Biomedical subjects

A McGoldrick

Publications and source records attributed to A McGoldrick.

12 recordsLinked to original sources

PubMed2004

Estrogen receptor alpha and beta profiling in human breast cancer.

BACKGROUND: The identification of a second estrogen receptor (ER-beta) has significant implications for therapeutic strategy in breast cancer management arising from the potential agonist effect of Tamoxifen at estrogen receptor sites and as such, antiestrogen therapy may be inappropriate in patients with a dominance of ER-beta. METHODS: To determine the proportion of breast cancer patients who may be so at risk, we developed a novel multiplexed RT-PCR technique to establish the relative ER-alpha and ER-beta levels in 53 primary breast cancers, 11 normal breast tissues and six cell lines. We further assessed the prognostic significance of receptor status relative to the Nottingham prognostic index (NPI). The ER-alpha and ER-beta status was also determined by immunohistochemistry using previously published and 'in-house' scoring systems. RESULTS: Using RT-PCR analysis, 46 tumours were hormone receptor positive (ER+) with 42 displaying ER-alpha predominance. Comparison with immunohistochemistry demonstrated 44/53 (ER-alpha) and 27/50 (ER-beta) concordance rates. There was no significant difference in the NPI between ER-alpha and ER-beta predominant cohorts or between ER+ and ER- cohorts. CONCLUSION: This study identifies the existence of a subgroup of ER+ patients in whom Tamoxifen therapy may be inappropriate and has significant implications for adjuvant therapy of primary breast cancer.

Adult↗

PubMed2001

Rapid pathotyping of Newcastle disease virus (NDV) using fluorogenic probes in a PCR assay.

Hybridisation of PCR fragments with fluorogenic probes specific for pathotype allowed an estimation of pathogenicity of Newcastle disease virus (NDV) isolates using a modified TaqMan procedure. Six probes were used, designed to recognise nucleotide sequences in the fusion protein gene sequence corresponding to the precursor protein F0 cleavage site of both virulent and avirulent viruses. Forty-three of the 45 isolates tested, including 18 examined in a blind study were pathotyped successfully and rapidly, with close correlation between cleavage site nucleotide sequences, TaqMan results and intracerebral pathogenicity index (ICPI) values. One isolate, which could not be pathotyped by nucleotide sequencing, was shown using the TaqMan system to be a mixture of virulent and avirulent NDV. The results of this study suggest that using this modified TaqMan protocol, the likely virulence of most ND isolates can be determined rapidly and reproducibly.

Animals↗

PubMed2000

Classical swine fever virus: a second ring test to evaluate RT-PCR detection methods.

Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.

Animals↗

PubMed2000

Genetic typing of classical swine fever virus.

Three regions of the classical swine fever virus (CSFV) genome that have been widely sequenced were compared with respect to their ability to discriminate between isolates and to segregate viruses into genetic groups. Sequence data-sets were assembled for 55 CSFVs comprising 150 nucleotides of the 5' non-translated region, 190 nucleotides of the E2 envelope glycoprotein gene and 409 nucleotides of the NS5B polymerase gene. Phylogenetic analysis of each data-set revealed similar groups and subgroups. For closely related viruses, the more variable or larger data-sets gave better discrimination, and the most reliable classification was obtained with sequence data from the NS5B region. No evidence was found for intertypic recombination between CSFVs. A larger data-set was also analysed comprising 190 nucleotides of E2 sequence from 100 CSFVs from different parts of the world, in order to assess the extent and global distribution of CSFV diversity. Additional groups of CSFV are evident from Asia and the nomenclature of Lowings et al. (1996) [Lowings, P., Ibata, G., Needham, J., Paton, D., 1996. J. Gen. Virol. 77, 1311-1321] needs to be updated to accommodate these. A tentative assignment, adapting rather than overturning the previous nomenclature divides CSF viruses into three groups with three or four subgroups: 1.1, 1.2, 1.3; 2.1, 2.2, 2.3; 3.1, 3.2, 3.3, 3.4. The expanding data-base of CSFV sequences should improve the prospects of disease tracing in the future, and provide a basis for a standardised approach to ensure that results from different laboratories are comparable.

Animals↗

PubMed2000

Classical swine fever virus: a ring test to evaluate RT-PCR detection methods.

Six laboratories participated in an exercise to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Two sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 34 samples of random primed cDNA. These had been synthesised from viral RNA representative of seven different genetic subtypes of CSFV. The other set comprised 40 clinical samples containing tonsil, spleen, whole blood or serum from a pig that had been experimentally infected with CSFV. Each laboratory tested the samples using one or more PCR/RT-PCR tests that they were accustomed to using. The methods and results of the laboratories were compared with one another. The RT-PCR results obtained from testing the clinical samples were also compared with those obtained by virus isolation and antigen ELISA.ELISA. Both RT-PCR and RT-nested PCR appeared to give some false positive results. Several of the PCR tests appear suitable in terms of specificity and sensitivity. Further trials are necessary to compare results when the same test is performed by different laboratories, and to show that improved control procedures can eliminate problems due to false positive reactions.A limited comparison of extraction and reverse transcription procedures showed similar results in each of three participating laboratories, even though the methods were not standardised.

Classical Swine Fever Virus↗

PubMed1999

Frequent loss of imprinting of PEG1/MEST in invasive breast cancer.

The human PEG1 gene is a newly identified imprinted gene on 7q32. Genetic aberrations of this chromosomal region are often detected in invasive breast carcinomas. In this study, we show monoallelic PEG1 expression in normal breast tissue, indicating the presence of a functional imprint, and more importantly, we demonstrate loss of imprinting (LOI) in all of seven informative invasive breast carcinomas. In contrast to this, in one case of atypical ductal hyperplasia (ADH) found in residual breast, imprinting was maintained. This raises the possibility that aberrant imprinting of PEG1 may be involved in the progression from hyperplasia to invasive breast cancer.

Alleles↗

PubMed1999

Genetic typing of bovine pestiviruses from England and Wales.

Using RNA purified directly from stored clinical specimens, a collection of 62 pestiviruses were typed by RT-PCR and sequencing within the 5'-untranslated region of the genome. All the specimens had been obtained in 1966/1967 from diary cattle in England and Wales. Eight further pestiviruses, grown in cell culture, were characterised in the same way. Seven of these viruses were representatives of a panel of British isolates, obtained from cattle ten years before. The eighth was the virus used in a British bovine viral diarrhoea (BVD) vaccine. Most of the viruses were genetically unique and were of BVDV type Ia. One recent isolate was BVDV type Ib, two others were intermediate between Ia and Ib. No BVDV type II or border disease virus (BDV) isolates were found. There was no overall association between geographical and phylogenetic clustering, suggesting long-distance virus dispersal, presumably via trading of infected cattle. The sequences of the recently obtained cattle viruses were very similar or, in one case, identical to the older isolates in the region studied. Their close similarity to some previously characterised pestiviruses from British sheep suggests that a common pool of BVDV Ia is shared by these two livestock species, although another pestivirus--BVDV--is confined to sheep. The British cattle viruses were mostly distinct from continental European isolates, but more similar to type Ia isolates from North American cattle.

5' Untranslated Regions↗

PubMed1999

Characterisation of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a.

Analyses of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) isolates have suggested that tropism and pathogenicity are influenced by the spike protein and ORF 3. In general, enteric viruses (TGEV) have been shown to contain intact spike and ORF 3 genes, whilst respiratory isolates (PRCV) have major deletions within both regions. Virulence has been correlated to a functional ORF 3. Here, sequence analysis of a recent isolate of virulent TGEV, revealed a variant with an intact spike gene, but a large deletion in ORF 3a. This suggests that ORF 3a is not essential for enteric virulence.

Animals↗

PubMed1999

Closed one-tube reverse transcription nested polymerase chain reaction for the detection of pestiviral RNA with fluorescent probes.

An assay was developed in which reverse transcription (RT), nested polymerase chain reaction (PCR) and accumulation of amplicon-specific fluorescence could take place in a single, closed reaction tube. The assay, which was classical swine fever virus RNA-specific, was compared with other methods for detection of this virus, including various RT-PCR configurations, virus isolation and ELISA. The new method was very sensitive, and less prone to giving false positive results compared to nested PCR carried out in separate reaction tubes. Substitution of different fluorescent probes resulted in specific tests for border disease virus and for bovine viral diarrhoea type II (BVD-II), and one that could detect all pestiviruses except for some BVD-II viruses.

Animals↗

PubMed1998

A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan).

Detection of classical swine fever virus (CSFV) and its discrimination from other pestiviruses can be achieved by virus isolation (VI) in cell cultures, antigen detection, or molecular analysis. To simplify the latter, a 5'-nuclease assay (TaqMan) was developed for the rapid and specific detection of CSFV with the minimum of downstream PCR processing. A pair of 5'-non-coding region, panpestivirus-specific PCR primers were assessed in a one-step reverse transcription-PCR with each of 36 diverse pestiviruses. The PCR products were subsequently reamplified, in conjunction with a CSFV-specific fluorogenic probe, in a nested-PCR with a second set of panpestivirus PCR primers. During nested PCR, when the target of interest was present, the CSFV probe annealed to the amplicon between the forward and reverse primers and was subsequently cleaved via the 5'-3' nucleolytic activity of the DNA polymerase resulting in the release of the fluorescent reporter dye. Each PCR tube was then placed directly into a luminescence spectrometer to monitor for any increase in fluorescence due to cleavage of the probe. This assay detected representatives of all genetic sub-groups of CSFV, but gave negative results for other pestiviruses. A preliminary assessment showed that the method could be used to detect CSFV RNA extracted from infected pig blood with a sensitivity greater than that of VI.

Age Factors↗

PubMed1997

Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus.

An RT-PCR method was developed that amplified genetic material from the 5' end of the S protein gene of both transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), but discriminated between the two by the size of the product generated. A number of restriction endonuclease enzymes were assessed for recognition of the amplicons so produced. The assay was shown to detect viral RNA from all of the 26 different TGEV and PRCV isolates examined, covering a period from 1946 to 1996. Detection of TGEV in clinical specimens was possible using a spin column method to extract RNA and sensitivity was compared to virus isolation and antigen detection ELISA. The method could provide a means of confirming positive results from immunological screening tests such as FAT and ELISA, reducing the need for virus isolation and convalescent serology.

Animals↗

PubMed1995

Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus.

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.

Cell Line↗