Alzheimer's disease (AD) and its transitional state mild cognitive impairment (MCI) are characterized by amyloid plaque and tau neurofibrillary tangle (NFT) deposition within the cerebral neocortex and neuronal loss within the hippocampal formation. However, the precise relationship between pathologic changes in neocortical regions and hippocampal atrophy is largely unknown.In this study, combining structural MRI scans and automated image analysis tools with reduced cerebrospinal fluid (CSF) Aß levels, a surrogate for intra-cranial amyloid plaques and elevated CSF phosphorylated tau (p-tau) levels, a surrogate for neocortical NFTs, we examined the relationship between the presence of Alzheimer's pathology, gray matter thickness of select neocortical regions, and hippocampal volume in cognitively normal older participants and individuals with MCI and AD (n = 724). Amongst all 3 groups, only select heteromodal cortical regions significantly correlated with hippocampal volume. Amongst MCI and AD individuals, gray matter thickness of the entorhinal cortex and inferior temporal gyrus significantly predicted longitudinal hippocampal volume loss in both amyloid positive and p-tau positive individuals. Amongst cognitively normal older adults, thinning only within the medial portion of the orbital frontal cortex significantly differentiated amyloid positive from amyloid negative individuals whereas thinning only within the entorhinal cortex significantly discriminated p-tau positive from p-tau negative individuals.Cortical Aβ and tau pathology affects gray matter thinning within select neocortical regions and potentially contributes to downstream hippocampal degeneration. Neocortical Alzheimer's pathology is evident even amongst older asymptomatic individuals suggesting the existence of a preclinical phase of dementia. Selective neurodegeneration of the cerebral cortex is a characteristic pathological feature of Alzheimer's disease (AD). Intracellular tau-associated neurofibrillary tangles (NFTs) and extracellular amyloid-ß (Aß) associated plaques show a characteristic laminar and regional pattern of distribution within gray matter, with greater involvement of the medial temporal lobe and heteromodal association areas than primary sensory or motor cortices in vivo assessment of neuropathologic changes underlying AD. Positron emission tomography (PET) studies examining fibrillar amyloid deposition with Pittsburgh Compound B (PiB) and MRI studies of functional and structural connectivity have observed a significant overlap in a number of neocortical regions that appear preferentially affected in the earliest stages of AD in vivo the effect of tau burden on atrophy in heteromodal and limbic cortices.Recent advances in neuroimaging and image analysis algorithms allow for the Cortical atrophy resulting from cellular shrinkage, dendritic spine loss, and axonal disruption is reflected as a loss of gray matter that diminishes cortical thickness www.loni.ucla.edu/ADNI). The ADNI is a large multi-site collaborative effort launched in 2003 by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, the Food and Drug Administration, private pharmaceutical companies and non-profit organizations as a public–private partnership aimed at testing whether serial MRI, PET, other biological markers and clinical and neuropsychological assessment can be combined to measure the progression of MCI and early AD. The Principal Investigator of this initiative is Michael Weiner, MD, and ADNI is the result of many co-investigators from a broad range of academic institutions and private corporations, with subjects recruited from over 50 sites across the US and Canada. For more information, please see http://www.adni-info.org.All 724 participants were selected from the Alzheimer's disease Neuroimaging Initiative (ADNI) database (http://www.adni-info.org/index.php?option=com_content&task=view&id=9&Itemid=43). The institutional review boards of all participating institutions approved the procedures for this study. Written informed consent was obtained from all participants or surrogates. Briefly, experienced clinicians conducted independent semi-structured interviews with the participant and a knowledgeable collateral source that included a health history, neurological examination, the Mini-Mental State Examination Each participant from the ADNI cohort was formally evaluated using eligibility criteria that are described in detail elsewhere (As illustrated in CN (n = 208) - Individuals who were cognitively normal at baseline and clinical follow-up (CDR 0).MCI (n = 353) – Individuals with mild cognitive impairment (MCI) defined using the revised MCI criteria AD (n = 163) – Individuals who met criteria for probable AD http://www.loni.ucla.edu/ADNI/Data/index.shtml). Parameter values vary depending on scanning site and can be found at http://www.loni.ucla.edu/ADNI/Research/Cores/.All ADNI MRI scans were acquired at multiple sites using either a GE, Siemens, or Philips 1.5T system. Multiple high-resolution T1- weighted volumetric MP-RAGE scans were collected for each subject and the raw DICOM images were downloaded from the public ADNI site , freely available at The neocortex of the brain on the MRI scans was then automatically subdivided into gyral-based regions of interest (ROIs). To accomplish this, a registration procedure was used that aligns the cortical folding patterns For longitudinal analysis, the baseline and one-year follow-up structural MRI scans were rigid body registered and an unbiased template volume was created From the current ADNI sample, a number of individuals (n = 338) underwent lumbar puncture for CSF biomarker evaluation. Methods for CSF acquisition and biomarker measurement have been reported previously for this sample Spearman's rank correlation coefficients were first used to examine relationships between the mean thickness of the individual ROIs and total hippocampal volume. Separate linear regression models were constructed for the amyloid positive, amyloid negative, p-tau positive and p-tau negative MCI and AD individuals, with the baseline thickness of the individual ROIs as predictors for hippocampal atrophy rate. Logistic regression models and multivariate analysis of variance (MANOVA) were used to compare whether baseline thickness of the individual ROIs significantly differentiated amyloid positive from amyloid negative as well as p-tau positive from p-tau negative CN individuals. In all of the analyses performed, age, gender, education, and APOE-ε4 carrier status were additionally included.Overall, the hippocampus demonstrated the largest magnitude of correlation with the entorhinal cortex , temporal pole , inferior temporal gyrus  and the medial portion of the orbital frontal cortex  . The infLinear regression models amongst the MCI and AD individuals demonstrated that decreased Aβ levels  = −0.01 to −0.003, p<0.01) and increased p-tau levels  significantly predicted hippocampal atrophy rate. Within the amyloid positive MCI and AD individuals, baseline thickness of the entorhinal cortex  and inferior temporal gyrus  significantly predicted the atrophy rate of the hippocampus. Similarly, within the p-tau positive individuals, baseline thickness of the entorhinal cortex  and inferior temporal gyrus  significantly predicted the atrophy rate of the hippocampus . Within Logistic regression models amongst the CN cohort demonstrated that APOE-ε4 carrier status (Odds Ratio (OR) = 0.08, 95% CI = 0.02 to 0.27, p<0.0001) and thinning in the medial portion of the orbital frontal cortex  significantly differentiated the amyloid positive from amyloid negative older individuals whereas thinning of the entorhinal cortex  significantly differentiated the p-tau positive from p-tau negative older individuals . None ofOur results demonstrate that 1) amongst cognitively normal older participants and individuals with MCI and AD, select heteromodal association cortices correlate with hippocampal volume, 2) amongst MCI and AD individuals, gray matter thinning within the same two neocortical regions predicts longitudinal hippocampal volume loss in both amyloid positive and p-tau positive individuals and 3) amongst cognitively normal older adults, thinning within the medial portion of the orbital frontal cortex significantly differentiates amyloid positive from amyloid negative individuals whereas entorhinal cortex thinning significantly discriminates p-tau positive from p-tau negative individuals. Taken collectively, this indicates that cortical Aβ and tau pathology is evident even among asymptomatic older individuals, affects gray matter thinning within select neocortical regions and potentially contributes to downstream hippocampal degeneration. These findings are in agreement with prior work indicating the value of CSF Aβ as an early biomarker The four cerebral cortical regions that best correlated with hippocampal volume follow an interesting neurodevelopmental pattern. On architectonic grounds, the regions identified on the lateral hemisphere trace evolutionary lineage from the so-called ‘paleocortical’ trend whereas regions identified on the medial hemisphere are derived from the ‘archicortical’ trend Correlations between thickness of the individual regions and hippocampal volume may reflect underlying patterns of neuropathology. Thickness of the medial portion of the orbital frontal cortex, inferior temporal gyrus, entorhinal cortex, and the temporal pole demonstrated the strongest magnitude of association with the hippocampus amongst each of the three participant groups. In contrast, parietal, occipital, and lateral frontal cortices, correlated with the hippocampus only amongst cognitively normal older adults and MCI individuals. With AD onset, these regions failed to associate with the hippocampus. Of interest, these regions showed the largest magnitude of association with each other amongst each of the three participant groups , 4 and 5One explanation for these findings may involve the evolution of the neuropathologic cascade in AD. Amyloid deposits are first noted in the medial portions of the orbital frontal, basal temporal and entorhinal cortices and tau-associated NFTs initially affect the medial temporal and temporopolar cortices Another possibility is that associations between regions reflect underlying anatomic connections and the decreased correlation coefficients noted with AD onset represent a selective disruption in cortico-hippocampal connectivity. Though several studies have noted disruptions in anatomic connectivity with AD Significant reductions in CSF Aβ levels and elevations in CSF p-tau levels coupled with thinning within select heteromodal regions predicts longitudinal hippocampal atrophy. Prior PiB studies have demonstrated a significant relationship between neocortical amyloid deposition and hippocampal atrophy in vivo evidence that amongst cognitively normal individuals tau pathology selectively affects the thickness of the entorhinal cortex. It is important to note that almost half (47%) of the p-tau positive individuals were additionally amyloid positive and future work will involve looking at the interaction between Aβ and tau pathology.Amongst cognitively normal older adults, cortical thinning of the medial orbital frontal cortex significantly differentiated amyloid positive from amyloid negative individuals whereas thinning of the entorhinal cortex significantly discriminated p-tau positive from p-tau negative individuals. Consistent with neuropathology studies, which demonstrate early amyloid deposition within the medial orbital frontal cortex and early NFT accumulation within the entorhinal cortex The finding that amyloid positive individuals show selective thinning within the medial orbital frontal cortex is in agreement with one prior study demonstrating that older individuals with subjective memory complaints show significant correlations between gray matter atrophy and neocortical PiB increase within the medial orbital frontal/anterior cingulate cortex These findings have important clinical implications. Subtle gray matter disturbances in specific regions of the cerebral neocortex are present amongst a subset of asymptomatic older individuals and can be quantified using sensitive structural neuroimaging techniques. In combination with CSF Aβ and tau, structural MRI measures can provide an indication of disease stage and have value in identifying those who will likely benefit from early therapeutic interventions. Our results also indicate the importance of administering anti-amyloid therapy early in the disease process (at a pre-MCI stage) in order to alter or delay hippocampal degeneration and thus progressive memory loss, the harbinger of clinical Alzheimer's dementia.Why does neurodegeneration affect select cerebral cortical regions? From a phylogenetic perspective, heteromodal and archicortical medial temporal regions constitute the oldest areas of the brain a priori anatomically defined regions with disease-based statistical methods to examine neocortical and subcortical areas in AD. Another limitation involves the use of CSF Aβ and p-tau to assess the presence of amyloid and neurofibrillary pathology in the neocortex. Though amyloid load as measured using PiB and CSF Aβ are highly correlated and likely reflect plaque deposition This study has several limitations. One issue involves the use of gyral-based neuroanatomic ROIs to examine specific neocortical regions. It is likely the case that Alzheimer's pathology does not follow the specific boundaries of these ROIs and affects areas within and across multiple ROIs. The use of regions of interest generated from a disease specific effect


Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter.in vitro it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods.By exploiting a new method of producing the retroviral genome in vitro transcription of the retroviral genome by T7 RNA polymerase.We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow in vitro produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression.When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the in vitro transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the in vitro transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with  Retroviral vector systems are routinely used as delivery vehicles for efficient and stable gene transfer into mammalian cells . Since tin vitro generated RNA to nucleate retroviral virions, completely circumventing packaging cell genomic transcription and translation   promoter . This ex) Figure . pT is sWhen transfected into packaging cells, the standard retroviral vectors (pC) produced high levels of GFP expression as measured by flow cytometry Figure . Howeverin vitro transcription system based vectors (pT) carrying the same CMV expression cassette readily generated infectious virus following transfection of in vitro transcribed RNA genomes into packaging cells. The pT vectors produced no detectable GFP expression in the packaging cells, due to the lack of requisite translation signals (e.g. 5' cap) on the RNA transcripts, a notable advantage of the system . Primarin vitro-transcribed retroviral RNA genomes can be utilized to transfer a high-level expression cassette which includes genetic elements not compatible with commonly used retroviral packaging systems. Using this cassette we observe an increase in expression level of up to 3.5 times in primary human cells, reflecting the provision of an intron and the use of strong enhancer/promoter and polyadenylation signals. The ability to circumvent packaging cell transcription, post-transcriptional editing and translation of the vector genome, while maintaining assembly into virions, effectively addresses the shortcomings of current retroviral systems. Thus, this approach is based on standard molecular biology techniques, yet allows virtually any genetic sequence constellation to be manifested by the integrated retroviral provirus: Strong enhancers/promoters, introns and polyadenylation signals for boosting gene expression; gene reporters that encompass multiple exon-introns; post-transcriptional signal sequences to improve gene trapping; cytotoxic gene expression or shRNA expression cassettes that target essential genes.In this report we demonstrate how Vectors pT-cGreen/neerGc L311/L312) were based on vector L139 pT7RU5mcsSIN, a derivative of LB-G  were bas. A T7 poTo generate the uncapped retroviral genomic transcripts, MluI-linearized pT-vector DNA was transcribed with T7 polymerase . RNA was quantified  and the quality checked by denaturing agarose gel electrophoresis   were transfected into 293-based Phoenix A packaging cells by the standard protocol ,15 whichin vitro transcribed genomic RNA was introduced into the packaging cells using the cationic lipid DMRIE-C . The RNA was mixed with lipid at a ratio of 1 μg RNA to 3.5 μl DMRIE-C in a final volume of 250 μl of serum-free OptiMEM  and incubated at room temperature for 20 minutes. Subconfluent (80%) Phoenix-A packaging cells in poly-lysine coated 12-well plates were prepared by rinsing with serum-free medium and 360 μl OptiMEM was added per well. The RNA-lipid mixture was added dropwise to the cells and incubated for 2 hours before the cells were washed and incubated in complete medium. pT virus-containing medium (1 ml) was harvested 18 hours later. Titers were typically approximately 1 × 105 gfu/ml for infection of primary human endothelial cells [The al cells .Primary human umbilical vein endothelial cells  were cultured according to the supplier's recommendations. Cells were infected as described  . Viral sAll authors participated in the overall study design and writing of the manuscript. MB/DM conducted the experiments in the laboratory of JBL. All authors have read and approved the final manuscript.


Fifty-three premenopausal patients presenting with advanced breast cancer have been treated with a potent new luteinising hormone-releasing hormone agonist Zoladex (ICI 118630) in a phase I clinical trial. On progression of disease 26 patients have undergone therapeutic oophorectomy. We present the clinical and endocrinological responses to treatment in 45 assessable patients. The response rate to Zoladex in this series was 31% and the ER status of the primary tumour was predictive of a response to the luteinising hormone-releasing hormone.


Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments showed that it had strong anti-inflammatory effects. Tumour necrosis factor (TNF) is an important inflammatory mediator. In order to study the mechanism of the anti-inflammatory effect of EsA, it was determined whether TNF production from macrophages was altered by EsA under lipopolysaccharide (LPS) stimulated conditions. EsA was found to decrease both extracellular and cell associated TNF production in a dose dependent manner at concentrations higher than 1 μmol/l EsA. Previous studies have showed that EsA reduced the releasing of platelet activating factor (PAF) from rat macrophages. The reducing effects of EsA on the release of TNF and PAF may explain its anti-inflammatory effect.


A technique for growing colonies from single-cell suspensions of human tumour xenografts using agar in diffusion chambers is described. Modified Millipore diffusion chambers containing tumour cells in semi-solid agar-medium were implanted into the peritoneal cavity of pre-irradiated mice and provided standard culture conditions for the study of colony-forming cells. All 11 xenograft tumours so far studied produced colonies. The incubation period for colony growth ranged from 12 to 28 days and the plating efficiency ranged from 0-3% to 16% for different tumours, but both parameters were constant for each individual tumour. The reproducibility of the system provides a colony-forming assay which can be used to study the effects of irradiation and cytotoxic drugs on human tumour clonogenic cells and may therefore have some advantages over similar assays based on experimental animal tumours.


Sister chromatid exchange (SCE) induction by two radiosensitizing platinum compounds (cis-dichloro-bis isopropylamine trans dihydroxy platinum IV (CHIP) and cis-platinum metronidazole2 Cl2 (FLAP] was studied in CHO cells in vitro. Both drugs induced SCE in a dose dependent manner. CHIP was a much more potent inducer of SCE than FLAP and produced almost 4 times as many SCE as FLAP at equimolar concentrations and twice as many at equitoxic dosage. Induction of SCE by a component of the FLAP molecule--metronidazole--was also examined. It did not cause any increase of SCE frequency over the control level when applied at 10 times the highest concentration of FLAP which was used.


Bovine herpesvirus 1. We used a bioinformatics approach to distinguish authentic mature miRNA sequences from other classes of small RNAs and short RNA fragments represented in the sequencing data. Using this approach we detected 219 out of 356 known bovine miRNAs and 115 respective miRNA* sequences. In addition we identified five new bovine orthologs of known mammalian miRNAs and discovered 268 new cow miRNAs many of which are not identifiable in other mammalian genomes and thus might be specific to the ruminant lineage. In addition we found seven new bovine mirtron candidates. We also discovered 10 small nucleolar RNA (snoRNA) loci that give rise to small RNA with possible miRNA-like function. Results presented in this study extend our knowledge of the biology and evolution of small regulatory RNAs in mammals and illuminate mechanisms of small RNA biogenesis and function. New miRNA sequences and the original sequencing data have been submitted to miRNA repository (miRBase) and NCBI GEO archive respectively. We envisage that these resources will facilitate functional annotation of the bovine genome and promote further functional and comparative genomics studies of small regulatory RNA in mammals.MicroRNA (miRNA) and other types of small regulatory RNAs play a crucial role in the regulation of gene expression in eukaryotes. Several distinct classes of small regulatory RNAs have been discovered in recent years. To extend the repertoire of small RNAs characterized in mammals and to examine relationship between host miRNA expression and viral infection we used Illumina's ultrahigh throughput sequencing approach. We sequenced three small RNA libraries prepared from cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with  MicroRNAs (miRNAs) are small 21–23 nucleotide regulatory RNAs that modulate gene expression in animals and plants. In animals regulation of gene expression by miRNAs is achieved by sequence-specific targeting of the 3′ untranslated regions of messenger RNAs by the RNA induced silencing complex (RISC). This results in translational repression of protein synthesis and, in some cases, destabilization of messenger RNA Identification of comprehensive sets of miRNAs and other small regulatory RNAs in different organisms is a critical step to facilitate our understanding of genome organization, genome biology and evolution. The recently completed bovine genome is the first sequenced genome of ruminant mammal that displays a broad range of phenotypic characteristics that reflect adaptation to different habitats and domestication. The large number of cattle breeds selected for their commercially valuable traits (e.g. meat and milk production) and their ability to thrive in different environments make the bovine genome an attractive model to study genetic and epigenetics variations underlying diverse cattle phenotypes The latest release of the miRNA database (miRBase 13.0 March 2009) contains 356 bovine miRNA genes that code for 326 distinct mature miRNAs and eight sequences originating from the RNA hairpin arm opposite to the annotated mature miRNA containing arm, the so-called miRNA* Bovine herpesvirus 1 (BoHV-1). Illumina's ultrahigh throughput sequencing approach has been used successfully to identify new miRNAs and other classes of small regulatory RNAs in several recent studies We aimed to extend the known repertoire of small regulatory RNAs expressed in bovine tissues and identify miRNA that could be disregulated upon viral infection. We used a cell line derived from the adult bovine kidney under normal conditions and upon infection of the cell line with the To simplify the sequencing data, all identical sequence reads in each small RNA library were grouped and converted into sequence tags–unique sequences with associated counts of the individual sequence reads. The resulting sets of non-redundant sequence tags for each library were mapped to the bovine and BoHV-1 reference genomes Following mapping of sequence tags to the reference bovine genome we analyzed small RNA sequences originating from known bovine miRNA loci listed in miRBase 13.0. We found that out of 356 known cow miRNAs 338 could be positioned onto assembled chromosomes; out of those 219 miRNA were detected in at least one of our three small RNA libraries, 190 amongst those were detected in either two or all three small RNA libraries  genomic loci annotated as known bovine miRNAs or as other classes of non-coding RNA were excluded; (2) to be considered for further analysis an individual locus had to be supported by at least two independent sequence reads originating from at least two small RNA libraries; (3) the loci lacking hairpin-like RNA secondary structures including the positions of the small RNA tags were eliminated. The resulting set of sequences and their respective RNA structures were analyzed further to distinguish genuine miRNA precursors from other RNAs that contain similar RNA structures , worms (Caenorhabditis elegans), and mammals (primates and rodents) de-novo identification of mirtrons we used methodology described earlier TPRG1L and DDX5 genes respectively and were expressed in all three small RNA libraries as evidenced by presence of sequence tags  is a class of evolutionary conserved non-coding RNAs present throughout the Eukaryotes Although analysis of snoRNA loci in the bovine genome is complicated by the current lack of comprehensive annotation of non-coding RNAs we analyzed bovine snoRNA loci that could be identified using human snoRNA annotations and syntenic alignment tool from UCSC genome browser . We idenThe source of bovine RNA, RNA sample preparation, and construction of small RNA sequencing libraries is described in supplemental methods . In brieApproximately 20 µg of total RNA isolated from MDBK cells either infected (MOI of 0.75 and 7.5) or mock inoculated with BoHV-1 was supplied to GeneWorks Pty Ltd  for construction of small RNA libraries and ultrahigh throughput sequencing. Sequencing was performed on the Illumina Genome Analyzer G1 according to the manufacturer's protocol.Bos Taurus v. 3 genome sequence assembly and annotations  http://www.ncbi.nlm.nih.gov/). To identify novel miRNA genes we identified all hairpin-like RNA structures encompassing small RNA sequence tags using RNAfold Individual sequence reads with the base quality scores were produced by GeneWorks Pty Ltd using Illimina's Data Analysis Pipeline software v.1.0. Subsequent sequence data analyses were carried out as described by Glazov et al. with some modification To identify the evolutionary conserved orthologs of the bovine miRNAs in other mammalian genomes we used bovine pre-miRNA sequences to search for highly similar sequences in human, mouse, dog, horse, and opossum genomes. The search was performed using BLAT Kent 2002 Known mammalian miRNA sequences were obtained from microRNA database at the Sanger center (miRBase 13.0 March 2009) http://microrna.sanger.ac.uk/registry/). Original sequence data generated in this study have been deposited to the NCBI Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE15450.All sequences identified as new miRNA precursors and the mature miRNA were submitted to the miRBase at the Sanger Centre (Table S1Summary of the expression data for known detected cow miRNA(0.16 MB XLS)Click here for additional data file.Table S2Sequence and genomic coordinates information for new miRNA* identified in this study.(0.03 MB XLS)Click here for additional data file.Table S3Summary of the expression data for newly detected cow miRNA(0.06 MB XLS)Click here for additional data file.Table S4Sequence and genomic coordinates information for new miRNA precursors(0.03 MB XLS)Click here for additional data file.Table S5Sequence and genomic coordinates information for new mature miRNA and miRNA*(0.03 MB XLS)Click here for additional data file.Figure S1Summary of phylogenetic relationships of the mammalian species discussed in this study.(0.06 MB PDF)Click here for additional data file.Figure S2Known miRNA with unusual small RNA processing pattern.(0.10 MB PDF)Click here for additional data file.Figure S3Bidirectional miRNA candidates.(0.10 MB PDF)Click here for additional data file.Figure S4Mirtrons(0.09 MB PDF)Click here for additional data file.Figure S5snoRNA-derived small RNAs(0.13 MB PDF)Click here for additional data file.Figure S6Bovine miRNA bta-mir-219(0.06 MB PDF)Click here for additional data file.File S1Supplemental methods.(0.03 MB PDF)Click here for additional data file.


Bombesin-like peptides and their receptors are widely distributed throughout the gut and are potential mitogens for a number of gastrointestinal (GI) cancers. We have analysed the expression of bombesin-like peptides and their receptor subtypes in normal and neoplastic colorectal tissue. Expression was analysed by reverse transcription polymerase chain reaction (RT-PCR) using receptor and ligand subtype-specific primers and then expression localized by in situ hybridization (ISH) with riboprobes synthesized by in vitro transcription of cloned PCR products. Colorectal cancer tissue and matched normal mucosa from 23 patients were studied. Two of these patients had synchronous adenomatous polyps and two had synchronous hepatic metastases which were also studied. An additional two patients with adenomatous polyps were studied along with matched normal mucosa. Gastrin releasing peptide (GRP) receptor and ligand expression was present in all samples but with overall greater expression in the tumour samples. Neuromedin B (NMB) receptor expression was not detectable. NMB ligand was detected in all but one mucosal sample with overall overexpression in the tumour samples. Bombesin receptor subtype 3 (BRS-3) receptor expression was not detectable. These data support the possibility that GRP may be an autocrine growth factor in colorectal cancer. © 2000 Cancer Research Campaign


Treatment of ruptured Achilles tendons currently constitutes of conservative early functional treatment or surgical treatment either by open or minimal invasive techniques. We hypothesize that an experimental Achilles tendon suture in an animal model significantly deteriorates Achilles tendon microcirculation immediately following suturing.Fifteen Achilles tendons of eight male Wistar rats (275–325 g) were included. After preparation of the Achilles tendon with a medial paratendinous approach, Achilles tendon microcirculation was assessed using combined Laser-Doppler and spectrophotometry (Oxygen-to-see) regarding:- tendinous capillary blood flow [arbitrary units AU]- tendinous tissue oxygen saturation [%]- tendinous venous filling pressure [rAU]The main body of the Achilles tendon was measured in the center of the suture with 50 Hz. 10 minutes after Achilles tendon suture (6-0 Prolene), a second assessment of microcirculatory parameters was performed.Achilles tendon capillary blood flow decreased by 57% following the suture . Tendinous tissue oxygen saturation remained at the same level before and after suture . Tendinous venous filling pressure increased by 33%  after suture.Achilles tendon suture in anaesthetised rats causes an acute loss of capillary perfusion and increases postcapillary venous filling pressures indicating venous stasis. The primary hypothesis of this study was confirmed. In contrast, tendinous tissue oxygen saturation remains unchanged excluding acute intratendinous hypoxia within the first 10 minutes after suture. Further changes of oxygen saturation remain unclear. Furthermore, it remains to be determined to what extent reduced capillary blood flow as well as increased postcapillary stasis might influence tendon healing from a microcirculatory point of view in this animal setting. Achilles tendon ruptures are currently treated either conservatively with an early functional rehabilitation programme or surgically with varying suture techniques. Besides the conventional open approach with its known inherited potential adverse effects such as infections and extensive scaring minimal-invasive percutaneous techniques are becoming popular. Current scientific studies adress biomechanical properties of different suture techniques for tendon ruptures. -3 FurtheAs tendon healing is often prolonged and associated with a less than optimal outcome, current studies aim at ameliorating the conditions for tendon healing. ,7 HoweveWe therefore designed a preliminary animal study to investigate our hypothesis that an Achilles tendon suture significantly deteriorates Achilles tendon microcirculation immediately following the tendon suture.Experiments were approved by the local animal right protection authorities and performed in accordance to the NIH guidelines for the Care and Use of Laboratory Animals .The study included 8 male Wistar rats (250–350 g body weight (bw); Charles River Laboratories, Sulzfeld, Germany), housed 1/cage at 21°C with a natural light/dark cycle as well as water and standard laboratory chow ad libitum. Under pentobarbital sodium anesthesia , the rats were tracheotomized and mechanically ventilated . Furthermore, animals' right carotid artery was cannulated with polyethylene catheters . The cannulation allowed heart rate and blood pressure to be monitored. During the procedure, body temperature was maintained at 36–37°C by means of a heating pad. Systemic blood parameters and blood gas analysis were assessed before starting measurements using a Coulter Counter .Fifteen gastrocnemius and soleus muscles as well as Achilles tendons were surgically exposed by a medial approach down to the calcaneal insertion. Only 15 Achilles tendons of 8 Wistar rats could be used because of carotid cannulation dislocation with consecutive fatal bleeding and exitus letalis after measurement of a single tendon in one case.Tissue was allowed to stabilize for 10 minutes before investigating the tendinous microcirculation using a non-invasive combined Laser-Doppler- and Photospectrometry-system . Each tendon was measured 10 minutes after preparation of the Achilles tendon as well as ten minutes after tendon suture at the same location. Measurements at baseline and 10 minutes after tendon suture always started at the right Achilles tendon. 15 minutes after initiation of measurement of the right Achilles tendon of one rat, the contralateral Achilles tendon was measured at baseline.We used a fixation apparatus for the probe in order to minimize measurement artefacts due to vibration figure . BaselinThe determination of hemoglobin and the principle of blood flow measurement are combined in the O2C system. The optical method for measuring both, blood flow by Laser-Doppler technique and hemoglobin oxygenation and hemoglobin concentration in tissue by spectrometric techniques, has been described in detail elsewhere.  The loca• tendinous capillary blood flow • tendinous tissue oxygen saturation [%]• tendinous venous filling pressure [AU]We have recently described the use of the O2C-system in measurements of increased capillary blood flow at the point of pain in patients with insertional and mid-portion tendinopathy of the Achilles tendon compared to healthy subjects as well as the influence of permanent and intermittent application of cooling and compression on microcirculation of Achilles tendons in healthy humans. ,9 A 5% iIndependent samples t-test was applied for comparison of baseline measurement of right vs. left Achilles tendon. Paired t-test was used for comparison of pre- vs. post-interventional microcirculatory changes. A p-value less than 0.05 was considered to indicate statistical significance. The SPSS statistical software package 16.0 for Windows  was used for statistical analysis.Achilles tendon mid-portion capillary blood flow was 70 ± 30 arbitrary units at rest without tendon suture. There were no significant differences in capillary blood flow between right and left Achilles tendon at baseline . 10 minutes after suturing the tendon's mid portion, capillary blood flow significantly decreased to 31 ± 16 arbitrary units by 57% . Oxygen saturation did not significantly change within ten minutes following the suture of the Achilles tendon and remained at 77 ± 22% . The postcapillary filling pressure significantly increased ten minutes after the Achilles tendon suture to 72 ± 20 AU  may exert positive effects on tissue healing, a process highly dependent upon adequate circulation. IPC is a treatment based on the passive increase of blood flow by cycling external pressure. The biomechanical effects include reduced venous stasis, decreased venous pressure and increased arterial blood flow.  IPC is uFurthermore, in vivo transient limb ischemia releases a low molecular weight, hydrophobic, circulating factor which induce a potent protection against myocardial ischemia/reperfusion injury in Langendorff perfused hearts and isolated cardiomyocytes in the same species. This cardioprotection is transferable across species, independent of local neurogenic activity, and requires opioid receptor activation. A commonAchilles tendon suture in anaesthetised rats causes an acute loss of capillary perfusion and increases postcapillary venous filling pressures indicating venous stasis. In contrast, tendinous tissue oxygen saturation remains unchanged excluding acute intratendinous hypoxia within 10 minutes after suture. Reduced capillary blood flow as well as increased postcapillary stasis might influence tendon healing from a microcirculatory point of view at least in this animal experiment. Nevertheless, the consequences of our findings remain unclear and must be an issue of further studies.Ethical Board Review statement: Experiments were approved by the local animal right protection authorities and performed in accordance to the NIH guidelines for the Care and Use of Laboratory Animals .The authors declare that they have no competing interests.RK carried out the data assessment. RR performed preparation of the tendons and supplied materials. RK, JL and KK drafted the manuscript. RK and KK participated in the design of the study. RK and JL performed the statistical analysis. PMV conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


However, repeat dearterializations for 60 min in portal deviatedrats induced irreversible liver damage and all rats died in a few days. Repeated dearterializationsfor 60 minutes alone retarded the tumour growth as efficiently (p = 0.007). Simultaneousocclusion of the hepatic artery and the portal vein for 30 minutes with a side-side porto-cavalshunted  did not affect tumour growth (p = 0.154). Liver aminotransferases(ASAT and ALAT) were substantially increased following dearterialization for 30 min inrats with either an end-side or a side-side porto-caval shunt. Dearterialization for 60 min in ratswith end-side porto-caval shunts gave a further release of ASAT and ALAT.As liver tumours receive some of their blood supply from the portal vein, we wanted to illustratethe influence of portal blood flow in combination with dearterialization in the treatment of livertumours. Forty male, inbred Wistar/Furth rats with an adenocarcinoma transplanted to the liverwere treated with various inflow occlusions repeated daily for 5 days. Deviation of the portalblood flow alone with an end-side porto-caval shunt did not alter the tumour growth (		In conclusion, portal deviation did not augment the therapeutic benefit of repeat dearterializationsfor the treatment of this experimental liver tumour. Repeat dearterializations aloneseemed to be a feasible and efficient therapy for liver tumours.


The monoclonal antibody CL21 recognizes a determinant present on the surface ofleukocytes, but not on erythrocytes or nonhemopoietic tissue. The CL21 antigen wasfirst expressed at 48 hr of development at 20°C (stage 28) on embryonic cells culturedfrom lateral plate mesoderm. Based on immunofluorescence staining and flowcytometric analysis, the distribution of fluorescence intensity of larval thymocytes andsplenocytes was unimodal. Distributions of dull and bright cells were detected in bothadult thymocytes and splenocytes. These different subpopulations appeared during thelate perimetamorphic period. Adult splenocytes were metabolically activated whencultured in the presence of mAb CL21 bound to a substrate but not in the presence ofmAb CL21 in suspension. Immunoprecipitation and SDS-PAGE under nonreducingconditions revealed that a single 180-kD molecule was expressed on thymocytes.Analysis of splenocytes demonstrated the presence of two molecules having similarmolecular mass that resolved to a single band under reducing conditions.


Hepatic artery thrombosis is a devastating complication after orthotopic liver transplantation often requiring revascularization or re-transplantation. It is associated with considerably increased morbidity and mortality. Acute cognitive dysfunction such as delirium or acute psychosis may occur after major surgery and may be associated with the advent of surgical complications.Here we describe a case of hepatic artery thrombosis after living-donor liver transplantation which was not preceded by signs of liver failure but rather by an episode of acute psychosis. After re-transplantation the patient recovered without sequelae.This case highlights the need to remain cautious when psychiatric disorders occur in patients after liver transplantation. The diagnostic procedures should not be restricted to medical or neurological causes of psychosis alone but should also focus vascular complications related to orthotopic liver transplantation. Hepatic artery thrombosis (HAT) is the most frequent arterial complication in orthotopic liver transplantation (OLT) occurring in 2.5-6.8% of adult transplant recipients including adult living donor liver transplant recipients -3 with aA 42-year-old male patient underwent a living-donor liver transplantation (LDLT) for progressive hepatitis B, cirrhosis with 7 points on the Child-Turcotte-Pugh scale and a model for end-stage liver disease score of 10. He was placed on the waiting list for transplantation because of suspected hepatocellular carcinoma. He had no history of psychiatric disorder. The donor was his 43-year-old brother who had no major medical problems. The clinical match was appropriate infection the patient also received 103 units of hepatitis B immunglobuline at reperfusion and intravenous infusion of piperacillin/tazobactam at surgery until the 3rd POD but no antimycotics or prophylactic CMV therapy (both donor and recipient had experienced CMV infection).At the time of transplantation the patient received induction immunosuppression consisting of 500 mg methylprednisolone and 20 mg basiliximab, the latter was repeated on the 4th post-operative day (POD). Immunosuppression was maintained with oral mycophenolat mofetil from the first POD at 2 × 1 g daily and oral prednisolone with an initial daily dose of 75 mg. The latter was tapered by 5 mg every two days. On the 5th POD oral tacrolimus was added to the immunosuppressive regimen starting with 0.5 mg on the first day and increasing daily dose by 1 to 2 mg to achieve trough plasma level of 4-10 In the postoperative course serum transaminases peaked on the first and 4th POD but then decreased continuously as prothrombin time increased (coagulation factors were not substituted). Albeit a gradual increase in total bilirubin plasma levels only a mild graft dysfunction was suspected because bile flow over the T-drain increased. Daily doppler sonography of hepatic vessels showed unaltered graft perfusion. Overall the patient had an uncomplicated postoperative without signs for infection or organ dysfunction. The clinical course and laboratory findings did show mild a graft dysfunction but neither rejection nor acute graft failure were indicated.On the 9th POD the patient suffered from acute paranoid psychosis, being alert but agitated and physically aggressive. He was mistrustful and afraid of being harmed by people around him. The patient became unmanageable and had to be sedated with an intramuscular injection of 5 mg haloperidol and an intravenous injection of 20 mg diazepam. Due to this unusual presentation of postoperative delirium a diagnostic workup was initiated. Patent hepatic artery could not be shown in doppler sonography and subsequent angiography. Explorative laparotomy on the same day revealed a complete thrombosis of the donor hepatic artery and gross ischemic infarction in all liver segments. Because progression of infarction and subsequent graft loss was to be expected the patient was placed on the transplant waiting list for re-OLT.An acceptable organ was offered from an 18-year-old heart-beating donor on the following day and the patient underwent re-OLT 10 days after the LDLT. The hepatic artery of the new graft was reanastomosed to the old conduit. The immunosuppressive regimen was reinitiated as described above including tacrolimus (see Figure Here we describe a case of HAT after LDLT preceded not by signs of an liver failure but by acute paranoid psychosis. At transplantation the patient required a complex arterial reconstruction with an interposition graft which is a known risk factor for HAT ,8. WhethPsychiatric disorders are not uncommon in the postoperative course of the liver transplant patient . Like otPsychiatric disorders may also be drug induced. Immunosuppressive drugs like tacrolimus and corticosteroids are known causative agents of psychiatric reactions -16. NeurBecause of elevated bilirubin we also suspected impaired graft function could cause acute psychosis in our patient. In chronic liver failure manifestations of encephalopathy are common and may reach from mild obtundation with dementia and movement disorders to deep coma . In the Besides infection and sepsis surgical complications are the most likely cause of graft dysfunction in the early post-transplant period. Therefore we initiated further diagnostic work-up. Abdominal sonography showed no flow in the hepatic artery and subsequently HAT was confirmed by angiography. During explorative laparotomy the liver was found to be with gross infarction and we decided to evaluate the patient for re-transplantation. Luckily we obtained a graft within 24 hours and after re-transplantation psychosis resolved without sequelae.We propose that in the described patient HAT is the most likely cause of an acute liver failure although laboratory findings indicated only mild graft dysfunction. Acute liver failure was accompanied by hepatic encephalopathy apparent as acute delusional psychosis.This case highlights the need to remain suspicious when psychiatric disorders occur in patients after liver transplantation. The diagnostic procedures should not be restricted to medical or neurological causes of psychosis but should also focus vascular complications of OLT.The authors declare that they have no competing interests.ADG and TL collected and analyzed the data and wrote the article. GR, HB and AO contributed substantially to the process of analyzing the data and writing the paper. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2482/10/7/prepub


Childhood neuroblastoma tumours of the sympathetic nervous system show a remarkable clinical heterogeneity ranging from spontaneous regression to unfavourable outcome despite intensive therapy. Favourable neuroblastomas often express high levels of trkA mRNA, encoding the tyrosine kinase receptor for nerve growth factor. We have investigated mRNA expression for the neurotrophin receptor trkC in 23 primary neuroblastomas using a sensitive RNAase protection assay. TrkC expression was detected in 19 of these tumours at highly variable levels with a 300-fold difference between the highest and lowest values. Significantly higher levels of trkC mRNA were found in tumours from patients with favourable features such as low age (P < 0.012), favourable tumour stage (P < 0.012) and favourable prognosis (P < 0.05). Children with intermediate or high trkC mRNA expression had better prognosis compared with those with low or undetectable levels . Further characterisation of trkC mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that mRNA encoding the full-length cytoplasmic tyrosine kinase domain of the receptor was only expressed in a subset of favourable tumours. These data show that favourable neuroblastomas may express the full trkC receptor while advanced tumours, in particular MYCN-amplified neuroblastoma, seem to either express no trkC or truncated trkC receptors of as yet unknown biological function. These data are suggestive of a role for trkC and its preferred ligand neutotrophin-3, NT-3, in neuroblastoma differentiation and/or regression.


During the last years the analysis of microRNA expression patterns has led to completely new insights into cancer biology. Furthermore, these patterns are a very promising tool for the development of new diagnostic and prognostic markers. However, most human tumour samples for which long term clinical records are available exist only as formalin-fixed paraffin-embedded specimens. Therefore, the aim of this study was to examine the feasibility of microRNA profiling studies in routinely processed formalin-fixed paraffin-embedded human breast cancer specimens using fluorescence labelled bead technology.A statistically highly significant correlation  was observed for the expression of 319 microRNAs in routinely processed FFPE breast cancer specimens and paired fresh frozen tissue samples (n = 5). Results were confirmed in a larger series analyzing a selection of 10 microRNAs reported to be deregulated in breast cancer (n = 12). The expression pattern of 3 microRNAs was independently validated in this cohort using real-time RT-PCR technology.Comprehensive microRNA expression patterns can be reliably derived from routinely processed FFPE breast cancer specimens using fluorescence labelled bead technology. Formalin-Fixed, Paraffin-Embedded (FFPE) tissue samples represent an invaluable source for the study of human disease. Millions of blocks are archived world wide with corresponding well-documented clinical histories and histopathological reports. The potential value of these archives for retrospective molecular studies has been well recognized . HoweverThe analysis of microRNA expression patterns in human tumour specimens promises to provide completely new insights into tumour biology. In addition, it may contribute to the development of new diagnostic or predictive markers ,3. But tThe quantification of microRNA expression levels using LNA probes coupled to fluorescence labelled beads offers several advantages: No amplification step is required which may introduce a potential bias and the hybridization of probes and target sequences takes place in a homogeneous system .So far, no systematic comparison of microRNA profiles obtained from fresh-frozen and corresponding FFPE samples using the fluorescence labelled bead technology is described.In this study we examined the expression pattern of 319 microRNAs in routinely processed formalin-fixed paraffin-embedded breast cancer specimens and paired fresh-frozen specimens from the very same tumours. For this purpose the fluorescence labelled bead technology from Luminex  was employed.The integrity and quality of the RNA preparations was analysed using the microcapillary fluid device form Agilent . The mean "RNA integrity number" (RIN) for all The mean fluorescence intensity, the highest fluorescence intensity, and the sum of all fluorescence intensities were not reduced in any of the FFPE samples compared to the corresponding fresh-frozen sample (data not shown). Therefore, no reduction in microRNA recovery due to formalin-fixation was observed.The expression level of 319 microRNAs (FlexMir panel version 8 from Luminex) was measured in 5 fresh-frozen breast cancer specimens and corresponding formalin-fixed paraffin-embedded tissue samples from the very same tumour. The measurements showed a very good correlation for all 5 paired samples Fig. . The Spe10 individual microRNAs were selected from the literature as most frequently deregulated in primary human breast cancer  was measured in all samples (12 fresh frozen and 12 FFPE) using the stem-loop primer real-time PCR methodology from ABI . Overall, the expression pattern (miR-10b versus miR-145 and miR-10b versus miR-21) obtained with this completely different technique is quite similar: The expression level of miR-145 is only slightly higher than the expression level of miR-10b whereas miR-21 is much stronger expressed than miR-10b  than using the bead-based methodology (mean: approx. 40-fold).The systematic comparison of paired fresh-frozen and formalin-fixed paraffin-embedded tissue specimens from the very same tumour shows that reliable microRNA expression pattern can be obtained from routinely processed breast cancer specimens using the fluorescence-labelled bead-based technology from Luminex. However, a disadvantage of the current microRNA profiling methodology from Luminex is the requirement of quite large amounts of total RNA (2.5 – 5 μg) which is clearly not available for every interesting case.The much stronger overexpression of miR-21 as measured by real-time PCR in comparison to the bead-based methodology could be due to slight differences in amplification efficiencies for these two microRNAs. These slight differences in efficiency create a considerable difference in expression level after 25 or 30 cycles due to the exponential nature of the amplification process. Additional sources of differences between the methods compared are the completely different RNA processing (cDNA synthesis versus direct biotin-labelling) and differences in analytical sensitivity . But nevertheless, the overall pattern is the same using both techniques.Leaving out all signals below 100 arbitrary units improved the correlation quite significantly for three out of five samples, clearly indicating that most of the variability between paired samples are due to fluctuations of the signals of very weekly expressed microRNAs.Nelson et al.  analysedLi et al.  publishe2 = 0.86 – 0.89). However, no corresponding pairs of routinely processed human tissue samples were analyzed. Only a single archival human colon cancer specimen for which no corresponding fresh-frozen material was available was analyzed.Xi et al.  studied Wang et al.  mention Lawrie et al.  showed iT) values and the shift in CT values, a direct comparison with the correlation coefficients obtained by us using a different methodology is not possible. However, both technical approaches seem to provide reliable data.The recently published study from Hoefig et al.  represenFinally, the evaluation of different microRNA isolation procedures recently published by Doleshal et al. , containThe data presented in this study convincingly show that routinely processed human FFPE tissue specimens are suitable for large-scale as well as small-scale microRNA profiling projects using fluorescence labelled bead technology. Therefore, this methodology can now be used for large retrospective studies utilizing stored archival FFPE samples together with the corresponding clinical and histopathological records.Note added in proof:During the revision of this manuscript Zhang et al. published the comparison of microRNA expression patterns from fresh-frozen and corresponding FFPE lymph node specimens (n = 7) using the Agilent microarray platform .  These au12 fresh frozen breast cancer specimens and corresponding formalin-fixed paraffin-embedded tissue samples from the very same tumours were retrieved from the archives of the Institute of Pathology, Medizinische Hochschule Hannover in an anonymous fashion following the guidelines of the local Ethics committee. Tumour cell content was controlled on an HE stained serial section and above 80% for all cases.Total RNA was isolated from fresh-frozen specimens using TRIZOL™ reagent  following the protocol supplied by the manufacturer. Isolation of total RNA from FFPE specimens was performed as described previously.  FollowinAn aliquot of 150 ng of every RNA preparation was checked for integrity in an Agilent 2100 Bioanalyzer . Sample preparation and analysis were performed following the manufacturer's instruction.Quantification of microRNAs hybridized to fluorescence labelled beads was performed with a BioPlex 200™ from Biorad  using the software Luminex IS™, version 2.3 from Luminex . All beads coated with LNA probes complementary to mature microRNAs were purchased from Luminex .Prior to hybridization total RNA is labelled with biotin which is later bound to a streptavidin-phycoerythrin conjugate. For this purpose the FlexmiR™ MicroRNA Labeling Kit from Luminex was used. For the comprehensive profiling of 319 microRNAs  2.5 μg total RNA were labelled, for the analysis of 10 selected microRNAs (FlexmiR™ Select) 0.5 μg were labelled following the protocol provided by the manufacturer. Subsequent washing, hybridization and analysis of samples were performed exactly as described in the protocols provided by Luminex.The system was calibrated using the xMAP™ calibration control reagents from Luminex following the recommendations of the manufacturer. Every run contained a negative control for background subtraction (water instead of RNA) and a positive control . For data normalization the background value for every individual microRNA bead was subtracted from every corresponding measurement. Subsequently, all corrected mean fluorescence intensities were normalised to pool 1 using the average correction factor for the four normalization controls included in all five pools as recommended by the manufacturer (The 319 different microRNA beads are divided into 5 pools because there are not enough different fluorescence labels available to distinguish more than 70 – 80 beads in a single tube.). These normalised net mean fluorescence intensities were directly compared between paired samples. All control and normalization beads  were left out for these comparisons.For the measurement of the expression levels of selected microRNAs  the real time-RT-PCR-based detection methodology from ABI  was used. Relative expression levels were calculated using the constitutively expressed small RNAs RNU24 and Z-30 as endogenous controls. All primers and probes were purchased from ABI. cDNA synthesis and real-time PCR were performed following the protocols supplied by the manufacturer.The distribution of the microRNA expression data did not pass the test for normality (α = 0.05) for any specimen. Therefore, Spearman rank correlation factors were calculated (two-tailed). For all calculations the software package GraphPad Prism  was used. p-values < 0.05 were considered statistically significant.FFPE: formalin-fixed paraffin-embedded; LNA: locked nucleic acid.UL and HK conceived the study, BH and UL performed all measurements and evaluated the data, MC and UL performed the statistical analysis and drafted the manuscript, HK selected and evaluated all cases, UL wrote the manuscript with support from HK and MC. All authors read and approved the final manuscript.Comparison of expression levels of 319 microRNAs in 5 paired fresh-frozen and formalin-fixed paraffin-embedded human breast cancer specimens. The data provided represent the correlation analysis of the microRNA expression levels measured in the paired specimens BrC1 – BrC5 (fresh-frozen versus FFPE). The results for BrC1 are also shown in Fig. Click here for fileComparison of expression levels of 319 microRNAs in 5 paired fresh-frozen and formalin-fixed paraffin-embedded human breast cancer specimens leaving out weak signals. The data provided represent the correlation analysis of the microRNA expression levels measured in the paired specimens BrC1 – BrC5 (fresh-frozen versus FFPE) leaving out all signals below 100 arbitrary units (see main text for further details).Click here for fileComparison of expression levels of 10 selected microRNAs in BrC1 – BrC6. The data provided represent the relative expression levels of 10 microRNAs reported to be deregulated in human breast cancer (see Table Click here for fileComparison of expression levels of 10 selected microRNAs in BrC7 – BrC12. The data provided represent the relative expression levels of 10 microRNAs reported to be deregulated in human breast cancer (see Table Click here for file


Background: Chronic intestinal schistosomiasis is rare in the United Kingdom. The symptoms arenonspecific and may mimic several other gastrointestinal conditions. We present a case of chronicintestinal schistosomiasis in a West Indian woman presenting to a genitourinary clinic.        Case: The patient presented with chronic lower abdominal pain and dysuria. A sexually transmitteddisease (STD) screen was negative and midstream urine cultures were sterile. A rectal biopsyrevealed a non-necrotizing granulomatous reaction around the ova of Schistosoma. Her symptomsresolved with anti-schistosomiasis therapy.            Conclusion: This case illustrates that physicians should be aware of chronic schistosomiasis in thedifferential diagnosis of chronic lower abdominal pain in women who have come from or visitedareas where schistosomiasis is endemic.


Growth factor activity which stimulates anchorage-independent growth (AIG) in a rat fibroblast line, was detected in human premalignant adenoma tissue from familial polyposis coli colectomy specimens and in serum-free culture supernatant from an adenoma cell line PC/AA. The activity extracted from adenoma tissue was compared quantitatively in the AIG bioassay with extracts of normal mucosa from split thickness colorectal tissue. Adenoma tissue yielded three times the amount of acid-extractable protein g-1 wet wt and adenoma extracts consistently had significantly greater specific activity over a wide protein concentration range. Activity extracted from adenoma tissue and from the derived cell line PC/AA were compared qualitatively after fractionation by gel filtration. Both extracts showed almost identical profiles of biological activity after assay of individual fractions for AIG stimulation, suggesting that the factor(s) originates from the epithelial component of the adenoma tissue since PC/AA is a pure epithelial cell line. Activity eluted as two major peaks with apparent mol. wts of 9 kd and 20-25 kd (relative to standards) in both cases. This report demonstrates for the first time that elevated production of a growth factor may be an early change in the evolution of human colorectal cancer from small, premalignant adenomas.


An 11-year-old girl with rapidly enlarging bilateral breast lumps is reported. It was diagnosed as a case of juvenile fibroadenoma following fine needle aspiration cytology and confirmed on histopathological examination of the excised specimens. Bilaterally symmetrical giant juvenile fibroadenomas are very rare in prepubertal girls.2 The lesAn 11-year-old premenarche girl presented with bilateral, rapidly increasing breast lumps for two months . There wFNAC showed aggregates of cohesive epithelial cells . A few bGiant juvenile fibroadenoma in the prepubertal age group is almost always benign and should be treated with breast conserving surgery.4 JuvenilVarious other conditions like lipoma, hamartoma, cysts should be kept in the list of differential diagnosis. Most of Giant juvenile fibroadenoma simultaneously occurring in both the breasts is rare. Isolated case reports are available in the English literature.11 Someti12


Vascular endothelial growth factor (VEGF) expression was examined by immunohistochemistry in 45 prostatic carcinoma specimens and ten benign prostatic tumours (BPH). The majority of carcinoma specimens exhibited cytoplasmic staining for VEGF and showed a trend of increasing expression with dedifferentiation (2p = 0.003). Immunoreactive VEGF was also seen in the prostatic carcinoma cell lines, the order of staining intensity was PC3 > DU145 > LNCaP. Intense granular cytoplasmic staining for VEGF was observed in neuroendocrine-like cells which were seen focally in many of the prostatic specimens. Consecutive sections were incubated with a chromogranin A antibody to confirm the neuroendocrine phenotype of these cells. A significant correlation (P < 0.0001) between the total number of intensely stained VEGF-positive cells and chromogranin A-positive cells was found. A subpopulation of neuroendocrine-like cells also showed intense immunoreactivity for transforming growth factor alpha . A correlation was observed (2p = 0.0092) between the intensity of VEGF and TGF-alpha immunostaining in carcinoma cells which were not of neuroendocrine differentiation. The presence of these two angiogenic factors may aid the neovascularisation of carcinomas and their increased expression in tumour-associated neuroendocrine cells may contribute to a more aggressive phenotype.


Mitomycin-treated transplantable Moloney virus-induced lymphatic leukaemia cells (YC8) not only failed to stimulate normal allogeneic lymphocytes in one-way mixed leucocyte culture (MLC) but also exerted a strong inhibitory effect on the proliferative response of normal lymphocytes, in MLC and after stimulation by mitogens. Potentially inhibitory factors which could be released in the culture fluids by the YC8 cells were not found, but a YC8-derived adherent cell subpopulation was identified as being responsible for the in vitro suppression.


The association of the atrophy-hypertrophy complex in monolobar Caroli’s disease (Type I) is reportedin a 30 year old male who presented with recurrent cholangitis. Ultrasound and CT scan showedlocalised, right sided, saccular biliary dilatation in a normal sized liver. Severe right lobar atrophy wasdetected at operation and the resected right lobe weighed only 140 gms. Distortion of the hilar vascularanatomy and posterior displacement of the right hepatic duct orifice were problems encountered atsurgery.


Inherited cardiomyopathy (CM) represents a diverse group of cardiac muscle diseases that present with a broad spectrum of symptoms ranging from benign to highly malignant. Contributing to this genetic complexity and clinical heterogeneity is the emergence of a cohort of patients that are double or compound heterozygotes who have inherited two different CM mutant alleles in the same or different sarcomeric gene. These patients typically have early disease onset with worse clinical outcomes. Little experimental attention has been directed towards elucidating the physiologic basis of double CM mutations at the cellular-molecular level. Here, dual gene transfer to isolated adult rat cardiac myocytes was used to determine the primary effects of co-expressing two different CM-linked mutant proteins on intact cardiac myocyte contractile physiology. Dual expression of two CM mutants, that alone moderately increase myofilament activation, tropomyosin mutant A63V and cardiac troponin mutant R146G, were shown to additively slow myocyte relaxation beyond either mutant studied in isolation. These results were qualitatively similar to a combination of moderate and strong activating CM mutant alleles αTmA63V and cTnI R193H, which approached a functional threshold. Interestingly, a combination of a CM myofilament deactivating mutant, troponin C G159D, together with an activating mutant, cTnIR193H, produced a hybrid phenotype that blunted the strong activating phenotype of cTnIR193H alone. This is evidence of neutralizing effects of activating/deactivating mutant alleles in combination. Taken together, this combinatorial mutant allele functional analysis lends molecular insight into disease severity and forms the foundation for a predictive model to deconstruct the myriad of possible CM double mutations in presenting patients. Inherited cardiomyopathies (CM) are the most prevalent genetic disorders of the cardiovascular system Some of the genetic complexity associated with CM can be attributed to environmental factors and genetic modifiers. However, emerging complexity in the form of multiple CM allele inheritance is also evident in vitro gene-dosage experiments One hypothesis explaining these findings is that a higher total dosage of CM mutant protein is causal for the increased disease severity or mitigate the functional cellular outcomes of either mutant allele when studied in isolation. A prediction of this model is that individuals inheriting two mutant CM alleles with opposing biophysical properties may paradoxically have a less severe outcome than either parent with only a single CM mutant allele. Adding to the complexity of CM is the realization there are ∼467 CM-linked mutant alleles that have been reported in eleven different sarcomeric genes 2)−467)/2]), with ∼75,000  of those totaling the number of double heterozygous genotypes. The total number of compound heterozygous combinations was derived by the following formula: ((X2)−X)/2, where X is the number of mutations identified for a given sarcomeric gene. This formula is calculated for total number of mutations within each sarcomeric loci and then summated to equal 33,737 possible genotypes.An alternative hypothesis explored here is that multiple CM mutant proteins cause allele-specific combinatorial primary effects that uniquely alter sarcomeric function and are not necessarily linked directly to total mutant gene dosage. Thus, our working hypothesis is that altered biophysical properties of double mutant sarcomeric proteins could interact to either heighten 2+ activating (thin filament activator) or Ca2+deactivating (thin filament deactivator) archetypes. Results derived from these archetypes could then inform a predictive model for extrapolation to all possible CM mutant combinations. The study employed CM mutant thin filament regulatory proteins as a proof-of-concept methodology to address this problem for three reasons. First, this family of sarcomeric proteins is highly amenable to acute genetic engineering and structure-function studies 2+ sensitivity and thin filament cooperativity thereby modifying myocyte force development and relaxation performance In light of the staggering number of potential compound/double genotypes, an examination of every possible double mutant combination is unfeasible. We reasoned that a predictive model, predicated on experimental data, could be implemented as an index of functional outcomes in multiple CM allele inheritance. To facilitate this undertaking we assumed that specific CM mutants could be categorized as CaTo derive a working model predicting the physiologic effects of multiple CM alleles, experiments combined CM alleles that separately cause mild, moderate or strong myofilament activation . A combi10–1012 pfu/ml.Adenoviral construction of AdGFP, AdLacZ, αTmE180G, αTmK70T, αTmA63V, cTnIR146G, and cTnIR193H were previously described 6 cells with 77% viability. The care and use of the laboratory animals for this study was in agreement with the guidelines set forth by the Internal Review Board of the University of Michigan Committee on the Use and Care of Animals. Veterinary care was provided by the University of Michigan Unit for Laboratory Animal Medicine.Rat hearts were excised from heparinized (1500 U/kg) and anesthetized  adult female Sprague Dawley rats . Ventricular cardiac myocytes were isolated via collagenase-hyaluronidase digestion as previously described Post digestion the myocytes were resuspended in DMEM (Dulbecco's Minimum Eagle Media) plus 5% fetal bovine serum (FBS) and approximately 20,000 cells were plated on laminin-coated coverslips and incubated for 2 hours. At which time the media is replaced with serum-free DMEM. Subsequently myocytes are transduced with recombinant adenoviral vectors. For single gene transfer myocytes were transduced with an experimentally predetermine optimal multiplicity of infection  based on the stoichiometric replacement for each vector and level of efficiency. For dual gene transfer two different recombinant adenoviruses were combined so that the total MOI was 500 and then simultaneously applied to the plated myocytes 6) were used for immunodetection. In some instances horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies were used for chemilumenescence detection  To assess the mutant cTnIs expression and incorporation into the sarcomere, cultured ventricular myocytes were scraped into Laemmli sample buffer and prepared for gel electrophoresis by boiling and sonication. As previously described, cardiac proteins were separated on a 4–20% gradient SDS-page gel  and prepared for Western blot analysis. A cTnI specific polyclonal antibody  and Tm specific monoclonal antibody  as previously described. A coverslip containing cultured myocytes was bathed in HEPES buffered medium 199  kept at 37°C. Myocytes were electrically paced at 40 volts with a frequency of 0.2 Hz. Ca2+ transient measurements were accomplished using the excitable Ca2+ indicator Sarcomere length measurements and CaTo validate the experimental design of dual mutant CM gene transfer, we determined the efficiency of adenoviral-based dual gene transfer using reporter genes . AcutelyWe tested three different CM mutant alleles shown previously and confirmed here to cause weak, moderate, or strong myofilament activation when tested individually . The pri2+ transients were simultaneously measured yielding results similar to mechanical relaxation  show evidence of a functional ceiling for some mutant combinations suggesting there are limits to mechanical dysfunction induced by compound mutations. We also studied in combination CM mutants with distinct activating and deactivating properties. A priori, such combination could result in a dominant effect or, alternatively, a hybrid functional outcome. Findings here show that combining CM mutants with opposing effects on myofilament function (activator + deactivator) produces a hybrid outcome and evidence of functional neutralization at the intact myocyte level. This finding raises the possibility that two CM mutant alleles, that in isolation markedly alter cell performance to cause CM, could in combination yield near normal cellular physiology. These data are used to develop a working model (detailed below) for predicting molecular outcomes on the >100,000 possible CM double mutant combinations.These results demonstrate that double cardiomyopathy mutants, with mild to strong myofilament activating properties combine to worsen cellular diastolic dysfunction. These additive effects on mechanical performance  plays in thin filament regulation.The cause and effect relationship for CM patients with multiple disease alleles is poorly understood due to a myriad of compounding factors including environment, genetic modifiers, increased mutant allele dosage, or allele-specific functional interactions that likely contribute to the severity of the disease presentation myocytes . It is w2+-mediated thin filament activation 2+ sensitivity and thus sarcomere relaxation 2+ concentrations when compared to their single mutant counterparts. This heightened activation manifests itself as a more severe cellular diastolic dysfunction, as the sarcomere remains contracted with decaying Ca2+ concentrations. While the A63V+R193H double activator combination slowed relaxation beyond that of either mutation studied in isolation, the net effect was not precisely additive on a one to one basis as the sum total of relaxation times for single R193H and A63V myocytes  was ∼17% greater than those measured in A63V+R193H dual gene transfer myocytes (493 ms). This suggests that the A63V+R193H combination may reach a functional threshold or a physiologic limit in which additional mutant incorporation no longer alters thin filament activation similar to previous reports where extracting only a fraction of troponin complexes (as little as 5%) results in nearly maximal Ca2+ independent thin filament activation 2+ activation beyond this saturation point. The functional threshold attained by the A63V+R193H myocytes in this case could be ascribed to the lower gene dosage of both mutants attained in the dual gene transfer model. Perhaps with increased A63V Tm incorporation the alterations in diastolic dysfunction could be precisely additive. Alternatively, A63V mutant Tm and R193H mutant cTnI may overlap in terms of their biophysical roles in thin filament regulation and thus only combine to fractionally worsen diastolic dysfunction leaving open the question of possible compound CM mutant combinations that may encroach on a physiologic limit where the thin filament attains complete Ca2+-independent dis-inhibition and maximal tension generation during relaxation conditions. The R193H cTnI mutant alone encroaches on this limit as it has a dominant effect in all combinations to cause a Ca2+ independent diastolic tone In this study we ascribe the worsening of myocyte mechanical performance to the combined effects that gain of function mutations in cTnI and Tm have on enhancing CaAn equally important factor that likely impacts the additive effects of two activating mutant alleles is the combined incorporation of each mutant into the sarcomere given the central role thin filament stoichiometry and cooperativity plays in the regulation of contraction 2+ sensitivity A critical insight garnered from this study is that two CM mutant alleles in combination may not necessarily result in a more severe cellular dysfunction. This is evident from the deactivator + activator combination (cTnIR193H + cTnCG159D) in which the diastolic dysfunction characteristic of single cTnIR193H mutant was neutralized in part by cTnCG159D producing an intermediate activating phenotype. The G159D mutation appears to play a structural role in the troponin complex, but also interacts with cTnI's N-terminus In contrast to the mutant Tm and cTnI pairings, the modification of the thin filament with cTnCG159D and cTnIR193H is modeled with a different distribution pattern and higher percentage of double mutant regulatory units . It is cUsing experimental data from activating and deactivating sarcomeric CM mutants obtained here and elsewhere as foundations, a working model was developed to promote predictive insights into myocyte function of the theoretically possible tens of thousands of compound CM combinations. The model was constructed by categorizing single mutant CM alleles by their functional properties as myofilament activators or deactivators, and by the strength with which these properties have been altered. The activation properties of numerous single sarcomeric mutants in both the thick and thin filament have already been characterized This model also incorporates the neutralizing effects of activator and deactivator alleles in combination . Here, tFor experimental purposes we did not employ the more prevalent double heterozygous genotypes, which predominantly consist of MyHC and MyBP-C mutant allele combinations. As myosin is a vital ligand required for maximal activation of tension development In conclusion, our findings show at the cellular/molecular level double mutant thin filament proteins have direct and combinatorial effects to alter cellular diastolic function. The mechanistic basis for additive/mitigating effects cannot be solely due to total mutant gene dosage alone, but also appears to be highly dependent on the population and location of double mutant regulatory units along the thin filament and the biophysical properties of the effected sarcomeric protein in terms of the activating/deactivating nature and the strength of these mutant alleles. These results also underscore that the primary outcome for double heterozygous patients may not necessarily cause a worsening of the disease phenotype particularly in cases where the genotype produces an activating and a deactivating mutant myofilament protein.


Orbital trauma usually affects the bony parts of the orbit; however, in rare cases foreign bodies are found within the orbit. In this report, we introduce a case with unusual large intraorbital foreign bodies (two parts of a brake lever) after a motorcycle accident. Although one of the foreign bodies was located in the posterior orbit, they required only one simple operation for retrieval. We will discus the management strategy. Intraorbital foreign bodies (FBs) usually occur after a high-velocity injury such as a gunshot or industrial accident.2 AlthougTraumatic eye injuries due to large foreign bodies are rare. There are few reports of unusually large intraorbital FBs such as an iron nut or wood.4 There iA 25-year-old man presented to our emergency clinic 6 h after a penetrating orbital injury with a brake lever in his right lower eyelid during a motorcycle accident . Both eyIntraorbital FBs can be associated with severe injuries leading to loss of vision or may lead to sight-threatening complications.2 A retai5Retained metallic intraorbital FBs are well tolerated and should be managed conservatively in the absence of specific indications for removal.2 When th125


Smoking is a known aetiological risk factor for cervical cancer. Smoking-related DNA damage (DNA adducts), in cervical epithelial cells, has recently been demonstrated to suggest a causal role in the development of cervical cancer. Human papillomavirus 16 (HPV 16) is a known oncogenic virus and is also implicated as a cause of cervical cancer. It has been suggested that both smoking and HPV may act synergistically in the development of cervical cancer. We have investigated the cervical DNA adduct level and the prevalence of HPV 16 (using polymerase chain reaction) in women who had normal cervical cytology. Both the DNA adduct assay and the HPV assay were carried out on exfoliated cervical cells recovered from cervical scrapes. In 87% of the cases there was enough DNA from the exfoliative cervical cells to analyse for DNA adducts. Smokers had higher DNA adduct levels than non-smokers (P = 0.002), confirming the previous data from cervical biopsy samples. Forty-two per cent of the specimens were found to be HPV 16 positive. There was no significant difference in smoking-related DNA damage (DNA adduct levels) between HPV-positive and HPV-negative smokers. This suggests that smoking DNA damage does not augment HPV infectivity. These results do not, therefore, support the molecular synergism theory.


Angina pectoris early after aortic valve replacement surgery in patients with previously normal coronary arteries may be life threatening and has to be assessed immediately.12 weeks after aortic valve replacement surgery, a 60-year-old female patient was referred for evaluation of recent onset of severe chest pain on mild exertion and at rest. Coronary angiography showed severe stenosis nvolving the left coronary ostium and the left main stem. The patient was urgently referred for bypass surgery and had an uneventful postoperative recovery.A high degree of suspicion is needed for early recognition and aggressive management of this rare but serious complication. Unstable angina is rare, but may be life-threatening in patients in the early postoperative period following aortic valve replacement with normal preoperative coronary arteries.We report the case of a 60-year-old female patient undergoing valve replacement surgery for symptomatic aortic valve stenosis. Preoperative echocardiographic assessment revealed a severely calcified aortic valve with a calculated aortic valve area of 0.8 ccm. Mean pressure gradient was 55 mmHg and left ventricular ejection fraction was well preserved.The patient was free of angina and reported dyspnoea on exertion.Preoperative coronary angiography revealed normal coronary arteries.Valve replacement surgery was performed using a Sorin 23 mm mechanical valve prosthesis. Early postoperative recovery was unremarkable.12 weeks after surgery the patient was referred for evaluation of recent onset of severe chest pain on mild exertion and at rest.ECG revealed severe ST-segment depression in leads V2-5 during episodes of chest pain.Coronary angiography showed a 90% diameter reduction involving the left coronary ostium and the left main stem.The patient was urgently referred for bypass surgery and had an uneventful postoperative recovery.Angina pectoris early after aortic valve replacement surgery in patients with previously normal coronary arteries may be life threatening and has to be assessed immediately. A high degree of suspicion is needed for early recognition and aggressive management of this rare but serious complication.Unstable angina early after aortic valve replacement in patients with normal coronary arteries in the preoperative angiography is rare.Generally, possible differential diagnoses of postoperative angina pectoris in patients undergoing mechanical aortic valve replacement are coronary embolism, progression of coronary heart disease in patients with coronary atherosclerosis, graft occlusion in patients with concomitant aortocoronary bypass (ACBP) and iatrogenic coronary ostial/main stem stenosis.Many cases of iatrogenic coronary ostial/main stem stenoses have been reported since the late 1960ies, all cases showing similar patterns – sudden onset of angina pectoris 3–6 months postoperatively in patients without or with only mild coronary artery disease – and similar histologic findings – intimal fibrous proliferation of one or both coronary ostia [We report the case of a 60-year-old moderately obese female patient, who was referred for evaluation of recent onset dyspnea on exertion. Physical examination revealed a 3/6 crescendeo-decrescendo systolic murmur at the aortic valve.Electrocardiography showed regular sinus rhythm without additional findings Fig. 2 and a mean pressure gradient of 55 mmHg.Echocardiographic assessment revealed a severely calcified aortic valve with a calculated valve area of 0,80 cmLeft ventricular ejection fraction was well preserved with left ventricular wall thickness of 14 mm .Preoperative coronary angiography revealed completely normal coronary arteries Fig .The patient underwent aortic valve replacement surgery. The procedure was performed using mini-sternotomy with a single 2-stage venous cannula and normothermic cardiopulmonary bypass. The use of normothermic techniques has been reported to confer several advantages over conventional hypothermia, such as reduced bleeding and requirements for electrical defibrillation, shorter intubation times, and improved hemodynamic parameters postoperatively. Myocardial protection with cold antegrade and retrograde St. Thomas' cardioplegic (II) solution was obtained immediately after aortic cross clamping. The aortic valve is exposed through an oblique aortotomy incision made well above the orifice of the right coronary artery. The severely calcified stenotic aortic valve was excised, replaced with a Sorin 23 mm mechanical valve prosthesis, attached with subannular mattress sutures of 2-0 Ethibond .Early postoperative recovery was unremarkable.12 weeks after surgery the patient was referred for evaluation of recent onset of severe chest pain on mild exertion and at rest. ECG at admission showed inverted T-waves in leads V2 to V5 and in lead aVL Fig. .During episodes of chest pain ECG revealed severe ST-segment depression in leads V2 to V5 Fig .Coronary angiography showed a 90% diameter reduction involving the left coronary ostium and the left main stem Fig .The patient was urgently referred for bypass surgery with a left internal mammary artery graft to the LAD and a left radial artery graft to the circumflex artery.Postoperative recovery was unremarkable. Four months later she is doing well without chest pain or signs of myocardial ischemia.The present case underlines the importance of early diagnosis and treatment if angina pectoris occurs after aortic valve replacement.Coronary ostial stenoses can be detected in 0,1% of coronary angiographies in unselected patients ,11. AparIatrogenic coronary ostial stenosis is a well recognized, but uncommon and potentially life threatening complication of aortic valve replacement. Symptoms include chest pain during exercise or at rest, sudden onset of acute heart failure and acute pulmonary edema. Usually, symptoms occur within the first 6 months, though they may occur up to 30 months after the operation .Main stem stenosis after aortic valve replacement was first recognised in 1969 by Trimble et al . who desSeveral pathogenic mechanisms have been suggested: aortic root fibrosis secondary to turbulent flow around the prosthesis ; the preThere may also be a genetic predisposition for developing this complication since 70% of the affected individuals as compared to 10–15% in a control group had an epsilon 4 allele apolipoprotein E genotype .Instrumentation with minimal trauma of the left main stem is most likely the cause of early postoperative stenosis after aortic valve replacement surgery ,8,14.Avoiding cannulation of the coronary ostia for antegrade cardioplegia, but instead using retrograde delivery as an alternative method for myocardial perfusion during open heart surgery may reduce the risk of postoperative coronary ostial or left main stem stenosis ,15.If recurrent or newly onset angina pectoris occurs early after aortic valveReplacement, a high degree of suspicion os warranted to recognize this rare but life threatening complication of coronary ostial stenosis, even if preoperative coronary angiography did not show coronary heart disease.New operative techniques that reduce manipulation and consequently avoid trauma of the coronary vessels may prevent postoperative coronary ostial stenosis. Future studies in patients undergoing this procedure using retrograde cardioplegia only will have to prove this hypothesis.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.SG was the main author and wrote the article. CKN was the surgical consultant, was involved in data collection and revised the manuscript. CS was the surgical consultant was involved in data collection and interpretation. JA was the cardiology consultant and gave final approval of the manuscript. All authors have read and approved the final manuscript.


We report three fatal cases of bacteremia  caused by Vibrio cholerae O1 (Ogawa), which occurred in the context of a community outbreak of cholera diarrhea in Blantyre, Malawi. Only four cases of invasive disease caused by V. cholerae O1 have previously been reported. We describe the clinical features associated with these rare cases and discuss their significance.


Blood lymphocytes from 47 patients with lung carcinoma have been tested for cytotoxicity against cells isolated from the autologous tumour. Significant cytotoxic potential was found in 15 cases. The effectors were also tested against allogeneic tumour targets from lung and other sites. Reactions were only rarely detected (2/32 positive against lung and 1/13 positive against non-lung cells). The restriction of cytotoxicity to the autologous combination was also apparent in in vitro-generated effectors. Blood lymphocytes were co-cultivated with autologous tumour and subsequently tested against autologous or allogeneic targets. Cytotoxicity was found in 13/17 lung tumours against autologous tumour, with no reactions recorded against allogeneic tumour targets, but one case positive against the K562 cell line. These data suggest either the expression of individually distinct antigens on human pulmonary neoplasms, or the requirement for histocompatibility between target and effector in cytotoxicity reactions in man, and therefore differ from previously described patterns of lymphocytotoxicity against human tumours.


Thirty-one cervical biopsies of invasive carcinoma have been studied by immunohistochemical means using the monoclonal antibody Ki67 to determine tumour cell proliferation rates. A wide range (10-50%) in the extent of Ki67 staining (expressed as the percentage of labelled tumour cells) was observed indicating considerable variation on tumour growth rates. There was no significant relationship between the percentage of positive cells and conventional histological parameters such as cell type or tumour differentiation. Immunostaining with monoclonal antibody Ki67 therefore provides a new approach to the assessment of cervical tumour biopsies which will require long term clinical follow-up to establish its prognostic significance.


A 58 year old woman with symptomatic severe bicuspid aortic valve stenosis underwent aortic valve replacement with a mechanical prosthesis. On post-operative day four, the patient developed sudden onset of sustained narrow complex tachycardia with a heart rate of 170 - 180 . She comInitial evaluation of the presenting arrhythmia  revealedAccelerated junctional tachycardia in the post-cardiac surgery period has been described in the pediatric and adult congenital populations, and has been associated with significant morbidity . HoweverIn conclusion, we report the case of a patient who developed incessant, accelerated junctional tachycardia few days after aortic valve surgery. This condition is self-limited and can be treated acutely with anti-arrhythmic medications such as amiodarone. Recognizing this condition and treating it conservatively are important to ensure a favorable clinical outcome.


The ultrastructure of two lymphoblastoid cell lines derived from Marek's disease lymphomata has been studied. The cells varied from 5 to 12 mum in diameter and had large round or oval nuclei. A nucleolus was occasionally present and about 3% of cells showed projections of the nuclear envelope. The cytoplasm contained many ribosomes and several mitochondria but endoplasmic reticulum was sparse. A small number of cells contained annulate lamellae and crystalline structures were occasionally seen. Cells with immature intranuclear herpesvirus particles were rarely present. The cells had many ultrastructural features in common with Burkitt's lymphoma-derived cell lines.


Post-partum choriocarcinoma is a rare complication of pregnancy. We have analysed a series of nine consecutive patients presenting with choriocarcinoma after a full-term non-molar pregnancy. All patients were managed at the Supraregional Trophoblastic Disease Screening and Treatment Centre at Weston Park Hospital, Sheffield between 1987 and 1996. All presented with persistent primary or secondary post-partum haemorrhage. Treatment with multiagent chemotherapy  was successful in all cases. Early diagnosis is important because this rare condition is potentially curable with appropriate chemotherapy. © 1999 Cancer Research Campaign


A slowly growing solid sarcoma was implanted subcutaneously on the anterior chest wall of mice. On reaching a predetermined size the tumours were locally irradiated using 240 kV x-rays with single doses of 0, 2000 or 5000 rad. The mice were sacrificed 12 weeks after irradiation and examined for lung metastases, which were found to be less frequent in those mice whose implanted tumours had received the most irradiation.


PMN-dependent plasma leakage was equally well reduced by the different sulphated polymers. Vascular permeability induced by histamine or thrombin acting via a PMN-independent mechanism was not reduced. Fucoidan was the only polysaccharide able to suppress IL-1-induced PMN infiltration for 60–70%. Local administration of dextran sulphate had no effect on PMN-dependent plasma leakage. Differential inhibition of PMN recruitment was determined after injection of dextran sulphate or fucoidan depending on the type of insult. Therefore, these results suggest that different adhesion pathways are utilized during PMN recruitment in vivo in response to chemoattractants and IL-1.The selectin-mediated rolling of leukocytes along the endothelial cells is a prerequisite step followed by firm adhesion and extravasation into the inflamed tissue. This initial contact can be suppressed by sulphated polysaccharides. We have studied the effect of sulphated polysaccharides on the ultimate polymorphonuclear leukocyte (PMN) recruitment and plasma leakage in rabbit skin in response to intradermal injection of various inflammatory mediators. PMN infiltration evoked by various PMN chemoattractants (FMLP, C5a desArg, LTB