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Biomedical subjects

A P Griffiths

Publications and source records attributed to A P Griffiths.

At least 19 recordsLinked to original sources
PubMed2007

Immunohistochemical study of nuclear factor-kappaB activity and interleukin-8 abundance in oesophageal adenocarcinoma; a useful strategy for monitoring these biomarkers.

AIMS: To determine if immunohistochemistry (IHC) could be used to monitor nuclear factor-kappaB (NF-kappaB) activity in oesophageal adenocarcinoma and pre-malignant (Barrett's) oesophageal tissues, relative to normal oesophageal mucosa. The pro-inflammatory cytokine interleukin-8 (IL-8), a transcriptional target of NF-kappaB, was also studied to better understand NF-kappaB functionality; its RNA and protein levels were assessed in oesophageal tissues. METHODS: IHC was employed using an antibody against the nuclear localisation sequence (NLS) of the p65 subunit as well as an antibody against IL-8. To assess NF-kappaB function, changes in gene expression of NF-kappaB controlled genes (IL-8 and I-kappaB) were also assessed in the histological sequence using real-time PCR. More global expression changes were also studied using membrane arrays. RESULTS: IHC was effective at monitoring overall NF-kappaB activity and IL-8 abundance. This method also allowed NF-kappaB activity and IL-8 abundance to be pinpointed in specific cell types. There were significant increases in nuclear NF-kappaB activity and IL-8 abundance across the histological series. Gene expression analysis also showed consistent up-regulation of IL-8, confirming the IHC data and showing enhanced transcriptional NF-kappaB activity. I-kappaB (another NF-kappaB target) showed down-regulation in dysplastic and adenocarcinoma tissues. Down-regulation of I-kappaB gene expression may partly explain increased NF-kappaB activity. CONCLUSION: IHC, using antibodies against the NLS of p65, may be useful in monitoring overall NF-kappaB activity in oesophageal tissues. As IHC is amenable to high-throughput screening (whereas traditional electrophoretic mobility shift assay methods are not), this may lead to the development of a better screening tool for early cancer risk.

Adenocarcinoma↗
PubMed2007

Severe proctitis, perforation, and fatal rectal bleeding secondary to cytomegalovirus in an immunocompetent patient: report of a case.

Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in immunocompromised patients. In immunocompetent individuals, the infection is usually subclinical but it can sometimes be life threatening. We describe a case of fatal CMV proctitis in a 71-year-old man following an Ivor-Lewis esophagectomy. After surgery he developed renal failure, methicillin-resistant Staphylococcus aureus pneumonia, and acute respiratory distress syndrome. He recovered but developed melena and massive fresh rectal bleeding. Sigmoidoscopy revealed severe proctitis and a biopsy was consistent with ischemia. Despite undergoing a proctectomy he continued to bleed and died despite every effort. The final histological examination of the rectum revealed a CMV infection.

Adenocarcinoma↗
PubMed2006

Mitochondrial DNA mutations in oral squamous cell carcinoma.

It has previously been demonstrated that mitochondrial DNA (mtDNA) mutations within the ND2 gene of histologically normal parotid salivary gland tissue of smokers may be molecular biomarkers for smoking-induced mtDNA damage. Oral squamous cell carcinoma (SCC) is strongly related to cigarette smoking; therefore, we used PCR and direct sequencing to establish whether mtDNA mutations were also present in oral SCC which could be used as additional biomarkers for smoking-associated DNA damage. In addition to searching for mutations in the ND2 gene, the mitochondrial D-Loop was also analysed. Three mutation hotspots were observed in the D-Loop at nt 146, 152 and 186, two of which (nt 146 and 152) have also been implicated in oesophageal SCC, another smoking-related cancer. The mutation hotspot observed at nt 186 has not previously been reported in other tumours. Furthermore, we show that the mutations previously reported within the ND2 gene in normal parotid tissue of smokers were not evident in these samples, but that a mutation hotspot occurs at nucleotide 4917 in oral SCC. We also show that D-Loop mutations occur predominantly in male smokers and female non-smokers and that this association with gender is statistically significant (P = 0.003). We conclude that the mtDNA mutation hotspots found in this study, in particular nt 186, are potential biomarkers for oral SCC. However, owing to gender-specific differences in occurrence in smokers and non-smokers, and a lack of environmental smoking history, in general, it is difficult to associate these mutations with mtDNA damage induced by smoking. If the mutations observed in the subset of male patients are smoking induced, given our previous findings, mutation hotspots in the ND2 gene may be tissue specific suggesting the causative mutagens for mtDNA damage within these tissues are likely to be different.

Aged↗
PubMed2005

Fluorescence in situ hybridisation analysis of chromosomal aberrations in gastric tissue: the potential involvement of Helicobacter pylori.

In this series of experiments, a novel protocol was developed whereby gastric cells were collected using endoscopic cytology brush techniques, and prepared, such that interphase fluorescence in situ hybridization (FISH) could be performed. In total, 80 distinct histological samples from 37 patients were studied using four chromosome probes (over 32,000 cells analysed). Studies have previously identified abnormalities of these four chromosomes in upper GI tumours. Using premalignant tissues, we aimed to determine how early in Correa's pathway to gastric cancer these chromosome abnormalities occurred. Aneuploidy of chromosomes 4, 8, 20 and 17(p53) was detected in histologically normal gastric mucosa, as well as in gastritis, intestinal metaplasia, dysplasia and cancer samples. The levels of aneuploidy increased as disease severity increased. Amplification of chromosome 4 and chromosome 20, and deletion of chromosome 17(p53) were the more common findings. Hence, a role for these abnormalities may exist in the initiation of, and the progression to, gastric cancer. Helicobacter pylori infection was determined in premalignant tissue using histological analysis and PCR technology. Detection rates were comparable. PCR was used to subtype H. pylori for CagA status. The amplification of chromosome 4 in gastric tissue was significantly more prevalent in H. pylori-positive patients (n=7) compared to H. pylori-negative patients (n=11), possibly reflecting a role for chromosome 4 amplification in H. pylori-induced gastric cancer. The more virulent CagA strain of H. pylori was associated with increased disease pathology and chromosomal abnormalities, although numbers were small (CagA+ n=3, CagA- n=4). Finally, in vitro work demonstrated that the aneuploidy induced in a human cell line after exposure to the reactive oxygen species (ROS) hydrogen peroxide was similar to that already shown in the gastric cancer pathway, and may further strengthen the hypothesis that H. pylori causes gastric cancer progression via an ROS-mediated mechanism.

Aged↗
PubMed2004

Generation of locus-specific probes for interphase fluorescence in situ hybridisation--application in Barrett's esophagus.

Despite the wide range of probes commercially available for interphase fluorescence in situ hybridisation (FISH), the supply of locus-specific probes is limited to genes or chromosomal regions commonly altered in genetic diseases or during carcinogenesis. Generation of these probes is therefore desirable to accommodate individual research requirements. Hence, we detail the methodology required to design and produce custom locus-specific interphase FISH probes for any human genomic region of interest and their application was illustrated in cytogenetic investigations of Barrett's tumourigenesis. Previously utilising FISH, we observed that Barrett's tissues demonstrated chromosome 4 hyperploidy [Gut 52 (2003) 623], but as centromeric probes were used in this analysis, it was not known if the whole chromosome was amplified. We consequently generated single-copy sequence probes for the 4p16.3 and 4q35.1 subtelomeric loci. Multicolour FISH was subsequently performed on interphase preparations originating from patients with Barrett's esophagus at varying histological grades, thus demonstrating the whole region of chromosome 4 was amplified within the tissues. Additionally, probes for the DNA methyltransferase genes were produced to determine if gene dosage alterations were responsible for increasing methylation activity during Barrett's neoplastic progression. No significant alterations at the DNMT1 and DNMT3a loci were detected. An increased copy number of these genes is therefore not the basis for the hypermethylation commonly observed in this premalignant lesion.

Adenocarcinoma↗
PubMed2003

The bile acid deoxycholic acid (DCA) at neutral pH activates NF-kappaB and induces IL-8 expression in oesophageal cells in vitro.

Barrett's oesophagus patients accumulate chromosomal defects during the histological progression to cancer, one of the most prominent of which is the amplification of the whole of chromosome 4. We aimed to study the role that the transcription factor NF-kappaB, a candidate cancer- promoting gene, present on chromosome 4, plays in Barrett's oesophagus, using OE33 cells as a model. Specifically, we wanted to determine if NF-kappaB was activated by exposure to bile acid (deoxycholic acid) in oesophageal cells. We employed pathway specific cDNA microarrays and real-time PCR, to first identify bile acid induced genes and specifically to investigate the role of NF-kappaB. An NF-kappaB reporter system was used, as well as an inhibitor of NF-kappaB (pyrrolidine dithiocarbamate) to confirm the activation of NF-kappaB by bile. We show that physiological levels of DCA (100-300 microM) were capable of activating NF-kappaB in OE33 cells and inducing NF-kappaB target gene expression (particularly IkappaB and IL-8). Other gene expression abnormalities were also shown to be induced by DCA. Importantly, preliminary experiments showed that NF-kappaB activation by bile occurred at neutral pH, but not at acid pH. Acidic bile did however cause over-expression of the c-myc oncogene, as reported previously. Hence, we present data showing that NF-kappaB may be a key mediator of carcinogenesis in bile exposed Barrett's tissues. In addition, neutral bile acids appear to play a significant part in reflux induced gene expression changes. We postulate that the activation of the survival factor NF-kappaB by bile may be linked to the previous cytogenetic data from our laboratory showing the amplification of NF-kappaB's chromosome (chromosome 4), during Barrett's cancer progression. Hence chromosome 4 amplification may provide a survival mechanism for bile exposed oesophageal tissues via NF-kappaB.

Adenocarcinoma↗
PubMed2003

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma.

p53 mutations and loss of heterozygosity have been commonly associated with oesophageal adenocarcinoma. In this investigation, the p53 status of a Welsh population of Barrett's-associated oesophageal adenocarcinomas were fully characterised at the gene sequence, chromosomal, mRNA and protein levels. In total, 31 tumours were examined for p53 gene sequence mutations using RFLP with sequencing, allelic loss of the gene was characterised by FISH, mRNA expression by p53 pathway signalling arrays and protein levels by p53 immunohistochemistry. In all, 9.6% of adenocarcinomas harboured p53 mutations, 24% displayed p53 allelic loss and 83% exhibited p53 protein accumulation. Point mutations and deletions of the gene did not coexist within the same samples. All samples containing p53 mutations also displayed positive immunostaining; however; in the majority of cases, p53 protein accumulation developed in the absence of mutations. The gene expression analysis demonstrated no differences in p53 and mdm-2 transcription levels between the p53 immunonegative and immunopositive samples, indicating other mechanisms underlie the proteins' overexpression. In conclusion, p53 mutations and deletions do not appear to be frequent events in oesophageal adenocarcinomas; however, abnormal accumulation of the protein is present in a vast majority of cases. P53 gene mutations are not the primary cause of protein overexpression--an alternative mechanism is responsible for the positive p53 immunohistochemistry detected.

Adenocarcinoma↗
PubMed2003

Detection of p53 mutations in precancerous gastric tissue.

Intestinal-type gastric cancer is preceded by gastritis and intestinal metaplasia. There is uncertainty regarding the stage at which genetic alterations in the p53 gene occur. Reactive oxygen species (ROS) may participate in the production of mutations and the inactivation of p53 is due to infection by the bacterium Helicobacter pylori. We have investigated whether alterations of the p53 gene can be detected in gastritis and intestinal metaplasia using the restriction site mutation assay. We also assessed the potential contribution of ROS to p53 inactivation using electron spin resonance spectroscopy (ESR) and correlated with the presence of H. pylori. In all, 35% of the gastritis samples and 45% of the intestinal metaplasia samples were found to contain mutations in exons 5-8 of the p53 gene. Electron spin resonance spectroscopy analysis showed a significant increase in free radical levels in gastritis samples compared with normal, intestinal metaplasia and cancer samples, suggesting that free radicals present in gastritis may contribute to p53 mutations. There was no significant difference in free radical levels between the H. pylori-positive and -negative groups. However, a small subpopulation of the H. pylori-negative patients had much higher levels of free radicals. This suggests a more prominent role for other factors in ROS production.

Acute Disease↗
PubMed2003

Early p53 mutations in nondysplastic Barrett's tissue detected by the restriction site mutation (RSM) methodology.

Barrett's oesophagus is a premalignant condition whose incidence is rising dramatically. Molecular markers are urgently needed to identify Barrett's patients at the highest risk of cancer progression. To this end, we have used a rapid molecular technique, restriction site mutation (RSM), to detect low-frequency mutations in the p53 tumour suppressor gene in premalignant Barrett's tissues of cancer-free patients. In total, 38 endoscopically diagnosed Barrett's patients with a range of histological stages of Barrett's progression, plus four control patients without Barrett's oesophagus, were analysed for early p53 mutations. Tissue samples taken from these patients (93 samples in total) were analysed for the presence of low-frequency p53 mutations at hotspot codons: 175, 213, 248, 249, 282. In total, 13 of the 38 Barrett's patients were shown to possess a p53 mutation in at least one sample (no mutations in the four control patients). Although no statistically significant associations were found, p53 mutations reflected histological progression in Barrett's patients with p53 mutations found in 30% of metaplasia patients (P=0.4) and low-grade dysplasia patients (P=0.33) and 45% of high-grade dysplasia patients (P=0.15). Detected p53 mutations were mainly GC to AT transitions at CpG sites.

Barrett Esophagus↗
PubMed2003

Ileitis due to Yersinia pseudotuberculosis in Crohn's disease.

We present the case of a young woman with fulminating terminal ileitis due to Yersinia pseudotuberculosis, in whom Crohn's disease was subsequently diagnosed. Serological testing was prompted by radiological, macroscopic and histological changes which were atypical of Crohn's disease. We speculate that Y. pseudotuberculosis ileitis may occasionally produce an acute exacerbation of Crohn's disease, but that its occurrence may not be diagnosed if the atypical features are not recognised. In this case, the classical features of Y. pseudotuberculosis may have been modified by pre-existing Crohn's disease. We propose that, because of defects in mucosal barrier function and immunological function, patients with Crohn's may have increased susceptibility to infection by Yersinia sp.

Adult↗
PubMed2003

Chromosome 4 hyperploidy represents an early genetic aberration in premalignant Barrett's oesophagus.

BACKGROUND AND AIMS: Characterisation of the underlying molecular mechanisms that promote Barrett's progression may ultimately lead to identification of potential predictive genetic markers that classify patients' malignant risk. In an attempt to understand these causative pathways, fluorescence in situ hybridisation (FISH) was used in this study to determine when specific genetic alterations arise during Barrett's associated neoplastic progression. METHODS: Endoscopic cytology brushings were obtained from 28 patients with Barrett's metaplasia, 28 with dysplasia (20 low grade dysplasia (LGD) and eight with high grade dysplasia (HGD)), and seven with adenocarcinoma, together with paired control brushings from regions of normal proximal squamous cell epithelium. The exfoliated epithelial cells were washed and deposited onto slides. Probes specific for the centromeres of chromosomes 4, 8, 20, and Y, and locus specific probes for the tumour suppressor genes p16, p53, and Rb were subsequently hybridised. RESULTS: Aneuploidy was found early in progression, with metaplastic tissues displaying increased copy numbers of chromosomes 4 and 8. Chromosome 4 hyperploidy was found in 89%, 90%, 88%, and 100% of metaplasias, LGD, HGD, adenocarcinomas, respectively, while chromosome 8 hyperploidy occurred in 71%, 75%, 100%, and 100% of patients with the respective staging. Loss of the p16 tumour suppressor gene also presented in metaplastic epithelium (7%) but most other genetic aberrations were only seen in HGD. CONCLUSIONS: Genetic instability arises well before dysplasia in Barrett's oesophagus, with chromosome 4 and 8 hyperploidy representing the earliest and most common alterations identified. As these aberrations are widespread at all the premalignant stages, there may be genes on chromosomes 4 and 8 that are involved in both the initiation and progression of Barrett's oesophagus.

Adenocarcinoma↗
PubMed2002

Mitochondrial DNA mutations in the parotid gland of cigarette smokers and non-smokers.

It has previously been demonstrated that mitochondrial DNA (mtDNA) mutations accumulate in the lung and increase in frequency with age. It has also been shown that the level of mtDNA mutations including deletions and base substitutions are elevated in lung tissue of smokers relative to non-smokers. We have previously shown that the 'common' 4977 bp mtDNA deletion is present in the parotid (salivary) gland of smokers and non-smokers and that there is a significant increase in the level of this deletion in Warthins tumour, an oncocytoma of the parotid gland. In this study we used semi-quantitative PCR to confirm the presence of 4977 bp mtDNA deletion in the parotid gland of non-smokers and smokers. Importantly, we show that the deletion accumulates with age regardless of smoking status and that there was no significant difference in the level of the 4977 bp deletion in parotid tissue of smokers and non-smokers. Using strand conformational polymorphism (SSCP) and direct sequencing we also found 5/23 smokers had parotid tissue specific base substitutions: either an A/T to G/C transition at A4767 or a G/C to A/T transition at G4853. These results are evidence of age related increase in the 4977 bp deletion and a higher level of mutations, probably due to oxidative damage, in the parotid gland of smokers.

Adenolymphoma↗
PubMed2002

Genetic pathways involved in the progression of Barrett's metaplasia to adenocarcinoma.

BACKGROUND: The prediction of which patients with Barrett's metaplasia will develop cancer is difficult. Better genetic characterization of the condition may aid clinicians in devising more effective management and follow-up strategies. METHODS: A review was undertaken of the accumulated genetic data relating to the progression of squamous epithelium to adenocarcinoma. The normal functions of a number of cancer-related genes are described and an explanation is given of how alterations in these genes interfere with normal cell processes and lead to cancer. RESULTS AND CONCLUSION: The main genetic alterations accompanying the progression through dysplasia to adenocarcinoma were collated from 135 papers. The principal genetic changes implicated are the loss of p16 gene expression (by deletion or hypermethylation), the loss of p53 expression (by mutation and deletion), the increase in cyclin D1 expression, the induction of aneuploidy and the losses of the Rb, DCC and APC chromosomal loci.

Adenocarcinoma↗
PubMed2000

Hepatocellular carcinoma arising in non-cirrhotic liver in genetic haemochromatosis.

The occurrence of primary hepatocellular carcinoma (HCC) in patients with genetic haemochromatosis (GH) with cirrhosis is well known. In the past it has been suggested that patients with GH without cirrhosis have the same risk of HCC and life expectancy as the general population. However, recently there have been cases of HCC arising in non-cirrhotic livers, and we report another such case. A review of the literature has been included.

Aged↗
PubMed2000

Consistency in the observation of features used to classify duct carcinoma in situ (DCIS) of the breast.

AIM: To determine interobserver and intra-observer agreement in the assessment of cytological grade and intraduct necrosis in pure duct carcinoma in situ (DCIS) of the breast. METHODS: Sixty unselected cases with illustrated diagnostic criteria were circulated to 19 practising histopathologists. RESULTS: Overall agreement was moderate for cytological grade in three categories: 71% agreement; weighted kappa (kappa w), 0.36; intraduct necrosis in three categories (absent, present, extensive): 76% agreement; kappa w, 0.57; and the Van Nuys classification system: 73% agreement; kappa w, 0.48. Agreement was no better among observers participating in the National External Quality Assurance Programme. Intra-observer agreement for cytological assessment (69.6% agreement; kappa w, 0.52) and intraduct necrosis (68.3% agreement; kappa w, 0.48) was moderate, suggesting that individual variation rather than precision of criteria contributes to the lack of agreement. CONCLUSIONS: Moderate agreement on observations can be achieved by non-specialist pathologists, with better agreement on necrosis than cytological grade. There was evidence of consistent individual bias towards over or under scoring cytological grade, which could be corrected with adequate and prompt feedback.

Breast Neoplasms↗
PubMed1999

Orbital metastasis due to interval lobular carcinoma of the breast: a potential mimic of lymphoma.

A 53-year-old woman had an orbital mass composed of a neoplastic small round cell infiltrate and no apparent extraorbital primary tumor. Although the initial diagnosis was primary orbital lymphoma, a combination of mucin histochemistry and immunohistochemical staining for cytokeratin and estrogen receptors led to the discovery of an impalpable lobular carcinoma of the breast. We discuss how detailed histopathological assessment can lead to beneficial therapy.

Biopsy, Needle↗