VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation.In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo.These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF  Macrophage-Colony Stimulating Factor (M-CSF) acts on mononuclear phagocytes, stimulating proliferation, driving differentiation of monocytes to macrophages, and protecting these cells against apoptosis in vivoVEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation M-CSF, mononuclear phagocytes, and VEGF are linked to poor prognosis in patients with solid tumors. For example, M-CSF is elevated in breast cancer patients with advanced disease and distant metastases compared to patients with localized tumors in vivo ability of M-CSF to induce VEGF production and cause subsequent angiogenesis.Here, we decipher the molecular mechanism of M-CSF-induced VEGF production. Further, we illustrate the Single donor human monocytes were isolated from leukocyte source packs obtained from the American Red Cross  as described previously 6 cells/condition) were left untreated, treated with DMSO or methanol, the MEK/ERK inhibitor, U0126 (10 µM), the JNK inhibitor, SP600125 (10 µM), the p38 MAP kinase inhibitor, SB203580 (10 µM), the PI3K/Akt inhibitor, LY294002 (1 µM or 10 µM), the mTOR inhibitor, Rapamycin , or mithramycin  for 30 minutes . Afterwards, the cells were stimulated with rhM-CSF (100 ng/ml) for 48 hours and the cell-free supernatants were frozen until assayed for VEGF by ELISA .Human monocytes  and subsequently transferred to nitrocellulose paper and probed with the following antibodies: phosphorylated p44/p42 MAPK (Thr202/Tyr204)  and equal loading was observed by blotting for total ERK2 .Human monocytes were either left non-stimulated or stimulated with rhM-CSF (100 ng/ml) for 30 minutes. Nuclear lysates were isolated from the cells using the CelLytic™ NuCLEAR™ Extraction Kit (Sigma). All samples were normalized for total protein using the Bio-Rad Protein Assay Kit. The amount of Sp1 in the nuclear lysates was analyzed using the NoShift™ Transcription Factor Assay Kit and NoShift™ Sp1 reagents as described by the manufacturer (Novagen).Human monocytes were left non-stimulated or stimulated with rhM-CSF (100 ng/ml) for 6 or 24 hours. Cells were fixed then permeablized with 1% Triton X-100 and subsequently blocked with 5% goat serum in PBS, incubated overnight with 1 µg/ml of either a primary antibody to human Sp1 (Upstate) or IgG control antibody (Santa Cruz). The cells were incubated with a 1∶5000 dilution of Alexa 594-conjugated secondary antibody (Molecular Probes). The slides were then viewed and imaged using the Zeiss LSM 510 confocal microscope. Images were quantified using Image J software. Cells were differentiated from debris by using the phase contrast photos and only those cells displaying monocyte morphology, horseshoe-shaped nuclei, were included in the analysis.All luciferase reporter constructs containing both wild type and mutant sequences based on the VEGF promoter were supplied by Dr. Amit Maity (University of Pennsylvania). 1 µg of each construct was transfected into freshly-isolated human monocytes using the Amaxa Nucleofector Transfection System as per the manufacturer's protocol (Amaxa). All cells were incubated in 100 ng/ml rhM-CSF for 16 hours, lysed using Promega Lysis Buffer on ice, and centrifuged to remove cell debris. 20 µl of each cell lysate was mixed with 90 µl luciferase substrate and read on the luminometer in triplicate.5′-AGGCCAGCACATAGGAGAGATG-3′), and reverse primer (5′-CAAGGCCCACAGGGATTTTC-3′). The primers for VEGF mRNA were designed based on the human VEGF-A sequence obtained from PubMed (Genbank accession no. NM003376). Reaction conditions were as follows: 10 µl SYBR® Green PCR Master Mix (Applied Biosystems), 0.4 µl each forward and reverse VEGF primers (10 µM) or primers targeting GAPDH (control), 8.2 µl RNase-free water, and 1.0 µl cDNA (0.5 µg) for a total reaction of 20 µl. The real-time PCR was completed on the ABI PRISM Sequence Detector 7700 (PerkinElmer) using Sequence Detector software. Reaction conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Quantification was performed as previously described Monocyte total RNA was collected using the Absolutely RNA™ RT-PCR Miniprep Kit Stratagene®  for total RNA purification as previously described C57BL/6 female mice purchased from Jackson Laboratories  were anesthetized with isoflurane and subcutaneously injected with 0.5 ml growth factor-reduced Matrigel™ matrix  supplemented with either PBS, rmVEGF (10 ng/ml), rmM-CSF (100 ng/ml), rmM-CSF (100 ng/ml)+anti-VEGF neutralizing antibody (5 µg/ml), or rmM-CSF (100 ng/ml)+isotype IgG antibody (5 µg/ml). All components added to the unpolymerized Matrigel™ were allowed to incubate at 4°C for at least 12 hours prior to injection. After 10 days, the mice were sacrificed and the Matrigel™ plugs removed, formalin-fixed, paraffin-embedded and sectioned.Mononuclear phagocyte recruitment within the plugs was identified using an anti-F4/80 antibody . VEGF was detected within the plugs by staining with a rabbit anti-VEGF antibody or the isotype IgG antibody . Endothelial cell infiltration into the plugs was determined by an anti-von Willebrand factor (vWf) antibody . Blood vessels within each plug are defined as complete circles of vWf(+)-stained cells. All staining was performed at the Pathology Core at The Ohio State University.p-value that tests if there is a difference between treatments was generated. All analyses were run on Stata 10.0, Stata Corporation, College Station, Texas. Groups were considered significantly different at p<0.05.Statistics were determined by The Ohio State University Medical Center for Biostatistics. For IHC of F4/80(+) cells and VEGF and the Sp1 binding assay, an independent samples Student's T-Test was used to assess differences between groups. An ANOVA with Tukey's post-hoc test was performed to determine differences between groups in the blood vessel counts, ELISA studies, confocal microscopy data and the VEGF promoter constructs data. For the inhibitor studies and the luciferase assays, difference in fold change over treatment was tested using a random-intercept mixed model. Fold change was log transformed for variance stabilization and a single overall We demonstrated M-CSF induces the production and release of biologically active VEGF from human monocytes M-CSF activates both the PI3 kinase/Akt pathway Sp1 and AP-2 are transcription factors phosphorylated and activated by ERK To verify whether the inhibitory effect of mithramycin was due to inhibition of Sp1 binding, we attempted to use siRNA against Sp1. Stealth™ RNAi targeting Sp1 was transfected into primary human monocytes using Lipofectamine and assayed for a reduction in total Sp1 protein by Western blot. We found that protein levels of Sp1 were not depleted from these cells until 48 hours after transfection. This created a challenge in VEGF determination as cultured, primary human monocytes differentiate into macrophages which become less responsive to M-CSF and therefore make it difficult to measure VEGF production from these cells. Additionally, transfection of monocytes with Lipofectamine alone induced the production of VEGF that masked induction of VEGF by M-CSF (data not shown). As a result, we attempted to transfect the siRNA using the Amaxa Nucleofector. Using this method, transfection alone induced the production of the soluble form of VEGFR-1, which sequesters VEGF from antigenic detection by ELISA. For these reasons, we chose to perform multiple techniques described below to verify the role of Sp1 in M-CSF-induced VEGF production from these cells.The activation of Sp1 during M-CSF stimulation has not been previously reported. To confirm that M-CSF induces Sp1 activation, we performed an Sp1 binding assay to measure levels of Sp1 translocation to the nucleus in monocytes stimulated with M-CSF for 30 minutes. Our results indicate that M-CSF increased the amount of Sp1 that migrated to the nucleus of human monocytes (p = 0.008)  or U0126 and left untreated or treated with M-CSF for 6 or 24 hours. The cells were fixed and stained using a fluorescently-conjugated anti-Sp1 antibody and DAPI to stain the nucleus  ; the identical region of the VEGF promoter with the two Sp1 regions (−85 to −80 and −74 to −69) mutated to inactivity by replacing GC to AA in both instances resulting in no Sp1 binding; an AP-2 mutant (−80 to −73) by changing the GG at −75 and −76 to TA; and an Sp1/AP-2 double mutant were spliced into the pGL3-basic vector to create the promoter constructs . Monocytes transfected with the Sp1 mutant or the Sp1/AP-2 double mutant produced levels of luciferase similar to cells transfected with the empty vector and significantly less than the cells transfected with the VEGF wild type construct (p<0.001 for both) , a glycoprotein present in blood plasma and produced by endothelial cells, and counted the number of vWf-positive cells lining blood vessel lumens (completely-lined circles) throughout the plug  by monocytes in vivo. In addition to inducing VEGF production from monocytes, M-CSF is also known to stimulate the production of other pro-angiogenic factors, such as IL-8 Finally, we demonstrate the ability of M-CSF to induce angiogenesis in vivo. The abundance of clinical data from cancer patients correlating high levels of M-CSF, mononuclear phagocytes, and VEGF with poor prognosis highlights the importance of increasing our understanding of this paradigm. We believe studies linking these three components may cultivate understanding and aid in the genesis of novel therapies for numerous cancers.In summary, we demonstrate the biochemical details of M-CSF-induced VEGF production. We propose a pathway involving M-CSF-induced ERK activation followed by ERK-induced phosphorylation of the transcription factor Sp1, which then translocates to the nucleus and binds to its specific sequence within the VEGF promoter to drive transcription of VEGF mRNA. Previous data on VEGF regulation has been obtained using cell lines; we demonstrate our phenomenon in primary human monocytes, enhancing the relevance of this observation. This manuscript describes the pro-angiogenic potential of M-CSF as it pertains to mononuclear phagocyte recruitment, VEGF production, and angiogenesis


Multivariate survival analysis showed nodal status, FPTC, mesenteric lymphangiosis, and lymph node metastasis to the compartment III to be independent prognostic factors with relative risks of 6.6, 4.5, 2.9 and 2.2 respectively. Recurrent disease occurred in 91% of FPTC-positive and in 38% of FPTC-negative patients. FPTC had a positive predictive value of 91% and a specificity of 97% for tumour recurrence. FPTC is a strong negative, independent prognostic indicator for survival in gastric carcinoma. © 1999 Cancer Research CampaignWe prospectively investigated the prognostic significance of free peritoneal tumour cells (FPTC) in a series of 118 patients with completely resected gastric carcinoma. Immunocytochemistry with the monoclonal antibody Ber-Ep4 was performed on cytospins from intraoperative peritoneal lavage specimens. Twenty-three patients (20%) had FPTC which was significantly correlated with pT and pN categories, stage, tumour size, lymphatic invasion, Laurèn and WHO classifications and perigastric adipose tissue metastases. The median survival time for all FPTC positive compared with negative patients was significantly shorter (11 compared with > 72 months), with estimated 5-year survival rates of 8% vs. 60%. None of the patients with FPTC had an early gastric cancer. In advanced tumour subgroups without and with serosal invasion (


Abnormal Spindle-like Microcephaly associated) over-expression was recently implicated in the development of malignant gliomas.ASPM (Aspm expression was strongly correlated with tumor grade and increased at recurrence when compared to the initial lesion, whatever the initial grade of the primary tumor. ASPM expression also increased over serial passages in gliomaspheres in vitro and in mouse xenografts in vivo. Lentivirus-mediated shRNA silencing of ASPM resulted in dramatic proliferation arrest and cell death in two different gliomasphere models.To better characterize the involvement of ASPM in gliomas, we investigated the mRNA expression in 175 samples, including 8 WHO Grade II, 75 WHO Grade III and 92 WHO Grade IV tumors. These data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and is an attractive therapeutic target in glioblastoma multiforme. Gliomas are the most common type of primary tumors in the central nervous system (CNS), and their usual fate is to recur, either as a similar or more frequently as a higher grade lesion, explaining their grim prognosis. The cellular origin of gliomas remains unknown, but accumulating data suggest that transformed, stem-like precursors (CSC) may represent the cells that drive their growth ,2.of ASPM gene  localizes to centrosomes, spindle poles, and the midbody [The product  midbody . By prom midbody . Mutatio midbody . In addi midbody , suggest midbody ,7.in vitro and in vivo. Furthermore, we found that shRNA-mediated inhibition of ASPM results in dramatic growth inhibition and extensive cell death in gliomaspheres.In this study, we report that ASPM expression is correlated with tumor grade and increases at recurrence or after consecutive passages Tumor samples frozen in liquid nitrogen were obtained from patients who underwent surgery for a glioma at our institution and gave informed consent for molecular research studies. Histological diagnoses were made according to the World Health Organization (WHO). BAC-array-based Comparative Genomic Hybridization analysis (CGH-array) was performed as previously described . Non-tumTotal RNA extraction was performed using RNAble blue solution  according to the manufacturer's instructions. Total RNA was systematically analyzed on both a spectrophotometer and an Agilent 2100 bioanalyzer . cDNA was prepared from 1 μg RNA using a combination of random primers  and MMLV reverse transcriptase .ALAS housekeeping gene (Human Erythroid 5-Aminolevulinate Synthase) using primers F: 5'-TGCAGTCCTCAGCGCAGTCT-3' and R: 5'-TGGCCCCAACTTCCATCAT-3'. The 2-ΔΔCT equation was applied to calculate the relative copy number of tumor samples versus non-neoplastic tissues.Gene expression was quantified by Syber Green real-time qPCR analysis . Real-time qPCR cycles were as follows: initial denaturation at 95°C for 15 minutes, 95°C for 30 seconds and annealing at 60°C for 1 min. Cycles were repeated 40 times. Primers were as follows: ASPM, Quantitect Primers Assay (ref: QT00020209 Qiagen). The amount of cDNA was normalized to the amount of the 5 cells/75 cm2 flask  in 10 mL of DMEM/F12 medium supplemented with Insulin (5 mg/mL), Penicillin - Streptomycin (50 mg/mL) and growth factor , epidermal growth factor  and B27 (1/50) as a stem cell-permissive medium and maintained in a 5.0% CO2/95% O2 atmosphere with 100% humidity. Cultures were fed every three days by changing half of the medium. Primary gliomaspheres were obtained after 5-10 days.Briefly, tumors were immediately collected after surgery in HBSS  and dissected after removal of gross necrosis, subjected to a trypsin digestion (5 mg/mL) for 10 min at 37°C, and then trituration and filtering. The cell suspension obtained was then plated at a density of 5.10in vitro experiments.The U87 and U251 glioblastoma cell lines and the HEK293T kidney cell line were purchased from ATCC , grown on DMEM 10% FCS- 1% Penicillin-Streptomycin, and used as controls for We used pGIPZ  lentiviral vectors expressing a short hairpin RNA  for ASPM silencing  as a control.Lentiviral vector stocks were produced by transient transfection of HEK293T cells with the p8.91 encapsidation plasmid , and the6 cell/mL with LV-sh-miR of ASPM and non-targeted shRNA at a multiplicity of infection of 2.5 (MOI: number of transducing units per cell) supplemented with DEAE dextran (20 μg/mL). After expansion of the culture, GFP-positive viable cells were separated by cell sorting and flow cytometry FACS  to ensure knockdown efficiency. After 7 days, ASPM RNA expression was measured in glioma stem cells infected with lentiviruses by real-time qPCR.Gliomasphere cultures were mechanically disaggregated in order to increase the efficiency and uniformity of infection and then transduced at 3-5.10+cell staining. The percentage of labeled cells was quantified by cell sorting on flow cytometry FACS .Proliferation was assessed by EDU incorporation , and cell death by 7-AAD5 cells suspended in 100 μl of HBSS medium were injected subcutaneously into nude mice. Before xenograft establishment, tumors were passed four times and genomic stability was checked by CGH array analysis.2 to 5 × 10® . Images were captured with a Zeiss microscope (Axiophoto) equipped with a cooled CCD camera .To detect the endogenous ASPM protein, cultured cells were fixed for 15 min in 4% paraformaldehyde, washed twice in PBS, treated for 30 min with 0.1 Triton X-100 -5% FCS, and incubated for 30 minutes at room temperature with Rabbit-anti-ASPM  in 0.1 Triton X-100 -1% FCS. After two washes in 0.1 Triton X-100 -1% FCS, samples were incubated for 30 minutes at room temperature with goat anti-rabbit IgG-FITC (dilution 1:1000) in 0.1 Triton X-100 -1% FCS. After two washes, samples were stained with Hoechst solution and rinsed with PBS. Samples were mounted with FluoromountLAS-1000 Pro ver. 2 .Total protein, obtained by lysing tumor tissue or cells with RIPA 1× buffer  was quantified using a Pierce BCA™ Protein Assay Kit , separated on Nu-Page 3-8% Tris-acetate polyacrylamide gels (Invitrogen), and electro-transferred to nitrocellulose membranes  in 25 mM Tris, 192 mM glycine, 20% ethanol, and 0.01% SDS for 2.5 - 3 hours at 80-150 volts. After blocking in 5% milk solution, the membranes were probed with anti- ASPM (K-17 sc-48883) and anti- GAPDH (V-18 sc-20357) both from Santa Cruz Biotechnology, Inc., U.S.A. Antibodies were diluted 1:500 for ASPM and 1:700 for GAPDH in TBST  Tween-20) containing 5% milk powder. After washing in TBST, the membrane was incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies diluted 1:5000 in TBST. The immunoreactive signals were detected using an ECL reagent Advance Western Blotting Detection Kit  and an Image Reader 2 test was used to compare qualitative variable distribution, and the Mann-Whitney test was used for continuous variables. A two-sided p-value < 0.05 was considered to be significant.The χWe measured ASPM expression via qRT-PCR in 175 gliomas  and in three non-neoplastic brain tissues (NT). ASPM expression level increased between NT tissue and Grade II gliomas (p < 0.05), between Grade II and Grade III (p < 0.005), and between Grade III and Grade IV (p = 0.0001). ASPM expression did not differ between astrocytic , oligodendroglial  or mixed glioma samples  Figure .Preliminary results with western blot analysis showed that ASPM was also expressed at the protein level in gliomas and overexpressed in Grade IV gliomas as compared to Grades III and II . As shown in Figure in vitro in spheroid cultures and in vivo on xenograftsBecause ASPM expression increases with the recurrence of glioma, and because ASPM is involved in tumor stem cells, we investigated ASPM expression over time We generated three gliomaspheres cultures, GBM1, GBM2, GBM3, all characterized by EGFR amplification and 10q loss on CGH-array analysis; this genetic profile remained stable throughout the passages , 500,000 dissociated cells from GBM1 gliomaspheres were injected subcutaneously into immunodeficient nude mice. ASPM mRNA, measured by RT-PCR, increased progressively with successive tumor passages Figure .Aspm. In GBM1, GBM2 and GBM3 gliomasphere models, ~80% of cells were successfully transduced with the sh-miR-RNA specific to Aspm. In transduced cells, ASPM expression measured by RT-PCR was dramatically reduced to less than 5% in GBM2 and less than 0.02% in GBM1 and GBM3  incorporation) and cell death (assessed by 7-AAD+ labeling). We noted an accumulation of dead cells in sh-miR-Aspm populations  compared to control  were EdU+ cells .To determine the role of ASPM, we infected gliomaspheres with lentivirus expressing a sh-miR-RNA that specifically target d Figure . The morl Figure . In addis Figure , compares Figure . For GBMAspm silencing on GBM cancer stem cells, we conducted a secondary gliomasphere-forming assay in GBM1. Seven days after silencing, GFP+ cells were sorted and re-incubated at 1,000 cell/10 mL. We observed a dramatic decrease in cell number in GBM1 sh-miR-Aspm compared to the non-silenced control (1000 cells vs. 2.8.105 cells at day 9 and 500 cells vs. 3.7.106 cells at day 15). At D15, no gliomaspheres were observed (vs. 91 gliomaspheres in the control), indicating that the knockdown of Aspm completely abrogated the development of secondary gliomaspheres.In order to evaluate more specifically the impact of in vitro.This study indicates that ASPM is tightly associated with the malignant progression of gliomas and that its knockdown produces extensive death of glioma cells in vitro and in mouse glioma xenografts in vivo. To explain such a tight correlation between ASPM expression and the malignant progression of tumors, an attractive hypothesis is that symmetric division of CSC-expressing ASPM leads to progressive proliferative expansion of this undifferentiated compartment. Although we found that ASPM was also expressed at the protein level in all glioma grades and overexpressed in Grade IV gliomas, it is worth noting that the results were less striking. This finding has been previously noted and led to the suggestion that ASPM could be in part regulated at the translational level [ASPM. Whereas siRNA transfection results in a transient effect, lentiviral-mediated expression of shRNA allows the transgene to integrate into the genome of the host cell, resulting in long-term expression. Production of secondary spheres was also inhibited after ASPM knockdown, a finding compatible with a sustained inhibition of CSC proliferation. Interestingly, stable inhibition of ASPM not only prevented further growth but also produced rapid and extensive cellular apoptotic death in our model. This finding may indicate that, following ASPM inhibition, CSC underwent active maturation and differentiation but could not survive in our experimental setting (serum-free medium without appropriate growth factors). However, experiments by Horvath et al. conducted in a similar serum-free medium resulted in growth inhibition and not massive cell death. On the other hand, the function of ASPM in neoplastic cells, particularly gliomas, has not been completely elucidated. In particular, the links between ASPM and key molecular alterations of malignant gliomas, such as EGFR amplification or mutation (EGFRvIII) , remain to be investigated. Nevertheless, some data suggest that these alterations are closely related. ASPM mRNA expression was increased in an EGFRvIII-expressing U87 glioblatoma cell line as compared to an isogenic U87 line, while ASPM expression was inhibited following the addition of erlotinib (an EGFR tyrosine kinase inhibitor) to the EGFRvIII-expressing U87 line. This observation led Horvath et al to suggest that ASPM (and genes from the same module) is involved downstream of the mutant EGFR. Thus, inhibition of ASPM could prove lethal by severely hampering one of the key signaling pathways of glioblastomas.As previously described , we founal level . Howeveral level . The effin vitro sensitivity of tumor cells to ASPM knockdown support continued research to specifically target ASPM from a therapeutic perspective, focusing on in vivo studies.Because ASPM is absent or expressed at very low levels in normal brain, the considerable differential expression levels between GBM and normal parenchyma and the extreme The authors declare that they have no competing interests.SNNB, CC and YM carried out most of the in vitro work. RV and PR produced the lentivirus. SNNB. AR and BB performed the immunohistochemistry experiments. AI performed the CGH-array experiments. PL and ML did the in vivo experiments. SEH contributed to the in vitro experiments. MS conceived the study. JYD and MS wrote the manuscript. All authors read and approved the final manuscript.Figure S1 - ASPM protein expression. ASPM western blot analysis of grade II, III and IV gliomas (n = 4 in each group). Relative protein expression is presented as the ratio of density value for ASPM over GAPDH signal (mean +/- SEM).Click here for fileFigure S2 - Tumor spheroid characterization. Genomic stability was examined with CGHa analysis . The chromosomes are indicated on the x axis and copy number is on y axis. Yellow indicates the normal genomic copy number, while green indicates a loss and red indicates a gain in copy number. As shown here, GBM1 carries EGFR amplification and loss of chromosome 10q. After CSC isolation in stem cell-permissive medium, gliomaspheres were differentiated in presence of fetal bovine serum and stained by Sox2 and O4 for stemness markers and by GFAP and Tuji1 to characterize the potential of multi lineage differentiation of GBM1.Click here for fileFigure S3 - Knock-down of Aspm gene. Tumor spheroids  were transfected either with non silencing (scrambled ShRNA) or with ASPM ShRNA expressing lentivirus, resulting in a dramatic drop of ASPM RNA in GBM1, GBM3 and to a lesser extent GBM2.Click here for file


To the Editor: Handwashing is the single most important prevention step in reducing transmission of gastrointestinal zoonoses  were also identified as C. parvum. Isolates could not be obtained from lambs because the lambs had died and were subsequently incinerated by the wildlife center. Although assessment of the private water supply revealed unacceptable levels of coliforms, oocysts were not detected.In total, 128 microbiologically confirmed cases were reported to the incident control team. An additional 252 clinical cases were reported among wildlife center visitors for whom no stool sample was taken. The illnesses of these persons had a similar implied incubation period  and their age profiles were the same as patients with laboratory-confirmed cases. Of 128 patients with confirmed cases, 117 visited the wildlife center, and infections of the remainder were attributed to secondary spread. Most case-patients were Tayside residents and were generally resident in towns and villages near the wildlife center. Of the 128 human isolates, 103 were identified as Cryptosporidium infection. In view of the strength and clarity of the association between visiting the wildlife center (petting lambs in particular) and being a case-patient . Moreover, no handwashing facilities were located near the lamb-petting area, and considerable effort was required to locate a handwashing basin in the wildlife center complex. Several alcohol hand sanitizers were located on site, but the microbicidal effects on Cryptosporidium spp. are insufficient to prevent infection, especially after direct contact with livestock (These results suggest that the outbreak was caused by direct contact with scouring lambs, a recognized risk factor for cryptosporidiosis, coupled with inadequate handwashing facilities (Escherichia coli O157 outbreaks in England have accentuated the unresolved issues for UK petting farms concerning hand hygiene and zoonotic infections (After publication of the outbreak report, an assessment of handwashing and hygiene facilities elsewhere in Scotland found them to be suboptimal and that stronger education, regulation, and other control measures were needed to protect the public. Recent


Multiple cultures from GM-CSF-/-and wild type mice were established and compared for cell production. GM-CSF-/- LTCdeveloped more slowly, but by 16 weeks produced cells resembling DC in numbers comparableto wild type cultures. LTC maintained distinct populations of small and large cells, the latterresembling DC. Cells collected from GM-CSF-/- LTC were capable antigen presentingcells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressedthe CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF toGM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cellsfrom GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintainedin LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells inincreasing the survival of mature DC.The production of dendritic cells (DC) from haemopoietic progenitors maintained in longterm stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independentlyof added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility thatcultures depend on endogenous GM-CSF produced in low levels was tested by attempting togenerate LTC from spleen and BM of GM-CSF


Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK. EGFR mutations are found in 10–15% of Caucasian and 30–40% Asian patients Growth factors and their cognate receptor tyrosine kinases (RTK) are key regulators of tumor cell initiation and progression SRC and other SRC family kinases (SFKs) are activated downstream of many different RTKs We previously demonstrated Few studies have examined the specific set of SRC substrates phosphorylated after RTK activation Using quantitative MS analysis, we examined CSF-1R-induced tyrosine phosphorylation using quantitative PhosphoScan® analysis. PhosphoScan® analysis is an MS approach that utilizes immunoaffinity purification of tyrosine phosphorylated peptides prior to MS analysis to enrich their representation in the sample 13C6-15N2-L-lysine and 13C6-15N4-L-arginine. Cells expressing CA-CSF-1R Y561F and parental MCF-10A cells were labeled with the corresponding “light” isotopic variants of the same amino acids  analysis using metabolic labeling with stable isotopes (SILAC) together with phosphotyrosine proteome analysis using samples derived from MCF-10A cells expressing either CA-CSF-1R or CA-CSF-1R Y561F no acids . NaturalWe quantitatively assessed the tyrosine phosphorylation of 275 peptides in the comparison of CA-CSF-1R MCF-10A cells to parental MCF-10A cells , and 324To guide our assignment of the threshold for significant fold changes phosphorylation, we first graphed the distribution of the non-phosphorylated peptides present in the immunoprecipitated samples and determined the mean of the quantitative fold change values. We assumed data normality based on these distributions; therefore, fold change values greater than two deviations from the mean are considered 95% likely to represent significant differences in tyrosine phosphorylation. Each comparison had a different mean value for the non-phosphopeptide distribution; however, the same statistical threshold was applied to both comparisons. For the CA-CSF-1R/parental MCF-10A comparison, fold change ratios >1.94 were defined as an upregulation in the levels of tyrosine phosphorylation, ratios 0.64–1.94 as unchanged, and ratios <0.64 as a downregulation of tyrosine phosphorylation.100 tyrosine sites on 74 proteins were phosphorylated above threshold level in cells expressing CA-CSF-1R relative to parental MCF-10A cells and were defined as “CSF-1R-induced” . The thrAn additional 38 tyrosine sites on 36 proteins were found to be phosphorylated in the CA-CSF-1R vs. CA-CSF-1R Y561F analysis, but not detected in the CA-CSF-1R vs. parental MCF-10A analysis. These sites were denoted “CSF-1R induced*” . It remaAs expected, CSF-1R activation in parental MCF-10A cells induced a strong increase in tyrosine phosphorylation. Proteins previously shown to be tyrosine phosphorylated following CSF-1R activation were detected in our analysis , thus vaGeneGO® enrichment analysis of the CSF-1R-induced and CSF-1R-induced* protein set revealed that CSF-1R induces phosphorylation of proteins associated with cellular processes including cell adhesion, developmental pathways, translation initiation, cell cycle regulation, and cytoskeletal dynamics . InteresMCF-10A cells over-expressing CA-CSF-1R are able to proliferate and survive in the absence of EGF, suggesting that CSF-1R and EGFR activate redundant signaling pathways to some degree To identify phosphorylated targets common to CSF-1R and other growth factor receptors, we compared our dataset with other available phosphoproteomic datasets. Heibeck and colleagues utilized phosphotyrosine peptide immunoprecipitation and high sensitivity capillary LC-MS/MS to identify 481 basal and EGF-induced tyrosine phosphorylation sites from 285 proteins in a non-transformed human mammary epithelial cell (HMEC) line Due to the frequent activation/over-expression of ERBB2/HER2 in mammary epithelial tumors, we also examined the overlap between CSF-1R-induced changes in tyrosine phosphorylation and changes induced by this RTK. Phosphoproteomic analysis of tyrosine-phosphorylated proteins in ERBB2-overexpressing breast and ovarian cancer cell lines identified 78 proteins with increased phosphorylation We identified three proteins with increased tyrosine phosphorylation in all the proteomic datasets. CSF-1R, EGFR and ERBB2 activation all increase the tyrosine phosphorylation of p120, caveolin and PI3-K p85-alpha, suggesting that they are key common substrates of RTK activation. In addition, phosphorylation of ACK, calmodulin, EPHA2, integrin β4, intersectin 2, KIRREL, HGF-R, GAB1, CDC2, ERK2, annexin A2, and PI3-K p85-beta were induced by CSF-1R and identified in at least one of the other three proteomic datasets analyzed for RTK-induced tyrosine phosphorylation indicating that their phosphorylation is not unique to CSF-1R.From the comparative analysis, we determined that 35 of the CSF-1R induced phosphoproteins did not overlap with those identified in any of the analyzed RTK studies . Of thesTo identify putative SRC targets, we examined whether the detected changes in tyrosine phosphorylation are dependent on Y561 by comparing the phosphotyrosine profile of cells expressing the CA-CSF-1R Y561F mutant, which is unable to recruit SRC, to that of cells expressing the CA-CSF-1R. For the CA-CSF-1R/CA-CSF-1R Y561F comparison, fold change ratios >1.55 were defined as an upregulation in the levels of tyrosine phosphorylation, ratios 0.63–1.55 as unchanged, and ratios <0.63 as a downregulation of tyrosine phosphorylation See . As Y561Earlier studies have demonstrated that the CSF-1R Y561F mutation impairs the ligand-dependent auto-phosphorylation of CSF-1R; however, the Y561F mutation did not compromise the auto-phosphorylation of CA-CSF-1R in this analysis as both the activation loop residue, Y809, and Y873 had equal levels of tyrosine phosphorylation between CA-CSF-1R and CA-CSF-1R Y561F Quantitative comparison of the differences in peptide tyrosine phosphorylation between CA-CSF-1R and CA-CSF-1R Y561F-expressing cells identified 22 tyrosine sites on 19 proteins dependent on CA-CSF-1R Y561 that were phosphorylated above threshold  of the peptides detected in the CA-CSF-1R vs. CA-CSF-1R Y561F comparison were classified as SRC-independent . Multipl35% (112/324) of peptides detected were classified as SRC-inhibited . These pTo determine whether the peptides that exhibited putative SRC-dependent phosphorylation sites were enriched for specific sequences flanking the tyrosine phosphoacceptor, we utilized an on-line extraction algorithm, Motif-X (motif-x.med.harvard.edu), developed by Schwartz and Gygi The extracted motif as shown in The motif analysis identified a high selectivity for proline at the +3 position in the SRC-dependent phosphopeptides. Peptides derived from p120, p130Cas, STAT5A, EPHA2, KIAA1217 and ZDHHC7 have a proline at the +3 position. pLOGos representations of SRC consensus motifs derived from annotated substrates in the databases PhosphoSitePlus®, Phospho.ELM and Scansite are shown in To derive information on the cellular functions of the SRC-dependent substrates, we performed an enrichment analysis. This analysis included all SRC-dependent phosphorylated proteins with a fold change >1.32 . Three oWhile it wasn't feasible to validate a large number of tyrosine phosphorylation sites identified in this screen, six of the proteins identified in the SILAC analysis overlap with SRC-dependent substrates identified in a pilot, non-quantitative MS analysis . CA-CSF-To address the relevance of the identified SRC substrates to tumorigenesis, we conducted comparative bioinformatics analysis on phosphoproteomic datasets generated from human tumor samples. Because we profiled CSF-1R-induced and SRC-dependent changes in tyrosine phosphorylation in a mammary epithelial cell line, we first analyzed a small breast cancer dataset, derived from only eight tumors, available publicly from CST's PhosphoSitePlus® EGFR are frequently found in NSCLC tumors, and EGFR activation can similarly induce the upregulation of endogenous c-SRC. We performed correlation studies to determine the substrates whose phosphorylation significantly correlated with SRC activity based on detection of the phosphorylation of Y418 SRC autophosphorylation site.Given the limited amount of phosphotyrosine proteome data available from breast tumor dataset, we also analyzed a larger set of phosphoproteomic data derived from human non-small cell lung cancer. Rikova and colleagues analyzed the phosphorylation of proteins in 150 human NSCLC lung tumors using PhosphoScan® analysis The comparison revealed that many SRC-dependent proteins were also tyrosine phosphorylated in human lung tumors. Five proteins  showed a significant correlation  between SRC activation and tyrosine phosphorylation . Three oThe quantitative MS analysis performed in this study identified proteins phosphorylated downstream of CSF-1R activation in mammary epithelial cells and defined a specific set of these proteins as candidate targets of SRC family kinases. While earlier studies have identified CSF-1R and SRC targets phosphorylated in other cell types, some of which were detected in this analysis, previously unrecognized targets of these kinases were also revealed in this study. Comparative analysis with other RTK tyrosine phosphoproteomic datasets revealed multiple overlapping pTyr substrates in CSF-1R-, EGFR- and ERBB2-expressing cells and highlighted the shared regulation of caveolin, p120 and PI3-K by these RTK pathways. Many of the putative SRC substrates including p120, Hrs and STAT5A are likely direct SRC targets based on the degree of overlap between the peptides detected and the known SRC consensus motif, as well as the observed downregulation of tyrosine phosphorylation by pharmacological inhibition of SRC kinase activity. In addition, the phosphorylation of several SRC substrates significantly correlated with SRC activation in human lung tumors. Substrates that correlated with SRC activation, such as p120, Hrs, p130Cas, FRK and KIAA0323, may be critical mediators of SRC activity in human tumors.The CSF-1 pathway is required for macrophage differentiation and survival, yet previous work suggests the CSF-1 pathway may also drive tumor cell progression. Our data delineate the changes in tyrosine phosphorylation induced by CSF-1R signaling in epithelial cells. Unlike substrates of EGFR and ERBB2, CSF-1R targets in epithelial cell have not been well characterized. Like other growth factor receptor tyrosine kinases, CSF-1R induces pleiotropic phenotypic alterations in epithelial cells. CSF-1R activation in MCF-10A cells induces cell-cell adhesion disruption, loss of contact inhibition, growth in soft agar, and enhanced cell motility Using comparative bioinformatic analysis, we found that CSF-1R modified a set of proteins which did not overlap with those phosphorylated downstream of other RTKs e.g., EGFR and ERBB2. From this set of 35 proteins, we identified 19 proteins, which had not been previously linked to CSF-1 signaling based on studies published to date; these proteins may represent substrates unique to CSF-1R signaling. These proteins regulate a number of different cellular processes including trafficking, cell cycle progression and cell polarity and exhibit such diverse functions as kinases, adaptors and structural proteins. Many of these proteins are not well characterized; thus studies of these putative novel targets may reveal new mechanistic insights into epithelial cell biology and tumorigenesis. For example, two of these proteins, ACLY and UQCRC2, play a role in the regulation of cellular metabolism. ACLY, ATP citrate lyase, is an enzyme involved in the conversion of glucose to cytosolic acetyl CoA, which is a part of the glucose-dependent lipogenesis pathway As the first characterized proto-oncogene, SRC has been extensively studied in human tumors; however, SRC itself is not frequently mutated, and on its own is not strongly transforming. The increased activation of SRC detected in tumor cells may therefore result from upstream activated RTKs SRC activity and substrate phosphorylation is tightly controlled in vivo by intramolecular interactions and protein localization, and such regulatory mechanisms are disrupted in SRC mutant variants Our study aimed to define the CSF-1R induced, SRC-regulated proteins in part to identify candidate SRC substrates that may regulate cell adhesion in MCF-10A cells http://www.phosphosite.org/home). siRNA-mediated depletion of p120 dramatically disrupts cell-cell adhesion and reduces the levels of E-cadherin and associated proteins at AJs p120 likewise regulates the stability of E-cadherin and is a well-characterized SRC target Plakophilin 4, a p120 family member that exhibits cell type-dependent localization to AJs and desmosomes SRC may also influence the dynamics of cellular endocytosis indirectly leading to the downregulation of E-cadherin. Hrs, β-adaptin, and intersectin 2 demonstrated a SRC-dependent increase in tyrosine phosphorylation. These proteins have been previously shown to regulate distinct steps in endocytic trafficking Analysis of the overlap between substrates phosphorylated in our study and in human tumors defined a group of proteins that may be most relevant to Src activity in human cancers: HIPK3, STAT5A, p120, Hrs and FRK in human breast tumors, and p130Cas, p120, FRK, Hrs and KIAA0323 in human lung tumors. Each of these proteins has been previously implicated in cancer onset or progression, and our data suggest that their tyrosine phosphorylation may play a role in oncogenic changes. SRC activity in human tumors may further coordinate the phosphorylation of multiple substrates to mediate downstream phenotypes. For example, the coordinate phosphorylation of p120 and Hrs could be required to significantly impact E-cadherin surface stability and disrupt cell-cell adhesion. The identification of a critical set of SRC substrates may lead to the definition of a SRC signature that could serve as a molecular flag that SRC is active in a given tumor. Thus, while monitoring the phosphorylation of one or two SRC targets may not predict response to a clinical SRC inhibitor like dasatinib, phosphorylation of a collection of proteins may be a more useful predictor of efficacy.13C6-15N2-L-lysine and 13C6-15N4-L-arginine were purchased from Cambridge Isotope Labs . Antibodies to SRC phospho-Y418, STAT5A phospho-Y694, and STAT5A, were purchased from Cell Signaling Technologies . The tubulin antibody was obtained from Abcam Inc. , and the Hrs antibody was procured from Alexis Biochemicals/Enzo Life Sciences . SRC inhibitor, SU6656,  was used at 2µM.Stable isotope containing amino acids CA-CSF-1R and CA-CSF-1R Y561F MCF-10A cells were generated using pMSCV human CSF-1R L301S/Y969F provided by O. Witte . The Y561F mutant receptor was generated previously by Carolyn Wrobel Phosphopeptides were prepared using PhosphoScan® Kit .Peptides in the immunoprecipitation eluate (40 µl) were concentrated and separated from eluted antibody using Stop and Go extraction tips (StageTips) To guide our assignment of the threshold for significant fold changes phosphorylation, we first graphed the distribution of the non-phosphorylated peptides present in the immunoprecipitated samples and determined the mean of the quantitative fold change values. We assumed data normality based on these distributions; therefore, fold change values greater than two deviations from the mean are considered 95% likely to represent significant differences in tyrosine phosphorylation. Each comparison had a different mean value for the non-phosphopeptide distribution; however, the same statistical threshold was applied to both comparisons. For the CA-CSF-1R/parental MCF-10A comparison, fold change ratios >1.94 were defined as an upregulation in the levels of tyrosine phosphorylation, ratios 0.64–1.94 as unchanged, and ratios <0.64 as a downregulation of tyrosine phosphorylation. For the CA-CSF-1R/CA-CSF-1R Y561F comparison, fold change ratios >1.55 were defined as an upregulation in the levels of tyrosine phosphorylation, ratios 0.63–1.55 as unchanged, and ratios <0.63 as a downregulation of tyrosine phosphorylation. All of the detected phosphopeptides were classified for CSF-1R and SRC regulation on the basis of the calculated relative fold differences .Cells were lysed in RIPA lysis buffer supplemented with protease inhibitors and phosphatase inhibitors. Lysates were run on an SDS 10% polyacrylamide gel and transferred onto PVDF membranes , and immunoblots were visualized with an enhanced chemiluminescence kit .−4, and the occurrence threshold was set to a minimum of four peptides.The Motif-X algorithm http://www.genego.com). The enrichment analysis computes the probability that a set of genes that are annotated within a specific category would occur by chance and the results are given in the form of p-values. The enrichment score provided on bar graphs is computed as −log.Proteins identified from the PhosphoScan® analysis were assessed for enrichment of Gene Ontology and GeneGO® categories using GeneGO® software (http://www.bioconductor.org/). SwissProt identifiers from the CSF-1R study were mapped to human and mouse Entrez Gene Identifiers and merged to Entrez Gene Identifiers mapped identifiers provided in published studies. Networks showing the degree of overlap were generated in Cytoscape using the Cerebral Plug-in. Overlap with published tumor data sets was performed with a custom Perl script that identifies overlapping peptides on the basis of peptide sequence. Correlation p-values were calculated using the right-sided Fisher's exact test. To determine whether the phosphorylated proteins from the CSF-1R/SRC PhosphoScan® analysis have been previously linked to CSF-1R signaling, a comprehensive CSF-1R protein interaction network was generated using the Grow Tool in Ingenuity Pathways Analysis . The genes from this network were cross-referenced to the proteins from the PhosphoScan® analysis.Phosphorylated proteins from the CSF-1R/SRC PhosphoScan® analysis were compared to published mass spectrometry datasets using BioConductor/R Click here for additional data file.Table S2Tyrosine phosphorylated peptides identified in the comparison of CA-CSF-1R to CA-CSF-1R Y561F MCF-10A cells.(0.12 MB XLS)Click here for additional data file.Table S3Classification of CSF-1R- and Src-regulated changes in tyrosine phosphorylation in MCF-10A cells.(0.07 MB XLS)Click here for additional data file.Table S4GeneGO® enrichment in the CSF-1R induced and CSF-1R induced* set of proteins for biological processes and diseases.(0.04 MB XLS)Click here for additional data file.Table S5Proteins with increased tyrosine phosphorylation downstream of CSF-1R and EGF-R activation.(0.03 MB XLS)Click here for additional data file.


Sir,et al.[2) can be harvested. Primary closure of the donor site with mobilization of surrounding skin is possible only in a few cases when the size of the defect is less than 8 × 8 cm2.[The perforator-based anterolateral thigh flap was first described by Song et al. Minimal et al. The manaThe anterolateral thigh flap is invariably used for reconstructing large defects, where a split skin graft is needed in a majority of the cases for closure of the donor site. Although the harvesting of the split skin graft is routine for a plastic surgeon, it still has its own complications such as pain and infection of the donor site in the early postoperative period with a probability of later hypertrophic scarring. For resurfacing of the flap donor site, a split skin graft is usually harvested from another anatomical region which leads to two wounds at different sites.th postoperative day without disturbing the split skin donor site tulle dressing. All the cases had total (n= 13) or near total (n= 2) graft take. The skin graft donor sites healed over 14 to 17 days (mean = 15.8 days). Thus, all the patients had dressing limited to one limb and there was no additional donor site morbidity in any of the cases. Late management included the use of a single compression garment, both for the skin graft as well as for the grafted flap donor sites.In our series of 15 consecutive cases of anterolateral thigh flap we harvested split skin grafts from the medial or posterior aspect of the same thigh. The skin graft donor site was dressed with antibiotic-impregnated tulle dressing and absorbent sponge. The flap donor site was resurfaced with the split skin graft and fixed using a tie-over dressing. The tie-over dressing was removed on the 5To conclude, we suggest that in the management of the donor site of an anterolateral thigh flap, split skin grafts can be harvested from the same thigh without any additional complications. This helps to limit the use of dressing material and time. Postoperative management of the skin graft and flap donor sites becomes simple and cost-effective as both donor and recipient sites can be managed with the same compression garment. In addition, this method helps to restrict the scarring to the same thigh .


Background Since long-term administrations of anti-hyperlipidemicagents result in reduction in  % stenosis or increase inminimum lumen diameter (MLD) of stenotic coronarysegments, it is generally believed that anti-hyperlipidemic agentsstabilize vulnerable coronary plaques. However, recent pathologicand angioscopic studies revealed that vulnerability of coronaryplaques is not related to severity of stenosis and the rims ratherthan top of the plaques disrupt, and therefore, angiography is notadequate for evaluation of vulnerability.Angioscopy enables macroscopic pathological evaluation of the coronary plaques.Therefore, we carried out a prospective angioscopic open trial for evaluation of thestabilizing effects of bezafibrate on coronary plaques.Methods From April, 1997 to December, 1998, 24 patients underwentcoronary angioscopy of the plaques in the non-targeted vesselsduring coronary interventions and 6 months later. The patientswere divided into control  andbezafibrat  groups. Oraladministration of bezafibrate  wasstarted immediately after the interventions and was continued for6 months. The vulnerability score was determined based onangioscopic characteristics of plaques and it was compared beforeand 6 months later.Results  Six months later, vulnerability score was reduced in bezafibrate group andunchanged  in control group. Inbezafibrate group, the changes in vulnerability score was notcorrelated with those in % stenosis or MLD.Conclusion The results indicate that bezafibrate can stabilize coronary plaques.


Hamilton-Norwood scale (HNS) has been largely used to assess clinically the severity of androgenetic alopecia (AGA), especially for therapeutical trials and even to establish its association with important diseases such as ischemic heart disease and prostate cancer.To study HNS reproducibility in the hands of dermatologists and dermatology residents.Seven dermatologists and 16 residents in dermatology classified 43 photographs of male heads with different degrees of AGA. In a second study, 8 appraisers (3 dermatologists and 5 residents in dermatology) examined 56 pictures with the same procedure and repeated the observation 3 months later. In the first study, the inter-rater agreement was estimated by calculating an intra-class correlation coefficient (ICC). In the second study, for intra-rater repeatability, each rater's scores from session 1 were paired with his/her scores for the same subjects in session 2, and the ordinary least products linear regression was calculated.In the first study, the concordance of appraisers was unsatisfactory (ICC = 0.63-0.68)]. In the second study, repeatability was poor, without any significant difference between dermatologists and dermatology residents.Reliability of HNS is unsatisfactory even in the hands of expert appraisers. To obtain better reliability, the number of classes should be reduced, but with such reduction HNS would be usable to classify patients only in a broad way. Hamilton-Norwood scale (HNS) has been largely used to assess clinically the severity of androgenetic alopecia (AGA), especially for therapeutical trials, and to establish its association with important diseases such as ischemic heart disease and prostate cancer.‐8 Its re9Seven dermatologists and 16 dermatology residents were recruited as raters. They classified 43 photographs of male heads showing different degrees of AGA by constantly checking a cartoon depicting HNS, except the anterior model. Each examiner independently scored the pattern of each photograph as I, II, III, III vertex, IV, V, VI or VII degree using ordinals between 1 and 7 plus a value of 3.5 corresponding to III vertex degree.In a second study, 3 dermatologists and 5 dermatology residents scored 56 randomly selected photographs on two separate occasions, 3 months apart, with the same procedure. The pictures were shown in the second session in the same order as in the former one.Statistical analyses were done using SPSS version 17.0 .2 were calculated.[2 is a measure of linear association between two variables and corresponds to 1 when correlation is perfect.In the first study, the inter-rater reproducibility was estimated by calculating the intra-class correlation coefficient (ICC) based on an ANOVA mixed model. The valulculated. R2 is a P < 0.001). Only a slight difference between dermatologists and dermatology residents  was observed [In the first study, data were greatly dispersed (coefficients of variation varied from 66% to 357%). ICC was 0.65  was unsatisfactory. In fact, only one dermatologist achieved 0.75 of adjusted Robserved .et al. studied the concordance between the assessment by each observer and that by the patients of the balding patterns of the patients at age 35. The latter proved to be only moderately accurate. Littmann and -colleagues[AGA severity and drug efficacy have been repeatedly assessed by HNS. HNS has been employed even to evaluate issues as important as the relationship of baldness with coronary artery disease and prostate cancer. In such cases, the assessment has often been done by non-dermatologists or even by the patients themselves. HNS reliability has been verified recently. Taylor and - colleagues examinedolleagues studied Two considerations should be done, however, when appraising those studies. The first is that to achieve a good agreement, HNS classes had to be reduced to 4 and 3, respectively.10 The seOn the other hand, if to obtain a good reliability the number of classes is reduced, the classification becomes usable, as Chamberlain and Dawber put it, only in the broader classification of patients who are likely to respond to therapies.ICC is a descriptive statistic that can be used when quantitative measurements are made on units that are organized into groups. It describes how strongly units in the same group resemble each other and is currently used to assess the reproducibility of quantitative measurements made by different observers measuring the same quantity.Assessing AGA severity is not easy, especially if reliable, inexpensive and minimally invasive means are required. In our hands, HNS proved unsatisfactorily reproducible. A new classification, universal for men and women, has been recently introduced but stil


Outcomes following bacterial meningitis are significantly improved by adjunctive treatment with corticosteroids. However, little is known about the levels and significance of intrathecal endogenous cortisol. The aim of this study was to assess cortisol as a biological and diagnostic marker in patients with bacterial meningitis.Forty-seven consecutive patients with bacterial meningitis and no prior treatment were evaluated. For comparison, a group of 37 patients with aseptic meningitis and a group of 13 healthy control individuals were included.r = 0.587, P < 0.001). Furthermore, CSF cortisol levels correlated with Acute Physiology and Chronic Health Evaluation II score , Sequential Organ Failure Assessment score , Glasgow Coma Scale score  and CSF lactate levels . CSF cortisol was only weakly associated with intrathecal levels of IL-6  and IL-8 . CSF cortisol levels in bacterial and aseptic meningitis significantly differed (P < 0.001). The CSF cortisol concentration of 46.1 nmol/l was found to be the optimal cutoff value for diagnosis of bacterial meningitis.The mean age of the bacterial meningitis patients was 42 years, and the mean Glasgow Coma Scale, Acute Physiology and Chronic Health Evaluation II, and Sequential Organ Failure Assessment scores on admission were 12, 13 and 4, respectively. Altogether, 40 patients (85%) were admitted to the intensive care unit, with a median (interquartile range) length of stay of 8 (4 to 15) days. A bacterial etiology was confirmed in 35 patients (74%). The median (interquartile range) cortisol concentration in cerebrospinal fluid (CSF) was 133 (59 to 278) nmol/l. CSF cortisol concentrations were positively correlated with serum cortisol levels (CSF cortisol levels in patients with bacterial meningitis are highly elevated and correlate with disease severity. Moreover, our findings also suggest that intrathecal cortisol may serve as a valuable marker in discriminating between bacterial and aseptic meningitis. Bacterial meningitis represents a serious disease that is associated with significant morbidity and mortality. Outcomes of bacterial meningitis has remained stable since the advent of antibiotics, with the case fatality being as high as 25% . FurtherHaemophilus influenzae in children [Streptococcus pneumoniae [It has been suggested that poor outcomes following bacterial meningitis are significantly influenced by exaggerated immune responses in the brain. The inflammatory brain injury has been associated with overproduction of reactive nitrogen species and tumour necrosis factor (TNF)-α in the intrathecal compartment . Becausechildren . Recentleumoniae .Although it is known that exogenous corticosteroids can improve the outcome of bacterial meningitis, less is known about the role played by important endogenous anti-inflammatory mediators, such as cortisol and IL-10, in CSF during the course of bacterial meningitis. It is assumed that high levels of IL-10, as were observed in CSF from children with bacterial meningitis, can suppress the intensity of intrathecal inflammation and limit its deleterious effects . AlthougThe aim of our study was therefore to evaluate cortisol levels, both in CSF and serum, in the initial phase of bacterial meningitis, and to assess their correlation with inflammatory cytokines as well as routinely examined laboratory parameters. Also, we evaluated relationships between these mediators and the severity of bacterial meningitis, as determined using the Glasgow Coma Scale (GSC), the Acute Physiology and Chronic Health Evaluation (APACHE) II and the Sequential Organ Failure Assessment (SOFA). We also tested whether CSF cortisol levels may correlate with long-term outcome of bacterial meningitis, which was assessed using the Glasgow Outcome Score (GOS). Finally, we tested whether CSF cortisol could be used as a sensitive marker of bacterial meningitis, facilitating distinction of acute bacterial meningitis from aseptic meningitis on admission.n = 3), administration of methylprednisolone before admission (n = 4), and diagnostic lumbar puncture performed elsewhere (n = 2). Demographic and clinical data for the 47 patients with bacterial meningitis enrolled in the study are presented in Table This prospective study was conducted, in accordance with the Declaration of Helsinki, once approval had been obtained from the local ethics committee, during the period from December 2002 to December 2005. Because we used only leftovers from clinical specimens, the committee waived the need for informed consent. During the study period, 56 patients presenting with suspected bacterial meningitis (in whom this was subsequently confirmed) were admitted to the infectious disease department of a tertiary care hospital. Nine patients were excluded for the following reasons: antibiotic treatment before admission  with serum separation gel in order to separate blood serum. For glucose and blood count determination, blood was drawn into S-Monovette® tubes with K3-EDTA. All samples were centrifuged immediately after the collection, aliquoted and stored at -80°C until further analyses were conducted.CSF samples were collected in polystyrene tubes closed with screw-caps . Venous blood was collected into S-MonovetteLeucocyte numbers were determined using a Fuchs-Rosenthal counting chamber  after staining with crystal violet (0.2%) and lysis of erythrocytes with 4% acetic acid. Absolute numbers of mononuclear and segmented cells were determined using the counting chamber. CSF concentrations of glucose, lactate and protein were defined colorimetrically using an automated clinical chemistry analyzer .WBC counts were determined using clinical analyzer Coulter STKS . Serum C-reactive protein levels were measured using a nephelometer  using a set Latex CRP Mono (Behring), with normal range between 0 and 8 mg/ml.Concentrations of IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α in CSF and serum  were analyzed using a cytometric bead array kit (BD™ Cytometric Bead Array – Human Inflammatory cytokine kit) and with a three-colour flow cytometer FACSCalibur™ . The detection limit for all cytokines was 20 pg/ml.The concentration of total cortisol was determined by radioimmunometric assay, using a commercial DSL-2000 kit . The detection limit for cortisol was 5 nmol/l. The intra-assay and interassay coefficients of variation were measured using patient serum samples and were 5% and 10%, respectively, in all tests.Statistical analyses were performed using SPSS software™ by a certified biomedical statistician. Data are presented as mean (standard deviation) or as median (interquartile range). Levels that were undetectable were assigned a value equal to the lower limit of detection for the assay. The differences between variables in CSF and serum were analyzed using the Mann-Whitney rank sum test. Differences in analyzed parameters between groups were tested by one-way analysis of variance. The analyses consisted of two-tailed tests with an α level below 0.05. Spearman's correlation test was employed to determine whether a correlation existed between clinical and laboratory parameters. Receiver operating characteristic (ROC) curves, which represent the probability that a test will yield false-positive results, were drawn to determine the optimal cutoff value of CSF cortisol for discriminating bacterial meningitis from aseptic meningitis and controls. The area under the curve was also evaluated.n = 3), a communication between sinuses and intracranial space (n = 1), brain abscess (n = 1) and abdominal surgery (n = 1). Altogether, 40 patients (85%) were admitted to the intensive care unit, with an median (interquartile range) length of stay 8 (4 to 15) days.Four bacterial meningitis patients presented with septic shock on admission. Favourable outcomes of bacterial meningitis were observed in 36 patients (77%), and seven patients (15%) succumbed to bacterial meningitis within 28 days after admission. Moreover, four patients (8%) exhibited severe neurological sequelae by day 28, and surgery was necessary in six patients after they had completed the antibiotic regimen for bacterial meningitis. The reasons for the surgery were spondylodiscitis (Neisseria meningitidis and 11 cases (31%) were due to Streptococcus pneumoniae. Other aetiological agents identified included Escherichia coli (n = 3), Staphylococcus aureus (n = 2), Listeria monocytogenes (n = 2), Streptococcus bovis (n = 1), Streptococcus haemolyticus (n = 1) and Haemophilus influenzae (n = 1).The bacterial aetiology of bacterial meningitis was confirmed in 35 patients (74%). Out of these 35 cases of bacterial meningitis, 14 (40%) were caused by Routine cytological and clinical chemistry parameters in serum and CSF in the bacterial meningitis group are summarized in Table r = 0.587, P < 0.001).Cortisol and cytokine CSF concentrations in all groups and levels of statistical significance are summarized in Table r = 0.763, P < 0.001; Figure r = 0.650, P < 0.001; Figure r = -0.547, P < 0.001). Also, we found a correlation between GOS score and CSF cortisol . Of six cytokines evaluated in CSF, only moderate correlations with CSF cortisol were found for IL-6  and IL-8 . Additionally, there was no correlation between all evaluated cytokines and clinical scores.The primary aim was to assess the relationship between the severity of bacterial meningitis and CSF cortisol as well as CSF levels of inflammatory cytokines. CSF cortisol concentration exhibited a positive correlation with APACHE II score  and protein . Also, associations were detected between CSF level of IL-6 and lactate , protein , IL-8  and WBC count in CSF . Finally, intrathecal levels of IL-8  and IL-10  correlated positively with CSF concentrations of TNF-α.Further r = 0.399, P = 0.014) and SOFA score . Of the cytokines evaluated, IL-8 correlated with APACHE II, SOFA and GCS scores , as did IL-6 . No correlation was found between other serum cytokines  and severity of bacterial meningitis. Also, analyses revealed that serum cortisol levels correlated positively with IL-6 , IL-10  and IL-8 .The initial aim of the study was to evaluate the association between serum cortisol, cytokines and severity of bacterial meningitis. As was expected, serum cortisol exhibited a positive correlation with APACHE II score  and IL-8  in serum. Finally, a relationship was observed between serum concentrations of IL-6 and IL-8 .In addition, further P < 0.001; Table The levels of CSF cortisol in bacterial meningitis and aseptic meningitis differed significantly (In this study we hypothesized that CSF cortisol levels would be elevated in patients with bacterial meningitis and that this increase might correlate with disease severity. Also, we aimed to identify a level of CSF cortisol that could serve as a marker of bacterial meningitis.In accordance with our assumption, CSF cortisol concentrations were significantly elevated in patients with bacterial meningitis as compared with concentrations in patients with aseptic meningitis as well as in healthy control individuals. We report here, for the first time, the cutoff value of CSF cortisol that separates bacterial meningitis from aseptic meningitis on admission, with good sensitivity and specificity. Moreover, we found strong correlations between high CSF cortisol and elevated APACHE II, SOFA and GCS scores in the 47 patients with acute bacterial meningitis. Similar associations were observed for serum cortisol. As expected, our results revealed highly elevated concentrations of the majority of detected cytokines in CSF, and confirmed the previously described compartmentalization of the inflammatory response in the subarachnoideal space as compared with peripheral blood .Interestingly, the increased CSF cortisol levels exhibited a strong correlation with the severity of bacterial meningitis, whereas highly elevated intrathecal cytokine concentrations – especially IL-6 and IL-8 – exhibited no relationship with clinical scores. Because this is the first study of CSF cortisol during the acute stage of bacterial meningitis, it raises concerns about the pathophysiological and clinical importance of this hormone. With regard to serum cortisol levels during the course of bacterial meningitis, van Woensel and coworkers  reportedde novo synthesis of cortisol, catalyzed by the brain enzyme 11β-hydroxysteroid dehydrogenase type 1 [Increased CSF cortisol levels have previously been reported in various CNS disorders, such as multiple sclerosis, Alzheimer's disease, depression and post-traumatic stress disorder -23; howee type 1 . Also, te type 1 . During Our study in patients with bacterial meningitis demonstrates that the brain is exposed to cortisol levels that are substantially greater than normal. Moreover, the correlation observed between CSF cortisol and GOS in our patients with bacterial meningitis suggest that the high level of intrathecal cortisol could exacerbate bacterial meningitis-related inflammatory brain injury. In a rabbit model of experimentally induced pneumococcal meningitis, Zysk and coworkers  reportedIt has previously been proposed that both inflammatory cytokines and lactate in CSF may represent reliable laboratory markers for discriminating between bacterial meningitis and aseptic meningitis . Our finCertain limitations of our study should be considered. The difference in CSF cortisol levels found between bacterial meningitis and aseptic meningitis patients might partly be influenced by differences in severity between these two central nervous system infections. Also, CSF cortisol levels were detected using the radioimmunoassay method, which is not suitable for use in the clinical setting. Therefore, the value of CSF cortisol as a diagnostic biomarker requires confirmation in a larger, prospective clinical study.Intrathecal levels of cortisol, as opposed to serum levels, may represent a valuable biomarker of the severity of bacterial meningitis. Moreover, CSF cortisol may help to discriminate bacterial meningitis from aseptic meningitis reliably. Finally, our findings support the pathophysiological importance of intrathecal cortisol during bacterial meningitis, and further studies are warranted to elucidate the role played by this mediator in brain.• CSF and serum cortisol levels are markers of the severity of bacterial meningitis.• CSF cortisol levels are significantly higher in bacterial meningitis than in aseptic meningitis, which may help in differentiating between them.• The pathophysiological effects of high CSF cortisol concentrations during bacterial meningitis are unclear.APACHE = Acute Physiology and Chronic Health Evaluation; CSF = cerebrospinal fluid; GCS = Glasgow Coma Scale; GOS = Glasgow Outcome Score; IL = interleukin; ROC = receiver operating characteristic, SOFA = Sequential Organ Failure Assessment; TNF = tumour necrosis factor; WBC = white blood cell.The authors declare that they have no competing interests.MH designed and coordinated the study, collected data and drafted the manuscript. OB participated in the design of the study, supervised the laboratory experiments and helped to write the manuscript. OD helped to collect patient samples and data. JH carried out the CBA experiments and helped to collect patient samples and data. ZL carried out the cortisol analysis. BP is a certified statistician and conducted statistical analyses. JP and MH helped to collect patient data. All authors read and approved the final manuscript.


Pleural effusion secondary to ventriculoperitoneal shunt insertion is a rare and potentially life-threatening occurrence.We describe a 14-month-old Caucasian boy who had a ventriculoperitoneal shunt inserted for progressive hydrocephalus of unknown etiology. Two and a half months post-shunt insertion, the patient presented with mild respiratory distress. A chest radiograph revealed a large right pleural effusion and a shunt series demonstrated an appropriately placed distal catheter tip. A subsequent abdominal ultrasound revealed marked ascites. Fluid drained via tube thoracostomy was sent for beta-2-transferrin electrophoresis. A positive test was highly suggestive of cerebral spinal fluid hydrothorax. Post-externalization of the ventriculoperitoneal shunt, the ascites and pleural effusion resolved.Testing for beta-2-transferrin protein in pleural fluid may serve as a useful technique for diagnosing cerebrospinal fluid hydrothorax in patients with ventriculoperitoneal shunts. Mechanical shunting of cerebrospinal fluid (CSF) is an effective treatment for non-obstructive hydrocephalus. In particular, ventriculoperitoneal (VP) shunting has become a preferred method in most clinical centers. Despite the wide acceptance of VP shunting, there are important complications associated with this technique. Common problems include obstruction, mechanical shunt failure and infections  while CSWe report a 14-month-old Caucasian boy with a symptomatic pleural effusion that developed two and a half months post-VP shunt insertion for hydrocephalus of unknown etiology. We will review potential mechanisms behind the development of this complication and discuss the use of beta-2-transferrin for the identification of CSF hydrothorax in children with VP shunts.A 14-month-old Caucasian boy with idiopathic non-obstructive hydrocephalus and a VP shunt presented to the emergency room with a 1-day history of mild respiratory distress without cough or fever. The patient had presented at 11 months of age with a history of enlarging head circumference and developmental delay. A computed tomography (CT) scan of his head at 11 months of age revealed enlargement of the lateral, third and fourth ventricles and a bulky choroid plexus. A right-sided programmable valve VP shunt was inserted at that time.On examination, the 14-month-old patient was found to be afebrile and tachypneic (34 breaths/minute) with mildly increased work of breathing and decreased respiratory sounds in the distal aspect of the right lung. The cardiovascular exam was within normal limits. An examination of the abdomen revealed distension but no hepatomegaly or signs of peritoneal irritation.A chest radiograph Figures  and 2 re9/L, respectively. Further blood work demonstrated an alanine aminotransferase (ALT) level of 46U/L, an aspartate aminotransferase (AST) level of 51U/L, a serum calcium level of 2.61mmol/L and an alkaline phosphatase (ALP) level of 1403U/L. Subsequently, ALP steadily decreased to 498U/L but remained elevated throughout the patient's admission.At the time of admission, venous blood gas, serum electrolytes and creatinine were within normal limits. Blood urea nitrogen and white blood count were mildly elevated at 5.1mmol/L and 12.5 × 10A thoracentesis was performed and a chest tube was inserted which drained >300cc/day of clear, yellow fluid. Around the time of chest tube insertion, the patient was noted to have an increasing abdominal girth, sizeable positive fluid balance and weight gain. An abdominal/pelvic ultrasound revealed marked ascites. Structurally normal major abdominal and pelvic viscera as well as normal vena caval and hepatic venous flows were reported.β2-transferrin assay and was found to be positive. Subsequent to this finding, the VP shunt was externalized followed by a dramatic decrease in chest tube drainage. A repeat chest radiograph revealed resolution of the pleural fluid collection and the chest tube was removed. The patient's abdominal girth decreased and a repeat abdominal/pelvic ultrasound demonstrated resolution of ascites. The patient's externalized ventricular drain was subsequently converted to a ventriculoarterial shunt.A head CT showed no change from a previous scan carried out at 11 months of age. A parallel pleural fluid and CSF analysis was also performed which is detailed in Table β2-transferrin for the diagnosis of CSF hydrothorax in a child with a transudative pleural effusion. Although used previously in other applications, to the best of our knowledge, this is the first reported use of β2-transferrin to detect a CSF pleural effusion in a child with a VP shunt.We report the use of A variety of techniques to investigate ascites and/or pleural effusion in patients with VP shunts have been reported in the literature. Among the described methods is the use of radiopaque contrast. This particular procedure involves the injection of radioactive dye at a point along the shunt pathway followed by subsequent imaging studies to trace the migration of contrast-injected CSF . An alteA novel diagnostic strategy was utilized in the case of our patient. A sample of fluid, drained via tube thoracostomy, was sent for beta-2-transferrin level. This desialated isoform of transferrin is almost exclusively found in the CSF with only minimal amounts present in cochlear perilymph and in the aqueous and vitreous humor of the eye. Multiple studies have validated the use of beta-2-transferrin as a specific marker for CSF leakage with sensitivity and specificity approaching 100% and 95%, respectively . FurtherFollowing externalization of the distal VP tip, our patient's ascites and pleural effusion promptly resolved. This finding, in combination with a positive beta-2-transferrin assay, was highly suggestive of a CSF hydrothorax.The findings of CSF hydrothorax and ascites, as demonstrated in our patient, are rare complications of VP shunts. Reported to occur anywhere from days to years postoperatively, CSF ascites usually develops within the first 2 years after VP shunt placement . In greaWe propose a pathological intraperitoneal process, such as subclinical peritonitis, to account for the development of CSF ascites in our patient. Inflammation of the peritoneal membrane may lead to intraperitoneal accumulation of fluid through the impairment of lymphatic flow  and/or bAs evidence to date suggests that modern shunt materials are inert, alternative sources of peritoneal irritation need to be considered. For example, an immune reaction to a foreign protein such as a vaccine may be enough to precipitate ascites in patients with VP shunts . As the Alternative explanations for CSF ascites in the literature such as excess CSF production, surgical procedures, and high CSF protein are less plausible explanations for our patient's CSF ascites. Firstly, it has been suggested that CSF ascites can result from excessive CSF production, with the excess fluid overwhelming the absorptive capacity of the peritoneum . DespiteLastly, high CSF protein has been proposed as a potential cause of CSF ascites . AccordiThe finding of CSF ascites in our patient can be used to explain the coincident finding of pleural effusion. Taub and Lavyne (2004) suggest three mechanisms to account for the development of CSF hydrothorax post-VP shunt placement . FirstlyConsidering that no surgical complications during VP shunt placement were reported in our patient and that his shunt series at the time of admission was normal, an alternative explanation is required. Thus, it is the third mechanism proposed by Taub and Lavyne, the development of hydrothorax secondary to CSF ascites that offers the most promising explanation for our patient's pleural effusion .In order for CSF ascites to result in pleural effusion, some degree of open communication must exist between the peritoneal cavity and the pleural space. Congenital diaphragmatic defects or weak points in the diaphragm such as the Foramen of Morgagni or Foramen of Bochdalek provide potential conduits. Furthermore, one or more microscopic diaphragmatic communications is likely to be sufficient for the transdiaphragmatic movement of peritoneal fluid . These pAlthough no gross diaphragmatic defect was identified in our patient, the presence of microscopic diaphragmatic communications may have been present. Notably, the tapped pleural fluid characteristics of our patient were found to be in keeping with a transudative effusion. The collected pleural fluid also demonstrated a marginally higher protein concentration than in the patient's CSF fluid. Differences in the solute composition of ventricular-extracted CSF and pleural fluid have been previously demonstrated in patients with pleural effusion secondary to VP shunts . The finIn summary, we present the case of a 14-month-old boy with CSF hydrothorax secondary to VP shunt. Pleural effusion following VP shunt insertion is a rare and potentially life-threatening event and hence requires prompt diagnosis and correction. We report a novel and non-invasive technique by which to identify the presence of CSF in pleural fluid. Beta-2-transferrin assay may serve as a useful new means for the identification of CSF hydrothorax in the context of patients with VP shunts.Written informed consent was obtained from the patient's mother for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.Both authors contributed to the conception of this case report. Dr. Jennifer Smith drafted the report while Dr. Eyal Cohen provided critical feedback. Both authors have reviewed and approve of the final version.


Premature ovarian failure (POF) remains a clinically challenging entity because in vitro fertilisation (IVF) with donor oocytes is currently the only treatment known to be effective.A 33 year-old nulligravid patient with a normal karyotype was diagnosed with POF; she had a history of failed fertility treatments and had an elevated serum FSH (42 mIU/ml). Oocytes donated by her dizygotic twin sister were used for IVF. The donor had already completed a successful pregnancy herself and subsequently produced a total of 10 oocytes after a combined FSH/LH superovulation regime. These eggs were fertilised with sperm from the recipient's husband via intracytoplasmic injection and two fresh embryos were transferred to the recipient on day three.A healthy twin pregnancy resulted from IVF; two boys were delivered by caesarean section at 39 weeks' gestation. Additionally, four embryos were cryopreserved for the recipient's future use. The sister-donor achieved another natural pregnancy six months after oocyte retrieval, resulting in a healthy singleton delivery.POF is believed to affect approximately 1% of reproductive age females, and POF patients with a sister who can be an oocyte donor for IVF are rare. Most such IVF patients will conceive from treatment using oocytes from an anonymous oocyte donor. This is the first report of births following sister-donor oocyte IVF in Ireland. Indeed, while sister-donor IVF has been successfully undertaken by IVF units elsewhere, this is the only known case where oocyte donation involved twin sisters. As with all types of donor gamete therapy, pre-treatment counselling is important in the circumstance of sister oocyte donation. Patient age and number of prior failed fertility treatments have trended upward in recent years, and both factors have an adverse impact on pregnancy rates with IVF. Among women presenting for fertility treatment, those with premature ovarian failure (POF) comprise an important subset as about 1% of all women will have this diagnosis  Ltd, Swords) plus 75 IU/d LH  superovulation regime. She received 10,000 IU hCG via subcutaneous injection on stimulation day eight with an uneventful transvaginal ultrasound-guided oocyte retrieval 36 h later. Ten oocytes were obtained and eight of these were metaphase II. Following ICSI with fresh sperm obtained from the recipient's husband, normal 2 pn fertilisation was noted in seven embryos.After pituitary down-regulation with GnRH-agonist, the donor began a combined 225 IU/d FSH (PuregonParallel to this, an appropriate endometrial response in the recipient was established by supplementary estrogen administration and verified by serial transvaginal ultrasound assessments. On day three, two fresh embryos were transferred to the recipient 11 mm inferior to the uterine fundus under ultrasound guidance; four embryos were cryopreserved the same day. Luteal support post-transfer was administered as previously described . PregnanThe patient had an unremarkable obstetrical course. Following a failed labour induction, she had a caesarean delivery at 39 weeks' gestation resulting in the birth of healthy male/male twins. Mother and babies were discharged from hospital five days post-partum in good condition; all continue to do well. Of note, an unassisted pregnancy was achieved by the sister-donor within six months of oocyte retrieval, which resulted in another singleton delivery of her own.In the setting of the advanced reproductive technologies, the difficult diagnosis of premature ovarian failure is often followed by discussions about donor oocyte IVF. This treatment modality has become an important component of the comprehensive assisted fertility treatment programme, and has been available in Ireland for several years. Patient survey results from Irish patients have generally agreed with findings reported elsewhere, showing a strong preference for sister-donor over anonymous-donor oocyte approach in IVF . Althougde novo mutations [Although most POF cases probably result from utations , the genutations ,12. Anonutations , and speutations . While cIt must be acknowledged that few POF patients will have a sister willing and able to serve as a known oocyte donor for IVF. Accordingly, for most POF patients reproductive success will be achieved clinically from IVF using oocytes from an anonymous oocyte donor. But against the background of "routine" donor oocyte therapy for POF, the current report describes an IVF approach for the exceptional case where a dizygotic twin oocyte donor is available.The authors declare that they have no competing interests.ESS, ABO, DJW, US and APHW were consultants; ACB was medical student associated to the case. All authors read and approved the final manuscript.


Mismatch DNA repair deficiency is associated with resistance to certain major groove alkylating agents including methylating agents and cisplatin. We have now studied the relevance of mismatch repair alterations to the cytotoxicity induced by drugs which alkylate N3 adenines in the minor groove of DNA. We have used the mismatch repair defective human colocarcinoma cell line HCT-116 which has a mutation in the hMLH1 gene, and a subline where hMLH1 expression is restored by chromosome 3 transfer (HCT-116+ch3). We have tested three alkylating minor groove binders  and one non-covalent minor groove binder (PNU 151807). The HCT-116+ch3 subline was more sensitive than the parental line to the treatment with the three alkylating minor groove binders, while the non-alkylating compound had a similar activity in both cell lines. Further support for mismatch repair being involved in sensitivity of the minor groove alkylators is that two cisplatin-resistant sublines of the human ovarian adenocarcinoma cell line A2780 (A2780/CP70 and A2780/MCP-1) are defective in hMLH1 expression and are more resistant to these agents than the parental mismatch repair proficient cells. Furthermore, the restoration of hMLH1 activity in the A2780/CP70 cell line, by introduction of chromosome 3, was associated with an increased sensitivity to the three alkylating minor groove binders. Again, the non-covalent minor groove binder was equally effective in mismatch repair deficient and proficient clones. The data indicate that mismatch repair deficiency mediated by loss of hMLH1 expression is associated not only with drug-resistance to major groove binders, but also to minor groove binders. However, loss of mismatch repair does not mediate resistance to the non-covalent minor groove binder PNU 151807. © 1999 Cancer Research Campaign


Anteromedial temporosphenoidal encephalocele is the least common type of temporal encephalocele. It commonly presents with spontaneous cerebrospinal fluid rhinorrhea in adults. This article presents the CT cisternography and MRI findings of one such case, which also had an associated clinically silent defect in the greater wing of the sphenoid on the same side. Basal encephaloceles are a rare cause of spontaneous cerebrospinal fluid (CSF) rhinorrhea. They are most commonly seen in the anterior cranial fossa. Temporal encephaloceles of the middle cranial fossa are unusual but do occur and have been divided into five subtypes, with the anteromedial temporosphenoidal encephalocele being the least common type. In this A 65-year-old, apparently healthy male presented with intermittent leak of clear fluid from his nostrils for the last 2 years. Initially, he had assumed that he had an attack of the common cold; however, when the leak persisted, becoming more frequent and increasing in amount, he sought medical opinion.He gave no previous history of trauma, hypertension, headache, or meningitis. The leaking fluid was examined and was found to be CSF. Detailed clinical evaluation of the patient did not reveal any other neurological abnormality. There were no features of meningeal irritation.CT cisternography was performed, which revealed an approximately 3-mm size defect in the left anterolateral wall of the sphenoid sinus, with a soft tissue density mass filling the adjacent part of the sphenoid sinus . DespiteMRI revealed a tongue of the anteromedial temporal lobe herniating through the bony defect. Abnormal T2 hyperintensity was also demonstrated in the adjacent temporal white matter. No herniation of brain or meninges was seen through the lateral defect in the greater wing of the left sphenoid Figure  and B.Taking into consideration the history of recurrent CSF rhinorrhea and the imaging findings, the patient was advised surgery. He was operated through the transnasal route. At surgery, the dural and bony defects and the associated prolapse of the anteromedial temporal lobe were confirmed. The herniated tiny lip of tissue was amputated and the dural defect was closed. The patient had complete resolution of CSF rhinorrhea and was discharged on the seventh postoperative day. He is on regular follow-up and has not shown any recurrence of symptoms over the last 2 years.CSF rhinorrhea indicates that there is an abnormal communication between the intracranial subarachnoid spaces and the nasal cavity. Spontaneous CSF rhinorrhea is much less common than traumatic CSF rhinorrhea. The nontThese basal encephaloceles of nontraumatic etiology are more common in the anterior cranial fossa than in the middle cranial fossa. The temporal encephaloceles in the middle cranial fossa are divided into five major subtypes. A lateraThe development of an anteromedial temporosphenoidal encephalocele, and its presentation as CSF rhinorrhea, imply that an osteodural defect has formed in the pneumatized part of the skull base, with the development of an abnormal communication between the temporal subarachnoid space and the sphenoid sinus cavity. Some authors have sug43The present case is unique as two bony defects were definitely documented. One defect was adjacent to the foramen rotundum at the junction of the body and the greater wing of the sphenoid bone, and the encephalocele had developed through this; the other, smaller, defect was located laterally in the greater wing of the sphenoid. Another interesting feature in this case was that, as compared with the contralateral side, there was significant remodeling of the greater wing of the left sphenoid in the form of thinning and bowing toward the infratemporal fossa, a finding that supports the theory that long-standing outward CSF pressure plays an important role in the development of these encephaloceles.All cases of anteromedial temporal encephaloceles reported in the literature have been treated surgically. Surgical treatment is necessary as the CSF rhinorrhea seldom resolves on its own; also, when there is continuous or recurrent CSF rhinorrhea, there is a high risk of meningitis. Both transnasal–13 and t46In conclusion, anteromedial temporosphenoidal encephalocele is a rare cause of spontaneous CSF rhinorrhea in adults and imaging with CT/MRI is particularly useful for its confident preoperative diagnosis.


Also, the cytotoxic response to a subsequent in vitro stimulation with lipopolysaccharide was determined. Upon daily injection of 6000–54 000 U rmGM-CSF over a 6-day period, the number of peritoneal cells increased over ten fold with the highest rmGM-CSF dose. Increases in cell numbers was mainly due to increases in macrophage numbers. Upon injection of three doses of 3000 U rmGM-CSF per day for 3 consecutive days, the number of macrophages remained elevated for minimally 6 days. Although the peritoneal cells from rmGM-CSF-treated mice were not activated to a tumoricidal state, they could be activated to high levels of cytotoxicity with an additional in vitro stimulation of lipopolysaccharide. Resident cells isolated from control mice could be activated only to low levels of tumour cytotoxicity with lipopolysaccharide. Tumour cytotoxicity strongly correlated with nitric oxide secretion. When inhibiting nitric oxide synthase, tumour cell lysis decreased. Thus, the expanded peritoneal cell population induced by multiple injections of rmGM-CSF has a strong tumour cytotoxic potential and might provide a favourable condition for immunotherapeutic treatment of peritoneal neoplasms. © 1999 Cancer Research CampaignWe studied the effect of recombinant murine granulocyte–macrophage colony-stimulating factor(rmGM-CSF) on the cytotoxic potential of murine peritoneal cells. Mice received rmGM-CSF intraperitoneally using different dosages and injection schemes. At different time points after the last injection, mice were sacrificed, peritoneal cells isolated and their tumour cytotoxicity was determined by a cytotoxicity assay using syngeneic [methyl-