Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo for complete chromatin decondensation. The euchromatin domain contains several degrees of chromatin compaction and the histone tails are hyperacetylated, enriched in H3K4 monomethylation and hypo trimethylated on H3K9, H3K27 and H4K20. The transcriptionally active RNA polymerases II molecules are confined in the euchromatin domain and are preferentially located at the vicinity of the interface with heterochromatin.We investigated the functional relationships between chromatin compaction, distribution of histone modifications and location of RNA polymerase II in intact murine rod photoreceptors using cryo-preparation methods, electron tomography and immunogold labeling. Our results show that the characteristic central heterochromatin of rod nuclei is organized into concentric domains characterized by a progressive loosening of the chromatin architecture from inside towards outside and by specific combinations of silencing histone marks. The peripheral heterochromatin is formed by closely packed 30nm fibers as revealed by a characteristic optical diffraction signal. Unexpectedly, the still highly condensed most external heterochromatin domain contains acetylated histones, which are usually associated with active transcription and decondensed chromatin. Histone acetylation is thus not sufficient in vivo. We also show that a combination of histone marks defines distinct concentric heterochromatin domains.Our results show that transcription is located in the most decondensed and highly acetylated chromatin regions, but since acetylation is found associated with compact chromatin it is not sufficient to decondense chromatin  In vitro reconstituted or purified 30 nm fibers are flexible and organized into imperfect helical structures Gene expression is regulated at the transcriptional level by a variety of trans-acting factors that bind to specific promoter elements to elicit transcription initiation in response to intra- and extra-cellular signals. Only a fraction of the genome is competent for factor binding and the way DNA is wrapped into chromatin regulates its accessibility thus participating actively in the regulation of gene expression This structural definition partially overlaps the biochemical and functional description of chromatin, which is separated into EC and HC on the basis of nuclease accessibility, histone modifications and transcriptional activity in vivo at the ultrastructural level.Sedimentation studies on recombinant nucleosomal arrays were performed to explore the link between chromatin condensation and histone modifications and showed that acetylation of H4K16 inhibits the formation of 30 nm fibers The aim of this study was to correlate the packing of chromatin, the transcriptional activity and the distribution of histone tail modifications in sections of cell nuclei. Here murine rod photoreceptors were investigated by electron tomography and immunolabelling to study these correlations. Mouse rod cells have an extremely dense HC domain located at the centre of the nucleus and a small EC territory placed at its periphery, close to the nuclear envelope 3. Chromatin appeared arranged in concentric layers; the center of the nucleus being composed exclusively of HC whereas a 500–600 nm wide rim adjacent to the nuclear membrane contained a mixture of HC and less dense EC. A fraction of HC was found in close contact with the nuclear membrane thus forming a thin rim that was connected through HC bridges to the central HC. In contrast to other cell types, the EC compartment was markedly reduced in size and was confined to the nuclear periphery close to the nuclear membrane. Stereological measurements performed on 20 different nuclei showed that the HC compartment represents 71% of the surface when the nucleus is sectioned through its centre which, assuming spherical symmetry corresponds to about 17 µm3. The thin layer of HC in close contact to the nuclear membrane (within 150 nm from the nuclear membrane) was estimated to 3 µm3 or 15% of the entire HC compartment. The EC compartment contacts the nuclear membrane and projects into the central HC through multiple invaginations thus forming cavities with an extended HC/EC interface. The staining of the EC compartment showed substantial variations indicating that variable degrees of chromatin condensation coexist in this area.Retinas of freshly sacrificed mice were dissected according to an optimized protocol that preserves the electrophysiological activity of the tissue and were instantly cryo-immobilized by high pressure freezing to prevent chemical fixation and changes in physicochemical conditions that could reorganize the cellular ultrastructure. The retinas were then freeze-substituted, epon embedded and sectioned at room temperature. Toluïdin blue stained sections displayed the multilayered structure of the retina in which the nucleus, inner segment and outer segment of the photoreceptor were recognized   thus validating the estimated chromatin compaction.Finally, area 4 corresponds to a characteristic HC domain where the boundaries of individual particles cannot be distinguished. A total of 61% of the volume has an intensity above the threshold assigned for the nucleosomes which, assuming a similar staining than for the EC territories, yields a concentration of about 540 10−1. This observation indicates that the periphery of the HC territory is formed by closely packed 30 nm chromatin fibres whereas such fibres are not detected in the most central domain.Despite its electron dense nature the HC compartment showed at least two distinct textures indicative of different nucleosomal arrangements. The periphery of the HC territory had generally a granular appearance whereas the most central domain showed a highly homogeneous and smooth texture (2 (p/µm2) in HC and of 42 p/µm2 in EC yielding a HC/EC labelling ratio of 2. This ratio is significantly lower than the HC/EC nucleosome density ratio of at least 5 found by tomography suggesting that the accessibility of this epitope is affected by the nucleosomal packing. To quantify the radial variation of the labelling density within the nucleus, the HC domain was contoured and the gold particles were counted in concentric rings of decreasing size, each separated by 150 nm . Ultra-thin cryo sections of the retina were therefore produced to gain equal access to all regions of the nucleus and to optimally preserve the antigenicity of the epitopes y 150 nm Figure 8The distribution of three histone trimethylation marks specific for transcriptionally silenced chromatin were investigated by using antibodies recognizing histone H3 modified on lys 9 (H3K9me3), on lys 27 (H3K27me3) or histone H4 modified on lys 20 (H4K20me3).2 in the most external HC ring (from 0 to 150 nm from the EC/HC interface) and increased regularly to reach 255 p/µm2 in the center of the nucleus  and 2.8 times higher than in the periphery (140 p/µm2)  and8. A fiand2 whereas it rises to 87 p/µm2 in the periphery.The H3K27me3 marks are also enriched in the HC region but in contrast to the previously analyzed trimethylations, show a decreased labelling from the periphery to the center of the HC territory and8. A ce2) but, unexpectedly, is found highly enriched in the condensed HC closest to the HC/EC interface (97 p/µm2) and in the EC compartment (41 p/µm2) (and2). An antibody directed against all acetylated forms of H4 gave the same labelling profile  and8. The The distribution of acetylated histone H3 was investigated by using an antibody specific for acetylated K9 and K14. Despite a slightly lower labelling density, the distribution of H3K9K14ac is similar to that of H4K8ac Figure 8 and Unlike the H3K9, H3K27 or H4K20 methylations described above, the methylation of lysine 4 of histone H3 is a post translational modification exclusively associated with actively transcribed genes and particularly with the early transcribed region In order to position RNA polymerase II (RNA Pol II) molecules an antibody targetting the repeated heptapeptide of the C-terminal domain (CTD) of the largest RNA Pol II subunit was used. This antibody recognizes all forms of RNA Pol II since it is directed against the non phosphorylated CTD. The RNA Pol II were found almost exclusively (79%) within the EC compartment where the labelling was 14.5 times higher than in the HC domain. Interestingly the labelling was not distributed randomly within the EC cavities but appeared to be stronger close to the EC/HC interface  in the EC compartment. The histogram representing the distance of the active polymerases to the EC/HC interface shows that 66% of the active enzyme peaks at 20+/−20 nm from the interface. Moreover no active RNA Pol II labeling could be detected in the most central parts of the EC cavities. These results show that the transcribing enzyme is located even closer to the interface than the total pool of RNA Pol II.Murine rod photoreceptors are terminally differentiated cells and contain a highly condensed HC domain placed at the center of the nuclei. The small size of the EC compartment, the peculiar chromatin organization and the availability of genome wide gene expression data turn these cells into unique models to study the correlations between chromatin compaction, transcriptional activity and histone modifications. At first sight, rod photoreceptor nuclei show two distinct nuclear domains that reflect the early structural definition of EC and HC based on regions differentially stained with basophilic dyes in vitro for dispersed chromatin, 30 nm fibers are more elusive in the cellular context except for isolated starfish sperm or chicken erythrocyte nuclei placed in hypotonic conditions −1 proposed to be generated by tightly packed chromatin fibers A widely accepted model for chromatin compaction involves successive folding events during which linear nucleosome arrays wrap into 30 nm wide helical fibers forming either a solenoid or a zig-zag assembly. Mostly observed −1 in the Fourier transform of nuclear images. The highly compact nature of the rod nucleus may have favoured the detection of the fibers and special care was taken to perturb as little as possible the nuclear organisation during specimen preparation. In this respect, the nuclei were not isolated from cells, the physiological ionic strength was preserved during dissection and the freshly dissected tissue was shown to be functional just prior to high pressure freezing. Interestingly 30nm fibers are only detected in the periphery of the HC whereas the central part appears uniform and does not show any periodic signal at 1/30nm−1. Our results thus favour a model in which rod HC is organized as closely packed 30 nm fibers which, consistent with the liquid crystal model, appear melted in the most central HC. However, even in these central parts of the nuclei, our immunolabelling data reveal 30 nm wide fibers, suggesting that the diffusion of nucleosomes around the positions they would adopt within a regular 30 nm fiber is limited. The transition between the homogeneous and the granular HC is unrelated to histone acetylation but correlates with a specific trimethylation pattern that could contribute either directly or through the recruitment of specific factors to interfiber interactions.Our studies revealed for the first time a signal at 1/30nmThe rod HC is organized into three concentric layers, each characterized by a specific combination of histone modifications and this arrangement reflects the functional layout of the nuclei. The central HC core has a radius of 0.8 µm corresponding to 42% of the nuclear radius and contains histones highly enriched in H4K20me3 and H3K9me3 marks, weakly trimethylated on H3K27 and deacetylated. The size of this domain correlates with Fluorescence In Situ Hydridization (FISH) experiments showing that telomeric, centromeric and subcentromeric satellite DNA are found between 0 and 45% of the nuclear radius A peripheral domain surrounds this central core and extends to about 0.2 µm to the EC/HC interface which corresponds to 42% and roughly 60–80% of the nuclear radius since the interface is folded. In this domain the histone tails are not acetylated and show reduced H4K20me3 and H3K9me3 and higher H3K27me3 levels than the central core. The H3K27me3 mark was associated in several systems to facultative HC defined here as genomic regions that have the opportunity to adopt an open or a compact conformation within defined temporal or spatial constraints Finally a 0.2 µm thick rim of heterochromatin at the EC/HC interface displays the same histone trimethylation pattern than the peripheral HC domain but is surprisingly highly acetylated. FISH experiments have located the gene rich regions of the genome, irrespectively of their transcriptional status, between 70 and 100% of the nuclear radius in vitro transcription experiments indicate that acetylation facilitates chromatin unfolding and transcription in vivo observations confirm that H3 and H4 acetylation on its own is not sufficient to elicit chromatin decondensation at the HC/EC interface. In vitro the extended 11 nm chromatin fibers showing the typical beads-on-a-string appearance were only observed in the absence of the histone H1, which is believed to stabilize the 30 nm fiber by acting on the path of DNA at the exit of the nucleosome Transcription, as probed by the presence of RNA Pol II, is strictly restricted to the EC compartment, which is characterized by the presence of decondensed chromatin, acetylated and monomethylated histone tails, and the absence of H3K9me3, H3K27me3 and H4K20me3 marks. Hyperacetylation of H3 and H4 tails is a hallmark of actively transcribed genes in higher eukaryotes 5/µm3) and the volume of the EC compartment (about 9 µm3). This fraction may correspond to genes that can rapidly switch between an activated and a repressed state.Electron tomograms revealed large variations in nucleosomal packing in the EC compartment ranging from extended 11 nm fibers to more densely packed chromatin regions. These variations suggest that all the euchromatin is not in the same transcriptional state and that significant transcriptional regulation may still occurs in the EC compartment through modulation of the chromatin structure. The fraction of the genome exposed in the EC territory can be roughly estimated to be 20 Mbp or less than 1% of the mouse genome from the average nucleosome density . The experiments involving animals were conducted under the supervision of a staff member holding an animal experimentation authorization from the Ministry of Agriculture and Fisheries: having followed the obligatory national training programs for animal handling, including procedures of euthanasia. The institution is guided by the International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences and all experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Their eyes were rapidly enucleated and placed in Artificial CerebroSpinal Fluid (ACSF) buffer  for the dissection. The retina were gently separated from the pigment epithelium and the sclera after removal of the lens, iris and vitreous humor. The dissection was completed in less than 5 min Thirteen to twenty-one weeks old C57/BL6 mice were sacrificed in accordance with the National Institutes of Health For High Pressure Freezing (HPF), retinas were punched  to obtain an oriented disk 1.5 mm in diameter that fits into the specimen carrier. After infiltration in a drop of cryoprotectant containing 10% dextran (Sigma # D1662) and 10% BSA (Sigma # A4503) v/v in ACSF buffer (5 sec), each disk was placed onto a 200 µm thick flat gold-plated specimen carrier in order to be vitrified in the HPF machine .4 and were then warmed up slowly (1°C/h) to −60°C in an Automated Freeze Substitution device . After 8–12 h the temperature was raised to −30°C (1°C/h) and the samples were kept at this temperature for 8–12 h before being rinsed several times in acetone. The samples were then infiltrated with gradually increasing concentration of Epon in acetone  for 2–3 h while raising the temperature. Addition of pure Epon was performed at room temperature. After polymerization of the resin at 60°C, 70–100 nm thin sections were produced using an ultramicrotome , collected on formvar-carbon coated hexagonal 50 mesh copper grids and post-stained for 5 min with 2% aqueous uranyl acetate, rinsed and incubated for 2 min with lead citrate. The quality of the vitrification was assessed by the absence of recognizable ice crystal ghosts.After HPF, the samples were freeze substitute To determine the proportion of the nuclear volume occupied by the different chromatin compartments we used stereological methods http://www.cgl.ucsf.edu/chimera) after having low pass filtered the tomograms to 1/10 nm−1. In order to position the electron dense particles the low pass filtered tomograms were searched for peaks of high intensity separated by more than 12 nm. For modeling, a 12 nm sphere was placed at each peak coordinate.Ten nanometer colloidal gold particles were applied on one side of the grid to be used as fiducial markers to align the tilt series. The specimen was inserted in a transmission electron microscope equipped with a field emission gun and operating at 200 kV . Digital images were recorded at a magnification of 29.000× on a Pelletier cooled 2k CCD camera leading to a pixel spacing of 3.6 Å . Each tilt series was recorded in bright-field mode over a tilt range of −65° to +65° with 1° increments giving a total of 131 images automatically collected with the Xplore 3D software (FEI Company). The recorded images were aligned on a common origin by means of cross correlation techniques and the 3-D reconstruction was performed using the Inspect3D tomography reconstruction package (FEI Company) according to the weighted-back projection methods or the Simultaneous Iterative Reconstruction Technique (SIRT). Surface rendering was performed by gray level thresholding using the Chimera software , H4K8ac , pan acetylated H4 (Millipore # 06-866) and H3K4me1 (Abcam # ab8895). Inactive marks of transcription were detected with rabbit polyclonal antibodies directed against H4K20me3 (Abcam # ab9053), H3K27me3 (Millipore # 07-449) and H3K9me3 (diagenode # pAb-056-050). Total Histone H3 was detected with a rabbit monoclonal antibody directed against the C-terminus of histone H3 (Millipore # 05-928). RNA polymerase II was detected with an IgG1-type mouse monoclonal antibody developed at IGBMC (1PB7C2). This immunopurified antibody reacts with the conserved heptapeptide repeat of the largest subunit of eukaryotic RNA polymerase II. The active form of RNA polymerase II was detected with an IgM-type mouse monoclonal antibody directed against the phosphoserine 2 in the heptapeptide repeat of Rpb1 (Covance # MMS-129R). The primary antibodies were revealed with goat anti-rabbit ultra-small gold-conjugates . For the detection at the light microscope level goat anti-rabbit IgG coupled to AlexaFluor 488 were used (Invitrogen # A11008).Sample preparation: Briefly, after dissection, retina were cut into small blocks of about 1mm3, fixed in 2.5% formaldehyde, 0.1% glutaraldehyde in 0.1 M Phosphate Buffer Saline pH 7.4 (PBS) for 1h. Free aldehydes were blocked with several washes in PBS supplemented with 50 mM glycine. The blocks were infiltrated with 2.3 M sucrose in PBS in an Eppendorf tube on a rotating wheel for at least 2 h at 4°C. The blocks were then mounted on the holders of the cryoultamicrotome and rapidly frozen in liquid nitrogen. Cryosectioning: Ultra-thin cryo-sections (100 nm) were cut using a cryo-ultramicrotome  at −130°C using diamond knives  and recovered with a drop of 2.3 M sucrose on formvar-carbon coated 50 mesh nickel grids or on coated light microscope slides for immunofluorescence. After thawing, immunolabelling was performed using the automatic Immuno Gold Labelling apparatus . Immunolabelling: After a blocking step of two times 10 mn in glycine  and another 10 mn in 1% BSA/PBS (blocking buffer), the sections were incubated 20 min with the primary antibodiy. After 4 times 2 min washes on 10× diluted blocking buffer bound antibodies were revealed using goat anti-rabbit conjugated to ultra-small colloidal gold (US) diluted 1∶25 PBS with 0.1% acetylated BSA (BSA-c™) . After several washes in PBS/0,1%BSA followed by PBS, the antigen-antibody complexes were stabilized on 1% glutaraldehyde in PBS for 5 min After several washes in PBS followed by water colloidal gold silver enhancement was performed using R-Gent™ (Aurion) for 30 min resulting in an average particle size of 10 nm. Thereafter, sections were carefully washed 5 times (5 min each) with water, stained and embedded in a solution containing 4 parts of 2% methyl cellulose (Sigma-Aldrich # M-6385) and one part of 2% uranyl acetate. Sections were observed using a Philips CM 120 transmission electron microscope at an accelerating voltage of 100 kV.Retina samples were prepared, sectioned and immunolabelled according to Tokuyasu For immunofluorescence the primary antibody was detected with a goat anti-rabbit IgG coupled to AlexaFluor 488 diluted in blocking buffer. Sections were counterstained with DAPI   and embedded in Aqua-Poly/Mount (Polysciences # 18606-20) medium. The results were examined using a DM400B Leica epifluorescence microscope connected to a Cool Snap camera (Roper).2 was quantified for 15–20 rod nuclei for each immunolabelling experiment. The regions of interest were delimited and their surfaces were measured using the ImageJ software (http://rsbweb.nih.gov/ij/). Gold particles were counted within each region of interest after high pass filtering of the images and by using an automated peak search algorithm .The number of particles per µm


The effect of localized hypothermia on microcirculatory and metabolic parameters in s.c. DS sarcomas on the hind foot dorsum of Sprague-Dawley rats was investigated. Tumours were cooled by superfusion of the tumour surface with cooled saline solution to 25 degrees C or 15 degrees C. Control tumours remained at 35 degrees C. These temperatures were maintained for 30 min. In tumour oxygenation measurements, hypothermia at 25 degrees C and 15 degrees C caused progressive decreases in the size of the fraction of pO2 measurements between 0 and 2.5 mmHg together with a reduction in pO2 variability. No significant changes in median or mean pO2 or in the fraction of pO2 measurements between 0 and 5 mmHg, and 0 and 10 mmHg were observed. Using laser Doppler flowmetry, red blood cell flux was found to decrease significantly upon 25 degrees C or 15 degrees C hypothermia treatment to 67% and 37% of starting values respectively, whereas no significant changes were seen in control tumours over the whole observation period. Viscosity was measured in blood and plasma samples over a range of temperatures and was found to increase with decreasing temperature. Assessment of tumour glucose levels showed an increased concentration of glucose following 15 degrees C hypothermia, an observation consistent with a 'slowing down' of glycolysis. No changes in lactate or adenylate phosphate levels were observed. As a way of improving tumour oxygenation, localized hypothermia may therefore be a useful means of radiosensitization.


KI and WU Polyomaviruses in Children, France To the Editor: Two new members of the Polyomaviridae family, provisionally named Karolinska Institutet virus (KIPyV) and Washington University virus (WUPyV), have been recently discovered ; influenza virus types A and B; parainfluenza virus types 1, 2, and 3; and adenoviruses (AdVs) by direct immunofluorescence assay. The aspirates were also tested for human metapneumovirus (HMPV) by an enzyme immunoassay  and for the human bocavirus (HBoV) by PCR  children. The viruses found were RSVs in 175 (32.6%), HBoVs in 54 (10.0%), HMPVs in 50 (9.3%), rhinoviruses/enteroviruses in 11 (2%), influenza A viruses in 8 (1.5%), human AdVs in 6 (1.1%), and parainfluenza type 3 viruses in 4 (0.7%) samples. Aspirates were not tested for coronaviruses; detection of rhinoviruses/enteroviruses was likely low because cell culture is less sensitive than molecular assays.A total of 13 (2.4%) samples were positive for WUPyV; of these 4 (30.8%) were co-infected with another virus. The 13 children with samples positive for WUPyV had a median age of 11.2 (2–48) months and the male/female sex ratio was 2.2. KIPyV DNA was detected in samples from 3 (0.6%) boys ; 1 of those samples was co-infected with RSV and HMPV.Sequences of WUPyV and KIPyV isolates varied little from each other and from other GenBank sequences, which suggests that these polyomaviruses are genetically conserved viruses. Clinical characteristics of children infected with WUPyV and KIPyV are retrospectively recorded . All chiOur data are in agreement with the 2 original reports that show that the new KI and WU polyomaviruses may be detected in respiratory secretions from patients with respiratory diseases (KIPyV and WUPyV were mainly detected in samples from children with lower respiratory tract disease, such as bronchiolitis or atypical pneumonia, and in samples from children with exacerbated asthma. These preliminary data on the likely role of these viruses as respiratory pathogens need to be interpreted with caution. Aspirates were obtained only from those with observed symptoms; no asymptomatic controls were tested. Detection of WUPyV and KIPyV in respiratory samples may simply reflect a respiratory transmission route as previously suggested for BK virus and JC virus (As with other polyomaviruses, WUPyV and KIPyV could establish persistent and latent infections with likely asymptomatic reactivations (


Between 1974 and 1984 69 adults with acute lymphoblastic leukaemia (ALL) were treated with two different protocols. Fifty-four (78%) of the patients entered complete remission (CR); 27 of these then received a consolidation protocol consisting of daunorubicin, cytosine arabinoside and 6-thioguanine, followed by two courses of intravenous methotrexate 500 mg m-2 with folinic acid rescue. All patients received intrathecal methotrexate and cranial irradiation (24 Gy) followed by maintenance therapy with 6-mercaptopurine and methotrexate for at least 2 years. The median survival for all patients was 23 months from the time of presentation with an actuarial 5-year survival of 21%. The actuarial chance of surviving 5 years in CR for patients receiving the consolidation protocol was 38% compared to 19% for patients receiving no consolidation (P = NS). Only patient age and white cell count at presentation were found to influence the chance of achieving CR and the chance of overall survival. The presence or absence of c-ALL antigen did not influence prognosis. Patients younger than 35 years with low white cell counts at presentation (less than 10 X 10(9)1(-1] had a particularly good prognosis but no patient with T-ALL and no patient older than 50 years old at diagnosis survived more than 18 months.


Mutations in these genes have been linked to hereditary non-polyposis colon cancer; they also occur in a variety of sporadic cancers. Western blot analysis and immunohistochemistry demonstrated that hMLH1 and hPMS2 are widely expressed nuclear proteins with a distribution pattern very similar to that previously described for hMSH2. These observations showing similar localization of hMLH1 and hPMS2 with hMSH2 are consistent with the biochemical function of these proteins in DNA mismatch repair.


The kinase Wee1 has been recognized for a quarter century as a key inhibitor of Cyclin dependent kinase 1 (Cdk1) and mitotic entry in eukaryotes. Nonetheless, Wee1 regulation is not well understood and its large amino-terminal regulatory domain (NRD) has remained largely uncharted. Evidence has accumulated that cyclin B/Cdk1 complexes reciprocally inhibit Wee1 activity through NRD phosphorylation. Recent studies have identified the first functional NRD elements and suggested that vertebrate cyclin A/Cdk2 complexes also phosphorylate the NRD. A short NRD peptide, termed the Wee box, augments the activity of the Wee1 kinase domain. Cdk1/2-mediated phosphorylation of the Wee box (on T239) antagonizes kinase activity. A nearby region harbors a conserved RxL motif (RxL1) that promotes cyclin A/Cdk2 binding and T239 phosphorylation. Mutation of either T239 or RxL1 bolsters the ability of Wee1 to block mitotic entry, consistent with negative regulation of Wee1 through these sites. The region in human somatic Wee1 that encompasses RxL1 also binds Crm1, directing Wee1 export from the nucleus. These studies have illuminated important aspects of Wee1 regulation and defined a specific molecular pathway through which cyclin A/Cdk2 complexes foster mitotic entry. The complexity, speed, and importance of regulation of mitotic entry suggest that there is more to be learned. Mitotic entry is the paradigmatic cell cycle transition and example of Cdk regulation. Yet, our understanding of this transition remains superficial. A long-term goal of research in this area is to design drugs that treat cancer by either blocking mitotic entry or driving cells into mitosis in the face of lethal DNA damage. Cyclin B/Cdk1 Cdc2/Cdc28) complexes direct many of the events of mitosis. These events must be launched in swift, coordinated fashion but only after DNA synthesis is completed and DNA damage is repaired. To effect such control, cyclin B/Cdk1 activity is regulated through dynamic post-translational modifications. Wee1 is a universal Cdk1 inhibitor that phosphorylates a tyrosine residue (Y15) in the ATP binding site, thereby blocking Cdk1 activity /Cdk2 complexes--help pave the way for mitosis by reducing cyclin B/Cdk1 Y15 phosphorylation. Microinjection of cyclin A was observed to drive cultured human cells into mitosis and injection of cyclin A/Cdk2 inhibitors could block mitotic entry . InductiWe asked whether cyclin A/Cdk2 complexes might antagonize Cdk1 Y15 phosphorylation by directly binding and inactivating Wee1. Wee1 was present in cyclin A and Cdk2 immunoprecipitates from U2-OS cells and associated efficiently with exogenously expressed Cdk2 . In contCyclin A/Cdk2 complexes are known to preferentially recognize some substrates via short sequence motifs termed 'Cy' or RxL' motifs -25. Humain vivo data, studies in human somatic cell extracts showed that recombinant cyclin A can direct the phosphorylation and inactivation of Wee1 more efficiently than cyclin B, and combined addition of cyclins A and B induced nuclear envelope breakdown more efficiently than addition of either cyclin alone [We mutated each of the RxL sequences in Wee1, singly and in combination, and examined the impact on cyclin A/Cdk2 binding and T239 phosphorylation. Mutations of the RxL sequences diminished stable association of Wee1 with cyclin A/Cdk2 complexes in vivo and in vitro . Mutatioin alone .The RxL1 mutant appeared to be modestly more potent than the T239A mutant in mediating G2 phase arrest under the conditions examined, suggesting that loss of the RxL1 site might impact more than phosphorylation of T239. We therefore examined other properties of Wee1. Wee1 has been described as being localized to the nucleus during interphase and the cytoplasm during mitosis. Whether this redistribution is a cause or effect of nuclear envelope breakdown has remained unclear. Early studies demonstrated that nuclear Wee1 could potently protect cells from premature mitosis, even in the presence of activated cyclin B/Cdk1 complexes in the cytoplasm . These sThe simplest explanation for the restricted nuclear localization of the RxL1 mutant was that this site directs cyclin A/Cdk2-mediated phosphorylation of another residue(s) that drives Wee1 cytoplasmic redistribution. This scenario may yet prove to be true, but examination of the Wee1 primary structure suggested another potential explanation. The RxL1 sequence overlaps with residues that match the loose consensus Crm1-dependent nuclear export signal (NES) -30. SubsOne can draw a number of conclusions from this recent work. First, redistribution of human somatic Wee1 to the cytoplasm is an active, temporally regulated event, rather than a passive byproduct of nuclear envelope breakdown. Based on conservation of NES consensus sequences among vertebrate somatic Wee1 proteins, nuclear export might also be conserved, suggesting the presence of selection pressure to maintain it. Given the lack of conservation of the NES in embryonic proteins, regulated export of Wee1 may exert an additional constraint on mitotic entry unique to somatic cells. In this light, it was somewhat surprising that expression of the NES mutant did not impose increased G2 arrest under the conditions tested . It seemMight cyclin A/Cdk1 complexes also bind RxL1 and inactivate Wee1? Cdk2 appears generally to be the preferred binding partner for cyclin A when Cdk1 and Cdk2 are expressed at their normal levels ,18,32. HThe conclusion that cyclin A/Cdk2 complexes drive mitotic entry raises an apparent conundrum. These complexes also drive DNA synthesis ,20,36, aThere are a number of issues that merit further study. The bifunctional nature of the NES/RxL1 site raises the question of whether physical occupancy of the site by cyclin A/Cdk2 complexes competes with that of Crm1. The stable association of Wee1 with both binding partners  suggestsThe authors declare that they have no competing interests.


Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of α2,3-linked and α2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAα2,6Gal and Maackia amurensis agglutinin (MAA) for SAα2,3Gal in the respiratory tract of normal adults and children.We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers.We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the α2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs.The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAα2,3 binding viruses, such as influenza or human parainfluenza are to be made. There are two main epithelial cell types in the bronchus – ciliated cells and goblet cells that secrete mucus. Within the submucosa there are also submucous glands present. The goblet cells have a glycoprotein which is acidic due to the presence of sialic sulphate groups and this secretion may vary with various diseases . SAα2,6GThe affinity of the attachment of the HA to cell surface receptors is believed to be an important determinant in tissue tropism of the virus and constitutes part of the species barrier that keeps avian influenza viruses from readily infecting humans. Pigs contain a respiratory epithelium that has been reported to contain both "avian-virus" binding SAα2,3Gal and "human-virus" binding SAα2,6Gal linkages supposedly explaining why they can be infected with both human and avian influenza viruses . Therefoin situ. The SAα2,6Gal and SAα2,3Gal expression on histological specimens in situ can be done using fluoresceinated lectins and by histochemistry. Since the early 1990's many histology laboratories have used unmasking or retrieval techniques to enhance immunohistochemical detection of antigens.Some of the studies on the types of SA expressed on cell surfaces have been done on sialic acids extracted from cell membrane homogenates. However, for the understanding of influenza pathogenesis and pandemic emergence, it is important to have methods that can define the profiles of SAα2,6Gal and SAα2,3Gal in histological tissue sections In this study we address three issues. The first was to investigate if antigen unmasking or retrieval would affect lectin-ligand expression in histological tissues. The second issue was to compare the findings obtained by using lectin fluorescence with cytochemistry for lectin-ligand analysis in the same tissue samples. Finally, we wanted to use optimized methods to re-evaluate the distribution of the SAα2,6Gal and SAα2,3Gal in human respiratory tissues and then correlate this with the reported presence or absence of influenza infection in different parts of the respiratory tract.Biopsy samples were collected from the archived files of the Histopathology Department of Queen Mary Hospital, Pok Fu Lam, Hong Kong. Five surgically removed lungs from children with congenital cystic adenomatoid malformation (CCAM), seven non-neoplastic bronchial biopsies from patients investigated for possible malignancy, eight normal nasopharyngeal biopsies from patients with suspected nasopharyngeal carcinoma and eight lung biopsy samples from 20–40 week abortuses were also used. Seven biopsy tissues of CCAM, representing paediatric lung tissues of ages 1 month to 7 years were also retrieved from the files of the Department of Histopathology, Adelaide Women's and Children's Hospital, Adelaide, South Australia. All tissues had been fixed in 10% neutral buffered formalin, processed into paraffin and stored at room temperature. Tissue blocks from the lungs of five patients who had died of acute bacterial pneumonia were used as a non-influenza comparison. Intestinal tissue from four ducks kindly provided by the Agriculture, Fisheries and Conservation Department, Government of HKSAR, were used as a positive control for SAα2,3Gal binding. The research was approved by the Ethics Committee of the University of Hong Kong/Hospital Authority Western Cluster.We used lectin histochemistry and fluorescence which is the standard method of detection of the SA linkages . Lectin For the initial trial of optimization for oligosaccharide ligand retrieval methods one lung block from a case of CCAM was used. The tissues were sectioned at 5 μm and deparaffinized. Control sections had no retrieval. Two different methods were used for retrieval: microwave and enzyme digestion. For microwaving, an Energy Beam Sciences microwave processor was used together with 2 types of buffer. 0.1 M EDTA and 10 mM citrate buffer was used and the sections were microwaved for 10,15, 20 and 25 minutes at 95°C. Two enzyme digestion methods were used: trypsin and pronase, and for both these methods sections were incubated for 15 mins at 37°C.Single fluorescent studies were performed as follows. The sections were microwaved in 95°C citrate buffer pH 6.0 for 15 minutes, washed with 0.05 M Tris Buffer Saline (TBS) pH 7.6 and then incubated with either 1/100 FITC conjugated SNA (EY Laboratories), or 1/100 FITC conjugated MAA (EY Laboratories) for 1 hour at room temperature in the dark. Double fluorescent studies were performed according to the method of Mason et al. . Briefly2O2 in TBS for 12 min and after washing with TBS 3 times, 5 minutes each were then incubated with 1/50 HRP conjugated SNA (EY Laboratories) and 1/50 HRP conjugated MAA (EY Laboratories) at room temperature for 1 hour respectively. After 3 further washes in TBS the sections were developed with AEC substrate kit (Vector Laboratories) at RT for 30 minutes followed by counterstaining with Mayer's haematoxylin and mounting with DAKO aqueous mount .Lectin horseradish peroxidase (HRP) detection: the sections were microwaved in 10 mM citrate buffer pH 6.0 for 15 min, blocked with 3% H2O2 in TBS for 12 min and with avidin/biotin blocking kit (Vector). They were then incubated with 1/100 biotinylated MAA1 (or 1/100 biotinylated MAA2) (Vector) for either 1 hour at RT or 4°C overnight, blocked with 1% bovine serum albumin for 10 mins at RT, and then incubated with strep-ABC complex  diluted 1/100 for 30 mins. at room temperature. Development was performed using the AEC substrate kit (Vector) at room temperature for 10 minutes. The nuclei were counterstained with Mayer's hematoxylin and then the sections were dried and mounted with DAKO aqueous mount (Dako Cytomation). Duck intestine sections were used as controls with and without pre-treatment with SAα2,3 specific neuraminidase from Glyko to ensure that sialic acids were specifically targeted. Stain intensity was measured semi-quantitatively using duck MAA as a control. Similar stain intensity to the duck intestine was graded as strong (++) and a weaker pattern as +.Lectin biotin detection: The sections were microwaved in 10 mM citrate buffer pH 6.0 at 95°C for 15 min then blocked with 3% HTo determine lectin binding profiles we used data from the Consortium for Functional Glycomics (CFG) web site  using glIn the absence of unmasking techniques and using the lectins SNA and MAA from EY Labs, there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin binding to the surface epithelial cells and mucus containing cells for both SNA and MAA Figure  and thisThe 8 samples of fetal tissue at 20 weeks gestation showed strong binding (++) of MAA to the bronchial epithelium and to the developing pneumocytes with absent SNA detected. With advancing development the SNA binding increased and there was a switch in the MAA1 and MAA2 binding between the alveoli and bronchi Table . It was When dual labelling was performed on the bronchial tissues of adults and of children using SNA and MAA, we identified a heterogeneous distribution of SNA and MAA binding in the epithelium with no clear distinction between ciliated and non-ciliated cells resulting in a mixed and occasionally dual expression of both SAα2,3Gal as well as SAα2,6Gal in the ciliated cells, goblet cells and basal cells Fig . We alsoBecause we found that there was a different binding pattern in the upper and lower respiratory tract for MAA1 and MAA2 using these lectins from Vector labs we then investigated whether the MAA from 2 other suppliers – EY Laboratories and Roche would identify one or both of the MAA isoforms. We therefore used serial sections from adult lung tissue and stained them using the Vector MAA1 and MAA2, digoxigenin labelled MAA (Roche) and biotinylated MAA from EY Laboratories. While EY Laboratories state that their MAA is a combination of 2 isoforms, this information is not evident from Roche. As expected using MAA1 Fig  the alveAnalysis of the glycan array binding data of MAA1, SNA and H5N1 submitted to the CFG showed that MAL did detect SAα2,3Galβ1,4GlcNac(glycan numbers 235–238) with detection of the sulphated forms (216 and 227) [see Additional file N-acetylneuraminic  acids – a series of 9-carbon sugars. Influenza virus infection of humans involves binding of the virus haemagglutinin (HA) to these sialyoligosaccharides on the surface of cells of the respiratory tract. In addition, the virus neuraminidase (NA) cleaves the sialic acid on the host cell and is important in releasing newly formed virus from the cell after virus replication is completed so these virions can spread out in search of other cells to infect. Since respiratory mucus is also rich in SA, this provides a potential barrier to the spread of newly formed virions. By cleaving these SA, the influenza virus NA facilitates the virus spread through this mucus layer [Cells of the respiratory tract express a number of glycan containing conjugates on the cell surface, many of which terminate with us layer . Thus thThe crystal structures of HA shows that the terminal sialic acids bind in a groove at the top of the HA molecule . PreviouUsing retrieval methods and selection of lectin conjugate we have demonstrated that the lectin binding to the SAα2,3Gal receptor for avian influenza viruses is more widely expressed in the respiratory tract of humans than previously documented . In partThe difference between our studies and previous ones on sialic acid expression in respiratory epithelial cells can be partially explained by the methods used for lectin analysis as well as the type of lectin conjugate used. Antigen retrieval or unmasking did not become an established procedure in many laboratories until the mid 1990's and the earlier publications used paraffin embedded tissues without retrieval ,8. LaterTwo isoforms of MAA have been recognized for many years and their binding profiles have been characterized. Since MAA1 bound to non-SAα2,3 glycans it has not been used as extensively by some researchers as MAA2. For instance, Shinya et al have recently demonstrated SAα2,3 Gal expression only in the lung but not the bronchus or upper respiratory tract . Since tBecause of this varied distribution of MAA1 and MAA2 throughout the respiratory tract, we hypothesised that this may shed new light on the distribution and binding of H5N1 viruses to the upper and lower respiratory tract. Using this information we therefore used ex-vivo cultures of the upper and lower respiratory tract and infected them with different H5N1, H1N1 and H3N2 viruses and found that contrary to previous suppositions, H5N1 viruses were able to replicate in the upper respiratory tract – a region which lacked MAA2 binding but had abundant MAA1 binding, thus indicating that the virus is perhaps binding to SAα2,3Galβ1,3/4GlcNac motifs or even to non-sialyated receptors .in vitro are only a partial representation of the true nature of sialic acid expression in the adult respiratory tract [in vitro model with cultured tracheobronchial epithelium showed more similarity to the receptor profile seen in the respiratory tract of children. Therefore it is possible that the human tracheobronchial epithelial culture model is more representative of the respiratory tract of children rather than that of adults, and represents a developmentally earlier model of the human respiratory tract.Our results also indicated that the recent findings of Matrosovich and colleagues who found SAα2,3Gal in ciliated cells and SAα2,6Gal in the goblet cells of tracheobronchial cells cultured In summary, we found that there was an overall increased detection of SAα2,3Gal in the respiratory tract when microwave unmasking is used and when a combination of different isoforms of MAA was used. We therefore advocate the routine use of this method for future investigations on the distribution of receptors for influenza viruses in the respiratory tract. We also urge attention to the exact isoform of MAA present in lectins supplied by different manufacturers . These rThe author(s) declare that they have no competing interests.Dr J Nicholls and Professor J S M Peiris designed the experiments and were responsible for primary analysis of tissues. Dr A J Bourne provided input to the CCAM cases from Australia. Dr H Chen and Y Guan assisted in the interpretation of results. All authors have read and approved the final manuscript.Maackia amurensis 1 (MAA1), Sambucus nigra agglutinin (SNA) and H5N1 binding affinity in glycan array Description: Summary of glycan binding profiles of Maackia amurensis 1 (MAA1), Sambucus nigra agglutinin (SNA) and H5N1 influenza (A/Vietnam/1203/04) (Viet04) in the glycan array. Significant binding sugars are shown in column 2 with their glycan numbers according to Printed Array 2.1 shown in column 1. Strong affinity is **, weak affinity is * and no significant affinity is blank. Many SAα2,3Gal oligosaccharides (underlined) bind to both MAA1 as well as H5N1 (A/Vietnam/1203/4).Click here for file


TNF-α induced endothelin-1 gene expression was associated with a parallel increase in the release of the corresponding peptide in the culture medium. These findings suggest that the enhanced synthesis and release of endothelin-1 occurring in conditions of increased generation of TNF, may act as a modulatory factor that counteracts the hypotensive effect and the excessive platelet aggregation and adhesion induced by TNF.We have studied the effect of human recombinant tumour necrosis factor-α (TNF-α) on gene expression and production of endothelin-1 in cultured bovine aortic endothelial cells. TNF-α (10 and 100 ng ml


BCL10 gene in 81 primary prostate carcinomas, 20 squamous cell cancers of the head and neck, 15 small-cell lung cancer cell lines, 24 renal carcinoma cell lines and 13 sarcoma cell lines. We failed to find evidence of somatically acquired mutations of the BCL10 gene suggesting that BCL10 does not play a major role in the development of these malignancies. © 1999 Cancer Research CampaignWe have used single-strand conformation polymorphism (SSCP) analysis to screen for mutations in the


Purpose and Method. Hyper-fractionated radiotherapy for treatment            of soft tissue sarcomas is designed to deliver a higher total dose of radiation without an            increase in late normal tissue damage. In a previous study at the Royal Marsden Hospital,           a total dose of 75 Gy using twice daily 1.25 Gy fractions resulted in a higher incidence of late           damage than conventional radiotherapy using 2 Gy daily fractions treating to a total of 60 Gy.           The current trial therefore used a lower dose per fraction of 1.2 Gy and lower total dose of 72           Gy, with 60 fractions given over a period of 6 weeks.           Subjects. A total of 37 patients  with a median           age of 56 years (range 19–88 years) were treated.           Results. Of eight patients treated pre-operatively, six showed a            partial response and in two the tumour was static. The maximum acute toxicities were grade            1 in eight, grade 2 in 14 and grade 3 in 15 patients. Late toxicities of the skin were            graded 1 in 10 and grade 2 in nine patients. Five patients complained of pain in the irradiated            bone and soft tissues, which was of moderate severity (grade 2). Stiffness was graded 2 in            three patients and severe (grade 3) in one.Three patients had moderate and one patient had            severe lymphoedema following treatment. The 5-year recurrence-free survival probability of           patients treated radically was 76%. Following excision of local recurrences the study group           had a disease-free survival probability of 86% at 5 years.           Discussion. The regime is well tolerated with comparable local control           and late complication rates to standard daily fractionated therapy.The potential benefit of this           regime needs to be defined in a prospective randomized trial.


The cyclin kinase inhibitor WAF1/CIP1, also termed CDKN1, mediates p53-induced cell cycle arrest in response to DNA damage. This property makes it an attractive tumour-suppressor candidate for a p53-associated tumour-suppressor gene. In order to investigate the role of WAF1/CIP1 in the pathogenesis of primary human brain tumours we performed single-stranded conformation polymorphism (SSCP) analysis and direct sequencing of exon 2 of the gene in a representative series of 158 brain tumours and corresponding blood samples. In addition, all tumours were examined for mutations in exons 5-8 of the p53 gene. Analysis of WAF1/CIP1 revealed multiple polymorphisms, the most abundant being AGC-->AGA (Ser-->Arg) at codon 31 with an allele frequency of 8.5%. Less common polymorphisms included GTG-->GGG  at codon 25, GCC-->ACC (Ala-->Thr) at codon 64, CGC-->CTC (Arg-->Leu) at codon 32, GGC-->AGC (Gly-->Ser) at codon 14 and GCG-->GTG  at codon 39 each with an allele frequency of 0.3%. These polymorphisms were all located in a conserved region of exon 2. Two of the polymorphisms were also seen in a group of 157 healthy controls indicating that WAF1/CIP1 polymorphisms do not predispose to cancer. None of the tumours included in our series showed a somatic mutation in WAF1/CIP1. All samples were also analysed for loss of heterozygosity on the short arm of chromosome 6 in the region of the WAF1/CIP1 locus. Allelic loss was observed in only one patient with a glioblastoma. Mutations in the p53 gene were found in 22 of 158 tumours. No association was found between any polymorphism of the WAF1/CIP1 gene, p53 mutations and histopathological tumour type. Our data indicate that WAF1/CIP1 mutations are probably not involved in the formation of primary human brain tumours.


To assess the efficacy of ultrasound (US)-guided percutaneous acetic acid  injection for small hepatocellular carcinomas (HCCs) for long-term prognosis, percutaneous acetic acid injection using 15% to 50% acetic acid was performed in 91 patients with one to four HCCs smaller than 3 cm during the past 6.5 years. During the series of treatment sessions for each patient, the same concentration of acetic acid was used. All tumors could be treated successfully with percutaneous acetic acid injection despite the differences in acetic acid concentration used. The number of treatment sessions to treat similar size of tumor was less when the higher concentration of acetic acid was used. No serious complications occurred as a direct sequela to percutaneous acetic acid injection. None of the tumor treated regrew. The 1-, 2-, 3-, 4-, and 5-year survival rates for 91 patients were 95%, 87%, 80%, 63%, and 49%, respectively. The 1-, 2-, 3-, 4-, and 5-year cancer-free survival rates of these patients were 83%, 54%, 50%, 37%, and 29%, respectively. Both liver function and size of tumor affected both survival rate and cancer-free survival rate significantly, but the number of tumors did not. The concentration of acetic acid did not affect the survival rate. Percutaneous acetic acid using 15% to 50% acetic acid will be effective therapy for small HCCs for long-term prognosis.


Helicobacter pyloriinfection and gastric cancer was observed in a short study out of these patients. The peculiargeography and some special dietary habits with a possible familial predisposition may havea bearing on the high risk of gastric cancer in the valley.The present study is a comprehensive retrospective analysis of 1341 gastric neoplasms out of10 733 individuals subjected to upper gastrointestinal endoscopy at the main teaching cumreferral hospital in the Kashmir Valley. Of these 78% were males and 22% females, majoritybeing in the age group of 41–60 years with 60% of the patients being smokers. On endoscopy,the commonest site of cancer was the body of stomach 40.7%, followed by the antrum 35.5%and the cardiac region 23.8%. Endoscopic features revealed nodular masses 39%, polypoidmasses 21%, malignant ulcers 11%, infiltrative masses 12%, rounded tumor masses 9%,linitus plastica 5% and early gastric carcinoma 3%. Histology revealed adenocarcinoma91%, , leiomyosarcoma 7%,and reticulum cell sarcoma 2%. No significant association between


The incidence of B cell leukaemia in 186 consecutive untreated patients with histologically defined B cell neoplasms is described. The lymphomas were classified by the Kiel convention. B cell leukaemia in the context of this paper refers to the situation where a neoplastic clone of B cells in the blood greatly outnumbers normal blood B cells. It is defined as an absolute blood B cell count greater than 0.75 X 10(9)1(-1) where either greater than 90% B cells express kappa immunoglobulin light chains or greater than 80% express lambda light chains. This was found in several patients where the total blood lymphocyte count was within normal limits. All patients with diffuse lymphocytic lymphoma with the histological appearances of B cell chronic lymphocytic leukaemia (ML-BCLL) were found to have B cell leukaemia. However, more than half these patients had blood B cell counts less than 10 X 10(9)1(-1). B cell leukaemia was also a feature in approximately 33% of patients with follicle centre cell tumours and 33% of those with lymphoplasmacytoid tumours. B cell leukaemia was not detected in 34/35 patients with myelomatosis. The 35th patient had plasma cell leukaemia. Only 3/22 patients with high grade lymphoma had B cell leukaemia. In the three principal tumour types associated with B cell leukaemia mu + delta was the most common immunoglobulin heavy chain phenotype. Spontaneous mouse red cell rosette formation also characterised leukaemic B cells in these three groups but high proportions of mouse rosetting cells were seen only in association with ML-BCLL. None of 4 cases of prolymphocytic leukaemia showed mouse red cell rosetting. HLA-DR alpha chain was found on the leukaemic cells of all patients except one with ML-BCLL. B cell lymphopenia was a frequent finding in all histological groups in those patients who did not have B cell leukaemia.


Patients with cardiac disease, chronic pulmonary disease, poor tissue perfusion, or metabolic abnormalities were excluded.Retrospective study of patients admitted to an intermediate care unit (InCU) at a tertiary care center over a 20-month period with moderate to severe respiratory distress secondary to asthma, bronchiolitis, or pneumonia. Patients with venous pCO2-vpCO2 paired values were available from 62 patients. The mean ± SD for EtCO2 and vpCO2 was 35.7 ± 10.1 mmHg and 39.4 ± 10.9 mmHg respectively. EtCO2 and vpCO2 values were highly correlated . The correlations for asthma, bronchiolitis and pneumonia were 0.74 (p < 0.0001), 0.83 (p = 0.0002) and 0.98 (p < 0.0001) respectively. The mean bias ± SD between EtCO2 and vpCO2 was -3.68 ± 4.70 mmHg. The 95% level of agreement ranged from -12.88 to +5.53 mmHg. EtCO2 was found to be more accurate when vpCO2 was 35 mmHg or lower.Eighty EtCO2 is correlated highly with vpCO2 in non-intubated pediatric patients with moderate to severe respiratory distress across respiratory illnesses. Although the level of agreement between the two methods precludes the overall replacement of blood gas evaluation, EtCO2 monitoring remains a useful, continuous, non-invasive measure in the management of non-intubated children with moderate to severe respiratory distress.EtCO This has had far reaching implications in patient care. End-tidal COsettings.,2 In thece 2000. -5 In botce 2000. -8 EtCO2 sedation -13 and isedation -172 monitoring has proven to be efficacious in diverse clinical areas, its utility in non-intubated patients with pulmonary disease remains undefined. In patients with significant pulmonary disease, it is generally believed that EtCO2 values will not accurately reflect blood gas pCO2 because of ventilation-perfusion mismatch, increased dead space, and/or increased shunt fraction. [Though EtCOraction. -21 In faraction. ,22-25 Ho2 monitoring is less accurate in patients with pulmonary disease, there is a paucity of data assessing its utility as a corollary to blood gas pCO2 in non-intubated pediatric patients with moderate to severe respiratory distress. In this study, we investigated the association of EtCO2 to vpCO2 in hospitalized non-intubated children with moderate to severe respiratory distress secondary to asthma, bronchiolitis, or pneumonia. We also examined the level of agreement between vpCO2 and EtCO2 to determine if EtCO2 could replace blood gas evaluation in the management of non-intubated pediatric patients with respiratory distress secondary to a pulmonary process.Because of the general assumption that EtCO2 monitoring is standard of care for all patients admitted to the InCU with respiratory distress. The study was approved by the institutional review board of CHB.We performed a retrospective chart review of pediatric patients admitted with moderate to severe respiratory distress secondary to asthma, bronchiolitis, or pneumonia to the intermediate care unit (InCU) at Children's Hospital Boston (CHB) between July, 2003-February, 2005. The InCU is designed for patients who are moderately to critically ill, who need close monitoring and increased nursing needs, but who do not need invasive monitoring, acute ventilatory support, or vasopressor therapy. Continuous nasal cannula EtCO2 measurement within 10 minutes of each other were eligible for inclusion. Moderate to severe respiratory distress was defined as tachypnea and oxygen saturation < 94% on room air with retractions and decreased aeration on physical examination. Patients with chronic pulmonary disease , cardiac disease, poor tissue perfusion (defined as capillary refill greater than 2 seconds), or underlying metabolic abnormalities were also excluded. Patients undergoing acute respiratory failure defined as immediate subsequent transfer from the InCU to the intensive care unit for invasive respiratory support were also excluded.All patients admitted to the InCU with moderate to severe respiratory distress secondary to above diagnoses and who had a blood gas evaluation and an EtCO2 requirement at time of EtCO2 reading. The patients' pulmonary status including severity of retractions and level of aeration as recorded by InCU nurses on InCU data tracking flowsheet were also included. Retractions and aeration were measured on a scale ranging from 0  to 3 . EtCO2 values were measured using Microstream® Capnoline™ H nasal cannula  attached to Invivo MDE Escort Prism® monitors .Data collected on each patient included patient demographics, vitals measurements, and O2 and vpCO2 values was analyzed using the Pearson product-moment correlation coefficient (r) and simple linear regression. Multivariate linear regression was used to assess for variation in bias between EtCO2 and vpCO2 according to clinical and patient characteristics. Bland-Altman analysis was performed to determine the level of agreement between EtCO2 and vpCO2 values.[2 and vpCO2 to the line of unity between the two measurements to further define the relationship between EtCO2 and vpCO2. Statistical analysis was performed using SAS Version 9.1 .The association between EtCO2 values.,27 We al2-vpCO2 values from 62 patients. 40 of the 80 paired measurements were simultaneous. The mean ± SD time difference between measurements was 0.67 ± 8.19 minutes. The bias between EtCO2-vpCO2 values was not significantly affected by the time difference between measurements (p = 0.6110).There were 80 paired EtCOAge ranged from 5.5 months-20 years with a median age of 5.7 years. Asthma was the admitting diagnosis for 41 patients, bronchiolitis for 9 patients and pneumonia for 12 patients. Other characteristics of our study sample are included in Table SD for EtCO2 and vpCO2 was 35.7 ± 10.1 mmHg and 39.4 ± 10.9 mmHg respectively. EtCO2 and vpCO2 were highly correlated . As shown in Figure The mean ± SD between EtCO2 and vpCO2 values from the Bland-Altman analysis , respiratory rate (p = .9305), oxygen requirement (p = 0.4222), or clinical assessment measurements such as aeration (p = 0.3876) or retractions (p = 0.4381).The mean bias ± 2 values in which accuracy would be maximized in relation to vpCO2. Figure 2 and vpCO2, which deviated significantly from the line of unity  = 35.5, p < 0.001). The regression line and the line of unity were within 3 mmHg of each other for vpCO2 values under 35 mmHg but parted increasingly in the higher range, differing by 5 mmHg above vpCO2 of 47 mmHg. This suggests that maximum EtCO2 accuracy is achieved when vpCO2 is 35 mmHg or below.Further analysis was conducted in order to identify the range of EtCO2 monitoring in the assessment and management of non-intubated pediatric patients with moderate to severe respiratory distress has remained largely undefined. Historically, studies have found EtCO2 monitoring to have limited accuracy in both intubated and non-intubated patients with pulmonary disease.[2 monitoring is mostly useful in following the trend in ventilatory status and not as a specific correlate to blood gas pCO2.[2 can serve as a direct corollary to blood gas pCO2 in patients without pulmonary disease[2 remains an accurate tool to assess blood gas pCO2.The utility of EtCO disease.,22-25 Begas pCO2.,28 Howevy disease, it rais2 to be highly correlated with venous pCO2 and that this relationship was significant across the range of common respiratory illnesses that cause moderate to severe respiratory distress in children. Only one prior study has evaluated the accuracy of non-invasive capnography in non-intubated pediatric patients with respiratory distress.[2 value to capillary pCO2 in pediatrics patients presenting to the emergency department with respiratory emergencies. Their study also found a high correlation between EtCO2 and blood gas pCO2. .In this study, we found EtCOdistress. Abramo e2 was still highly correlated to blood gas pCO2 and EtCO2 monitoring proved useful in the clinical management of the patients beyond just 'following the trend.'Important differences between this prior study and our current study exist. Specifically, there were differences in the severity and underlying causes of respiratory distress in the study populations. 73% of study participants in the prior study were discharged home from the emergency department and the study included patients with signs of upper airway obstruction that may not have had any direct pulmonary involvement. In our study, patients were admitted with moderate to severe respiratory distress secondary to significant lower tract disease. All patients were determined by the admitting physician not to be safe for floor management and therefore admitted to the InCU. Even in this setting, EtCO2 can replace blood gas pCO2 in the management of non-intubated pediatric patients with moderate to severe respiratory distress. This decision should be based on the level of agreement between the two methods.[2 and venous pCO2, correlation is not a measure of agreement but instead is a measure of association. Perfect agreement exists between two methods only when pairs of measurements lie along their line of unity with a slope of 1 and an intercept of 0. Conversely, perfect correlation between two methods exists when pairs of measurements approximate any straight line.[One of the main objectives of our study was to determine if EtCO methods. Though wght line. For examSD between EtCO2 and vpCO2 in our study was -3.68 ± 4.70 mmHg. This bias did not vary across the averaged values of EtCO2 and vpCO2. Abramo et al[2 and EtCO2 of 3.2 ± 2.4 mmHg. Physiologically, there exists a difference between ideally measured EtCO2 concentration and blood gas pCO2, whether arterial or venous. Assuming a difference between arterial and mixed-venous pCO2 of 2–5 mmHg[2 and venous pCO2. Therefore, the observed mean difference of -3.68 closely approximates the true physiologic difference.The Bland-Altman analysis allows for an overall assessment of agreement between two methods. From the Bland-Altman analysis, the bias ± amo et al found a  2–5 mmHg one woul2 and vpCO2 ranged from -12.88 to +5.53 mmHg. This range of EtCO2 values is clinically too imprecise for EtCO2 to replace vpCO2 in the clinical management of patients with respiratory distress. However, knowing the range of EtCO2 values can inform clinical decisions in regards to the management of these patients.The 95% limits of agreement between EtCO2 to accurately reflect the true value of vpCO2, we found on further analysis that the regression line of EtCO2 in Figure 2 and vpCO2 measurements when vpCO2 was below 35 mmHg. At higher vpCO2 values, EtCO2 was not as accurate. Therefore, for values of vpCO2 of 35 mmHg or less, EtCO2 may have an acceptable level of accuracy to serve as indirect measurement of vpCO2 and thereby be a therapeutic guide.In an effort to further define the ability of EtCO2 in assessing the accuracy of EtCO2 to reflect blood gas pCO2 content. Venous pCO2 levels reflect many factors beyond just the ventilatory status of the patient including global oxygen consumption, O2 delivery, tissue perfusion, level of metabolic acidosis, and hypoxia.[2 is not regarded as the gold standard to which EtCO2 should be compared.Our study has several limitations. First, no prior study has used venous pCO hypoxia. Therefor2 in this study was determined mainly by our patient population. Arterial blood gas evaluations in non-intubated pediatric patients are only performed when absolutely necessary secondary to their invasive nature, risk of complication, and high level of pain associated with the procedure.[2 and arterial pCO2 have found a high level of correlation and agreement between the two measures, especially in patients who have good tissue perfusion. [2 values would most accurately reflect the ventilatory status of the patient. Furthermore, by using venous samples for blood gas pCO2 measurement we feel that our study more accurately reflects current practice patterns in the management of pediatrics patients with respiratory distress who are not receiving sedation for invasive ventilatory supportThe use of venous pCOrocedure.,34 In thrfusion. -37 The p2 and blood gas pCO2 were recorded as simultaneous, a number of paired values were not done instantaneously. We limited the time difference between the two measurements to 10 minutes for inclusion in our study. Under ideal circumstances, blood gas evaluations and EtCO2 measurements should be done simultaneously because of the minute to minute fluctuations in CO2 levels. A final limitation of the study was its retrospective nature which may have introduced bias.Another limitation of our study was the timing of measurements. Though most evaluations of EtCO2 and EtCO2. We have also further defined the role of EtCO2 in the management of non-intubated pediatric patients with moderate to severe respiratory distress.Despite these several limitations, we still demonstrated a high correlation and moderate amount of agreement between both vpCO2 as a correlate to vpCO2 in the clinical assessment of pediatric patients with moderate to severe respiratory distress. Though EtCO2 monitoring does not replace blood gas assessment it can serve as an important adjunct in the clinical management of pediatric patients even with significant pulmonary disease. Further study should continue to define the utility of EtCO2 as a measure of blood gas pCO2 in pediatrics patients with moderate to severe respiratory distress.This study demonstrates the utility of EtCO2: End-tidal Carbon Dioxide; vpCO2: Venous Blood Gas Carbon Dioxide; InCU: Intermediate Care Unit; PICU: Pediatric Intensive Care Unit; ED: Emergency Department; CHB: Children's Hospital Boston; SD: Standard Deviation; SE: Standard Error; OR: odds ratio; CI: confidence interval.EtCOThe authors declare that they have no competing interests.JM was the principal investigator, largely responsible for data collection, data analysis, manuscript preparation and revision. JA served the role of research assistant, playing a significant role in data analysis and figure preparation. MA was the primary mentor, specifically involved in study design, data analysis, manuscript preparation and revision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Renal cell carcinomas (RCC) are among the most common and most lethal genitourinary malignancies. GOLPH2  has recently been proposed as a biomarker for hepatocellular and prostate cancer. In this study we analysed the expression patterns and the prognostic and diagnostic value of GOLPH2 in RCC.GOLPH2 protein expression was analysed by immunohistochemistry in 104 clinically well characterized RCC cases in comparison with matched normal kidney tissue and in association with clinico-pathological parameters. Statistical analyses including Kaplan Meier analyses were performed with SPSS version 15.0.GOLPH2 was highly expressed in normal renal tubules and in almost half of RCC with a statistically significant predominance in the papillary and chromophobe histological subtypes. No other associations with clinico-pathological parameters were detectable. The Kaplan-Meier curves showed a weak trend for unfavourable prognosis of tumours with high GOLPH2 expression, but failed significance.GOLPH2 protein is expressed in normal renal tissue  and is down-regulated in the majority of clear cell RCC. In papillary and chromophobe RCC GOLPH2 expression is consistently present. In contrast to its diagnostic value in hepatocellular and prostatic carcinomas, a prognostic or diagnostic value of GOLPH2 in RCC appears to be unlikely. Renal cell cancer (RCC) is one of the most common genitourinary malignancies and causes of cancer associated death in the United States of America in 2008 . AlthougGOLM1 gene on chromosome 9q21.33, first described by Kladney et al. in liver tissue of a patient with giant-cell hepatitis [GOLPH2 is a golgi phosphoprotein  of yet unknown function. The 73 kDa Golgi apparatus associated protein is coded by the Until now only few studies on GOLPH2 exist. In liver diseases GOLPH2 has been described as a potential serum marker of hepatocellular carcinoma . RecentlIn this study, we carefully analysed the GOLPH2 protein expression in a well characterized renal cell cancer cohort with matched normal tissue. Central aim was to evaluate the potential diagnostic and prognostic value of GOLPH2. We found GOLPH2 differentially expressed between normal and malignant renal tissue and between the different RCC subtypes, but a prognostic value could not be detected.th September 2004.One-hundred-four patients  diagnosed for renal cancer at the Institute of Pathology, Charité – Universitätsmedizin Berlin between 2003 and 2005 were enclosed in this study. The study has been approved by the Charité University Ethics Committee under the title "Retrospective Untersuchungen von Gewebeproben mittels immunhistochemischer Färbung und molekularbiologischer Methoden"  on 20Patient age ranged between 28 and 92 years with a median of 62. Histological diagnosis was established according to the guidelines of the World Health Organization. Cases were selected according to tissue availability and were not stratified for any known preoperative or pathological prognostic factor. 83 (79.8%) patients had a clear cell RCC (ccRCC), 16 (15.4) a papillary RCC and 5 (4.8%) a chromophobe RCC. Twenty-one patients had systemic disease (M1) at the time of diagnosis. Clinical follow-up data, as annually assessed survival time was available for all patients. The median follow-up time of all cases was 30 months, ranging from one to 47 months. 21 of the patients died from renal cancer. The pT status was as follows: pT1 – 53 (51.0%), pT2 – 3 (2.9%), pT3 – 45 (43.3) and pT4 – 3 (2.9%). Ten patients (9.6%) had pathologically confirmed nodal metastases . 50 (48.1%) patients had no nodal metastases (pN0). For 44 (42.3%) patients no lymph nodes were histologically examined (pNx). Tumour grades were G1 – 11 (10.6%), G2 – 74 (71.2%), G3 – 15 (14.4%) and G4 – 4 (3.8%) respectively.A tissue-micro-array (TMA) was constructed to represent 108 cases, as previously described . The tisThe TMA blocks were freshly cut (3 μm) and mounted on superfrost slides (Menzel Gläser). Immunohistochemistry was conducted with the Ventana Benchmark automated staining system  using Ventana reagents for the entire procedure. To detect GOLPH2, a commercially available antibody . To achieve a greater uniformity of the evaluation, the first step was to construct a panel with four illustrative examples pictures, of which a hardcopy lay next to the microscope. We used a 10% threshold to determine positivity, irrespective of the intensity grade. Only tumours without any GOLPH2 immunoreactivity or with staining of less than 10% of the tumour cells were considered negative. To delineate between low and high levels of GOLPH2 expression, tumours with moderate to strong GOLPH2 expression (2&3) and tumours with none to weak (0&1) staining intensity were lumped.2-tests were applied to assess the statistical significance of the associations between GOLPH2 expression and clinico-pathological parameters. Univariate survival analysis was carried out according to Kaplan-Meier, differences in survival curves were assessed with the Log rank test. P values < 0.05 were considered significant.Statistical analysis was performed using SPSS, version 15.0. Fisher's exact test, χGOLPH2 immunostaining was easy to evaluate with the typical cytoplasmic and in most cases perinuclear accentuated staining pattern. In normal renal tissue we detected GOLPH2 with its characteristic fine granular staining pattern more often and with a stronger staining intensity in the distal tubules than in proximal tubules Figure . For 97 Staining intensities of GOLPH2 in RCC were: negative – 29 (27.9%), 1+ – 30 (28.8%), 2+ – 34 (31.5%) and 3+ – 11 (10.6%). GOLPH2 positivity was significantly higher in papillary and chromophobe RCC if compared to clear cell RCC , GOLPH2 had higher serum levels if compared to healthy individuals and has been proposed as a novel serum marker of HCC ,15. HoweEven though in a previous study from our study group (manuscript in preparation) we were able to confirm the GOLPH2 expression in HCC, we also detected GOLPH2 in various other malignancies on a multi-tissue-micro-array which argues against a liver-specific relevance of this biomarker.In summary, this first systematic analysis of GOLPH2 in renal cell cancer describes that GOLPH2 expression is not restricted to liver or prostate cancer but is also found in a higher proportion of RCC, although no direct diagnostic or prognostic value of GOLPH2 as a tissue biomarker could be confirmed.In this tissue-micro-array-based immunohistochemistry study a differential expression of GOLPH2 in normal and malignant renal tissue was demonstrated. While distal tubular epithelia were mainly strongly positive for this marker, other parts of the nephron and the majority of the clear cell RCC were negative. The diagnostic use of the rather constant positivity of papillary and chromophobe RCC is limited since one third of the clear cell carcinomas were also positive for GOLPH2. No other associations with conventional clinico-pathological characteristics were found and no prognostic value of GOLPH2 was demonstrated which limits the diagnostic value of GOLPH2 in RCC.The authors declare that they have no competing interests.FRF coordinated the study, performed immunohistological and statistical analyses and wrote the paper. MOR contributed to statistical analyses and revised the paper. MD and KJ provided samples and clinico-pathological data and supported statistical analyses. HM provided logistical support for the study and revised the paper. GK conceived and coordinated the study, performed immunohistological and statistical analyses and revised the paper.The pre-publication history for this paper can be accessed here:


A mouse of monoclonal cell line (L1) was produced by fusing the mouse myeloma P3X63/Ag8 with CD2F1 spleen cells immunized with a highly immunogenic subline of L1210 leukaemia (L1210/DTIC). A very few positive clones (1%) were isolated and one of these was chosen for detailed study. The monoclonal antibody L1 is an IgM immunoglobulin strongly reacting in a complement-dependent cytotoxicity assay against L1210/Cr leukaemia and its more or less immunogenic sublines. The specificity of the L1 antibody against L1210 leukaemia was studied by extensive screening with normal adult and foetal tissues, lymphoid tissues from several independent strains and a panel of the most common experimental tumours, to all of which it was unreactive. Attempts at immunotherapy were carried out in DBA/2 mice challenged with L1210 leukaemia and treated with L1 (ascites) and complement. Although the in vitro cytotoxic titre of ascites fluid from mice bearing hybridoma was very high (10(-7)), no therapeutic effect was obtained in vivo.


P = 0.004 and 0.019 respectively). These results suggest that loss of these regions is associated with recurrence of TCC. Further investigation is required to identify genes in these regions, which might be responsible for driving recurrence in TCC of the urinary bladder. © 2001 Cancer Research Campaign http://www.bjcancer.comApproximately 2/3 of patients diagnosed with superficial transitional cell carcinoma of the urinary bladder (TCC) will recur within 2 years. Loss of chromosome 9 and loss of heterozygosity (LOH) at 9q34 in index TCCs identify a subset of patients at high risk of recurrence. This study explores genetic alterations on chromosomes 4, 8, 11 and 17 as predictors of recurrence. A total of 109 carcinomas were investigated at 26 loci. DNA was extracted from microdissected archival normal/tumour tissue and was analysed for loss of heterozygosity (LOH). Fluorescent PCR was performed and genotyping carried out on a Perkin Elmer ABI377 sequencer. LOH of D11S490 or D17S928 was significantly more frequent in index carcinomas of patients who experienced recurrence compared to those with no recurrence (


RARRES3 is a retinoid-inducible class II tumour-suppressor gene. This study analysed the expression of RARRES3 protein in normal, adenoma and carcinoma tissues of the colorectum and its correlation with tumour differentiation. The expression of RARRES3 protein in 151 paraffin-embedded colorectal tissues  was determined by immunohistochemistry. RARRES3 protein was expressed in all 11 distal normal, 120 adjacent normal and 20 adenoma tissues. In distal normal tissues, RARRES3 protein was expressed at the highest levels in differentiated mucosal epithelial cells. Among 120 carcinoma tissues, RARRES3 protein was detected in 97.6% (40 out of 41), 79.4% (54 out of 68) and 17.3% (three out of 11) of well-, moderately and poorly differentiated tumours, respectively. The expression of RARRES3 protein was positively correlated to tumour differentiation . Also, levels of RARRES3 protein were found to be higher in the normal tissues adjacent to 14.6% (six out of 41), 51.5% (35 out of 68), and 90.1% (10 out of 11) of well-, moderately and poorly differentiated tumours, respectively. The decreases in tumour differentiation and RARRES3 expression were significantly correlated compared to the adjacent normal tissues . The prognostic implication of RARRES3 protein expression was studied in 107 tumour, and no statistical difference in survival was observed. The expression of RARRES3 protein was positively correlated to cellular differentiation of normal and adenocarcinoma tissues of the colorectum, which supports the role of RARRES3 in normal and malignant epithelial differentiation of colorectum. RARRES3 expression was decreased only in carcinoma tissue, which suggests that altered RARRES3 expression occurs late in colorectal carcinogenesis. RARRES3), also named as TIG3  and mutations of DNA mismatch repair genes coincided with the transition to malignancy  and consisted of 100-μg RARRES3 peptide mixed with the incomplete Freund's adjuvant. Titres of the antiserum were determined using an enzyme immunoassay. The specificity of the antiserum was determined by Western blotting of cytosol extracts prepared from cells expressing the RARRES3-fusion protein  were obtained from 68 male and 52 female patients with a mean age of 64.2 years. The distribution of tumours according to the level of differentiation and Dukes' stages are listed in Table 1 tumours . Primary tumours .μg of RARRES3 peptide at 4°C overnight. Samples were spun at 100 000 g at 4°C for 10 min before adding the absorbed antisera to the tissue section. Sections were also stained with p21WAF1 and Ki-67 monoclonal antibodies obtained from DAKO Co.Tissue sections were air-dried, deparaffinised, and then boiled twice for 2 min in 10% DAKO ChemMate™ solution  containing 0.05% NP-40 (Nonidet P-40). The DAKO LSAB®2 Peroxidase kit was used to stain the expression of RARRES3 protein in tissue sections. Tissues were incubated with RARRES3 antiserum or preimmune serum at a dilution of 1 to 800 at room temperature for 1 h. The sections were lightly counterstained with Mayer's haematoxylin. To verify antibody specificity, RARRES3 antisera were preincubated with 8–30 Patterns of staining, cellular RARRES3 localisation, staining intensity and percentage of RARRES3 expressed cells were recorded. The evaluation of staining patterns was performed using the immunoreactive score (IRS) proposed by The nonparametric Kruskal–Wallis tests were applied to compare IRS of RARRES3 associated with tumours at various levels of differentiation as well as Dukes' stages. Further, Dunn's procedure was applied to compare the IRS between groups. Logistic regression analyses were used to assess the association and trend between tumour differentiation and chance of positive RARRES3 staining and chance of adjacent normal tissues having RARRES3 expression higher than that of tumours, while controlling for potential confounding factors, that is, subject's gender and age. Survival rates were calculated using the Kaplan–Meier method. Significance was calculated by the log-rank test. To further validate the effect of RARRES3 staining on survival, a multivariate Cox proportional hazard method was used to adjust for age and stage.μg of RARRES3 peptide resulted in the suppression of staining . The better the tumour tissue differentiation, the higher the IRS was rated. A significant linear trend was found in tumour differentiation and RARRES3 expression (P<0.0001). No significant association was found between RARRES3 IRS and Dukes' stages.Levels of RARRES3 protein varied among 120 tissues of colorectal adenocarcinoma . When RAP for trend <0.0001).We also compared levels of RARRES3 protein between adjacent normal and tumour tissues within the same tissue slide among 120 carcinoma tissues (Table 2P=0.883). Similarly, multivariate analysis showed no difference in survival between patients with negative and weak (P=0.929) or between negative and strong (P=0.292) RARRES3 staining in tumours.Altogether, 107 patients with colorectal adenocarcinoma were examined for prognosis related to RARRES3 expression. A total of 23 tumours were stained negative for RARRES3 protein, 25 tumours had weak RARRES3 expression and 59 tumours had strong RARRES3 expression. Kaplan–Meier survival curves for the different groups are presented in Figure 3RARRES3 is a retinoid-inducible tumour suppressor. This study shows that RARRES3 protein is expressed in mucosal tissues of normal colon and rectum, which is correlated to epithelial differentiation. The premalignant adenoma tissues expressed the RARRES3 protein at high levels. Within tumour tissues, the expression of RARRES3 protein is positively correlated to tumour differentiation. Compared to the adjacent normal mucosal tissues, tumours with moderately and poorly differentiated adenocarcinoma had significantly reduced RARRES3 expression. However, survival analysis showed that RARRES3 protein was not a prognostic marker for patients with colorectal adenocarcinoma.WAF1 . In this study, RARRES3 protein was expressed at high levels in tissues of normal colorectal mucosa and the precancerous lesion adenoma. The decrease in RARRES3 expression was only observed in carcinoma tissues, which are unclassified by the Dukes' system, and also observed in tissues at Dukes' stages A and B. Therefore, the decrease or loss of RARRES3 expression may occur in the early stages of colorectal carcinoma. To our knowledge, deletion at chromosome 11q12, where RARRES3 is localised, has not been reported in tissues of colorectal carcinoma. Also, downregulation instead of mutation of the RARRES3 gene was found during progression of B-cell lymphocytic leukaemia (Genetic alterations have been studied extensively during steps of colorectal carcinogenesis. Mutations in the adenomatous polyposis coli and KRAS oncogenes arose in precancerous lesions. Aberrations in TP53, DCC and SMADs coincide with transition to malignancy (In conclusion, this and previous studies have demonstrated the close association of RARRES3 expression in differentiated epidermis and colorectal epithelium. Progressive loss of RARRES3 expression in tissues of moderately and poorly differentiated colorectal adenocarcinoma supports the tumour-suppressive role of RARRES3 in moderately and poorly differentiated colorectal cancer. However, RARRES3 alone was not a prognostic marker for colorectal adenocarcinoma. RARRES3 protein is shown to suppress the downstream signal pathway of Ras. The significance of the loss of RARRES3 expression along with mutations in Ras will be investigated in the future.


P = 0.009). There was a parallel increase with severity of lesions for the trend in proportions of cases demonstrating p16 inactivation or cyclin D1 overexpression . For Ki67, a marker of cell proliferation, this trend was not significant (P = 0.08). Human papillomavirus infection was only found in 4 cases among the 48 samples tested (8.3%). In conclusion, progression of disease is accompanied by a parallel and continuous increase in telomerase activity and alterations in cell cycle regulators , as proposed by in vitro models. © 2001 Cancer Research Campaign http://www.bjcancer.comAlteration of the p16/pRb pathway may cooperate with telomerase activation during cellular immortalization and tumour progression. We studied p16 expression status by immunohistochemistry and telomerase activity using the TRAP assay in 21 premalignant lesions of the head and neck epithelium as well as 27 squamous-cell carcinomas. We also examined expression of other components of the pathway (cyclin D1 and pRb) as well as presence of human papillomavirus genomes which can target these molecules. 4 of 9 mild dysplastic lesions (44%), 8 of 12 moderate/severe dysplastic lesions (67%), and 25 of 27 squamous-cell carcinomas (92%) demonstrated high telomerase activity (


Intracardiac injection, in hooded Lister rats, of syngeneic MC28 sarcoma cells never induced tumour growth in normal bowel. Tumour growth occurred at the site of a colonic anastomosis if surgery preceded tumour injection but not if it followed tumour injection, even by as little as 1 h. Maximum enhancement of tumour growth occurred when the healing process had progressed between 2 and 8 days, with a peak at 5 to 7 days. The enhancing effect was largely over by the time the healing had progressed 14 days. The syngeneic OES5 breast carcinoma also grew at colonic anastomoses when surgery preceded tumour injection by 5 days, but not in normal colon. The MC28 sarcoma also grew at ileal anastomoses but not in the normal ileum after intracardiac injection. By injecting radiolabelled sarcoma cells, an estimate of the probability of a single bloodborne tumour cell lodging at a colonic anastomosis and leading to a tumour deposit was calculated to be of the order of 1:43 whereas the probability of the cell lodging in normal colon and causing a deposit is less than 1:4 x 10(4).