A computer image analysis system for microvessel density measurement in solid tumours

Microvessel density (MVD) is a widely used surrogate measure of angiogenesis in pathological specimens and tumour models. Measurement of MVD can be achieved by several methods. Automation of counting methods aims to increase the speed, reliability and reproducibility of these techniques. The image analysis system described here enables MVD measurement to be carried out with minimal expense in any reasonably equipped pathology department or laboratory. It is demonstrated that the system translates easily between tumour types which are suitably stained with minimal calibration. The aim of this paper is to offer this technique to a wider field of researchers in angiogenesis.

Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor

Gemcitabine is indicated in combination with cisplatin as first-line therapy for solid tumours including non-small cell lung cancer (NSCLC), bladder cancer and mesothelioma. Gemcitabine is an analogue of pyrimidine cytosine and functions as an anti-metabolite. Structurally, however, gemcitabine has similarities to 5-aza-2-deoxycytidine (decitabine/Dacogen®), a DNA methyltransferase inhibitor (DNMTi). NSCLC, mesothelioma and prostate cancer cell lines were treated with decitabine and gemcitabine. Reactivation of epigenetically silenced genes was examined by RT-PCR/qPCR. DNA methyltransferase activity in nuclear extracts and recombinant proteins was measured using a DNA methyltransferase assay, and alterations in DNA methylation status were examined using methylation-specific PCR (MS-PCR) and pyrosequencing. We observe a reactivation of several epigenetically silenced genes including GSTP1, IGFBP3 and RASSF1A. Gemcitabine functionally inhibited DNA methyltransferase activity in both nuclear extracts and recombinant proteins. Gemcitabine dramatically destabilised DNMT1 protein. However, DNA CpG methylation was for the most part unaffected by gemcitabine. In conclusion, gemcitabine both inhibits and destabilises DNA methyltransferases and reactivates epigenetically silenced genes having activity equivalent to decitabine at concentrations significantly lower than those achieved in the treatment of patients with solid tumours. This property may contribute to the anticancer activity of gemcitabine.

Targeting histone deacetylases for the treatment of immune, endocrine and metabolic disorders

The 'histone code' is a well-established hypothesis describing the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as histone acetyltransferases or HATs, and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. The proinflammatory environment is increasingly being recognised as a critical element for both degenerative diseases and cancer. The present review will discuss the current knowledge surrounding the clinical potential & current development of histone deacetylases for the treatment of diseases for which a proinflammatory environment plays important roles, and the molecular mechanisms by which such inhibitors may play important functions in modulating the proinflammatory environment. © 2009 Bentham Science Publishers Ltd.

Prostate radiotherapy treatment plan quality : effects of hip prostheses

Introduction This study aimed to examine the geometric and dosimetric results when radiotherapy treatment plans are designed for prostate cancer patients with hip prostheses. Methods Ten EBRT treatment plans for localised prostate cancer, in the presence of hip prostheses, were analysed and compared with a reference set of 196 treatment plans for localised prostate cancer in patients without prostheses. Crowe et al.’s TADA code [1] was used to extract treatment plan parameters and evaluate doses to target volumes and critical structures against recommended goals [2] and constraints [3, 4]. Results The need to avoid transmitting the radiation beam through the hip prostheses limited the range of gantry angles available for use in both the rotational (VMAT) and the non-rotational (3DCRT and IMRT) radiotherapy treatments. This geometric limitation (exemplified in the VMAT data shown in Fig. 1) reduced the overall quality of the treatment plans for patients with prostheses compared to the reference plans. All plans with prostheses failed the PTV dose homogeneity requirement [2], whereas only 4 % of the plans without prostheses failed this test. Several treatment plans for patients with hip prostheses also failed the QUANTEC requirements that less than 50 % of the rectum receive 50 Gy and less than 35 % of the rectum receive 60 Gy to keep the grade 3 toxicity rate below 10 % [3], or the Hansen and Roach requirement that less than 25 % of the bladder receive 75 Gy [4]. Discussion and conclusions The results of this study exemplify the difficulty of designing prostate radiotherapy treatment plans, where beams provide adequate doses to targeted tissues while avoiding nearby organs at risk, when the presence of hip prostheses limits the available treatment geometries. This work provides qualitative evidence of the compromised dose distributions that can result, in such cases.

Mesenchymal to epithelial transition in development and disease

Cellular plasticity is fundamental to embryonic development. The importance of cellular transitions in development is first apparent during gastrulation when the process of epithelial to mesenchymal transition transforms polarized epithelial cells into migratory mesenchymal cells that constitute the embryonic and extraembryonic mesoderm. It is now widely accepted that this developmental pathway is exploited in various disease states, including cancer progression. The loss of epithelial characteristics and the acquisition of a mesenchymal-like migratory phenotype are crucial to the development of invasive carcinoma and metastasis. However, given the morphological similarities between primary tumour and metastatic lesions, it is likely that tumour cells re-activate certain epithelial properties through a mesenchymal to epithelial transition (MET) at the secondary site, although this is yet to be proven. MET is also an essential developmental process and has been extensively studied in kidney organogenesis and somitogenesis. In this review we describe the process of MET, highlight important mediators, and discuss their implication in the context of cancer progression.

A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery

The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9 ppt which showed best growth performance. Total sequence data generated was 467.8 Mbp, consisting of 4,116,424 reads with an average length of 112 bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

GATA3 : a promising marker for metastatic breast carcinoma in serous effusion specimens

Background The utility of GATA3 as a marker for metastatic breast carcinoma in serous effusion specimens was investigated. Methods Cell block sections from 74 serous effusion specimens (32 ascitic, 2 pericardial and 40 pleural fluids) were stained with an anti-GATA3 murine monoclonal antibody. The specimens included 62 confirmed metastatic carcinomas from breast (30), female genital tract (13), gastrointestinal tract (7), lung adenocarcinoma (9), pancreas (1), kidney (1) and bladder (1). The breast carcinoma cases included 15 ductal carcinomas, 8 lobular carcinomas and in 7 the histology sub-type was not available. Twelve cases containing florid reactive mesothelial cells were also stained. The breast carcinoma cases were also stained for mammaglobin and GCDFP-15 to compare sensitivity with GATA3. Results Positive nuclear staining for GATA3 was present in 90% (27/30) of metastatic breast carcinoma specimens. All non-breast metastatic carcinomas tested were negative with the exception of the single case of metastatic urothelial carcinoma. No staining was observed in any of the benign reactive cases or in benign mesothelial cells present in the malignant cell block preparations. Two cases showed weak positivity of benign lymphoid cells. Staining results were unambiguous as all positive cases showed intense nuclear staining in >50% of tumor cells. Mammaglobin (57%; 17/30) and GCDFP-15 (33%; 10/30) were less sensitive markers of breast carcinoma. If used in a panel, mammaglobin and GCFP-15 staining would have identified only one additional case to those stained with GATA3. Conclusions GATA3 may be a useful addition to immunostaining panels for serous effusion specimens when metastatic breast carcinoma is a consideration.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

The automation of radiotherapy treatment planning assessment

Aim The assessment of treatment plans is an important component in the education of radiation therapists. The establishment of a grade for a plan is currently based on subjective assessment of a range of criteria. The automation of assessment could provide a number of advantages including faster feedback, reduced chance of human error, and simpler aggregation of past results. Method A collection of treatments planned by a cohort of 27 second year radiation therapy students were selected for quantitative evaluation. Treatment sites included the bladder, cervix, larynx, parotid and prostate, although only the larynx plans had been assessed in detail. The plans were designed with the Pinnacle system and exported using the DICOM framework. Assessment criteria included beam arrangement optimisation, volume contouring, target dose coverage and homogeneity, and organ-at-risk sparing. The in-house Treatment and Dose Assessor (TADA) software1 was evaluated for suitability in assisting with the quantitative assessment of these plans. Dose volume data were exported in per-student and per-structure data tables, along with beam complexity metrics, dose volume histograms, and reports on naming conventions. Results The treatment plans were exported and processed using TADA, with the processing of all 27 plans for each treatment site taking less than two minutes. Naming conventions were successfully checked against a reference protocol. Significant variations between student plans were found. Correlation with assessment feedback was established for the larynx plans. Conclusion The data generated could be used to inform the selection of future assessment criteria, monitor student development, and provide useful feedback to the students. The provision of objective, quantitative evaluations of plan quality would be a valuable addition to not only radiotherapy education programmes but also for staff development and potentially credentialing methods. New functionality within TADA developed for this work could be applied clinically to, for example, evaluate protocol compliance.

Role of capsule and O antigen in the virulence of uropathogenic Escherichia coli

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC) the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.

Functional heterogeneity of the UpaH autotransporter protein from Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTI). To cause a UTI, UPEC must adhere to the epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is mediated primarily by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contributes to UPEC-mediated UTI is autotransporter (AT) proteins. AT proteins possess a range of virulence properties, such as adherence, aggregation, invasion, and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large adhesin-involved-in-diffuse-adherence (AIDA-I)-type AT protein that contributes to biofilm formation and bladder colonization. In this study we characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaHCR) and a hydrophobic region (UpaHHR). Deletion of these domains reduced biofilm formation but not the binding to ECM proteins. Despite variation in the upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and functions of the UpaH AT protein.

Identification of genes important for growth of asymptomatic bacteriuria Escherichia coli in urine

Escherichia coli is the most important etiological agent of urinary tract infections (UTIs). Unlike uropathogenic E. coli, which causes symptomatic infections, asymptomatic bacteriuria (ABU) E. coli strains typically lack essential virulence factors and colonize the bladder in the absence of symptoms. While ABU E. coli can persist in the bladder for long periods of time, little is known about the genetic determinants required for its growth and fitness in urine. To identify such genes, we have employed a transposon mutagenesis approach using the prototypic ABU E. coli strain 83972 and the clinical ABU E. coli strain VR89. Six genes involved in the biosynthesis of various amino acids and nucleobases were identified (carB, argE, argC, purA, metE, and ilvC), and site-specific mutants were subsequently constructed in E. coli 83972 and E. coli VR89 for each of these genes. In all cases, these mutants exhibited reduced growth rates and final cell densities in human urine. The growth defects could be complemented in trans as well as by supplementation with the appropriate amino acid or nucleobase. When assessed in vivo in a mouse model, E. coli 83972carAB and 83972argC showed a significantly reduced competitive advantage in the bladder and/or kidney during coinoculation experiments with the parent strain, whereas 83972metE and 83972ilvC did not. Taken together, our data have identified several biosynthesis pathways as new important fitness factors associated with the growth of ABU E. coli in human urine.

Molecular characterization of the EhaG and UpaG trimeric autotransporter proteins from pathogenic Escherichia coli

Trimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagic Escherichia coli (EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenic E. coli (UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from several E. coli genomes revealed grouping of the proteins in clades almost exclusively represented by distinct E. coli pathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in an hns isogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage : genome analysis and virulence mechanisms

Escherichia coli strains causing urinary tract infection (UTI) are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC). The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI.

Molecular characterization of UpaB and UpaC, two new autotransporter proteins of uropathogenic Escherichia coli CFT073

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of UPEC are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. The genome-sequenced prototype UPEC strain CFT073 contains 11 putative AT-encoding genes. In this study, we have performed a detailed molecular characterization of two closely related AT adhesins from CFT073: UpaB (c0426) and UpaC (c0478). PCR screening revealed that the upaB and upaC AT-encoding genes are common in E. coli. The upaB and upaC genes were cloned and characterized in a recombinant E. coli K-12 strain background. This revealed that they encode proteins located at the cell surface but possess different functional properties: UpaB mediates adherence to several ECM proteins, while UpaC expression is associated with increased biofilm formation. In CFT073, upaB is expressed while upaC is transcriptionally repressed by the global regulator H-NS. In competitive colonization experiments employing the mouse UTI model, CFT073 significantly outcompeted its upaB (but not upaC) isogenic mutant strain in the bladder. This attenuated phenotype was also observed in single-challenge experiments, where deletion of the upaB gene in CFT073 significantly reduced early colonization of the bladder.