal involution, the key feature of dry AMD, because it leads to down-regulation of COX2 expression (and subsequently reduced VEGF expression) in the RPE. Researchers now need to find out whether this mechanism for the development of dry AMD holds in people—what happens in animals does not necessarily happen in people. If it does, pharmacological activation of CD36 or restoration of CD36 expression in the RPE might eventually provide a way to treat dry AMD.http://dx.doi.org/10.1371/journal.pmed.0050039.Please access these Web sites via the online version of this summary at macular degeneration and an encyclopedia page on macular degeneration (in English and Spanish)MedlinePlus provides links to information on NIH SeniorHealth site provides text and spoken information about AMDPages on the US National Institutes of Health US National Eye Institute and the UK Royal National Institute of Blind People also provide information about AMDThe the retina, photoreceptor cells, retinal pigment epithelium, and choroid Wikipedia has pages on  Age-related macular degeneration (AMD) is the leading cause of vision loss among older adults in industrialized countries . The moscyclooxygenase 2 COX2 [prostaglandin-endoperoxide synthase 2 [PTGS2]), which controls VEGF expression in various cells ), were stained by toluidine blue, examined with a light microscope, and measurements photoreceptor layer thickness were made. Ultra-thin sections (80 nm) were contrasted by uranyl acetate and lead citrate and were observed with an electron microscope JEOL 100 CX II (JEOL) with 80 kV.The eyes were enucleated, fixed in Bouin's fixative for 24 h, and embedded in paraffin. Sagittal sections (7 μm) were cut in parallel to the optic nerve and stained with periodic acid Schiff (PAS) and hemalun. Photoreceptor layer thickness was measured on four sections containing the optic nerve 14 μm apart from one another. using digitalized images and Image J Software. The data were averaged for each eye, and the mean values from the individual eyes were statistically analyzed. Investigators performed measurements unaware of the provenance of the samples.Total RNA was isolated with RNeasy Mini Kit (Qiagen). Single-stranded cDNA was synthesized from total RNA (pretreated with DNaseI amplification grade) using oligo-dT as primer and superscript reverse transcriptase (Invitrogen). Subsequent real-time polymerase chain reaction (RT-PCR) was performed using cDNA, qPCR SuperMix-UDG Platinum SYBR Green (Invitrogen), and the following primers (0.5 pmol/μl): Actin sense, 5′-AAA GAA AGG GTG TAA AAC GCA G-3′; actin antisense, 5′-AAA GAC CTC TAT GCC AAC ACA G-3′; CD36 sense: 5′-GAC AAT CAA AAG GGA AGT TG-3′; CD36 antisense: 5′-CCT CTC TGT TTA ACC TTG AT-3′; VEGF sense: 5′-TGG GAT GGT CCT TGC CTC-3′; VEGF antisense: 5′-TCG CTG GAG TAC ACG GTG GT-3′; COX2 sense: 5′-TGC TAC CAT CTG GCT TCG GGA G-3′; COX2 antisense: 5′-ACC CCT CAG GTG TTG CAC GT-3′.PCR reactions were performed in 40 cycles of 15 s at 95 °C, 45 s at 60 °C. Product was not generated in control reactions in which reverse transcriptase was omitted during cDNA synthesis.Ten day-old pups (Wistar rat and SHR) were humanely killed and eyes dissected and enucleated. Eyes were maintained at room temperature overnight in Dulbecco's Modified Eagle's Medium  then incubated 45 min with 2 mg/ml trypsin/collagenase I at 37 °C. After trypsin inhibition with DMEM containing 10% fetal calf serum (FCS), the RPE layer was harvested. The RPE was plated in 12-well plates at a rate of RPE from one eye per well in DMEM containing 10% FCS, 1% penicillin/streptomycin, and 0.2% fungizone. Cells were maintained for 12 d before the phagocytosis assay.2, 0.13 mM NaCl (pH 7.2). Retina samples were centrifuged on a sucrose gradient  1 h at 38,000 rpm, and OS were harvested at the ring interface and diluted in DMEM. Centrifugation 10 min at 8,000 rpm was performed and the pellet was resuspended in DMEM to obtain a stock solution of 108 OS/ml.OS were isolated following established protocols . Briefly8 OS/ml. After 1h 850 μl of complete medium was added. Cells were washed and 350 μl of RLT lysis buffer  was added for RNA extraction at 0 and 6 h.Confluent RPE monolayers were challenged with 150 μl of 10The CD36 antibody FA6–152 antibody (Abcam) can activate CD36 by dimerization of the receptor as previously shown . CD36 wa−6 M DUP697; this concentration was maintained throughout the experiments.COX2 inhibition was achieved by a 30 min preincubation with 102 inhalation followed by a thoracotomy. Venous catheter was introduced into the aorta through the left heart ventricle, and the right auricle was cut to allow evacuation of injected products. A perfusion was performed with a mixture of red Mercox resin and catalyst (Ladd Research). Eyes were extracted and lenses were removed. Tissues were conserved overnight at 37 °C in PBS to allow complete polymerization, and then digested by 5% KOH for 2 wk at 37 °C until only the vascular corrosion casts remained. Distilled water was used to remove salt and the mold was dried. Only corrosion casts with completely filled iris vessels were used, to exclude corrosion casts from incomplete perfusion. Retinal vasculature was removed using forceps. The specimens were mounted on SEM stubs, coated with gold palladium, and scanned at an accelerating voltage of 117 kV. In order to measure the thickness of the choriocapillary lumen, corrosion casts were cut paracentrally (1 mm from the aperture of the optic nerve) and positioned for perpendicular views of the choriocapillaries. To analyze the intercapillary space (avascular area), the casts were positioned for frontal views of the choriocapillaries. Electron micrographs were scanned and analyzed using Image J Software (http://rsb.info.nih.gov/ij/). The avascular area was measured on frontal views and expressed as the percentage of intercapillary surface (space between the plastic capillary casts) of the whole area. Thickness of choriocapillaries was measured on perpendicular views of the cast from the retinal to scleral side of the choriocapillary cast.Animals were killed by COP values of less than 0.05 were considered significant.Variance was analyzed by Kruskal-Wallis test. Data between two groups were compared with nonparametric Mann Whitney U-test. All analysis and graphic representation were performed with Prism software , and values are represented as mean ± standard error of the mean (SEM). P values were calculated for a confidence interval of 95% and −/− mice [In vitro participation of CD36 in the phagocytosis of OS by human  and rat −/− mice .Western blot analysis of RPE/choroid complexes showed greatly diminished CD36 protein expression in the eyes of SHRs compared to the Wistar rat control strain in vivo A. CD36 l−/− mice (+/+ mice showed regularly shaped photoreceptors (p < 0.05) at 12 mo of age; this was not yet observed at 4 mo of age , OS were in tight contact with RPE villi and were longitudinal or slightly oblique throughout the sagittal eye sections; perpendicularly (cross)-sectioned OS were never observed in control animals. These differences might be due to OS overgrowth in CD36 deficiency secondary to RPE phagocytosis deficiency. The elongated OS in these animals could explain the accumulation of periodic acid Schiff material  of 10-mo-old SHRs J and 12-−/− mice K showed material G inset amaterial J and 1K.−/− mice.Choroidal involution is a main feature of dry AMD . Little −/− mice showed capillary dropout and a moth-eaten appearance  were not altered (unpublished data). Interestingly, in contrast to CD36−/− mice, COX2−/− mice did not develop degeneration of photoreceptors by 12 mo of age On the basis of observations presented above, we surmised that COX2 expression in RPE also affected choroidal homeostasis. We proceeded to study choroidal morphology in COX2+/+ mice B, vascul−/− mice A were ex−/− mice C. Cross-ice  in COX2 expression in macrophages . In macrAll in all, our results show a novel molecular mechanism of photoreceptor degeneration and choroidal rarefaction, key cardinal features of dry AMD. Furthermore, our results suggest that pharmacological activation of CD36 or restoration of CD36 expression in the RPE of patients with dry AMD could be used therapeutically to prevent photoreceptor cell death by boosting SE renewal and to maintain a healthy choroid and retinal oxygenation by enhanced COX2 expression. These findings reveal a pathogenic process, to our knowledge previously unknown, with important implications for the development of new therapies for dry AMD.


Current models of clonal expansion in human Barrett’s oesophagus are based upon heterogenous, flow-purified biopsy analysis taken at multiple segment levels. Detection of identical mutation fingerprints from these biopsy samples led to the proposal that a mutated clone with a selective advantage can clonally expand to fill an entire Barrett’s segment at the expense of competing clones (selective sweep to fixation model). We aimed to assess clonality at a much higher resolution by microdissecting and genetically analysing individual crypts. The histogenesis of Barrett’s metaplasia and neo-squamous islands has never been demonstrated. We investigated the oesophageal gland squamous ducts as the source of both epithelial sub-types.p16 and p53 point mutations were carried out on a crypt-by-crypt basis. Cases of contiguous neo-squamous islands and columnar metaplasia with oesophageal squamous ducts were identified. Tissues were isolated by laser capture microdissection and genetically analysed.Individual crypts across Barrett’s biopsy and oesophagectomy blocks were dissected. Determination of tumour suppressor gene loss of heterozygosity patterns, p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett’s dysplasia.Individual crypt dissection revealed mutation patterns that were masked in whole biopsy analysis. Dissection across oesophagectomy specimens demonstrated marked clonal heterogeneity, with multiple independent clones present. We identified a By studying clonality at the crypt level we demonstrate that Barrett’s heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We suggest that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett’s metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands. Barrett’s oesophagus is the replacement of the normal oesophageal stratified squamous epithelium with metaplastic glandular epithelium in response to inflammation and ulceration provoked by duodeno-gastroesophageal reflux.2cyclin-dependent kinase N2 (p16) and genetic inactivation of TP53 (p53) tumour suppressor genes occur early in the MCSp16 lesion.p16 and p53 lesions throughout long lengths of Barrett’s oesophagusp16 alleles predisposes to a selective sweep, and that p16 mutation fixation occurs early in the progression of Barrett’s oesophagus.p16 loss of heterozygosity (LOH), methylation and point mutation patterns in biopsy material taken from different levels of long Barrett’s oesophagus segments supports widespread clonal expansion and fixed mutations.8Serial biopsies and molecular analysis of a cohort of patients with Barrett’s oesophagus has enabled researchers to study the evolution of common tumour suppressor gene mutation patterns as the MCS progresses. These longitudinal clonal ordering studies have shown that genetic and epigenetic inactivation of ase N2 p1 and genease N2 p1 and geneet alDespite years of active research the histogenesis of Barrett’s oesophagus has never been demonstrated. Different theories have been proposed: proximal migration of the gastric cardia; re-differentiation of the squamous epithelium and colonisation of cells from the oesophageal gland ducts.To date, most clonality studies have been carried out on heterogenous flow-purified whole biopsy samples. In this work our aims were to (1) study clonality at a crypt-by-crypt level, avoiding problems associated with contaminating normal stroma; and (2) examine the oesophageal gland squamous ducts as the potential source of Barrett’s columnar epithelium and neo-squamous islands.www.bsg.org.uk), by at least two pathologists. Serial 5 μm sections were cut. Sections 1–3 and 5–7 were mounted onto P.A.L.M. membrane slides  and were stained with methylene green. Section 4 was stained with haematoxylin & eosin (H&E).Paraffin-embedded biopsy (six biopsies from five patients) and oesophagectomy blocks  were obtained from the pathology archives of Leicester General Hospital. Tissue was independently assessed for Barrett’s metaplasia and dysplasia according to British Society of Gastroenterology 2005 guidelines . After individual crypt dissection residual epithelial tissue was catapulted into a single tube and immersed in 30 μl proteinase K for 2O2 in methanol for 10 min. Antigen retrieval was achieved by 10 min microwaving in sodium citrate buffer at pH 6. Slides were incubated in 3% bovine serum albumin in phosphate-buffered saline (PBS) for 15 min. Slides underwent primary antibody incubation with mouse monoclonal antibodies against cytokeratin 7  or cytokeratin 13 . This was followed by biotinylated rabbit anti-mouse secondary antibodies before application of a 1:500 dilution of the tertiary layer of peroxidase-conjugated streptavidin . Each layer was applied for 45 min and three 5 min PBS washes were performed between layers. Sections were then developed with 3,3-diaminobenzidine tetrahydrochloride solution  for 2 min, followed by rinsing in tap water and light haematoxylin counterstaining. The positive control tissues used were duodenum (CK 7) and tonsil (CK 13). Negative controls underwent all steps but were incubated with PBS instead of the primary antibody solution.Cytokeratin 7 and 13 staining was used to demonstrate glandular and squamous epithelial differentiation respectively.p53 and exon 2 of p16, using the primer 3 website . First round oligonucleotide primer pairs were specifically designed to amplify a region that included the amplicon covered by the primers used in the second round of the polymerase chain reaction (PCR). Primer optimisation determined the optimum reagent concentration and annealing temperature for each primer pair. Primer sequences are tabulated in the supplementary information (supplementary table 1A–C). Both PCR steps were carried out in an Omni PCR UV hood to minimise contamination . Only products with an uncontaminated negative control tube went forward for sequencing. PCR product was sequenced using BigDye terminator cycle sequencing on an ABI 3100 DNA sequencer . The sequences obtained were directly compared to the revised Cambridge reference sequence, and any identified mutations were checked against the Catalogue of Somatic Mutations in Cancer (COSMIC) database (www.sanger.ac.uk) . Sequencing was repeated twice from dissection lysate for the oesophageal gland squamous duct work.First and second round primers were designed to amplify exons 5–9 of APC gene), D9S932 (p16 gene) and D17S786 (p53 gene), were used for LOH analysis. Constitutionally homozygous markers were scored as non-informative. Forward oligonucleotide primers were labelled at the 5′ end with the carboxy fluorescein (FAM) fluorescent marker (Sigma). PCR amplifications were performed using the LA TAKARA kit . The PCR product was analysed on an ABI 3100 sequencer (Applied Biosystems) and genotyper 2.5 software . At each marker, loss of heterozygosity was considered present if the area under one allelic peak in the affected crypt was less than 0.5 times or greater than 2 times that of the other allele, after correcting for the relative areas using constitutional DNA (microdissected areas of lamina propria tissue).Three microsatellite markers; D5S346 .p16, p53 and adenomatous polyposis coli (APC) tumour suppressor genes which is not demonstrable when the whole biopsy section was digested and analysed. This is likely to be a consequence of a diluting effect of the normal stroma in the whole biopsy sample  between p53 point mutations and allelic loss of all three tumour suppressor genes. This is consistent with functional loss of the cell cycle checkpoint activity of TP53 protein, which is then permissive for widespread, large-scale genetic changes. Additionally, p53 point mutations, although not seen in every crypt, were often present in multiple blocks from a single oesophagectomy specimen, suggesting widespread and far-reaching clonal expansion as a consequence of the strong selective advantage that p53 loss would provide (supplementary et al,Dissection across large oesophagectomy blocks also revealed considerable phenotypic and genotypic heterogeneity in all cases. p53 point mutations.p16 inactivation. Bi-allelic loss of p16 in some crypts means that epigenetic methylation is unlikely to be an undetected founder mutation. p16 inactivation by allelic loss, point mutation or methylation is one of the proposed earliest lesions in the Barrett’s MCS pathway and is said to predispose towards a selective sweep.Phenotypic and genotypic heterogeneity in Barrett’s tissue has been well described,p16 point mutation originating in microdissected squamous duct tissue that was also found in the adjoining metaplastic crypt. The presence of an identical mutation in the two different epithelial types is strong evidence to suggest that the origin of the metaplastic tissue in human Barrett’s oesophagus is a progenitor located in the oesophageal gland squamous ducts. Additionally, the presence of a wild-type squamous island seen emerging from a wild-type squamous duct in the midst of, and completely surrounded by, a p53 mutant field, strongly indicates a new clone development. This supports the hypothesis that neo-squamous islands can arise de novo from glandular tissue after Barrett’s ablation therapy,et alIt has been proposed, but never proven, that submucosal oesophageal gland ducts may be the origin of metaplastic tissue in Barrett’s oesophagus.The demonstration of multiple competing independent clones and the identification of the origin of Barrett’s oesophagus from the oesophageal gland squamous duct, structures present throughout the length of the oesophagus, allows the development of a new model of clonal evolution in Barrett’s oesophagus. We suggest that duodeno-gastro-oesophageal reflux-induced ulceration and inflammation can induce tumour suppressor gene mutations in some of the stem cell populations located in oesophageal gland squamous ducts. This gives rise to multiple distinct clones of metaplastic tissue that then compete to colonise the oesophagus, creating a mosaic pattern of clones across the segment. Clonal expansion of populations with greater selective advantage leads to dominant and widespread clones; however, no single mutation is fixed since competing wild-type clones are also identifiable. Non-mutated squamous ducts are likely to be the source of wild-type squamous islands . These dThe cell biology of the oesophageal epithelium has been inadequately studied despite the high mortality and increasing incidence of cancer at this site. The interpapillary basal layer has been suggested as a possible stem cell zoneIn conclusion, we have demonstrated two important features of Barrett’s oesophagus. First, clonal heterogeneity arises from multiple independent clones, previously undetectable by whole biopsy analysis; and second, Barrett’s metaplasia and neo-squamous islands can arise from the oesophageal gland squamous ducts.


Primary cutaneous follicle center lymphoma (PCFCL) is characterized by a proliferation of follicle center cells in the skin. A definitive diagnosis is frequently delayed because of difficulties in interpretation of the histopathologic findings. It has an excellent prognosis with a 5-year survival over 95% and its risk of transformation has not been established. We describe a case report of man with a gastric diffuse large B-cell lymphoma (DLBCL) referred to our clinic because of nodules in the back that had gradually developed over a period of 10 years. A biopsy performed 3 years before was interpreted as reactive follicular hyperplasia. A new skin biopsy revealed a diffuse large B-cell lymphoma and immunoglobulin heavy chain gene rearrangements from the initial skin biopsy (PCBCL) and the DLBCL gastric biopsy were studied by polymerase chain reaction and an identical clonal rearrangement was detected which was highly suggestive of a transformation lymphoma. While the annual risk of transformation of systemic FL is 3%, PCFCL has an excellent prognosis with a 5-year survival over 95% [Primary cutaneous follicle center lymphoma (PCFCL) is characterized by a proliferation of follicle center cells (centrocytes and centroblasts) with a follicular, follicular and diffuse, or diffuse growth pattern . It showWe describe a case report of a 44-year-old white man with a gastric diffuse large B-cell lymphoma (DLBCL) referred to our clinic by the hematology department because of multiple erythematous to purple, sharply demarcated pruritic nodules in the back that enlarged and coalesced in the previous year originating a tumor measuring 11 × 7 cm in diameter . These l(IPI): 2.Recently, he was referred for an upper endoscopy because of epigastric pain and weight loss. Endoscopy revealed two gastric ulcers (H. Pylori positive)  and a coH-JH rearrangement was detected using FR1 and FR2 primers: 328 base pairs (bps) and 258 bps, respectively  chemotherapy, and omeprazole (20 mg once a day), with no improvement. Afterwards, he had undergone three different chemotherapy regimens  with no response.,s lymphoma [H-JH rearrangement in both biopsies (skin and gastric) obtained at different time points and from different locations is compatible with the presence of the same B cell clone and strongly suggests transformation of the PCFCL into a DLBCL. PCFCL morphologic and phenotypic features can be similar or identical to those observed in secondary cutaneous involvement by systemic FL . In our lymphoma , 6. In fPCFCL can infrequently progress into extracutaneous, but transformation into DLBCL has been rarely described . The current case emphasizes that indolent cutaneous PCFCL can transform into high-grade lymphoma with poor outcome. An early diagnosis and proper treatment are essential in the management of PCFCL.


Viruses that maintain their genomes as extrachromosomal circular DNA molecules and establish infection in actively dividing cells must ensure retention of their genomes within the nuclear envelope in order to prevent genome loss. The loss of nuclear membrane integrity during mitosis dictates that paired host cell chromosomes are captured and organized by the mitotic spindle apparatus before segregation to daughter cells. This prevents inaccurate chromosomal segregation and loss of genetic material. A similar mechanism may also exist for the nuclear retention of extrachromosomal viral genomes or episomes during mitosis, particularly for genomes maintained at a low copy number in latent infections. It has been heavily debated whether such a mechanism exists and to what extent this mechanism is conserved among diverse viruses. Research over the last two decades has provided a wealth of information regarding the mechanisms by which specific tumour viruses evade mitotic and DNA damage checkpoints. Here, we discuss the similarities and differences in how specific viruses tether episomal genomes to host cell chromosomes during mitosis to ensure long-term persistence. While the specific mechanism of genome segregation during mitotic cellular division employed by each DNA virus studied has subtle or sometimes major distinctions, the overall theme of the way in which viruses have evolved to ensure genome persistence in dividing cells remains strikingly similar. Viral genomes are replicated along with the cellular DNA and are attached to host cell chromosomes during cellular division. This ensures retention in the nuclear envelope and in some cases may facilitate a roughly even distribution of episomal DNA molecules to each daughter cell . To faciin vivo not by immortalizing infected B cells, but by establishing a true latency in normal resting memory B cells (Epstein–Barr virus (EBV) is a human γ-herpesvirus (genus lymphocryptovirus) that persists benignly in approximately 90 per cent of individuals worldwide. In developing countries, infection occurs in early childhood and is generally asymptomatic. Owing to better hygiene standards, infection is delayed until early adolescence in developed countries where it can result in infectious mononucleosis . Althoug B cells .OriP and the viral EBNA1 protein  element and four low-affinity binding sites within the dyad symmetry (DS) element  and three latent membrane proteins are expressed. These proteins are required to stimulate proliferation of the host cell and maintain a stable viral copy number within dividing cells . Replican et al. . OriP coThe viral genome partitioning function of EBNA1 has been directly attributed to its ability to associate with host cell chromosomes during mitosis. EBNA1 localizes to discrete foci during mitosis that colocalize with host cell mitotic chromosomes . The intIn contrast to the studies described above, it has been suggested that EBNA1 associates directly with mitotic chromosomes in human cells independently of its association with EBP2 . This inOriP-containing plasmids are also defective in genome tethering function  is a γ-herpesvirus. KSHV belongs to the genus Rhadinovirus and is the only member of the genus known to infect humans. The incidence of KSHV infection is low in Asia, northern Europe, Australia and the Americas (less than 5%) but higher in Mediterranean countries (4–35%) and sub-Saharan Africa (20–70%) . Kaposi'The KSHV genome comprises a long unique region (140 kb) flanked by two terminal repeat (TR) regions, which consist of tandem repeats of 803 bps long  and LANA-binding site 2 (LBS2) . In addiAlmost a decade ago, two independent research groups suggested a role for LANA in the tethering of viral episomes to host chromosomes during mitosis . As prevper se  and DEK  while bovine herpesvirus 4 (BoHV-4) ORF73 is the shortest (253 amino acids). The acidic internal repeat region is the least conserved region and varies in length and in some species is completely absent (murine γ-herpesvirus 68 (MHV-68), rhesus rhadinovirus (RRV) and BoHV-4). Conversely, the N- and C-terminal regions of proteins translated from ORF73 are highly conserved  types have been defined, which infect the basal cells of either mucosal or cutaneous epithelia in a variety of biological niches .All papillomaviruses contain an approximately 8000 bp double-stranded circular DNA genome that encodes eight or nine ORFs including six early ORFs  and two late ORFs (L1 and L2). Upon infection of basal cells within the epidermis, the viral genome is established as a stable episome and maintained at a low copy number. Viral genomes are replicated along with the cellular DNA during the S-phase, which requires the viral E1 and E2 proteins. E2 is a multifunctional DNA-binding protein that specifically recognizes the consensus motif (AACCG(N4)CGGTT) . E2 alsoIn addition to its role in viral genome replication and transcriptional regulation, E2 is required for the stable maintenance of viral genomes by facilitating an intricate segregation mechanism of viral genomes in dividing cells. This mechanism has been extensively studied in both human and animal papillomaviruses with subtle differences between genera reported. Initial studies were carried out using bovine papillomavirus type 1 (BPV-1). The BPV-1 E2 protein associates with mitotic chromosomes throughout mitosis in a manner that requires sequences within the N-terminal transactivation domain of E2  have been shown to stably interact with mitotic chromosomes only in early (prophase) and late (telophase) mitosis, but not at the point of chromosomal separation (anaphase)  has been described and shown to enhance the transcriptional and replication activities of E2  was isolated  targeted. Such a small molecule will ensure that viral oncoproteins are held under strict transcriptional control and episomal genomes replicate normally. This will prevent any possibility of forced integration, as has been suggested for papillomaviruses (