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Biomedical subjects

R L Ward

Publications and source records attributed to R L Ward.

At least 19 recordsLinked to original sources
PubMed2003

CpG island methylation is a common finding in colorectal cancer cell lines.

Tumour cell lines are commonly used in colorectal cancer (CRC) research, including studies designed to assess methylation defects. Although many of the known genetic aberrations in CRC cell lines have been comprehensively described, no studies have been performed on their methylation status. In this study, 30 commonly used CRC cell lines as well as seven primary tumours from individuals with hereditary nonpolyposis colorectal cancer (HNPCC) were assessed for methylation at six CpG islands known to be hypermethylated in colorectal cancer: hMLH1, p16, methylated in tumour (MINT-)-1, -2, -12 and -31. The cell lines were also assessed for microsatellite instability (MSI), ploidy status, hMLH1 expression, and mutations in APC and Ki-ras. Methylation was frequently observed at all examined loci in most cell lines, and no differences were observed between germline-derived and sporadic cell lines. Methylation was found at MINT 1 in 63%, MINT 2 in 57%, MINT 12 in 71%, MINT 31 in 53%, p16 in 71%, and hMLH1 in 30% of cell lines. Overall only one cell line, SW1417, did not show methylation at any locus. Methylation was found with equal frequency in MSI and chromosomally unstable lines. MSI was over-represented in the cell lines relative to sporadic CRC, being detected in 47% of cell lines. The rate of codon 13 Ki-ras mutations was also over three times that expected from in vivo studies. We conclude that CpG island hypermethylation, whether acquired in vivo or in culture, is a ubiquitous phenomenon in CRC cell lines. We suggest that CRC cell lines may be only representative of a small subset of real tumours, and this should be taken into account in the use of CRC cell lines for epigenetic studies.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Caring for the chronically ill: a clinic for final-year medical students.

In 2000 the Diabetes Centre and the Medical Oncology Department of St Vincent's Hospital, Sydney established a joint clinic for the teaching of final-year medical students. The clinic was established amid concerns that hospitals are increasingly focused on acute care and have few resources available for teaching about chronic conditions. The clinic aimed to improve both patient care and learning opportunities by engaging students in useful activities with chronically ill patients. The students met with their patients regularly to monitor progress, adjust medication (under supervision) and arrange support services. The students and staff from both units met once a week in a coordination meeting where cases were reviewed and learning issues discussed. Students had informal interactions with medical and allied health staff at other times. Overall the clinic provided a rich learning environment for students with a focus on the development of the integrated skills required in the care of chronically ill people, rather than on the specific medical disciplines involved.

Adult↗
PubMed2002

The null oncogene hypothesis and protection from cancer.

Tumour progression involves the inactivation of tumour suppressor genes and the activation of proto-oncogenes. Inactivation of both copies of a tumour suppressor gene is required for carcinogenesis, while germline deletion or inactivation of one copy results in an increase in the risk of cancer and is responsible for many of the known hereditary cancer syndromes. In contrast, activation of only one copy of a proto-oncogene is required for carcinogenesis. Germline deletion or inactivation of one copy of a proto-oncogene halves the risk of activation at this locus. We propose that studies of high risk cancer patients will show such "null oncogene" mutations.

Alleles↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2001

Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas.

BACKGROUND: Microsatellite instability (MSI) is seen in 10%-15% of sporadic colorectal cancers mostly in the right colon, but the precursors of cancers with MSI remain unknown. We examined whether sporadic cancers with MSI arise from pre-existing benign proliferative lesions (such as hyperplastic polyps or serrated adenomas [together denoted as "serrated polyps"]). METHODS: The frequency of benign epithelial lesions (serrated polyps and conventional adenomas) was determined by histologic review of resection specimens from individuals (n = 29) with sporadic colorectal cancer with MSI and from a matched control group (n = 29) with cancer showing microsatellite stability (MSS). MSI status, expression of mismatch repair enzyme (product of the human mut-L homologue 1 [hMLH1] gene), and hMLH1 gene promoter methylation in the benign lesions were determined. Data were analyzed by the chi-square test, by Wilcoxon's rank-sum test, and by conditional logistic regression as appropriate, and a two-sided probability less than.05 was considered to be statistically significant. RESULTS: Individuals with cancers showing MSI were more likely to harbor at least one serrated polyp than individuals with cancers showing MSS (odds ratio = 4.0; 95% confidence interval = 1.1 to 14.2; P =.03), but the frequency of conventional adenomas was the same in both groups (P =.52, Mann-Whitney test). Loss of hMLH1 protein expression was seen in lesions from 10 of 13 patients with MSI, but no loss was seen in lesions from four patients with MSS (P =.02, Fisher's exact test). Loss of hMLH1 protein expression was associated with MSI in assessable lesions. The hMLH1 promoter was methylated in all assessable serrated polyps from patients with cancers showing MSI but in none of the lesions from patients with MSS cancers. CONCLUSIONS: Some right-sided hyperplastic polyps may give rise to sporadic colorectal carcinomas with MSI. Methylation of the hMLH1 gene promoter within neoplastic cell subpopulations may be a critical step in the progression to carcinoma. The frequency with which benign lesions progress to cancer with MSI is unknown.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Identification of epitope regions recognized by tumor inhibitory and stimulatory anti-ErbB-2 monoclonal antibodies: implications for vaccine design.

The self-oncoprotein ErbB-2 is overexpressed in a number of malignancies. The presence of endogenous anti-ErbB-2 Ab and T cell immune responses to this protein in cancer patients has made ErbB-2 an attractive target for active immunization. However, the finding that murine anti-ErbB-2 Abs can have stimulatory, inhibitory, or no effects on cancer cell growth suggests that an inappropriately induced immune response may have an adverse effect. To ensure the induction of a beneficial Ab response, it is important to identify the epitopes recognized by these Abs. In this study we have used phage-displayed ErbB-2 gene fragment libraries and synthetic peptides to epitope-map a panel of anti-ErbB-2 mAbs. The epitopes of three mAbs, N12, N28, and L87, were successfully located to C531-A586, T216-C235, and C220-C235 of ErbB-2, respectively. It was found that while N12 inhibited tumor cell proliferation, N28 stimulated the proliferation of a subset of breast cancer cell lines overexpressing ErbB-2. The peptide region recognized by N12, (C531-A586; EP531), was used as an immunogen to selectively induce an inhibitory immune response in mice. Mice immunized with the GST fusion peptide (GST-EP531) recognized the peptide region EP531 as well as native ErbB-2. More importantly, Igs purified from mouse sera were able to inhibit up to 85% of tumor cell proliferation. In conclusion, our study provides direct evidence of the function-epitope relationship of anti-ErbB-2 Abs and also emphasizes the value of inducing a potent tumor inhibitory polyclonal Ab response by rationally selecting regions of ErbB-2 used for immunization.

Amino Acid Sequence↗
PubMed2001

Role for T cell-independent B cell activity in the resolution of primary rotavirus infection in mice.

We examined the importance of T cell-independent B cell activity in the resolution of primary murine (EDIM) rotavirus infection in adult mice. We showed that Rag 1 (C57BL / 6 background) and Rag 2 (BALB / c background) knockout mice, which lack both T and B cells, chronically shed high levels of rotavirus Ag in stool samples following oral inoculation. However, nude mice (BALB / c and C57BL / 6 backgrounds) and alpha beta TCR knockout mice (C57BL / 6 background) chronically shed 100-fold lower levels of virus in stool samples. Thus, B cells appeared to sharply reduce the level of chronic rotavirus shedding by a T cell-independent mechanism. C57BL / 6 mice depleted of CD4(+) cells or both CD4(+) and CD8(+) cells were also unable to resolve primary rotavirus infection but chronically shed equally low levels of rotavirus Ag in stool samples, whereas mice depleted of only CD8(+) cells resolved infection. Similar results were obtained with a second rotavirus strain (EC(w)) in which virus was shed chronically in stool samples at low levels in alpha beta TCR knockout mice and at high levels in Rag 1 knockout mice. Virus-specific intestinal IgA was readily detected in mice lacking thymic T cells and alpha beta T cells and in mice depleted of CD4(+) cells but levels were 95 % reduced in comparison to immunocompetent control mice. Together, these results show that B cells lacking CD4(+) T cell help have the capacity to substantially reduce rotavirus shedding, possibly through the production of T cell-independent IgA to rotavirus, but full resolution requires alpha beta T cells.

Animals↗
PubMed2001

Microsatellite-stable diploid carcinoma: a biologically distinct and aggressive subset of sporadic colorectal cancer.

Chromosomal instability and microsatellite instability represent the major pathways for colorectal cancer (CRC) progression. However, a significant percentage of CRC shows neither pattern of instability, and thus represents a potentially distinctive form of the disease. Flow cytometry was used to determine the degree of DNA aneuploidy in 46 consecutive sporadic colorectal cancers. Microsatellite status was determined by PCR amplification using standard markers, while immunostaining was used to examine the expression of p53. K- ras status was determined by restriction-mediated PCR assay. Twenty-five (54%) tumours were aneuploid, 14 (30%) were diploid and microsatellite-stable and seven (15%) were diploid and microsatellite-unstable. Tumours with microsatellite instability were more likely to be right sided, to occur in women and to be associated with an improved survival. Aneuploid tumours were significantly more common in men and were likely to be left sided. The diploid microsatellite-stable (MSS) tumours did not show a sex or site predilection, but were strongly associated with the presence of metastatic disease at the time of diagnosis. Our data suggests that diploid, MSS tumours represent a biologically and phenotypically distinct subset of colorectal carcinoma, and one that is associated with the early development of metastases. We suggest that the genetic stability that characterizes these tumours may favour the maintenance of an invasive phenotype, and thus facilitate disease progression. These findings may have important implications for treatment options in this disease subset.

Aged↗
PubMed2001

Safety and immunogenicity of tetravalent rhesus-based rotavirus vaccine in Bangladesh.

BACKGROUND: Rotavirus is the most common cause of severe gastroenteritis among children worldwide. OBJECTIVES: To compare the safety, immunogenicity and shedding patterns of rhesus rotavirus (RRV)-tetravalent vaccine vs. placebo among infants in rural Bangladesh. METHODS: A double blinded, placebo-controlled trial was conducted in which infants (n = 120) were randomly assigned to receive three doses of either vaccine or placebo administered at approximately 6, 10 and 14 weeks of age together with routine immunizations. Data on possible adverse effects of vaccinations were collected daily for 7 days after each dose. Stool samples were collected after each dose, and serum samples were obtained before the first and after the third vaccination. RESULTS: Fever (> or = 38 degrees C), as measured by study assistants, was noted more frequently among vaccinees (15%) than among placebo recipients (2%) during the 7 days after vaccination but was not reported more frequently by parents of vaccinees vs. placebo recipients. Overall 87% of vaccinees had an antibody response (measured by IgA or anti-RRV-neutralizing antibodies) after vaccination compared with 32% of placebo recipients. Rates of seroconversion were higher among subjects with lower levels of prevaccination antibodies and those who shed rotavirus after vaccination. Vaccine strain viruses were detected in stools from placebo vaccine recipients who had evidence of IgA seroconversion. CONCLUSIONS: In this population RRV-tetravalent vaccine was comparably immunogenic and safe as in trials conducted in developed countries, where this vaccine has been proved effective in preventing severe rotavirus diarrhea. These data support continued evaluation of rotavirus vaccines in developing countries.

Antibodies, Viral↗
PubMed2001

Identification and characterisation of a platelet GPIb/V/IX-like complex on human breast cancers: implications for the metastatic process.

The glycoprotein (GP) Ib /V/IX receptor complex is an important adhesion molecule, originally thought to be unique to the megakaryocytic lineage. Recent evidence now indicates that GPIb /V/IX may be more widely expressed. In this study we report the presence of all subunits of the complex on four breast cancer cell lines, and 51 / 80 primary breast tumours. The surface expression of GPIb /V/IX was confirmed by flow cytometry, and by immunoprecipitation of biotin surface-labelled tumour cells. Western blotting of cell lysates under reducing conditions revealed that tumour cell-GPIb alpha had a relative molecular weight of 95 kDa as compared to 135 kDa on platelets. Despite the discrepant protein size, molecular analyses on the tumour cell-GPIb alpha subunit using RT-PCR and DNA sequencing revealed 100% sequence homology to platelet GPIb alpha. Tumour cell-GPIb /V/IX was capable of binding human von Willebrand factor (vWf), and this binding caused aggregation of tumour cells in suspension. Tumour cells bound to immobilised vWf in the presence of EDTA and demonstrated prominent filapodial extensions indicative of cytoskeletal reorganisation. Furthermore, in a modified Boyden chamber assay, prior exposure to vWf or a GPIb alpha monoclonal antibody, AK2, enhanced cell migration. The presence of a functional GPIb /V/IX-like complex in tumour cells suggests that this complex may participate in the process of haematogenous breast cancer metastasis.

Blotting, Western↗
PubMed2001

The application of phage display in allergy research: characterization of IgE, identification of allergens and development of novel therapeutics.

The ability to display IgE antibody fragments, allergens and peptides upon filamentous phage has increasingly been used in allergy research. This technique offers the opportunity to isolate and produce IgE antibody fragments specific for allergens. These antibody fragments can then be used to address fundamental issues regarding the development of IgE antibodies in allergic patients, at both the molecular and structural level. Random peptide display has greatly facilitated the discovery of epitopes recognized by serum IgE antibodies from allergic patients, and it is a definitive tool for investigating the IgE-epitope interaction. Whole allergens can also be displayed on phage. Selecting IgE binding phage from diverse cDNA libraries of allergens has assisted in the identification of new allergens and provided a source of purified allergens for the diagnosis of allergic diseases. Finally, phage display of antibody fragments and random peptides is currently providing a means by which the IgE antibody can be targeted as a potential treatment for allergy. This review highlights several studies which have utilized phage display methodology in the area of allergy research, and it discusses how the therapeutic potential of this approach may be exploited.

Allergens↗
PubMed2001

Isolation of human antibodies against the central DNA binding domain of p53 from an individual with colorectal cancer using antibody phage display.

PURPOSE: Serum anti-p53 antibodies are detected in approximately 30% of patients with cancer, and most of these antibodies are directed against the NH2- and COOH-terminal domains of p53. The aim of this study was to identify individuals with serum reactivity against the central and COOH-terminal domains of p53 and to isolate and characterize these recombinant human antibodies. EXPERIMENTAL DESIGN: The serum of individuals with colorectal cancer was screened for the presence of anti-p53 antibodies. Antibody phage display libraries were constructed from four immunoreactive individuals, and the libraries were panned against the central and COOH-terminal domains of p53. RESULTS: An antibody fragment (1159.8) that was specific for the whole molecule as well as the central domain was isolated from one of the libraries. The serum from the original individual inhibited the binding of this Fab fragment to p53, thus indicating that the antibody reflected the specificity of the in vivo immune response. The VL and VH genes from Fab 1159.8 matched germ-line genes from the VH3 and VK2 families, respectively. Competition analysis with monoclonal antibodies showed that Fab binding could be inhibited most effectively with DO11 and, to a lesser extent, Pab240, indicating an epitope within or adjacent to residues 181-190 of p53. CONCLUSIONS: The successful isolation of this human anticentral domain Fab provides additional insight into the nature and specificity of the tumor-specific immune response. This antibody could be used as a reagent for functional studies of p53, or it may be a candidate for use as an anticancer idiotypic vaccine.

Adult↗
PubMed2000

Molecular characterization of serotype G9 rotavirus strains from a global collection.

Between 1992 and 1998, serotype G9 human rotavirus (RV) strains have been detected in 10 countries, including Thailand, India, Brazil, Bangladesh, Malawi, Italy, France, the United States, the United Kingdom, and Australia, suggesting the possible emergence of the fifth common serotype worldwide. Unlike the previously characterized reference G9 strains (i.e., WI61 and F45), the recent G9 isolates had a variety of gene combinations, raising questions concerning their origin and evolution. To identify the progenitor strain and examine the on-going evolution of the recent G9 strains, we characterized by genetic and antigenic analyses 16 isolates obtained from children with diarrhea in India, Bangladesh, the United States, and Malawi. Specifically, we sequenced their VP7 and NSP4 genes and compared the nucleotide (nt) and deduced amino acid sequences with the reference G9 strains. To identify reassortment, we examined the products of five gene segments; VP4, VP7, and NSP4 genotypes (genes 4, 9, and 10); subgroups (gene 6); electropherotypes (gene 11); and the genogroup profiles of all of the recent G9 isolates. Sequence analysis of the VP7 gene indicated that the recent U.S. P[6],G9 strains were closely related to the Malawian G9 strains (>99% nt identity) but distinct from G9 strains of India ( approximately 97% nt identity), Bangladesh ( approximately 98% nt identity), and the reference strains ( approximately 97% nt identity). Phylogenetic analysis identified a single cluster for the U.S. P[6],G9 strains that may have common progenitors with Malawian P[6],G9 strains whereas separate lineages were defined for the Indian, Bangladeshi, and reference G9 strains. Northern hybridization results indicated that all 11 gene segments of the Malawian P[6],G9 strains hybridized with a probe derived from a U.S. strain of the same genotype and may have the same progenitor, different from the Indian G9 strains, whereas the Bangladesh strains may have evolved from the U.S. G9 progenitors. Overall, our findings suggest that much greater diversity among the newly identified G9 strains has been generated by reassortment between gene segments than through the accumulation of mutations in a single gene.

Antigens, Viral↗
PubMed2000

Colorectal carcinomas arising in the hyperplastic polyposis syndrome progress through the chromosomal instability pathway.

The hyperplastic polyposis syndrome is characterized by the presence within the colon of multiple large hyperplastic polyps. We describe a case of hyperplastic polyposis syndrome associated with two synchronous carcinomas, one of which arises within a pre-existing hyperplastic lesion. Comparative genomic hybridization was used to determine genetic changes in both carcinomas and several associated hyperplastic lesions. Microsatellite analysis at five loci was performed on carcinomas and representative hyperplastic polyps, and p53 status was analyzed by immunohistochemistry. Both carcinomas showed multiple genetic aberrations, including high level gains of 8q and 13q, and loss of 5q. These changes were not seen in the hyperplastic polyps. Microsatellite instability was not seen in the carcinomas, four separate hyperplastic polyps, the hyperplastic polyp with mild adenomatous change associated with the carcinoma, or a separate serrated adenoma. Allelic imbalance in the cancers at D5S346 and D17S938 suggested allelic loss of both p53 and APC, as well as at the loci D13S263, D13S174, D13S159, and D18S49. An early invasive carcinoma in one hyperplastic polyp stained for p53 protein, but the associated hyperplastic polyp was negative. In this case, neoplastic progression followed the typical genetic pathway of common colorectal carcinoma and occurred synchronously with mutation of p53.

Adenomatous Polyps↗
PubMed2000

Prevalence of Helicobacter pylori infection in 160 patients with Barrett's oesophagus or Barrett's adenocarcinoma.

BACKGROUND: The role of Helicobacter pylori infection in the development of Barrett's oesophagus and its complications is uncertain. The aim of the present study was to determine the importance of H. pylori infection in this disease by comparing the frequency of oesophageal and gastric H. pylori infection in a group of patients with Barrett's oesophagus or adenocarcinoma, with the frequency of infection in a control group without Barrett's disease. METHODS: The study group included 160 patients (123 male, 37 female; mean age: 61.2 years) who were classified (according to the highest grade pathological lesion in the oesophagus) as having Barrett's intestinal metaplasia (IM; 88 patients), Barrett's oesophagus with low-grade dysplasia (LGD; 28 patients), high-grade dysplasia (HGD; five patients), Barrett's indefinite for dysplasia (n = 4), and Barrett's adenocarcinoma (33 patients). A total of 91 of these patients had gastric antral specimens available for study. The control group consisted of 214 consecutive, prospectively enrolled symptomatic patients (122 male, 92 female; mean age: 57.2 years) who underwent upper gastrointestinal endoscopy and in whom Barrett's oesophagus or Barrett's adenocarcinoma was not found. A modified Warthin-Starry method was used to detect H. pylori infection. RESULTS: Oesophageal H. pylori infection was found in eight of 160 (5%) patients with Barrett's oesophagus or Barrett's adenocarcinoma. Holicobacter pylori organisms in the oesophagus were found only on non-intestinalized cardiac or oxyntocardiac mucosa. All patients with oesophageal H. pylori infection and an antral biopsy available for study had antral H. pylori infection. Gastric antral H. pylori infection was significantly less prevalent in patients in the Barrett's study group (15/91, 16.5%) than in the non-Barrett's control group (67/214, 31.3%; Fisher's exact test, P = 0.01). Patients from the control group with an endoscopic diagnosis of duodenal ulcer, gastric ulcer, gastritis, or duodenitis had a significantly higher prevalence of infection compared with the Barrett's group, but there was no difference in the infection prevalence in patients in the Barrett's group and patients with reflux oesophagitis, hiatal hernia, no endoscopic abnormality, or any other diagnosis. CONCLUSIONS: Oesophageal H. pylori infection is uncommon in patients with Barrett's IM, dysplasia, or adenocarcinoma, and may be restricted to non-intestinalized columnar epithelium. Gastric H. pylori infection may have a protective effect for the development of Barrett's oesophagus.

Adenocarcinoma↗
PubMed2000

K-ras codon 12 mutations in Barrett's oesophagus and adenocarcinomas of the oesophagus and oesophagogastric junction.

BACKGROUND: Activation of the ras oncogene is commonly found in gastrointestinal tract cancers, but the role of ras in the development and progression of Barrett's oesophagus and associated cancers is uncertain. METHODS: The frequency of K-ras codon 12 point mutations was assessed in 52 paraffin-embedded tissues from 44 patients with oesophageal pathology. The specimens were classified pathologically as follows: adenocarcinoma of the oesophagus or oesophagogastric junction (n = 23), Barrett's high-grade dysplasia (n = 5), low-grade dysplasia (n = 14), intestinal metaplasia (n = 4), normal oesophagus (n = 5) or normal stomach (n = 1). DNA was extracted from three consecutive sections of each paraffin block and mutations at bases 1 and 2 of K-ras codon 12 were identified using a novel restriction endonuclease-mediated selective polymerase chain reaction method. RESULTS: Mutations were found in 7 of 23 (30.4%) adenocarcinomas and in 2 of 5 (40%) high-grade dysplasia specimens. No mutations were found in specimens of low-grade dysplasia, intestinal metaplasia without dysplasia, or normal oesophagus and stomach. There were no significant associations between the presence of mutations and clinicopathologic features in the patients with cancer. One patient who progressed from low-grade to high-grade dysplasia was found to have developed mutant K-ras in the course of this transformation. CONCLUSION: These results suggest that K-ras codon 12 mutations may occur frequently in patients with Barrett's oesophagus with high-grade dysplasia or adenocarcinoma of the oesophagus or oesophagogastric junction. K-ras mutation may be a late event in the Barrett's metaplasia-dysplasia-adenocarcinoma sequence.

Adenocarcinoma↗