There are three major B-cell compartments in peripheral lymphoid organs: the germinal center (GC), the mantle zone (MNZ) and the marginal zone (MGZ). Unique sets of B-cells reside in these compartments, and they have specific functional roles in humoral immune response. MNZ B cells are naïve cells in a quiescent state and may participate in GC reactions upon proper stimulation. The adult splenic MGZ contains mostly memory B cells and is also known to provide a rapid response to particulate antigens. The GC B-cells proliferate rapidly and undergo selection and affinity maturation. The B-cell maturational process is accompanied by changes in the expression of cell-surface and intracellular proteins and requires signals from the specialized microenvironments.CCL20 and CCL18 respectively. The MGZ was characterized by high expression of many chemokines genes e.g. CXCL12, CCL3, CCL14 and IFN-associated genes, consistent with its role in rapid response to infections. A stromal signature was identified including genes associated with macrophages or with synthesis of extracellular matrix and genes that influenced lymphocyte migration and survival. Differentially expressed genes that did not belong to the above categories include the well characterized BCL6 and CD10 and many others whose function is not known.We performed laser microdissection of the three compartments for gene expression profiling by cDNA microarray. The transcriptional program of the GC was dominated by upregulation of genes associated with proliferation and DNA repair or recombination. The MNZ and MGZ showed increased expression of genes promoting cellular quiescence. The three compartments also revealed distinct repertoires of apoptosis-associated genes, chemokines and chemokine receptors. The MNZ and GC showed upregulation of Transcriptional profiling of B-cell compartments has identified groups of genes involved in critical molecular and cellular events that affect proliferation, survival migration, and differentiation of the cells. The gene expression study of normal B-cell compartments may additionally contribute to our understanding of the molecular abnormalities of the corresponding lymphoid tumors. Appropriate T- and B-cell migration and timely interaction with antigen presenting cells (APC) are essential for the development of humoral immune responses ,2. Specihigh, CD27- [high, IgDlow and CD27+ phenotype, suggesting that this zone contains mainly memory B-cells [In follicles with developing GCs, the resting B-cells that are not the part of the GC response are pushed outward to form the mantle zone (MNZ) or corona around the GC B-cells. The mantle cell is a pre-GC, immunologically naïve B-cell that is also the putative cell of origin of mantle cell lymphoma . These Bh, CD27-  and are h, CD27- . In the h, CD27- . Unlike  B-cells .Many previous studies ,16 have We used the Lymphochip cDNA microarray  to inves+ CD27-, similar to FACS-sorted naïve B-cells. The GC was easily recognizable and generally contained a higher percentage of non B-cells, including T-cells, macrophages and follicular dendritic cells (FDC). The MGZ was obtained from a spleen with a morphologically clearly defined MGZ containing mostly IgM+ CD27+ B-cells, corresponding to the phenotype of FACS-sorted memory B-cells [GC and MNZ could be readily dissected from tonsillar frozen sections, but MGZ could only be reliably obtained from the spleen Figure . Immunos B-cells . The MGZ B-cells ,21. The Fifteen data sets corresponding to the five sample groups were generated. Different hybridizations were correlated through a correlation matrix plot, and replicated hybridizations were shown to be closely related (R ≥ 0.85). The plots allowed us to check reproducibility of the microarray assay among different samples of each tissue Figure . The numARK2 in GC, CCL20 in MNZ and CMRF-35H in MGZ. Other transcripts were expressed in all compartments with a relatively high differential expression in one, such as SET and FAIM in GC, Cyclin G2 in MNZ, and NM23-H1 and CARD11 in MGZ . The results corresponded well with both microarray and semi-quantitative RT-PCR.Some of the results of the semi-quantitative RT-PCR were further validated by the SYBRCCNB1, PCNA, Ki67), kinetochore association , functional components of mitotic checkpoints  and regulators of cell-cycle related events, including centrosome separation/segregation and cytokinesis , as expected from the known high proliferation rate of centroblasts. GC also highly expressed genes involved in DNA repair , as expected from the frequent physiological double-strand DNA breaks associated with somatic hypermutation and isotype switching. The GC profile also showed increased levels of transcripts involved in DNA replication  and in transcription and translation .Comparing the gene expression profiles of LCM GC and FACS-sorted GC B-cells  revealedCCNA, CCNB1and CCNF and of CDC2  is consistent with the resting state of the MNZ and MGZ B-cells. Characteristically CCNA expression is very low in Go phase and begins to increase in early G1. For the cell to enter the G2/M phase, an association with CDC2 is required [Cyclin G2 (CCNG2) was highly expressed in MNZ cells as compared to either GC or MGZ. The function of CCNG2 differs from the conventional cyclins in negatively regulating the cell cycle [CARD11 in MNZ may also be part of the program in maintaining the quiescent state. CARD11 has been shown to be critical for immune receptor signaling of both T and B-cells through the activation of JNK and NF-κB [CMRF-35H [CBL-B [GAS2 [The low expression of the cyclins required . The trarequired . Howeverll cycle . Studiesnd NF-κB . The incnd NF-κB . InteresCMRF-35H , CBL-B [H [CBL-B , and GAS-B [GAS2 , which mBCL2 expression in GC B-cells makes them vulnerable to undergo apoptosis unless rescued by survival signals [BIK, FASL and PDCD8 (AIF) suggests a further increase in susceptibility to apoptosis in the GC. However, FAIM is overexpressed in the GC and may represent a protective mechanism in GC B-cells that have appropriate BCR signaling and CD40L stimulation with resultant upregulation of FAIM and increased resistance to FASL-mediated apoptosis [BCL2, but have different profiles of other apoptosis/survival genes that may represent specific adaptation of these cells to their unique physiological states and microenvironment. The expression of BNIP3, encoding a proapoptotic protein of the BCL2 family[TCL1 was upregulated in MNZ only and may have an antiapoptotic role in that population.The markedly decreased  signals . An incrpoptosis . Presumapoptosis  showed ipoptosis . This suL2 family, is markSuppressor of Death Domains (SODD) in MGZ cells, suggesting a complex regulation of signaling through the TNFR superfamily. SODD is associated with TNFR1 in vivo, maintaining the receptor in an inactive monomeric state. The release of SODD from TNFR1 permits the recruitment of proteins such as TRADD and TRAF2 to the activated TNFR1 signaling complex [SODD may be a major mechanism to dampen TNFR1 signaling in MGZ B-cells in the resting state. The higher expression of CARD11 in MGZ may have a pro-survival function, but it may also have a role in MGZ organization. It has been shown that loss of CARD11 in mice resulted in the complete loss of CD5+ peritoneal cells and reduced number of IgD high IgM low mature splenic B-cells, indicating its role in B-cell development [NM23-H1 and NM23-H2, which share an amino acid identity of 88%, were highly expressed in MGZ. NM23 H1 is a granzyme A-activated DNase (GAAD) that is inhibited by SET [NM23-H1 and the low expression of its inhibitor SET was opposite in their expression profile in the GC, suggesting that this expression may influence apoptosis in opposite directions in these two B-cell compartments.There was increased expression of  complex . It has elopment . Two clod by SET . The higCCL18, encoding a chemokine secreted by immature dendritic cells (DC), is specifically upregulated in the GC compartment. Our finding was supported by a recent report showing that CCL18 is produced by GC DC and can attract MNZ B-cells towards GC [CCL18 may be especially important during the onset of a GC reaction, the time point to recruit antigen primed pre-GC B-cells, which then interact with GC DC to initiate and maintain the GC reaction. The GC compartment showed increased expression of CXCL10 (IP-10). which has pleiotropic effects, including stimulation of monocytes and T cell migration [Chemokines attract primary B-cells and play an important role in the homing and localization of B-cell subsets at different stages of antigen-independent and dependent reaction . Our micwards GC . The higigration .MIC1, also known as PLAB) [The GC also showed increased transcriptional level of genes that may suppress or control inflammatory responses; e.g., SOCS1 limits cellular response to IFNγ, IL-2 and IL-6,43. Macras PLAB) , is onlyCCL20 was observed in MNZ cells. Human naïve and memory B-cells express the cognate receptor for CCL20, CC-chemokine receptor 6 (CCR6) [CCL20 may play a vital role in naïve B-cell migration and localization in the MNZ. The chemokine gene CXCL12  is highly expressed in MGZ cells. The receptor for CXCL12 is CXCR4, which is present on CD34+ cells, myeloid cells, CD4+ T cells, B-cells, epithelial cells, endothelial cells, and dendritic cells. In the bone marrow, stromal cells secrete CXCL12, which is involved in the emigration of hematopoietic precursors to the marrow during embryogenesis [CCL14  and CCL3  were more highly expressed in the MGZ. CCL14 can activate human monocytes via receptors that also recognize CCL3 [CXCL13 and CCL5 were upregulated in both microdissected MGZ and MNZ compared with the FACS-sorted B-cell populations. Previous studies have established an important role for CXCL13 (BLC) in the development of Peyer's patches (PP) and many peripheral lymph nodes. It also controls B-cell migration and thus the organization of B-cell areas [Increased expression of the chemokine 6 (CCR6) . The higogenesis . In periize CCL3 . CCL3 isize CCL3 . Thus, tll areas . CCL5  were also preferentially expressed and may reflect the unique function of the MGZ to provide the first rapid response to particulate or T cell-independent antigens. The MGZ also showed increased expression of many members of G protein pathways consistent with more active chemotaxis, cell motility and secretory functions. In addition, MGZ cells showed higher expression of IL-7Rα, consistent with the role of the IL-7R in MGZ organization [IL-7Rα expression is also required for the recruitment of precursor cells to develop in secondary lymphoid organs and for the proper structural organization of these organs [A number of IFN-induced genes , encoding a metalloproteinase that preferentially degrades elastin and takes part in the remodeling of extracellular matrix. No collagen-specific gene was up-regulated in the MNZ.Cells function within the context of a three-dimensional (3D) extracellular matrix (ECM) that participates in regulating cellular motility, proliferation and survival. In the GC, lagen IX , and COLlagen XI , were unOsteonectin (SPARC), upregulated in LCM samples, encodes a matrix-associated protein that elicits changes in cell shape, inhibits cell-cycle progression, and influences the synthesis of extracellular matrix [MafB, and c-Maf) were also part of the stromal signature. The Maf family of genes encode bZip nuclear transcription factors and play an important role in morphogenesis and cellular differentiation [ICAM1 and VCAM1. MGZ B cells express the integrin LFA1 which binds to its ligands ICAM1 and VCAM1, and this interaction may control the localization of these B cells [VCAM1, ITGAL (LFA-1) and ITGA6 in the MNZ, suggesting a role for these adhesion molecules in mantle cell localization as well. The kruppel-like transcription factor BCL11a , which is essential for normal B-cell lymphopoiesis, was upregulated in LCM cells only. Interestingly, bone marrow from BCL11a-/- mouse can induce thymic lymphoma in wild type mice. Thus, the increased expression of BCL11a in the MNZ and MGZ may be physiologically relevant to the function of lymphocytes in these regions [Microdissected compartments contained a minor component of stromal T cells, macrophages, dendritic cells and fibroblasts whereas FACS-sorted cells from lymphoid tissues comprise almost exclusively B-cells. Thus, an insight into the gene expression profile of the stromal elements can be obtained by comparing the expression profile of FACS-sorted and microdissected cells. We found a set of genes that likely represent the stromal signature. r matrix . It regur matrix . Two memntiation . These g B cells  in this  regions .BCL6, CD10, GCET1, GCET2, JAW1 and CD38. A number of genes were clearly upregulated in the MNZ or MGZ but their functional significance is largely unknown. Some of these would be interesting targets for further investigation. Among the genes encoding surface molecules, CD59 was highly expressed in the GC. CD59 antigen is a small protein that inhibits complement-mediated pore formation or lysis by preventing the formation of membrane-embedded C9 multimers [CIITA was markedly down regulated in GC cells, associated with a general low expression of MHC transcripts.Many genes know to be specifically expressed in GC B-cells are found to be upregulated: e. g., ultimers . It is lultimers -63. NotaThe gene expression profiles of the three B-cell compartments reflect distinctive functional attributes of the resident B-cell populations. They also showed different molecular microenvironments that allow the different B-cell populations to differentiate and function properly. GC B-cells have a high proliferation gene signature, whereas MNZ and MGZ cells are characterized by signals that help to maintain the quiescent state. Genes involved in the apoptosis pathway are differentially expressed in the three B-cell compartments, reflecting different adaptations for survival in different B-cell populations. Expression of different chemokines, their receptors, and stromal molecules have been detected. Many of these have been implicated in the establishment of the normal lymphoid architecture in peripheral lymphoid organs and in attracting distinct immune-cell populations to specific lymphoid areas. The expression of unique sets of genes may also reflect the functional adaptation of cells in a specific location, such as genes involved in DNA repair in the GC and genes that are active in innate immune response to infection in the MGZ. Gene expression profiling of B-cell compartments has allowed us to obtain a global survey of the molecular signals that are functionally important in B-cell subpopulations as well as the respective microenvironments. One of the major challenges is to delineate the functions of the uncharacterized genes that are unique to each of the compartments. Another challenge is to exploit these normal transcriptional profiles to further our understanding of the normal immune response and the derangements resulting in the corresponding lymphoid tumors.Tissue blocks of tonsils and spleens were snap frozen in O.C.T immediately after surgery. Four micrometer thick frozen sections of reactive tonsils or spleens on plain glass slides were fixed with 70% ethanol for 30 seconds, rinsed in DEPC water and stained with hematoxylin for 30 seconds, followed by another water rinse. The sections were then dehydrated with 70%, 95% and 100% ethanol for 10 seconds each. Finally, the slides were passed through xylene twice, each for 30 seconds. A consecutive section was immunostained for CD3 to guide the dissection. The three B-cell compartments  were isolated using LCM with the Arcturus PixCell II system . To avoid contamination, only well-defined GC, MNZ and MGZ were dissected. Cells were captured at the 15-μm laser setting on CapSure LCM Caps (Arcturus). The laser was set to pulse at 60 mW for 200 ms. The Institutional Review Board of the University of Nebraska approved the usage of tissues for this study.7 B-cells were stained with IgM-Cy-chrome, IgD-FITC and CD27-PE  at 4°C for 30 min. MGZ B-cells were isolated from the splenic B-cells gated on the IgMhighIgDlowCD27+ fraction, whereas MNZ B-cells were selected based on IgMlowIgDhighCD27- using the BD FACSVantage™ SE high-speed cell sorter Tissue from fresh spleens or tonsils was cut into small pieces in cold RPMI-1640, and cells released by grinding with a glass tissue homogenizer. The crude cell suspension was passed through a nylon mesh  to generate a single-cell suspension. B-cells were firstly isolated using the Human B-cell Isolation Kit  and the Midi Macs system . The highly enriched B-cell population (negative fraction) was subjected to 3-color cell sorting. Briefly, 1 × 10in vitro transcription (IVT) using the Ampliscribe™ High yield Transcription Kit  containing 1000 units AmpliScribe T7 enzyme at 37°C for 8–12 hours, as per the manufacture's instruction. Second-round amplification and IVT were performed as described previously [Total RNA was extracted from each sample of microdissected cells with Trizol™  and further purified with the RNeasy Mini Kit . RNA amplification was performed using a modified Eberwine protocol . Brieflyeviously . The qua. The aminoallyl group on the dUTP reacts with the ester group on the cyanine dyes. Cy3 dye was used to label the standard cDNA and Cy5 dye the test probe, and hybridization was performed as previously described [Analysis of gene expression was performed using the Lymphochip cDNA microarray, which contained 15,132 cDNA clones representing 7399 known or uncharacterized genes . Labeledescribed .© , which provided an overview of the similarity of expression profiles between multiple samples. Only genes with at least two values out of the triplicate experiments showing similar behavior were included for analysis. The expression data for each gene from an individual compartment was median/mean centered with weighted variance across the two or three replicates showing similar behavior. The initial data reduction was performed using the two-tailed student t-test to compare the differences in gene expression levels between individual compartments. Genes differentially expressed between the two compartments with a p-value of less than 0.05 were selected for further analysis using the Significance Analysis of Microarrays (SAM) approach, as described previously [ or ) and viewed by TreeView.Each tissue type was independently isolated, amplified and profiled in three separate experiments to enhance the reliability of the gene expression data. Images of hybridized microarrays were obtained and processed using GenePix 4000B microarray scanner . Spots or areas of an array with obvious blemishes were flagged and excluded from subsequent analyses. Fluorescence ratios were normalized for each array by applying a single scaling factor to all fluorescent ratios from the array . The coreviously . SAM ass- reverse transcriptase as per the manufacturer's instructions . Five-fold serially diluted cDNAs from GC, MNB and MZB were amplified with gene-specific primers for 30 cycles with the following cycling conditions: A denaturation step at 94°C for 2.5 minutes and then each PCR cycle at 94°C for15 sec, 52°C for 30 sec, and 72°C for 30 sec followed by a final extension at 72°C for 5 min. The human HPRT transcript was used as the comparative standard. The products were analyzed by electrophoresis in 2% agarose gel. The primers were designed to amplify the cDNA close to the 3' end of the transcript, and all the PCR products were less than 200 bp in length.To confirm the differential mRNA expression of the genes identified by the Lymphochip in different B-cell compartments, a semi-quantitative RT-PCR was employed. In brief, 200 ng aRNA was reversely transcribed into cDNA with 200 ng random primer using MMLV-RNase H® Green qPCR Kit  on DNA Engine OPTICON2  as per the manufacturer's instructions. The PCR protocol used an initial denaturation of 95°C for 15 minutes followed by 35 cycles . The plate was read at 70°C according to the melting point of the amplicon. Serial dilutions of cDNA from the lymphoid standard [FAIM, CCL3, SODD, NM23-H1, CARD11, Cyclin G2 and CIITA-8) and the endogenous reference genes (HPRT). For each unknown sample, the relative amounts of target cDNAs and reference cDNAs applied to the PCR reaction system were calculated using linear regression analysis from the corresponding standard curves [Some of the results from the semi-quantitative RT-PCR were also validated by the real-time quantitative PCR with DyNAmo™ HS SYBRstandard  were used curves . Then thin vitro RNA amplification and semi-quantitative PCR. JI participated in the design of the study, microarray procedure, data analysis, and drafted the manuscript. LX, RL and SS participated in data analysis. JE provided the microarray facility and various technical assistance and advice. LS and AR provided the reference standard, the Lymphochip and helpful discussions. KD, GZ, TM participated in helpful discussions and interpretation of the data. WCC conceived, organized and supervised the study, and participated in the analysis and interpretation of the data. All authors have read and approved the final manuscript.YS carried out the LCM,


Tumours arising from odontogenic tissues are rare and constitute a heterogenous group of interesting lesions. The aim of this study was to determine the relative frequency of odontogenic tumors (OT) among Nigerian children and adolescents 19 years or younger.The histopathology records were retrospectively reviewed for all the tumors and tumor-like lesions of the oral cavity and the jaws seen in children and adolescents ≤ 19 years seen between January 1980 and December 2003. Hematoxylin and eosin-stained sections were re-evaluated and the diagnosis in each case was confirmed or modified according to World Health Organization (WHO) classification, 1992; and were subjected to analysis of age, sex, site of tumor and histopathologic type.A total of 477 tumors and tumor-like lesions were seen in patients ≤ 19 years during the period of the study. Of these, 92 (19.3%) were odontogenic tumors. Benign odontogenic tumors constituted 98.9% of the cases seen, while only 1 case (1.1%) of malignant variety was seen during the period. The mean (SD) age of patients was 14.9 (± 3.1) years . Male-to-female ratio was 1:1; and mandible-to-maxilla ratio was 2.7:1. OT's were most frequently seen in patients aged 16–19 years (46.7%) and the least number (2.2%) were found in patients aged 0–5 years. Among nine histologic types of OT seen, ameloblastoma (48.9%), adenomatoid odontogenic tumor (19.6%) and odontogenic myxoma (8.7%) were predominant. Multicystic/solid and unicystic variants of ameloblastoma were diagnosed in 40 (89%) and 5 (11%) cases respectively.Odontogenic tumors are relatively common in children and adolescents in Nigeria. One out of every 5 children and adolescents with tumors and tumor-like lesions of oral cavity and the jaws seen in this study had a diagnosis of odontogenic tumor. Tumors and tumor-like growths arising from the odontogenic tissues constitute a heterogenous group of particularly interesting lesions, as they display the various inductive interactions that normally occur among the embryologic components of the developing tooth germ . In humaThere are few reports especially from Africa, which have specifically reported the high frequency of odontogenic tumors (OT) in children and adolescents in the literature. In most previous African reports, odontogenic tumours in children were presented as part of orofacial  or oral The histopathology records of the Department of Oral Pathology and Biology, College of Medicine, University of Lagos, Lagos, Nigeria, were reviewed for all the tumors and tumor-like lesions of the oral cavity and the jaws seen in children and adolescent ≤ 19 years from January 1980 to December 2003. Hematoxylin and eosin-stained sections was re-evaluated and the diagnosis in each case was confirmed or modified according to World Health Organization (WHO) 1992 classification . The datA total of 477 tumors and tumor-like lesions were seen in patients ≤ 19 years during the period of the study. Of these, 92 (19.3%) were odontogenic tumors. Benign odontogenic tumors constituted 98.9% of the cases seen, while only 1 case (1.1%) of malignant variety was seen during the period. Table Table Ameloblastoma constituted almost half (48.9%) of the odontogenic tumors with female-to-male and maxilla-to-mandible ratios of 1:1.7 and 1:8 respectively. The mean (SD) age of patients in this group was 15.1 (± 3.0) years  with most patients (49%) in age group 3. Multicystic/solid and unicystic variants were diagnosed in 40 (89%) and 5 (11%) cases respectively.Adenomatoid odontogenic tumor (AOT) was the second most common tumor in this series (Table es Table  accountiOdontogenic myxoma accounted for 8 cases (8.7%) of odontogenic tumors with a male-to-female ratio of 1:3. Only 1 case occurred in the maxilla, the rest (7) were found in the mandible. The patients were found between 10 and 19 years.Odontogenic fibroma accounted for 7 cases (7.6%) of tumors in this series. More cases were found in males and in the mandible. Patients were seen between 5 and 19 years in this group.Ameloblastic fibroma accounted for 5.4% of cases seen. The lesion occurred exclusively in the mandible with a male-to-female ratio of 1:4.Odontomas accounted for 4.3% of OT seen. They were exclusively found in the maxilla with equal sex predilection.Table We present a report of odontogenic tumours in children and adolescents aged ≤ 19 years. This report represents the largest series of odontogenic tumors in children and adolescents in Africa. We found that 19.3% of tumors and tumor-like lesions of the oral cavity and the jaws in children and adolescent in this study were odontogenic tumors. This is similar to the findings of Arotiba . Howeveret al [et al [A major problem in comparing our report with previous studies is the lack of uniformty in the age group studied in those reports. Some studies were restricted to children under the age of 14 years ,15, or 1et al . Al-Khatl [et al ,13; thesOdontogenic tumors were less frequently seen in patients aged 0–5 years in this study in agreement with most reports in the literature ,11-14,17et al [et al [et al [Odontogenic tumors in children are known to have predilection for the mandible -13. Thiset al  however l [et al ; whereasl [et al  reportedet al [Ameloblastoma was the most common tumor in this study in agreement with reports from Africa ,11. Otheet al  found odet al ,11-14. Aet al . Previouet al ,21. Unicet al ,22,23.Adenomatoid odontogenic tumor (AOT) was the second most common OT in this study and 50% of this tumor was found in patients >15 years. Asamoa  reportedet al [In children, the reported incidence of myxoma ranges from 1.2% to 39% ,6,9,13. et al  reportedet al ,26,27. Tet al ,27 reporet al [et al [Odontogenic fibroma was reported to be rare in children with incidence ranging from 0% to 1.3% of OT ,9,11,13.et al . Arotibaet al  reportedl [et al ; whereasl [et al  have repl [et al ,26 have et al [et al [et al [Odontomas are often regarded as dental hamartomas, rather than odontogenic neoplasms ,28,29. Oet al , Tanaka l [et al  and Satol [et al  that repl [et al . This mal [et al ,17, othel [et al ,14 and sl [et al ,25. OdonMalignant odontogenic tumors are rare, most especially in children ,12,15,30Odontogenic tumors are relatively common in children and adolescents in Nigeria. One out of every 5 children and adolescents with tumor and tumor-like lesions of oral cavity and the jaws seen in this study had a diagnosis of odontogenic tumor.The author(s) declare that they have no competing interests.OFA conceived the study, coordinated the write-up and submission of the article, and also reviewed the slides.ALL, WLA and MOO did the literature search and participated in the writing of the manuscript.All the authors read and approved the final manuscript.None declared


A conditional-lethal recombinant virus was constructed in which the expression of the vaccinia virus I7L gene is under the control of the tetracycline operator/repressor system. In the absence of I7L expression, processing of the major VV core proteins is inhibited and electron microscopy reveals defects in virion morphogenesis subsequent to the formation of immature virion particles but prior to core condensation. Plasmid-borne I7L is capable of rescuing the growth of this virus and rescue is optimal when the I7L gene is expressed using the authentic I7L promoter. Taken together, these data suggest that correct temporal expression of the VV I7L cysteine proteinase is required for core protein maturation, virion assembly and production of infectious progeny. The cysteine proteinase encoded by the VV I7L gene, was originally identified based on a sequence comparison with the African Swine Fever virus proteinase and an ubiquitin-like proteinase in yeast ,2. We haresidues ,4. To delac operator and driven off of the T7 promoter, was responsible for cleaving the A17L membrane protein as well as the L4R core protein precursor. In this work, we show that I7L proteinase, in a different inducible mutant virus, this one regulated by the tetracycline (TET) operator/repressor system and driven off of the I7L native promoter, is responsible for cleaving the other core protein precursors (p4a and p4b). We also demonstrate that expression of the I7L gene from its native promoter appears to be important for optimal viral assembly and replication.While this work was in progress, Ansarah-Sobrinho and Moss  publisheTo investigate the role of the I7L proteinase in the viral life cycle, an inducible mutant virus was constructed in which the expression of the I7L gene could be regulated by the presence or absence of TET using the components of the bacterial tetracycline operon ,7. This 40 cells [40 cells. At an MOI of 0.1 or 0.5 there was an average reduction of 99.1% of infectious virus particles  and infected with vtetOI7L at an MOI of 0.2 plaque-forming units per cell in the absence of TET. Cells were harvested 24 hours post infection (hpi) and the titer determined on BSCene Fig. . This waene Fig. , suggest35S-met and harvested after 24 h. Extracts were immunoprecipitated with I7L antisera and protein detected by autoradiography. With wild type virus, I7L protein was expressed at each TET concentration (data not shown). However, in the mutant virus, expression of I7L enzyme was repressed in the absence of TET and increased with the addition of TET.We have previously shown through transient expression assays that the I7L proteinase is capable of cleaving the p4b, p4a, and p25k core protein precursors ,4 which To determine the effect of TET concentration on p4b core protein precursor processing, cells were infected in the presence of 0 to 5 μg/ml TET, harvested 24 hpi, and the extracts immunoblotted with anti-4b antisera. With wild type virus p4b was processed at each TET concentration as expected, however with the mutant virus, p4b processing was repressed in the absence of TET (data not shown). The slight processing in the absence of TET is likely due to slight leak-through of I7L gene expression in this system. The same results were seen for the processing of p4a, with processing in each of the wild type virus lanes, repressed processing with the mutant in the absence of TET and increased processing in the presence of TET (data not shown). Kane and Shuman  have preet al [The morphogenesis of vtetOI7L under nonpermissive conditions was analyzed via electron microscopy. TREx-293 cells were infected with vtetOI7L at an MOI of 1 in the presence or absence of TET and harvested 24 h later. In the presence of TET, cells contained a variety of both immature and mature forms of the virus  declare that there are no competing interests.CMB conducted all the experiments and wrote the manuscript. DEH conceived the study, coordinated the research efforts and edited the paper. Both authors read and approved the final manuscript.


The objective of this study was to investigate the relationship between perceived walkability and overall self-rated health among patients who use community-based clinics.A cross-sectional survey was distributed to a convenience sample in three community clinics. Forms were completed by 793 clinic patients. Multiple logistic regression analysis was to control for the effects of demographic variables and lifestyles.Perceiving the availability of places to walk was related to better self-rated health. The most important places were work (OR = 3.2), community center (OR = 3.12), park (OR = 2.45) and day care (OR = 2.05). Respondents who said they had zero (OR = .27) or one (OR = .49) place to walk were significantly less healthy than persons who said they had five or more places to walk.Persons who perceived that they had no place to walk were significantly less healthy than persons who thought they had at least one place to walk (OR = .39). Support for walkable neighborhoods and education of patients about options for walking may be in the best interests of community medicine patients. Much recent interest and research has been directed at the relationship between one's health and where one lives . A numbeNeighborhoods where walking is convenient might encourage their inhabitants to exercise. Indeed, having convenient places to walk in the neighborhood has been related to the proportion of persons in the neighborhood who met current activity recommendations  and withThe hypothesis that perceived walkability is directly related to an individual's overall self-rated health has not previously been investigated. The purpose of this project was to test that hypothesis in patients attending community-based clinics. Self-rated health has been shown to accurately predict overall health as measured by other more traditional measures of health .A cross-sectional survey was used to test the hypothesis that patients with good self-rated health perceived the neighborhoods in which they live as having good walkability, and that this effect was independent of demographic characteristics and lifestyle habits that would predict good health. The sample was drawn from three clinics that primarily serve low-income populations. Clients, parents (if the patient was a child) and accompanying visitors were asked to complete surveys and drop them into a box. Participation was voluntary. A total of 1471 surveys were distributed with an overall return of 825 (56.1%) surveys of which 793 (54%) met eligibility parameters. Pregnant women and persons under age 18 were excluded. Assuming 80% power, p < .05, 20% poor health among the persons inhabiting neighborhoods with good perceived walkability and 30% poor health among persons inhabiting neighborhoods with poor perceived walkability, 626 cases were needed to test the hypothesis. Completed forms were received from a total of 793 persons.Return rates varied by clinic. Clinic 1 is a university-based family medicine clinic providing a full range of primary care services to cross-generational clients. It is staffed by family medicine physicians and residents. Census was approximately 85 clients daily, of which, less than 5 % were non-English speaking. Clinic personnel distributed 500 survey forms over an eight week period with an 80.8% return rate.Clinic 2 serves women and children, providing obstetrical, well care (including immunizations), and acute care services to a targeted high-risk, low socioeconomic sub-population. It is staffed by pediatric and OB-GYN physicians and residents. Approximately 30% of the clinic clients do not speak English. A total of 471 surveys were distributed over a period of 18 weeks with a return of 37.4%. Both the large number of obstetrical patients (ineligible for survey) and percentage of non-English speaking clients contributed to the low return rate.Clinic 3 provides primary care services to a population of indigent adults meeting residential and income screening requirements. It is staffed by internal medicine physicians and residents. A total of 500 surveys were distributed in this clinic over a ten week period with a return of 42.6%.The dependant variable for the study was self-rated health. Subjects were asked whether in general they would say their health was excellent, very good, good, fair or poor. Excellent, very good, and good responses were combined to form a category called 'good health' while fair and poor comprised 'poor health'.Leyden's scale of walkability was modified for a US population for this study by droppBoth demographic characteristics and lifestyle variables were used to adjust the associations between perceived walkability and self-rated health. Lifestyle variables were: numbers of fruits and vegetables eaten per day , smoking status , days of physical activity per week that involve at least 20 minutes of exercise , and obese (yes vs no). Obese was defined as body mass index (BMI) >30. BMI was computed from self-reported height and weight.Demographic variables were race/ethnicity , gender, age category, marital status (married vs. not), and highest level of education achieved.Chi square tests were performed to test for any unadjusted associations between self-rated health and each categorical independent variable. Multivariate logistic regression modeling was employed to determine if associations between perceived walkability and self-rated health remained significant after adjustment for demographic and lifestyle variables. Separate logistic regression models were run for each variable. Statistical analysis was performed using EpiInfo 3.2.2.The question about self-rated overall health was answered by 793 respondents. Of these, 67 percent were classified as healthy because they said their health was excellent, very good, or good. The remainder was classified as having poor health because they said their health was fair or poor.The typical respondent was non-Hispanic white, female, had at least a high school education. The sample was evenly spread across age groups. About half were married and about half were not married Table .Unadjusted relationships between demographic variables and self-rated health are shown in Table The most common number of days of exercise per week was zero (34.9 percent). Most people said they ate at least one fruit or vegetable each day, though only less than half had three or more. The percent obese was 43.6. Over 70 percent were non-smokers . Meeting recommended physical activity guidelines is related to better self-rated health . CertainIf promotion of intensive physical activity is problematic, then more moderate activity might be a more reasonable goal. Simple walking, for example, reduces the cost of medical care for the elderly  and inteIn addition to valuing leisure-time walking, researchers and clinicians should take into account the benefits of work related activity and housework . So callOne approach to encouraging moderate physical activity is to increase walkability. The convenience of places to exercise is widely recognized to be important; adults generally support local policies that increase the availability of places to exercise -28. WalkOur purpose was to investigate the effect of perceived walkability on the health of patients attending community based clinics. The results should not be interpreted as being relevant to all persons in the community or even to all persons who live in neighborhoods they perceive to be unwalkable, but only to patients attending clinics that serve a disproportionate share of disadvantaged persons. This is important to public health because the success of strategies designed to improve in overall community health will have to focus on the disadvantaged people who bear the greatest burden of poor health. We believe that more studies of disadvantaged clinic populations such as this one are needed in the public health literature.The study reported here of low income primary care patients confirms the relationship between perceptions of convenient walking locations and self-rated health. Health status is measured by just one question, a practice which has become increasingly common in the public health literature,30. HoweAnother limitation of the study is the subjective nature of the walkability measure. Perceptions of walkability may not be accurate and thus objective assessment of walkability might have led to a different conclusion. However, since our purpose was to investigate the relationship between perceptions of walkability and self-rated health, the results are valid for this sample. If community medicine patients are incorrect in their perceptions of neighborhood walkability, then public health education campaigns and personal health education in clinics can inform them about options for walking in their neighborhoods and how they might overcome any perceived barriers.Our results also may have been influenced by the context from which subjects were drawn. Amarillo is located in the Panhandle of Texas. Most of the year the climate is mild, but during the summer season temperatures can average over 90 degrees. In some neighborhoods, crime rates are above the national average and walking after dark might be worrisome. Furthermore, cities in West Texas are designed for automobile traffic, to the detriment of pedestrians and bicyclists. Furthermore, the vast open spaces in the region make it impossible to reach many locations by foot. Accordingly the, culture of the area has not evolved to be supportive of walking. Therefore, our results may not be generalizable to communities where walking has historical been a visible aspect of the culture.Despite these limitations, the results of this analysis are intriguing and warrant further investigation using a prospective study design. The evidence presented suggests that support for walkable neighborhoods and health education about options for walking in their neighborhoods may be in the best interests of community medicine patients.The author(s) declare that they have no competing interests.JER planned the project, analyzed the data and wrote the first draft of the manuscript. JRP contributed comments and revisions to the manuscript. AD organized data collection and wrote a portion of the manuscript.The pre-publication history for this paper can be accessed here:


SDHD that is frequently mutated in paraganglioma and pheochromocytoma, which are, like neuroblastoma, tumours originating from the neural crest. In this study, we sought for evidence for involvement of SDHD in neuroblastoma.Deletions in the long arm of chromosome 11 are observed in a subgroup of advanced stage neuroblastomas with poor outcome. The deleted region harbours the tumour suppressor gene SDHD was investigated on the genome, transcriptome and proteome level using mutation screening, methylation specific PCR, real-time quantitative PCR based homozygous deletion screening and mRNA expression profiling, immunoblotting, functional protein analysis and ultrastructural imaging of the mitochondria.SDHD mutations causing hereditary paraganglioma. No evidence for hypermethylation of the SDHD promotor region was observed, nor could we detect homozygous deletions. Interestingly, SDHD mRNA expression was significantly reduced in SDHD mutated cell lines and cell lines with 11q allelic loss as compared to both cell lines without 11q allelic loss and normal foetal neuroblast cells. However, protein analyses and assessment of mitochondrial morphology presently do not provide clues as to the possible effect of reduced SDHD expression on the neuroblastoma tumour phenotype.Analysis at the genomic level of 67 tumour samples and 37 cell lines revealed at least 2 bona-fide mutations in cell lines without allelic loss at 11q23: a 4bp-deletion causing skip of exon 3 resulting in a premature stop codon in cell line N206, and a Y93C mutation in cell line NMB located in a region affected by germline SDHD in the pathogenesis of neuroblastoma. Also, although a haplo-insufficient mechanism for SDHD involvement in advanced stage neuroblastoma could be considered, the present data do not provide consistent evidence for this hypothesis.Our study provides no indications for 2-hit involvement of  MYCN amplification and 17q gain, also have a profound predictive power [MYCN amplification and 1p-deletion in subgroup 2B and 11q-deletion often in combination with 3p-deletion in subgroup 2A [Neuroblastoma (NB) is the most frequent extra-cranial solid tumour in children, originating from immature neural crest cells of the sympathetic nervous system . The tumve power ,3. Preseve power . Subgrougroup 2A -9.SDHD, which encodes the small subunit D  of the mitochondrial respiratory chain complex II (succinate-ubiquinone oxidoreductase) [The first evidence for the occurrence of 11q-deletions in NB was obtained in 1991 . Howeverductase) ,18 and wSDHD in tumour development was obtained by the discovery of germline mutations in this gene as the cause for familial paraganglioma (PGL) [SDHD mutations were also detected in patients with apparently sporadic pheochromocytoma (PC) [SDHD mutations in the 5' portion of the gene causing complete disassembly of complex II, whereas PGL are associated with mutations in the 3' region of the gene causing partial inactivation of its catalytic activity [SDHD in oxidative phosphorylation, SDHD has also been presumed to contribute to the function of the mitochondria as oxygen sensors. It was shown that SDHD inactivation leads to a pseudo-hypoxic state and upregulation of hypoxia responsive genes, possibly through increased production of reactive oxygen species (ROS) [SDHD mutations or reduced activity of SDHD might lead to impaired oxidative phosphorylation and hypoxia and thus contribute to NB oncogenesis.The first evidence for a role of ma (PGL) . Somaticoma (PC) ,21. It sactivity -28. PGL es (ROS) . A hypoxes (ROS) . ConsequSDHD as a positional and functional candidate for the presumed NB tumour suppressor gene on 11q23. In order to search for evidence for involvement of SDHD in NB development, an extensive series of investigations was performed on the DNA, RNA and protein level.In view of the above, we considered MYCN amplification. For all NB patients constitutional leukocyte DNA was available. In addition, 31 NB cell lines were included in the analysis of which karyotypes were available. For 20 of these cell lines, comparative genomic hybridization (CGH) data and/or M-FISH (multicolour fluorescence in situ hybridization) results have been published [Neuroblastoma (NB) tumour samples (at least 70% tumour cells) were collected at the Ghent University Hospital  n = 32) and in the Molecular Oncology Unit  (n = 35). Ethical approval was obtained for the collection of the tumour samples. The latter group includes selected patients with stage 3 or 4 NB without ublished -32. For  and in tublished .SDHD transcripts. RNA of the cell line pellets (treated and untreated) was extracted with the RNeasy Mini kit (Qiagen) according to the manufacturer, followed by RNase free DNase treatment on column (Qiagen). A fraction of the untreated NB cell line cultures was also used for functional enzyme assays.Cultures of NB cell lines N206, SK-N-AS, SK-N-SH, NMB, SK-N-FI, CLB-GA, LA-N-2 and NGP were treated with puromycine (100 μg/ml) during 6 hours in order to prevent possible nonsense mediated RNA decay of variant MLL (11q23.3) SpectrumOrange probe (Vysis) and BAC clone RP11-93E4 for the CRTAM gene (11q24.1) in combination with a centromeric probe for chromosome 11. Labelling and FISH was performed as described [The 11q status of the cell lines was evaluated using FISH. FISH was performed using the LSI escribed . For eacSDHD gene. In order to discriminate between whole chromosome loss, and unbalanced 11q loss , two microsatellite markers on 11p (D11S922 on 11p15.5 and D11S1324 on 11p14.1) were analyzed in patients that showed allelic imbalance for all 11q markers . Scoring of loss of heterozygosity (LOH) was performed by calculation of the allelic imbalance factor (AIF) [All NB patients were analyzed with 4 microsatellite markers on 11q23: D11S1986 (11q23.1), D11S1998 (11q23.3), D11S1356 (11q23.3) and D11S1299 (11q23.3), of which D11S1986 and D11S1998 are immediately flanking the SDHD using Primer Express v2.0 (Applied Biosystems)  Table . Exon 1 [ . Primers were designed in a CpG island close to the start of the gene (putative SDHD promotor) (chr11: 111495002–111495330: UCSC Genome Browser freeze version July 2003) . Amplification mixtures (50 μl) for the PCR reaction contained 50 ng template DNA, 1× Platinum Taq PCR reaction buffer (Invitrogen), 6 mM MgCl2, 200 μM of each dNTP, 1.25 U Platinum Taq polymerase (Invitrogen), 3% DMSO and 300 nM of each primer. The cycling conditions comprised 4 min polymerase activation at 93°C, 40 cycles with denaturation at 93°C for 30 sec, annealing at 64°C (methylated primers) or 65°C (unmethylated primers) for 30 sec and extension at 72°C for 30 sec, and a final extension for 5 min at 72°C. SssI methylase (New England Biolabs) treated DNA, following the manufacturer's instructions and normal human genomic DNA were used as a positive and negative control respectively after bisulfite modification.On the 31 NB cell lines and on another series of 50 NB tumours of which 15 were included in the mutation analysis, methylation-specific PCR (MSP) was performed as described, with minor modifications . MSP pri and checSDHD exons were designed using Primer Express v2.0 (Applied Biosystems), based on the publicly available SDHD genomic sequence (accession number AB026906)  and 500 nM of each primer. The cycling conditions comprised 3 min polymerase activation at 94°C, 35 cycles with denaturation at 92°C for 20 sec, annealing at 60°C for 20 sec and extension at 72°C for 2 min, a final extension for 5 min at 72°C and a slow decrease in temperature to 25°C over 30 minutes. One μl of the PCR products was analyzed on a Ready-To-Run Agarose Gel (1.2%) (Amersham Biosciences).Intronic primers flanking the m(= 62.1°C)+0.7°C and Tm+1.5°C. Exon 2 fragments were eluted at a temperature of Tm(= 55.7°C)+4.8°C. Exon 3 fragments were eluted at a temperature of Tm(= 57.4°C)-0.4°C, Tm+1.1°C and Tm+3.8°C. Exon 4 fragments were eluted at a temperature of Tm(= 56.4°C)-0.4°C, Tm+1.6°C and Tm+3.2°C. Elution of the fragments was performed using standard conditions according to the manufacturer. Elution profiles were analyzed using the Wavemaker software.Denaturing high-pressure liquid chromatography (DHPLC) was performed using the Wave system (Transgenomic). The melting profile of each fragment was determined using the Wavemaker software v4.1 (Transgenomic). Crude PCR product was injected into a preheated, fully equilibrated chromatographic column for the DHPLC analysis. Exon 1 fragments were eluted at a temperature of TSequencing was performed on all cell lines and on tumour samples with aberrant DHPLC elution peaks .Amplified fragments were purified using the Montage PCR96 filter plates (Millipore) or by excision of the fragment of interest from a 1.5% TBE-agarose gel and purification on a GenElute Minus EtBr Spin Column (Sigma-Aldrich). Cycle sequencing was performed using purified amplicons (3–10 ng), the above-mentioned primers Table  at a conPCR primers and minor groove binder (MGB) probes for sequence variant IVS4-32T>C were designed using Primer Express v2.0 following the user bulletin guidelines for the design of MGB probes (Applied Biosystems): forward primer 5'TTTTTTGCAGCCAAGTTATCTGTATAG3', reverse primer 5'TGTCCAAGGCCCCTAAAGAA3', MGB probe allele 1 5'TGTGGTTTTTtATTGATG3' labelled with 6-FAM and MGB probe allele 2 5'TGTGGTTTTTcATTGAT3' labelled with VIC. To address the frequency of the sequence variant g.7876A>G (Y93C) in a normal population, the following primers and probe were designed: forward primer 5'GGCTGCTTATTTGAATCCTTGCT3', reverse primer 5'ACTTGCCAGTGACCATGAAGAGT3' and MGB probe variant allele 5'ATGGACTgTTCCCTG3' labelled with VIC. The reaction mixture contained 10 ng of DNA, 100 nM of each MGB probe, 300 nM of each primer and 1× qPCR Mastermix (Eurogentec). For the screening of the g.7876A>G variant, multiplex PCR was performed using primers and probe of the normal allele of the above-mentioned SNP (IVS4-32T>C) and primers and probe for the variant allele g.7876A>G. Reactions were performed on the iCycler Thermal Cycler (Bio-Rad) with the following thermocycling conditions: an initial activation step at 95°C for 10 min, 50 cycles of 95°C for 15 sec and 60°C for 1 min. Allelic discrimination data analysis was performed on the iCycler IQ Optical System Software v3.0a (Bio-Rad).SDHD_IVS4-32).The SNP info for the IVS4-32T>C variant was submitted to NCBI's SNP database  as reported previously [Relative CR assay  with minCR assay . The comeviously .Enzyme activities were determined spectrophotometrically as previously described .Protein amount was determined with immunoblot analysis of complex II Fp fragment as previously described . Relativ4 for 1 h at 4°C. Block staining in uranylacetate (UAc) in 70 % ethanol was followed by dehydration in ethanol and propylene oxide, and embedding in Epon. Ultrathin sections were counterstained with UAc and lead. At least 2 cells in each culture were photographed at a magnification of 20,000 in a Zeiss electron microscope operating at 50 KV. Per culture, 25–63 mitochondria were examined. Their projected length (largest straight distance) was measured and matrix electron density was compared to the surrounding cytosol.Mitochondria of NB cell lines LA-N-2, SK-N-AS, CLB-GA, NGP, CHP-901, SK-N-SH, SK-N-FI, N206, SJNB-12, SJNB-8, NMB, SJNB-10 and IMR-32, breast carcinoma cell line MCF-7 and, Ewing sarcoma cell line SK-N-MC were analysed by electron microscopy. Monolayers from these cell lines were briefly rinsed twice in PBS and then immersed at room temperature in 3 % glutaraldehyde buffered with Na-cacodylate at pH 7.3 for 1 h. After rinses in this buffer with 1 % bovine serum albumin, cells were scraped off with a rubber policeman, and centrifuged with 3 % glutaraldehyde. After washing, the pellets were postfixed in 2 % buffered OsOElectron microscopic files were made on 11 archived neuroblastic tumours. In addition, previously published cases were examined for mitochondria morphology.MLL locus (11q23.23) (not shown) . No previously unnoticed submicroscopic 11q23-deletions were detected. Screening for homozygous deletions in all SDHD exons was negative for the 31 NB cell lines.Previous karyotyping, comparative genomic hybridization (CGH) and/or M-FISH revealed 11q-deletions in 9 out of 31 neuroblastoma (NB) cell lines  -32. In tMYCN amplification, in contrast to the other patient subgroup for which samples were unselected.In 20 of the 67 NB tumour samples, loss of heterozygosity (LOH) or allelic imbalance (AI) (AIF > 2) in the 11q23 region was found  analysis and subsequent sequencing of the Two variants were considered as bona fide mutations Figure . The firIn one patient without 11q allelic loss (F11) we observed in both tumour and constitutional DNA a TCTA insertion at position IVS2+37. However, no additional tumour material nor parental material was available for further analysis. So, it remains unclear whether this is a true mutation or a rare polymorphism.In addition, 1 new and 4 known polymorphisms were observed. The H50R variant found in cell line LA-N-2 was described as a polymorphism in several studies -45. ThisSDHD alleles has been deleted, indicates that all three variants are located on the same allele, representing a low frequent haplotype.The presence of the IVS3-29A>G, S68S and IVS4-32T>C variants in a cell line (NGP) and two tumours (F18 and F35), in which one of both SDHD promotor hypermethylation was tested for 31 NB cell lines and 50 NB patients using methylation-specific PCR (MSP). No evidence for methylation was obtained in any of the analyzed NB cases.SDHD cDNA showed that a 4 bp deletion in the intron-exon boundary in cell line N206 caused skipping of exon 3 leading to a premature stop codon.Amplification of the full-length No alternative transcripts could be detected in cell lines NMB, SK-N-FI, NGP and LA-N-2 carrying basepair variants  when grown with or without puromycin Figure .SDHD is bi-allelically expressed, thus supporting recent observations in lymphoblastoid cell lines, adult kidney and adult and fetal brain [The above-mentioned cDNA transcript sequencing revealed that al brain ,22, but SDHD expression levels were measured using real-time quantitative PCR in 31 NB cell lines, normal foetal neuroblast cells  and 7 normal adult tissues   and normal adult tissue mRNA samples (Mann-Whitney test: P = 1.49E-05).) Figure . The SDHSDHD mRNA levels were significantly reduced in cell lines with 11q allelic loss and SDHD mutated cell lines (i.e. NMB and N206) (N = 9) compared to cell lines without 11q allelic loss (N = 22) (Mann-Whitney test: P = 1.49E-03).SDHD gene encodes the small subunit D of the mitochondrial respiratory chain complex II we decided to assess the effect of the basepair variants on the activity of complex II of the respiratory chain by spectrophotometrical measurements in 5 NB cell lines  and 3 control NB cell lines without sequence variants . No significant differences in complex II enzyme activity could be demonstrated. Although, in LA-N-2 a slight decrease of complex II activity was observed (data not shown).As the On above-mentioned cell lines and NB cell lines CHP-901, SJNB-12, SJNB-8, SJNB-10 and IMR-32, breast cancer cell line MCF7 and Ewing sarcoma cell line SK-N-MC, immunoblotting of the Fp fragment of complex II showed no significant variation in abundance among the tumour cell lines (data not shown).Electron microscopic analysis of NB cell lines revealed that the morphology of the mitochondria is heterogeneous between the different cell lines, with respect to length, dilated intracrista spaces and condensation of the matrix Table . In mostIn order to examine whether dilated mitochondrial cristae are a feature of many, or all NBs, we studied the mitochondria in electron micrographs from 11 archived and 22 previously published neuroblastic tumours -53. DilaSDHD in neuroblastoma (NB) tumourigenesis. In a first step, mutation and methylation analyses were performed on a large panel of NB cell lines and tumours. A total of seven sequence variants (in nine different samples) were detected of which two could represent bona fide mutations, i.e. missense mutation Y93C in cell line NMB and a 4 bp deletion in cell line N206.In this study, we investigated the possible involvement of SDHD protein frequently altered due to germline mutations in paraganglioma (PGL) families  and consequently the structure of the transmembrane subunit and/or association of the catalytic domain subunits SDHA and SDHB to the membrane would be disrupted .The functional consequence of one sequence variant located within an intronic sequence (IVS2+37ins(TCTA)) is more difficult to evaluate due to lack of fresh tumour material, and parental DNA. Further analysis is needed in order to reveal a possible effect on splicing or RNA stability.Finally, one new and 4 known polymorphisms were detected in 5 NB cell lines and 3 tumour samples. Additional screening for homozygous deletions in all cell lines and methylation in cell lines and tumours were negative.SDHD as a classical tumour suppressor gene in NB. However, the finding of two apparently bona fide SDHD mutations in NB without allelic loss of distal 11q leaves the possibility open that the gene contributes to NB oncogenesis due to haplo-insufficiency, rather than functional inactivation of both alleles. In order to investigate this possibility, we decided to perform further studies at transcript and protein level. Interestingly, SDHD expression was shown to be consistently lower in cell lines with 11q allelic loss versus NB cell lines without loss and also significantly decreased in NB cell lines as compared to normal foetal adrenal neuroblast cells of 16, 18 and 19 weeks gestational time with a mean fold difference of 3.61 between neuroblast cells and NB cell lines. A similar correlation between 11q LOH and reduced SDHD expression was recently described in colorectal and gastric cancer [SDHD mutation. However, measurement of complex II activity might only reflect part of the functional properties of SDHD. Also, measurement of differences in protein quantity is far less sensitive than Q-PCR at transcript levels. Therefore, these observations at present do not fully exclude SDHD involvement in NB. Finally, we also looked at the morphologic characteristics of the mitochondria as a possible clue to SDHD dysfunction. In keeping with, at best, partial loss of function of SDHD, we did not observe similar gross morphologic changes as reported for PGL with SDHD mutations , the latter being characterized by destabilization of complex II with loss of enzymatic activity [Based upon these results, we can exclude a role for c cancer . Our finactivity . Howeveractivity . Subsequactivity ,58. ThisSDHD involvement in NB. However, the finding of, albeit rare, bona fide mutations and reduced expression of SDHD in NB with 11q allelic loss hints at a possible haplo-insufficient contribution to tumour development. A better understanding of the different functions of SDHD, in particular its possible contribution to energy independent apoptosis involving the release of cytochrome c and procaspases, will allow further functional assays to asses how this gene contributes to tumour development in general, and the high stage NB phenotype in particular [In contrast to previous findings in PGL and PC, this study excludes a classical two hit Knudson model for rticular ,60.CDKN1B (p27Kip1) [TP53 (p53) [DMP1 [PTEN [APC [NKX3.1 [SDHD in NB, a bipartite mechanism as tumour suppressor gene for the SDHD gene, as described for the APC gene can at present not be fully excluded. Following this hypothesis, germline mutations in SDHD would predispose to PGL or PC development. Rare somatic mutations and more typically loss of one allele could contribute to the metastasizing NB tumour phenotype , not as an initiating step but rather as later event in tumour development. However, further evidence is needed to support the haplo-insufficient involvement of SDHD in cancer. Ultimately, knockout mice for the SDHD gene leading to haplo-insufficiency for SDHD in neuroblast progenitor cells, would be the appropriate test to evaluate this hypothesis.Evidence for contribution to a cancer phenotype through haplo-insufficiency has recently been obtained for a number of loci, including p27Kip1) ,62, TP5353 (p53) , DMP1 [63) [DMP1 , PTEN [6P1 [PTEN , APC [66 [NKX3.1 . In mous [NKX3.1 . AlthougAIF = allelic imbalance factorCGH = comparative genomic hybridizationDHPLC = denaturing high performance liquid chromatographyLOH = loss of heterozygosityMGB = minor groove binderMSP = methylation specific PCRNB = neuroblastomaPC = pheochromocytomaPGL = paragangliomaROS = reactive oxygen speciesSDHD = succinate dehydrogenase, subunit DSNP = single nucleotide polymorphismSRO = shortest region of overlapThe authors declare that they have no competing interests.KDP carried out the genomic and transcriptomic studies, and drafted the manuscript. JH performed the methylation studies. JS carried out the immunoblottings and spectrophotometric analysis that was evaluated by RVC. AN performed the ultrastructural analysis that was screened and discussed by CV, FR and MP. GL and NVR collected the tumour material. JV and FS participated in the study's design and coordination. All authors have reviewed the manuscript and FS and ADP were the final editors of the manuscript.The pre-publication history for this paper can be accessed here:


The problems associated with the ageing immune system coupled with possible solutions were discussed recently at the British Society for Immunology Annual Congress in Harrogate in December 2004. The session "Ageing and the Immune System in vivo" dealt in details with the immune risk phenotype and the potential methods of reversing the problems of an ageing immune system. This is a commentary on that session. From an early age our immune system is introduced to a variety of infectious agents through contact with infected individuals. We also deliberately present our immune responses either to attenuated organisms or to components of infectious pathogens in order to provoke a response. A successful response to these organisms when first encountered provides us with immunological memory to enable our immune systems to make more rapidly responses to the same potentially infectious agents at a later date. In theory the longer we live the better our memory responses and our ability to cope with any potential pathogen seen previously. Since there are no completely sterile environments, individuals who have survived to reach a ripe old age must have combated many possible infectious agents, and have an immune system with a prodigious memory component. Only part of this is true. As we age the memory component of our immune system, as measured by the number of memory cells, does increase. Unfortunately this is not accompanied by improved immunity even to infectious agents that have been overcome earlier in life -4.The problems associated with the ageing immune system coupled with possible solutions were discussed recently at the British Society for Immunology Annual Congress in Harrogate in December 2004. The session "Ageing and the Immune System in vivo" dealt in details with the immune risk phenotype and the potential methods of reversing the problems of an ageing immune system.+CD28- cells (or CD8+CD28-CD27- in the very old), low numbers of B cells and the presence of CD8 T cells which were cytomegalovirus (CMV) tetramer positive. Within both the OCTO and NONA groups Dr Anders was able to show that the immune risk phenotype had some predictive input towards morbidity. This was even more apparent when cognitive impairment was included in the calculations.The concept of the immune risk phenotype was described by Dr Anders Wikby (Jönköping University) who carried out a longitudinal immunological study in order to establish predictive factors for longevity. Dr Anders first worked on a group of normal octogenarians in the OCTA study in Sweden which later became the NONA study as these individuals entered their nineties. Within this group he was able to describe the immune risk phenotype which he characterised by a CD4:CD8 ratio of <1, poor in vitro T cell proliferation, an increase in the number of CD8+ and show an increased incidence with age, so far it seems that clonal expansion is not due to malignant transformation but may follow antigen stimulation. The control of CMV within the body is mainly attributed to CD8+ T cells and Professor Moss revealed that in older individuals up to 50% of CD8+ T cells may be specific for CMV as judged by tetramer staining. It is possible that this increased response to CMV may lead to the impairment of responses to other viruses in the aged. This theme of CMV infection and ageing was then continued by Professor Graham Pawelec (University of Tuebingen) who introduced the use of the KLRG-1 marker as a marker of senescence. T cells which are KLRG-1+ do not proliferate in vitro when provided with a stimulus which would induced proliferation in T cells which lack this marker. In elderly individuals fewer CMV tetramer positive CD8+ T cells secreted interferon-γ in Elispot assays when compared with similar cells from younger individuals. Furthermore between 96 and 99%of the CD8+ T cells which stained with the CMV tetramer also expressed the KLRG-1 marker. The theme of CMV infection in the elderly was further continued by Professor Arne Akbar (University College London) whose research group has recently been analyzing telomere lengths in different T cell subsets using a fluorescent staining technique. In this method telomeres are stained with a fluorescently labeled probe and the brighter the fluorescent labeling the longer the telomere. Using this technique Professor Akbar reported that CMV specific CD4+ T cells in older individuals had very short telomeres indicating their limited replicative capacity and that more of these CMV specific CD4+ T cells expressed interferon-γ.The effect of CMV infection on the immune system in the elderly was also discussed by Professor Paul Moss (University of Birmingham) with particular emphasis on the clonal expansion of T cells. Clonally expanded T cells are usually CD8This view of a bleak future for displaying the immune risk phenotype was countered by the last two presentations which discussed different approaches to reversing the defects seen in the immune system with age. Both of these methods centered around reversing the atrophy of the thymus seen with age. Dr Jayne Sutherland, currently at the Anthony Nolan Research Institute, detailed her work carried out whilst with Professor Richard Boyd (Monash University) on reversing age associated thymic atrophy by surgical or chemical castration. Studies in mice revealed that reversal of thymic atrophy followed surgical castration and in humans chemical castration produced changes in the TREC levels which indicated improved thymic output. Functional studies showed that this treatment improved immune function in the treated aged subjects. This theme of improved immunity following reversal of thymic atrophy was carried further in work reported by Dr Richard Aspinall . He showed how therapeutic intervention with interleukin 7 and derivates could reverse atrophy of the thymus in old animals and also lead to improved immune function compared with age and sex matched controls. In addition he described work done in the Gambia which suggested a link between the development and enlargement of the thymus after birth and the level of interleukin 7 in breast milk.Two speakers from the posters presentations were also chosen to present their results. K. Wang from Professor Janet Lord's group (Birmingham) presented work on dehydroepiandrosterone, (DHEA). This steroid hormone declines with age and treatment of NK cells with DHEA increases NK cytotoxicity and further analysis shows that DHEA induces PKC-β translocation and upregulates perforin expression in these cells. D. Silva from Donald Palmers laboratory  presented work on the expression of neuropeptides in epithelial cells from the thymuses of different species.The meeting revealed that much needs to be done in characterizing the changes in the immune system which are associated with ageing, and members of the audience were asked whether they were interested in receiving more details about the newly formed "Differentiation and Immunosenesence Affinity Group" associated with the BSI.


One of the central problems for much of the 20th century was how to reconcile genetic stability with evolutionary change. Genomic fidelity was thought to arise from an inherent invariability in the DNA structure itself. Biologists now know that DNA constantly undergoes modifications as it unwinds, replicates, condenses, twists, and untwists. This dynamic interplay produces both stability and variation—and occasionally genetic damage. If DNA damage goes unrepaired, it can disrupt chromosomal integrity and may lead to cancer and other diseases. When the DNA double helix breaks, the cell must enlist a number of proteins to repair the broken DNA ends, but much remains to be learned about the molecular mechanisms involved. Tracking a protein that binds to single strands of DNA during replication and recombination in living yeast cells, Xuan Wang and James Haber report that this protein plays a role in at least two key steps in the repair of double-strand breaks in DNA.When double-strand breaks occur, the cell mounts a search for similar (homologous) sequences that can be used as a template to repair the damaged sequence. If successful, the broken DNA molecule basepairs with the homologous region and forms a complex, ultimately replacing the damaged sequence with a similar sequence. In yeast—which serves as a stand-in for higher eukaryotes, including humans—this “strand invasion” process requires both an exchange protein, called Rad51, and a single-stranded DNA-binding protein, called RPA (replication protein A). Single-stranded binding proteins bind to regions of DNA that are opened up during replication. They also bind to strands when broken ends of DNA are cut by enzymes that leave long single-stranded tails. RPA proteins are thought to facilitate the formation of Rad51 polymers, or filaments, on single-stranded DNA by clearing away structures that block Rad51's path. The growing filament searches for homologous DNA sequences and promotes the invasion of the single strand, preparing it to copy the homologous template by “repair DNA synthesis,” which patches up the lesion.To investigate how RPA functions in double-strand break repair in a living organism, Wang and Haber created cells with a double-strand break at a specific site and monitored the activity of proteins recruited to repair the damage. With this approach, the researchers could observe these interactions in living yeast to determine what role RPA plays in repairing DNA damage and how it works with the Rad51 protein.The authors show that as soon as a double-strand break occurs, the RPA protein binds to the exposed strand ends, before the Rad51 protein does. This is not unexpected, because this binding order supports the model that RPA prepares the way for Rad51, perhaps by stabilizing the strand long enough for Rad51 filaments to establish themselves. The surprise was that RPA appears to be necessary even after Rad51 binds to the DNA strand, perhaps by stabilizing the interaction with homologous DNA sequences. That RPA is required for successful repair is supported by evidence that a particular mutated form of RPA can stimulate Rad51 DNA binding normally, but inhibits strand exchange and template copying, thus preventing repair of DNA damage.Wang and Haber's work highlights the complex repertoire of DNA–protein and protein–protein interactions that manage and manipulate the genome in the service of genomic stability. The study of DNA repair mechanisms in living cells—a daunting task—promises to lend valuable insights into the truly dynamic nature of maintaining genome stability.


In situ apoM mRNA hybridization demonstrates that apoM is exclusively expressed in the hepatocytes and in the tubule epithelial cells in kidney. Expression of apoM could be regulated by platelet activating factor (PAF), transforming growth factors (TGF), insulin-like growth factor (IGF) and leptin in vivo and/or in vitro. It has been demonstrated that apoM expression is dramatically decreased in apoA-I deficient mouse. Hepatocyte nuclear factor-1α (HNF-1α) is an activator of apoM gene promoter. Deficiency of HNF-1α mouse shows lack of apoM expression. Mutations in HNF-1α (MODY3) have reduced serum apoM levels. Expression of apoM is significantly decreased in leptin deficient (ob/ob) mouse or leptin receptor deficient (db/db) mouse. ApoM concentration in plasma is positively correlated to leptin level in obese subjects. These may suggest that apoM is related to the initiation and progression of MODY3 and/or obesity.Apolipoprotein M (apoM) is a 26-kDa protein that is mainly associated with high-density lipoprotein (HDL) in human plasma, with a small proportion present in triglyceride-rich lipoproteins (TGRLP) and low-density lipoproteins (LDL). Human apoM gene is located in p21.31 on chromosome 6 . Human apoM cDNA (734 base pairs) encodes 188-amino acid residue-long protein. It belongs to lipocalin protein superfamily. Human tissue expression array study indicates that apoM is only expressed in liver and in kidney and small amounts are found in fetal liver and kidney.  Human apolipoprotein M (apoM) was found and initially isolated from chylomicrons by Xu and Dahlbäck in 1999 . Gel filThe identified human apoM cDNA (734 base pairs) encoded 188-amino acid residue-long protein. The 5'-untranslated region was 33 nucleotides and the 3'-untranslated region 120 nucleotides, not including the poly (A) tail. Southern blot analysis of different species gave positive signals in all mammalian genomes but not in DNA from chicken and yeast . Sub. Sub1]. in situ hybridization. In day-15 embryos, apoM was expressed in both liver and kidney. During human embryogenesis, apoM was strongly expressed in livers of 3–5 month-old human embryos and continued to be strongly expressed throughout embryogenesis. In the kidney, apoM expression was highest in 5–9 month-old embryos. There was some expression of apoM in small intestine, particularly in later stages of embryogenesis. In skeletal muscle, minute apoM expression was found in 3–5 months-old embryos, and some apoM expression was found in stomach in earlier stages of embryogenesis [in situ apoM mRNA hybridization demonstrated that apoM is exclusively expressed in the hepatocytes and in the tubule epithelial cells in human kidney [in vivo, which may be related to the hepatic lipid and/or lipoprotein metabolism.Northern blot analyses of multiple tissues  showed that apoM was mainly expressed in kidney and liver . Furtherogenesis . These fIn vitro, several biological factors have been tested to examine their influences over the transcription and secretion of apoM in hepatic cell line (HepG2 cells). Like apoB, apoM is highly hydrophobic and must co-circulate with lipoprotein particles in the blood stream. It has been demonstrated that apoB could be down-regulated by transforming growth factor-beta (TGF-β) [in vivo. In another study, Xu et al demonstrated that platelet-activating factor (PAF) could up-regulate apoM expression in HepG2 cells, whereas, lexipafant, a PAF-receptor antagonist significantly suppressed the mRNA levels and the secretion of apoM in HepG2 cells in a dose-dependent manner. Neither tumor necrosis factor-α (TNF-α) nor interleukin-1α (IL-1α) influences apoM expression or secretion in HepG2 cell cultures [ (TGF-β) ,8. Xu et (TGF-β) . It suggcultures . It indiin vivo and in vitro [in vivo. Richter et al. reported that apoM gene expression could be regulated by HNF-1α. Mutant HNF-1α-/- mice completely lack expression of apoM in liver and kidney. Serum apoM levels in HNF-1α+/- mice are reduced by 50% compared with wild-type animals. By analyzing the apoM promoter and identifying a conserved HNF-1 binding site, they showed that HNF-1α is a potent activator of apoM promoter, that a specific mutation in the HNF-1 binding site abolished transcriptional activation of apoM gene. HNF-1α protein can bind to the HNF-1 binding site of apoM promoter in vitro [ob/ob mice [ob/ob mice and in leptin-receptor deficient db/db mice than in control mice. Furthermore, leptin administration significantly increased plasma apoM levels and apoM mRNA levels in liver and in kidney in ob/ob mice [in vivo.Administration of adrenocorticotropic hormone (ACTH) has beneficial effects on plasma lipoproteins -15. A coin vitro ,19, indiin vitro . Liang a/ob mice , suggest/ob mice . Xu et a/ob mice . It is c2, n = 51). In univariate analysis, apoM correlated significantly to leptin , BMI , fasting insulin , total cholesterol , and LDL-cholesterol . The correlations between apoM and cholesterol and between apoM and leptin remained significant after adjustment for the influence of BMI. Forward stepwise multiple regressions when leptin, BMI, insulin and cholesterol were entered in a model as independent variables and apoM as the dependent variable showed that cholesterol and leptin were independent predictors of circulating apoM. These two parameters yielded an r2 of 0.28, thereby explaining approximately 30% of the variance in apoM. Hence, apoM is positively correlated to leptin and negatively correlated to cholesterol levels in humans [Xu et al. investigated the relationship between plasma apoM levels and leptin levels, body mass index (BMI), fasting glucose, fasting insulin as well as lipoprotein concentrations in females displaying a wide range in BMI (18.9–57.1 kg/mn humans . Richtern humans . Alzheimn humans -26. Incrn humans ,28, suggn humans . With thin vivo and in vitro. HNF-1α is one of the most important activator of apoM gene promoter. Plasma apoM concentration is positively correlated to leptin levels and negatively related to plasma cholesterol levels. Both leptin and leptin receptor are essential for apoM expression in vivo. Plasma apoM levels may be used as the marker for identification of MODY3. The detailed relationship between apoM and MODY3 as well as obese needs further investigation.In conclusion, apoM is a novel HDL apolipoprotein. Like apoB apoM could be regulated by several cytokines


Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds.Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB has a dual role, both in the formation of rod cell shape, and in chromosome segregation, however, its function in cell shape is distinct from that of MreC. Additionally, MreB is important for the localization of the replication machinery to the cell centre, which becomes aberrant soon after depletion of MreB. 3D image reconstructions suggest that frequently, MreB filaments consist of several discontinuous helical filaments with varying length. The localization of MreB was abnormal in cells with decondensed chromosomes, as well as during depletion of Mbl, MreBH and of the MreC/MreD proteins, which we show localize to the cell membrane. Thus, proper positioning of MreB filaments depends on and is affected by a variety of factors in the cell.In this work, we show that Our data provide genetic and cytological links between MreB and the membrane, as well as with other actin like proteins, and further supports the connection of MreB with the chromosome. The functional dependence on MreB of the localization of the replication machinery suggests that the replisome is not anchored at the cell centre, but is positioned in a dynamic manner. In vitro, actin filaments can deform vesicles and thus push membranes, providing the force to elongate cellular extensions such as pseudopods  complemented with 1% casamino acids. Fluorescence microscopy was performed on an Olympus AX70 microscope. Cells were mounted on agarose gel pads containing S750 growth medium on object slides. Images were acquired with a digital CCD camera; signal intensities and cell length were measured using the Metamorph 4.6 program . For and 3D reconstruction, 10 to 12 images (spacing between 0.2 to 0.38 μm) were taken through the focal plane, and processed in Metamorph 6 program. DNA was stained with 4',6-diamidino- 2-phenylindole  and membranes were stained with FM4-64 .For microscopic analysis, H J D S performed all experiments, PLG helped with 3D image reconstructions, conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.


We correlated p53 protein expression with pathological subtype and clinical outcome in 75 embryonal brain tumors. The presence of JC virus, which results in p53 protein accumulation, was also examined.p53 protein levels were evaluated semi-quantitatively in 64 medulloblastomas, 3 atypical teratoid rhabdoid tumors (ATRT), and 8 supratentorial primitive neuroectodermal tumors (sPNET) using immunohistochemistry. JC viral sequences were analyzed in DNA extracted from 33 frozen medulloblastoma and PNET samples using quantitative polymerase chain reaction.p53 expression was detected in 18% of non-anaplastic medulloblastomas, 45% of anaplastic medulloblastomas, 67% of ATRT, and 88% of sPNET. The increased p53 immunoreactivity in anaplastic medulloblastoma, ATRT, and sPNET was statistically significant. Log rank analysis of clinical outcome revealed significantly shorter survival in patients with p53 immunopositive embryonal tumors. No JC virus was identified in the embryonal brain tumor samples, while an endogenous human retrovirus (ERV-3) was readily detected.Immunoreactivity for p53 protein is more common in anaplastic medulloblastomas, ATRT and sPNET than in non-anaplastic tumors, and is associated with worse clinical outcomes. However, JC virus infection is not responsible for increased levels of p53 protein. INI1 mutations, and cause particularly grim clinical outcomes [The current World Health Organization classification for tumors of the nervous system includes medulloblastoma, medulloepithelioma, ependymoblastoma, supratentorial primitive neuroectodermal tumor (sPNET) and atypical teratoid/rhabdoid tumor (ATRT) in the category of embryonal brain neoplasms . These toutcomes . Medullooutcomes . Global outcomes .Little is known about the differences in p53 expression and function among the various embryonal brain tumor subtypes. Initial reports on the p53 tumor suppressor gene suggested it was mutated in 10% or less of medulloblastomas . HoweverSeveral other lines of evidence also support a role for the p53 pathway in medulloblastomas. First, medulloblastomas sometimes arise in the context of Li Fraumeni syndrome, in which p53 germline mutations predispose patients to a wide range of neoplasms . Second,It has been suggested that viral infection of human CNS tissues could promote formation of brain tumors by inhibiting p53 and Rb activity . Some reIn order to confirm the association between p53 immunopositivity, clinical outcome, and embryonal tumor subtype, we stained a tissue array containing representative cores from 80 embryonal brain tumors for p53. We also investigated JC virus infection as a possible mechanism for accumulation of p53 protein by searching for viral sequences using a highly sensitive quantitative real time polymerase chain reaction (PCR) assay. We found an association between p53 immunoreactivity, clinical outcome, and tumor subtype, but did not detect JC virus in medulloblastoma or supratentorial PNET.Medulloblastomas and other embryonal brain tumors diagnosed at the Johns Hopkins University Department of Pathology were identified through review of departmental records. Classic, desmoplastic/nodular and large cell/anaplastic medulloblastomas were classified using World Health Organization guidelines . NuclearThe tissue array was sectioned at four microns, deparaffinized, and subjected to antigen retrieval by steaming (20 minutes at 80°C). Slides were then incubated at room temperature for 45 minutes with monoclonal antibody directed towards p53 . Primary antibody was detected using the avidin-biotin complex (ABC) method with diaminobenzadine serving as the chromagen. We semiquantitatively graded staining intensity as negative, weak, or strong. Carcinomas with mutations leading to p53 stabilization were used as positive controls. No staining was seen in the absence of primary antibody (negative control).JC virus sequences were amplified from DNA using the forward primer PEP-1 (5'-AGT CTT TAG GGT CTT CTA CC-3') and reverse primer PEP-2 (5'-GCC AAC CTA TGG AAC AG-3') . Additio1/N was used to calculate the fiducial (exact) 95% upper bound of JC virus prevalence.p53 statistical analyses were performed using GraphPad PRISM4 software . A two tailed Fishers Exact test was used to compare immunohistochemical staining profiles between groups. Significance of survival differences was assessed using log-rank analysis of Kaplan-Meier curves. The formula 1-We used immunohistochemistry to examine p53 protein expression in representative cores from 80 embryonal brain tumors on a tissue array. Cores from 5 of the cases could not be evaluated due to crush artefact, cautery, or low cellularity. Of the remaining 75 tumors, 64 were medulloblastomas; 31 of these medulloblastoma were of the classic subtype, 13 were nodular/desmoplastic, and 20 were large cell/anaplastic. The tissue array used in this study also contained 11 other CNS embryonal tumors, including 8 sPNET and 3 ATRT. Among the sPNET were 3 with the long epithelial surfaces characteristic of medulloepithelioma.Immunoreactivity for p53 was present in a minority (35%) of the 75 embryonal tumors, and a relatively small subset of cells stained in most of the positive cases. In all positive cases the majority of the immunoreactivity was in tumor cell nuclei. An example of a nodular medulloblastoma with weak, scattered p53 immunoreactivity is shown in Figure We next examined whether p53 immunopositivity was associated with worse clinical outcomes in embryonal brain tumor patients. Of the 75 tumors from 73 patients scored on the array, survival data was available for 66 individuals. 61 percent of patients were alive at last contact, with follow up times ranging from 3 to 215 months (median 47 months). When survival of the entire embryonal brain tumor cohort was analyzed, 67% (31/46) of patients with p53 immunonegative tumors were alive, as compared to 45% (9/20) of patients with immunopositive tumors. Log rank analysis of Kaplan Meier survival curves confirmed the significance of this difference (P = 0.02). When only the 56 medulloblastoma patients with clinical follow up were analyzed, 71% of individuals with p53 negative tumors survived, compared to 58% of individuals with p53 positive tumors. However, these differences were not significant on log rank analysis (P = 0.25). The 6 patients with intensely immunoreactive tumors  all died from their disease in less than two years.3 - 2 × 105 per PCR assay (mean 3.4 × 104). Because two copies of ERV-3 are present per human genome, DNA from at least 1000 cells was assayed in each PCR reaction. In contrast, all of the specimens tested were completely negative for JC virus. The quantitative Taqman assay was able to detect a minimum of ~10 copies of JC virus per PCR reaction as determined using a standard dilution curve. Thus the number of JC virus genomes should not exceed 10 per 1,000 tumor cells. Given our 0% detection rate, the 95% confidence limit for the largest value of the 'true' underlying JC virus prevalence is no more than 7.6% (1-(0.05)1/38).To address the possibility that p53 accumulation in medulloblastoma and other embryonal brain tumors is due to viral infection, we used quantitative RT PCR to search for viral sequences in tumor DNA. Of the cases used on the tissue array, 19 had material frozen suitable for high-quality DNA extraction. Four of these cases were p53 positive. Also available in our frozen tumor bank was tissue from 14 additional embryonal lesions . We did not detect JC virus sequences in any of these 33 tumors. We repeated the analysis on DNA extracted from a different tumor tissue fragment in 5 cases, but these spatially distinct regions also failed to contain viral DNA. ERV-3, an endogenous human retroviral element, served as a control and was easily amplified from tumor DNA samples  as compared to non-anaplastic ones (18% positive). The percentage of p53 immunopositive medulloblastomas in our study (27%) fell within the previously reported range of 3% to 53% ,17,33,34Interestingly, supratentorial PNET and ATRT were also more commonly p53 immunopositive than non-anaplastic medulloblastoma in our study. While extracerebellar PNET were included in several earlier studies, p53 immunoreactivity was not reported separately for these lesions ,15. Ho aClinical outcomes were significantly worse for embryonal brain tumor patients in our study whose lesions were p53 immunopositive. However, cases most commonly positive for p53  are all more clinically aggressive than non-anaplastic medulloblastomas, making it difficult to infer causality resulting from p53 accumulation. p53 expression did not predict outcome within the group of medulloblastoma patients, although all 3 strongly p53 immunopositive tumors were severely anaplastic and associated with quite short survival. Interestingly, in a recently published report, Ray and colleagues found that p53 immunoreactivity was the only biological marker predictive of poor outcome on both univariate and multivariate analyses in a group of 112 medulloblastoma patients .We also examined the potential role of viral infection as a mechanism for p53 protein stabilization in medulloblastoma. None of the four p53-positive cases from our tissue array with frozen material available contained viral sequences. We also tested nine additional samples of histological subtypes that were more commonly associated with p53 immunopositive staining (anaplastic medulloblastomas and sPNETs) where p53 staining was not performed. None of these tumors were JCV positive. While sample availability precluded complete testing of all confirmed p53 positive tumors for JCV, the available data do not support a causal role for JCV infection in p53 accumulation and development of embryonal brain tumors.The impact of viruses on medulloblastoma pathogenesis is controversial. The most commonly implicated agents are the polyomavirus family members JC virus, BK virus and SV40 virus. Of these, JC virus, which infects approximately 80% of the pediatric population and can cause CNS disease in immunosuppressed patients, has been most studied. It was shown decades ago that inoculation of JC virus into rodents resulted in formation of medulloblastoma-like cerebellar tumors ,22,42,43We failed to detect JC virus sequences in the 33 cases examined, using a sensitive and specific technique which should identify as few as 10 viral genomes per PCR reaction. This suggests the p53 immunopositivity we observe is not caused by JC virus infection in the majority of cases. Our data are also not consistent with the hypothesis that ongoing JC virus infection is common in medulloblastoma. Other recent studies have also reported a lack of JC virus DNA in medulloblastomas. Hayashi and colleagues failed to identify JC viral sequences in 13 medulloblastomas . Kim andIn summary, we find significantly increased p53 protein levels in anaplastic medulloblastomas, sPNET, and ATRT as compared to classic and nodular medulloblastoma. JC virus was not detected in the 33 tumors examined, suggesting that T-antigen binding does not appear to be an ongoing factor in the pathobiology or p53 protein accumulation of embryonal brain tumors.The author(s) declare that they have no competing interests.Drs. Eberhart, Shah and Gravitt planned the study and wrote the initial manuscript draft. Dr. Eberhart, Ms. Chaudhry and Ms. Khaki collected samples, isolated DNA, and performed immunohistochemical staining. Dr. Gravitt supervised and R Daniel performed the JC virus copy number analysis in tumors. All authors reviewed and commented on the final manuscript.The pre-publication history for this paper can be accessed here:


The Angiotensin Converting Enzyme (ACE) insertion/deletion (I/D) polymorphism has received much attention in pharmacogenetic research because observed variations in response to ACE inhibitors might be associated with this polymorphism. Pharmacogenetic testing raises the hope to individualise ACE inhibitor therapy in order to optimise its effectiveness and to reduce adverse effects for genetically different subgroups. However, the extent of its effect modification in patients treated with ACE inhibitors remains inconclusive. Therefore our objective is to quantify the effect modification of the insertion/deletion polymorphism of the angiotensin converting enzyme gene on any surrogate and clinically relevant parameters in patients with cardiovascular diseases, diabetes, renal transplantation and/or renal failure.Systematic Review. We will perform literature searches in six electronic databases to identify randomised controlled trials comparing the effectiveness and occurrence of adverse events of ACE inhibitor therapy against placebo or any active treatment stratified by the I/D gene polymorphism. In addition, authors of trials, experts in pharmacogenetics and pharmaceutical companies will be contacted for further published or unpublished data. Hand searching will be accomplished by reviewing the reference lists of all included studies. The methodological quality of included papers will be assessed. Data analyses will be performed in clinically and methodologically cogent subgroups. The results of the quantitative assessment will be pooled statistically where appropriate to produce an estimate of the differences in the effect of ACE inhibitors observed between the three ACE genotypes.This protocol describes a strategy to quantify the effect modification of the ACE polymorphism on ACE inhibitors in relevant clinical domains using meta-epidemiological research methods. The results may provide evidence for the usefulness of pharmacogenetic testing for individualised ACE inhibitor therapy. Research in genetics and genome sequencing has led to a better understanding of the molecular genetic mechanisms and to the detection of inter-individual genetic differences, so-called polymorphisms, which may have a functional consequence on the response to drugs. Pharmacogenetic tests provide information to better predict and prevent therapeutic failures and adverse drug reaction and raise the hope for an individualised pharmacotherapy -3. AlthoThe ACE polymorphism identified in 1990 by Rigat and co-workers  is one oTherefore, our objective is to systematically review all randomised controlled trials that assessed to what extent the insertion/deletion polymorphism of the angiotensin converting enzyme gene influences the effect and adverse events of angiotensin converting enzyme inhibitors on any surrogate and clinically relevant parameters in patients with cardiovascular disease, diabetes, renal transplantation and/or renal failure.We will perform literature searches in (Pre-) MEDLINE , EMBASE , Biosis , the Cochrane Controlled Trials Register  and the Science citation index. A preliminary literature search in Medline has been carried out to estimate the range of relevant literature. Out of the citations of the pilot searches (172 citations) we identified articles that met our inclusion criteria. Keywords of these articles were used to refine our search strategies. In collaboration with an information specialist we designed the final search strategies for the six databases avoiding any language restrictions Click here for file