Id-1 induces cell invasiveness in immortalized epithelial cells by regulating cadherin switching and Rho GTPases.

Epithelial-mesenchymal transition (EMT), characterized by cadherin switching, contributes to cancer metastasis. Our recent study showed that Id-1 (inhibitor of differentiation-1) promotes metastasis in esophageal cancer cells, but whether the invasive and metastatic dynamics can be induced early in the carcinogenesis process is still unclear. Immortalization is regarded as the initial stage in the malignant transformation of normal cells. In this study, we investigated the role and mechanisms of Id-1 in inducing EMT and cell invasiveness in immortalized esophageal epithelial cells. We found that immortalized epithelial cells expressed higher endogenous levels of Id-1 compared with normal cells. Ectopic Id-1 expression inhibited the differentiation of immortalized esophageal epithelial cells and promoted cadherin switching, which was accompanied by increased adhesiveness to extracellular matrix, cell motility, migratory potential and matrix metalloproteinase-dependent invasiveness. GTPase activity assays showed that over-expression or short-hairpin RNA knockdown of Id-1 led to corresponding changes in Rac1 activity, whereas RhoA activity was significantly decreased with Id-1 depletion. Inhibitors targeting Rac1, RhoA, and Rho kinase suppressed the invasiveness of Id-1-expressing NE2-hTERT cells. Knockdown of N-cadherin in Id-1-over-expressing cells inhibited cell invasiveness and down-regulated RhoA activity. These data suggest that the Id-1-induced invasive potential may be regulated through the N-cadherin-RhoA axis and Rac1 activation.

Regulation of p63 expression in primary and immortalized nasopharyngeal epithelial cells.

Mutation of the p53 gene is a common event in human cancer. Interestingly, p53 mutation is uncommon in nasopharyngeal carcinoma (NPC). The DeltaNp63 has been postulated to have a dominant-negative effect on the function of the p53 gene and may play a role in the pathogenesis of nasopharyngeal carcinoma. Immortalization is a common property of cancer cells and is believed to be an early event in carcinogenesis. At present, the relationship between DeltaNp63 and immortalization is poorly understood. In this study, we defined the expression profile of p63 and its various isoforms in primary and immortalized nasopharyngeal epithelial cells. Also, we elucidated some events regulating the expression of p63. Elevated expression of p63 was generally detected in both primary and immortalized nasopharyngeal epithelial cells at their proliferation stage and the predominant isoform of p63 expressed was DeltaNp63alpha. p63 expression was suppressed upon cellular senescence of primary nasopharyngeal epithelial cells and induction of terminal differentiation in immortalized nasopharyngeal epithelial cells. Expression of DeltaNp63 alone was able to drive clonal proliferation in primary nasopharyngeal cells in culture while downregulation of DeltaNp63 induced cellular apoptosis. All these results support a role of DeltaNp63 in proliferation and immortalization which facilitates pathogenesis of nasopharyngeal carcinoma. TGFbeta and retinoic acid downregulated the expression of p63 in immortalized nasopharyngeal epithelial cells and may play a role in regulating differentiation in squamous epithelial cells with potential applications in prevention and treatment of nasopharyngeal carcinoma.

CD103+ intraepithelial lymphocytes--a unique population in microsatellite unstable sporadic colorectal cancer.

Colorectal cancers with microsatellite instability (MSI) typically show increased numbers of intraepithelial lymphocytes (IEL) in comparison to microsatellite stable (MSS) cancers. The aim of this study was to determine the phenotype of this unique lymphocyte population in MSI and MSS colorectal cancers. Twenty-four individuals with sporadic colorectal cancer (17 MSI, 7 MSS) were included in this study. Intraepithelial and stromal lymphocytes were detected using immunohistochemistry with anti-CD8 and anti-CD103 antibodies, and two observers independently quantified the numbers of lymphocytes. CD103+ (alpha E beta 7+) IELs detected within tumour tissue co-expressed CD8+ while the stromal lymphocytes were phenotypically heterogeneous, with respect to CD8+ and CD103+ expression. MSI colorectal cancers harboured increased numbers of CD8+ CD103+ IELs, as well as CD8+ CD103- and CD8+ CD103+ stromal lymphocytes, when compared with MSS colorectal cancers. CD103+ IELs were found at 27-fold greater numbers in the tumour epithelium than in normal epithelium from the same patient (P = 0.001, Wilcoxon matched pairs test). From our findings, we have proposed a mechanism for the homing of these alpha E beta 7+ lymphocytes to tumour tissue in MSI and MSS colorectal cancers.

Biodistribution of filamentous phage-Fab in nude mice.

In vivo panning of peptide libraries in mice has allowed the isolation of peptides which target the vasculature of specific organs. The application of this approach to phage displaying Fab fragments (phage-Fab) could lead to the isolation of antibodies which recognize novel tumor antigens. In this study, we have evaluated the biodistribution of phage-Fab in nude mice. Balb/c nude mice were injected intravenously with 10(9) TU of phage displaying the anti-colon cancer Fab c30.6. Blood samples were collected at nine time points over a period of 72 h and three groups of four mice were sacrificed at 4 min, 24 h and 72 h. Normal tissues (liver, colon, spleen, kidneys, lungs, skeletal muscle) and faeces were collected at these time points and the number of viable phage in each sample was determined. The distribution of phage in tissues was also examined by immunohistochemical analysis of paraffin-embedded tissues. Regression analysis of plasma kinetic data showed that the half-life and the volume of distribution of phage was 3.6 h and 1 ml, respectively. Phage uptake occurred predominantly in lungs, kidneys, spleen and liver. Relatively few phage were distributed to colon and muscle, and phage were eliminated from the circulation by 72 h. Immunohistochemical analysis showed phage to be mainly within the vasculature at 4 min, whereas notable phage extravasation was observed at 24 h and 72 h. In conclusion, this study provides information on the in vivo behavior of phage-Fab which will be useful in the design of in vivo panning strategies. By choosing appropriate time points for tissue collection, it may be possible to isolate novel Fabs against both intra- and extravascular targets.

Comparison of phage pIII, pVIII and GST as carrier proteins for peptide immunisation in Balb/c mice.

Carrier proteins are important for improving the efficiency of synthetic peptide vaccines. Recently, genetically-based systems such as filamentous phage display or glutathione S-transferase (GST)-fusion proteins have been employed for immunisation. Whilst these carrier systems can facilitate the evaluation of a potential vaccine by reducing the time and cost of production, their relative efficacy and the kinetics of the immune response to each carrier has not been directly compared. In this study, we have displayed the epitopes of the anti-ErbB-2 Mabs N12 (C531-A586, EP531) and N28 (T216-C235, EP216) on phage minor coat protein pIII, major coat protein pVIII and GST. Balb/c H-2(d) mice were immunised with the constructs and the sera were tested after the initial, the 3rd, 5th and 6th immunisations for an anti-peptide, an anti-ErbB-2 and an anti-carrier response. The specificity of the antibody response was also mapped using synthetic peptides. It was found that GST was the best of the three carriers, both in terms of the magnitude and the kinetics of the induced anti-peptide and anti-ErbB-2 response. Multiple (five) administrations of the immunogens were necessary to obtain a high titre of antibodies specific for ErbB-2. It was further noted that whilst an anti-EP531 response was induced using all three carriers, EP216 was not immunogenic irrespective of the carrier used. The lack of immunogenicity of EP216 implies it does not contain a H-2(d) T cell recognition site. All three carriers provide a useful system for vaccination and consequently facilitate the identification of T-cell epitopes in Balb/c inbred mice.

Epitope discovery using monoclonal antibodies and phage peptide libraries.

Phage display is a biological system which facilitates the cloning and rapid selection of peptides from large combinatorial libraries. In comparison to the chemical combinatorial approach, the advantages of phage display lie in its simplicity and replicability. While phage display has many diverse applications, this review will focus on the use of phage peptide libraries to discover epitopes recognised by monoclonal antibodies. As monoclonal antibodies are useful tools for the detection of proteins and for the investigation of molecular interactions, the identification of their epitopes will serve to elucidate the structure and function of proteins, as well as aid in the discovery of new drugs and the development of vaccines.

5 year statistics of animal and insect bites in Hong Kong.

Statistics of bites of patients in a rural district hospital in Hong Kong were accumulated over a 5 year period from 1984-1989. Analysis revealed a predominance of dog bites. Seasonal variations were noted for snake and insect bites, but not for dog bites. Comparison was made with some UK data.

Unlocking the potential of electronic health records for translational research. Findings from the section on bioinformatics and translational informatics.

OBJECTIVES: To review current excellent research and trend in the field of bioinformatics and translational informatics with direct application in the medical domain. METHOD: Synopsis of the articles selected for the IMIA Yearbook 2012. RESULTS: Six excellent articles were selected in this Yearbook's section on Bioinformatics and Translational Informatics. They exemplify current key advances in the use of patient information for translational research and health surveillance. First, two proof-of-concept studies demonstrated the cross-institutional and -geographic use of Electronic Health Records (EHR) for clinical trial subjects identification and drug safety signals detection. These reports pave ways to global large-scale population monitoring. Second, there is further evidence on the importance of coupling phenotypic information in EHR with genotypic information (either in biobank or in gene association studies) for new biomedical knowledge discovery. Third, patient data gathered via social media and self-reporting was found to be comparable to existent data and less labor intensive. This alternative means could potentially overcome data collection challenge in cohort and prospective studies. Finally, it can be noted that metagenomic studies are gaining momentum in bioinformatics and system-level analysis of human microbiome sheds important light on certain human diseases. CONCLUSIONS: The current literature showed that the traditional bench to bedside translational research is increasing being complemented by the reverse approach, in which bedside information can be used to provide novel biomedical insights.

Genome, and beyond. Findings from the section on bioinformatics.

OBJECTIVES: To summarize current excellent research in the field of bioinformatics, with an emphasis on those that have direct application in the medical domain. METHOD: Synopsis of the articles selected for the IMIA Yearbook 2011. RESULTS: The selection process for this yearbook's section on Bioinformatics results in six excellent articles highlighting the continuous progress towards a better understanding of human phenotype. Compared to the selection in Yearbook 2010, several key advancements can be noted. First, year 2010 marked the inaugural use of a complete human genome in a clinical context. This proof-of-principle study represents a large step towards personalized medicine. Second, there is a clear trend to understand diseases beyond the genome level, namely to include environmental and epigenetic information. Third, an innovative framework making use of the web to harness participant-driven genotype-phenotype information sets a new scene for conducting research in an era where social media plays an increasingly important role. CONCLUSIONS: The current literature showed that all pieces are now present to enable a much more comprehensive understanding of human diseases and traits, beyond the highly focused genetic or genomic studies seen previously.

Closing the genotype-phenotype gap. Findings from the section on bioinformatics.

OBJECTIVES: To summarize current excellent research in the field of bioinformatics. METHOD: Synopsis of the articles selected for the IMIA Yearbook 2010. RESULTS: The selection process for this yearbook's section on Bioinformatics results in five excellent articles highlighting the progress made in advancing the understanding of genotype-phenotype relationship, and their concrete application in clinical settings. First, next generation sequencing techniques have allowed the discovery of an ever larger number of genetic variations at a greater resolution, and methods were developed to ensure accurate data analysis. Second, innovative approaches were applied to gene expression data to allow its link to a wider phenotypic spectrum and to enhance its use for disease understanding. Third, there is a notable trend in visualizing diseases as network rather than individual entities, and this has provided new insights for disease interpretation. The progress mentioned above is further aided by continual development in bio-ontologies which provide means for semantic, and thus phenotype, comparison. CONCLUSIONS: The current literature showed a tightening link between genotype and phenotype, placing us one step closer to a better disease classification, patient stratification as well as the development of personalized medicine.

Accelerating knowledge discovery through community data sharing and integration.

OBJECTIVES: To summarize current excellent research in the field of bioinformatics. METHOD: Synopsis of the articles selected for the IMIA Yearbook 2009. RESULTS: The selection process for this yearbook's section on Bioinformatics results in six excellent articles highlighting several important trends First, it can be noted that Semantic Web technology continues to play an important role in heterogeneous data integration. Novel applications also put more emphasis on its ability to make logical inferences leading to new insights and discoveries. Second, translational research, due to its complex nature, increasingly relies on collective intelligence made available through the adoption of community-defined protocols or software architectures for secure data annotation, sharing and analysis. Advances in systems biology, bio-ontologies and text-ming can also be noted. CONCLUSIONS: Current biomedical research gradually evolves towards an environment characterized by intensive collaboration and more sophisticated knowledge processing activities. Enabling technologies, either Semantic Web or other solutions, are expected to play an increasingly important role in generating new knowledge in the foreseeable future.

The promise of systems biology in clinical applications. Findings from the Yearbook 2008 Section on Bioinformatics.

OBJECTIVES: To summarize current excellent research in the field of bioinformatics. METHOD: Synopsis of the articles selected for the IMIA Yearbook 2008. RESULTS: Current research in the field of Bioinformatics shows that the emergent field of systems biology is starting to offer innovative solutions to clinically-relevant problems. The approach used can be top-down, where models are created based on hypotheses to describe previously unexplained phenomena and then tested against experimental or clinical evidence. It can also be bottom-up, where mathematical models are built by harnessing existing information about the components (e.g. protein entities, interaction networks) of a system in order to discern critical system-level mechanisms that can be relevant for clinical applications. Progress in this area is aided by the ongoing development in data integration and management, whose current focus is on better semantics for facilitating translational research. Advances in other important areas, such as microarray technology, text mining and ontologies, are also noted. CONCLUSIONS: The best paper selection of articles on bioinformatics gives examples of original research that exploits mathematical modeling to tackle medical problems and of improved semantic solutions for data integration. As new directions are explored and the technologies mature, these approaches are expected to be increasingly integrated into clinical practice.

Id1 overexpression induces tetraploidization and multiple abnormal mitotic phenotypes by modulating aurora A.

The basic helix-loop-helix transcription factor, Id1, was shown to induce tetraploidy in telomerase-immortalized nasopharyngeal epithelial cells in this study. Using both transient and stable Id1-expressing cell models, multiple mitotic aberrations were detected, including centrosome amplification, binucleation, spindle defects, and microtubule perturbation. Many of these abnormal phenotypes have previously been reported in cells overexpressing Aurora A. Further experiments showed that Id1 could stabilize Aurora A, whereas knocking down Aurora A expression in Id1-expressing cells could rescue some of the mitotic defects. The mechanisms by which Aurora A could be modulated by Id1 were explored. DNA amplification of the Aurora A locus was not involved. Id1 could only weakly activate the transcriptional activity of the Aurora A promoter. We found that Id1 overexpression could affect Aurora A degradation, leading to its stabilization. Aurora A is normally degraded from mitosis exit by the APC/C(Cdh1)-mediated proteasomal proteolysis pathway. Our results revealed that Id1 and Cdh1 are binding partners. The association of Id1 and Cdh1 was found to be dependent on the canonical destruction box motif of Id1, the increased binding of which may compete with the interaction between Cdh1 and Aurora A, leading to stabilization of Aurora A in Id1-overexpressing cells.

Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase.

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase-immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16(INK4A) locus and downregulation of RASSF1A expression. The deletion of the p16(INK4A) locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array-based CGH revealed a gain of a 17q21-q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16(INK4A) locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF-kappaB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development.

Electrohydraulic lithotripsy of bladder stones. A Hong Kong experience.

Forty transurethral electrohydraulic lithotripsy procedures for bladder stones have been done. The rigid cystoscope was adequate in most cases but the rigid nephroscope was advantageous in large stones because of its larger bore and range of accessories. Use of the flexible cystoscope for stone extraction was difficult but it could be used with a few friable stones of less than 1 cm or for follow-up.

Quantitative analysis of the accuracy of linear array transrectal ultrasound in measurement of the prostate.

In 61 prostate autopsy specimens, linear array transrectal ultrasound and electronic vernier calipers were used to measure 3 maximal diameters along the longitudinal, anterior-posterior and transverse axes. The results were comparable. We assessed the reliability of empirical formulae, derived from various combinations of these 3 diameters, for the prediction of prostatic volume and weight. Formulae using all 3 diameters were accurate. The combination of longitudinal and anterior-posterior diameters, which was used clinically for prostate measurement, was less accurate.

Sigmoidoscopy with flexible electronic endoscopy: a preliminary experience in Hong Kong.

Eighty-two cases of electronic flexible sigmoidoscopy were performed from October 1984 to October 1985 in a rural hospital in Hong Kong. The scope is a relatively new type of flexible endoscope, which has no optical fibre bundle for imaging. The image is taken with a solid state television camera and relayed via a videoprocessor to a television monitor. The image quality and ease of handling were satisfactory. Acceptance by the endoscopy team and the patients was good. The differences in comparison with conventional fibrescopes and television systems are discussed.

Evaluation of different lymphoid tissue sources for the construction of human immunoglobulin gene libraries.

BACKGROUND: Phage display technology allows the isolation of novel human monoclonal antibodies. The technology relies on the construction of a recombinant antibody library and its display on phage particles. The quality of an antibody library is affected by several factors including the size, diversity and source of immunoglobulin genes. OBJECTIVE: The aim of the project was to determine the best tissue source for the construction of antibody libraries. STUDY DESIGN: Three tissue sources were used in this study: peripheral blood mononuclear cells from a healthy donor, Epstein-Barr virus (EBV) transformed peripheral blood mononuclear cells and lymph node tissue from individuals with breast cancer. The quality of each tissue source was assessed using two criteria: (1) the number of mature and activated B cells in each source; (2) the amount of immunoglobulin heavy and light chain genes amplifiable by polymerase chain reaction (PCR). RESULTS: EBV-transformed peripheral blood mononuclear cells and lymph node tissue were shown to contain more B cells than peripheral blood mononuclear cells. A relatively larger amount of immunoglobulin gene products could be amplified from EBV-transformed peripheral blood mononuclear cells and the lymph node. However, immunoglobulin containing gamma 1 chains could not be amplified from EBV-transformed mononuclear cells, and the resultant pattern of gene amplification suggests a possible selection bias. CONCLUSION: This study indicates that among the three tissue sources examined, lymph node tissue is the most suitable source for the construction of antibody libraries.