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Timothy J O'Leary

Publications and source records attributed to Timothy J O'Leary.

14 recordsLinked to original sources
PubMed2006

A liposome-PCR assay for the ultrasensitive detection of biological toxins.

We describe an ultrasensitive immunoassay for detecting biotoxins that uses liposomes with encapsulated DNA reporters, and ganglioside receptors embedded in the bilayer, as a detection reagent. After immobilization of the target biotoxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporters, which are quantified by real-time PCR. Assays for cholera and botulinum toxins are several orders of magnitude more sensitive than current detection methods.

Botulinum Toxins↗
PubMed2005

Conversions of formaldehyde-modified 2'-deoxyadenosine 5'-monophosphate in conditions modeling formalin-fixed tissue dehydration.

Formalin-fixed, paraffin-embedded specimens typically provide molecular biologists with low yields of extractable nucleic acids that exhibit extensive strand cleavage and covalent modification of nucleic acid bases. This study supports the idea that these deleterious effects are promoted by the first step in formalin-fixed tissue processing--i.e., tissue dehydration with a graded series of alcohols. We analyzed the conversions of formaldehyde-modified 2'-deoxyadenosine 5'-monophosphate (dAMP) by reverse-phase ion-pair, high-performance liquid chromatography and found that dehydration does not stabilize N-methylol groups in the modified nucleotide. Furthermore, spontaneous demodification in a dry state or in anhydrous ethanol can be as fast as it is in aqueous solutions if the preparation is contaminated with salts of orthophosphoric acid. In ethanol, orthophosphates also catalyze formation of abundant N6-ethoxymethyl-dAMP, as well as cross-linking and depurination of nucleotides present in the mixture. Identification of the products was performed using ultraviolet absorbance spectroscopy and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry. Alternatives to the traditional processing of formalin-fixed tissues are discussed.

Acetals↗
PubMed2005

Food and Drug Administration regulation of in vitro diagnostic devices.

The Food and Drug Administration regulates the sale and distribution of laboratory devices under a statutory and regulatory framework that is unfamiliar to most clinical laboratory scientists. In this article we briefly describe the criteria that are used to classify and review in vitro diagnostic devices. We discuss the similarities and differences between devices that are not subject to premarket review, and those that are required to undergo either a premarket application or premarket notification [510(k)] pathway. We then discuss the methods that the Food and Drug Administration uses to assess the performance of in vitro diagnostic devices in the marketplace as a component of the total life cycle approach to medical device regulation.

Device Approval↗
PubMed2004

State of the Art Symposium: prescreening and rescreening.

The effectiveness of prescreening and rescreening cervical cytology preparations-whether used as quality assurance measures or in the attempt to improve sensitivity-has engendered broad discussion within the cytology community. This paper forms an introduction to some of the issues surrounding prescreening and rescreening in cervical cytology.

Automation↗
PubMed2004

Modeling formalin fixation and antigen retrieval with bovine pancreatic RNase A II. Interrelationship of cross-linking, immunoreactivity, and heat treatment.

In this study, gel electrophoresis and capture enzyme-linked immunosorbent assay were used to assess the effect of formaldehyde treatment on the structural and immunological properties of bovine pancreatic ribonuclease A (RNase A). Prolonged incubation of RNase A in a 10% formalin solution leads to the formation of extensive intra- and intermolecular cross-links. However, these formaldehyde cross-links do not completely eliminate the recognition of RNase A by a polyclonal antibody. Comparative immunotitration of monomers, dimers, and oligomers greater than pentamers isolated from formalin-treated RNase A demonstrated that reduction of immunoreactivity due to intramolecular modifications prevails over the excluded volume effect of intermolecular cross-links. The latter only becomes important for intermolecular cross-links involving four or more molecules. The restoration of RNase A immunoreactivity during heating correlates with the reversal of formaldehyde cross-links if the incubation temperature does not exceed the denaturation temperature of the formalin-treated RNase A preparation. We conclude that formaldehyde cross-links stabilize antigens against the denaturing effects of high temperature, but the reversal of these cross-links is necessary for the restoration of immunoreactivity.

Animals↗
PubMed2004

Modeling formalin fixation and antigen retrieval with bovine pancreatic ribonuclease A: I-structural and functional alterations.

Understanding the chemistry of protein modification by formaldehyde is central to developing improved methods to recover proteins from formalin-fixed paraffin-embedded tissues for proteomic analysis and to improve protein immunoreactivity for immunohistochemical studies. We used biophysical techniques to investigate the effects of formaldehyde treatment on bovine pancreatic ribonuclease A (RNase A). Treatment of RNase A with formaldehyde was shown by gel electrophoresis to lead to the rapid formation of intra- and intermolecular protein cross-links. Thermal studies revealed that these protein cross-links significantly increased the thermal denaturation temperature of RNase A preparations. Analysis of formaldehyde-treated RNase A oligomers isolated by gel chromatography revealed that intramolecular protein cross-links are primarily responsible for the increase in protein thermostability. Formaldehyde treatment also lowered the isoelectric point of the enzyme from 9.45 to the 6.0-7.4 range. Optical spectroscopic studies demonstrated that the formaldehyde-induced modifications did not significantly alter the secondary or tertiary structure of RNase A. Heating formaldehyde-treated RNase A at 65 degrees C resulted in a significant reversal of the protein intra- and intermolecular cross-links and led to a partial restoration of enzymatic activity.

Animals↗
PubMed2003

Image quality issues in a static image-based telepathology consultation practice.

Field selection and image quality have often been identified as impediments in the successful employment of static-image telepathology. One thousand seven hundred fifty-three electronic consultations using static images were performed at the Department of Telemedicine, Armed Forces Institute of Pathology (AFIP) between November 1994 and September 2001, with 98.3% receiving a telepathology diagnosis. In 47.9% of cases, imagery was considered good by AFIP consultants, 38.5% were considered adequate, and 14.6% of cases were considered to have poor-quality imagery. Deficiencies in image quality were recorded for each case. Cases with imagery rated as good averaged significantly fewer deficiencies per case (0.45, range: 0 to 3) than cases with imagery rated adequate (0.95, range: 0 to 6) or poor (2.4, range: 0 to 7). Deficiencies in focus were most commonly identified in this series of cases (28.1%), followed by improper white balancing of the capture device (14.1%) and inadequate resolution (10%). Cases in which images were of inadequate resolution showed an increased likelihood for discordance between the telepathology diagnosis and the diagnosis rendered on follow-up material ("truth diagnosis"). Inadequate field selection, although only cited in 6.7% of cases overall, was seen with a significantly higher frequency in cases in which there was discordance between the telepathology and truth diagnosis. A review of common image deficiencies in static-image telepathology and possible causes is presented.

Diagnostic Errors↗
PubMed2002

Diagnosis of gastrointestinal stromal tumors: A consensus approach.

As a result of major recent advances in understanding the biology of gastrointestinal stromal tumors (GISTs), specifically recognition of the central role of activating KIT mutations and associated KIT protein expression in these lesions, and the development of novel and effective therapy for GISTs using the receptor tyrosine kinase inhibitor STI-571, these tumors have become the focus of considerable attention by pathologists, clinicians, and patients. Stromal/mesenchymal tumors of the gastrointestinal tract have long been a source of confusion and controversy with regard to classification, line(s) of differentiation, and prognostication. Characterization of the KIT pathway and its phenotypic implications has helped to resolve some but not all of these issues. Given the now critical role of accurate and reproducible pathologic diagnosis in ensuring appropriate treatment for patients with GIST, the National Institutes of Health convened a GIST workshop in April 2001 with the goal of developing a consensus approach to diagnosis and morphologic prognostication. Key elements of the consensus, as described herein, are the defining role of KIT immunopositivity in diagnosis and a proposed scheme for estimating metastatic risk in these lesions, based on tumor size and mitotic count, recognizing that it is probably unwise to use the definitive term "benign" for any GIST, at least at the present time.

Antineoplastic Agents↗
PubMed2002

Serial analysis of gene expression in murine fetal thymocyte cell lines.

FTL-1, -3 and -10 are three murine day 14 fetal thymocyte cell lines produced in order to model developmental stages within early (CD3-CD4-CD8-) thymocyte differentiation. In this study, we used the serial analysis of gene expression (SAGE) method to perform a systematic analysis of transcripts present in these three cell lines. A total of 77,313 SAGE tags were sequence identified from the three cell lines, representing 24,645 unique transcripts. Differentially expressed mRNA transcripts representing different gene classes were identified, including T cell functional genes, cytokine receptors, adhesion molecules and transcription factors. These results may serve as a model of the transcriptome of early thymocyte differentiation. A large number of unknown expressed sequence tags were also found to be differentially expressed. In order to validate the SAGE data, selected differentially expressed transcripts identified by SAGE were analyzed by quantitative RT-PCR in normal murine double-negative stage DN1-4 thymocytes. Expression of the transcription factors RUNX2 and PHD finger protein 2 and of the IGF type 1 receptor was shown to have differentially regulated expression patterns in sorted DN1-4 cells. These genes, and others identified by this analysis, are likely to play important roles in the development of T cells.

Animals↗
PubMed2002

Salpingitis, salpingoliths, and serous tumors of the ovaries: is there a connection?

We have observed luminal and mucosal calcifications frequently surrounded by a mantle of bland epithelium in the fallopian tubes ("salpingoliths") of women with serous tumors of the ovaries. These lesions resemble noninvasive peritoneal "implants" in women with advanced stage atypical proliferative serous tumors (APSTs) and micropapillary serous carcinomas (MPSCs). The presence of salpingitis and salpingoliths was prospectively evaluated in 358 women with a variety of nonneoplastic and neoplastic ovarian conditions and compared with 87 previously reported women with APSTs/MPSCs in an effort to determine whether these lesions were specifically associated with serous tumors. The frequency of chronic salpingitis among women without ovarian pathology was 27%, and the frequency of salpingoliths was 4%. Serous epithelial tumors (cystadenomas, APST/MPSC, and carcinomas) were significantly more often associated with chronic salpingitis (53%) and salpingoliths (32%) than all other cases with or without ovarian neoplasms (p<0.01). APSTs/MPSCs were associated with salpingoliths significantly more frequently than all other groups (p<0.001). For patients with APSTs/MPSCs, salpingoliths were found significantly more often in advanced stage (FIGO II and III) patients (51%) than stage I patients (24%) (p<0.01), but salpingitis, present in 60% of these patients, was not stage-dependent (p>0.05). Chronic salpingitis was identified in 66% of women with endometriosis, which was significantly more frequent than those with normal ovaries (27%) (p<0.001). In conclusion, fallopian tube abnormalities may be related to both the high frequency of infertility and the noninvasive peritoneal implants in women with APSTs/MPSCs. Whether the fallopian tubes with salpingoliths are the source of the peritoneal "implants," the recipient of implants, or are independent is unknown. In addition, the high frequency of salpingitis in women with endometriosis may be related to the mechanism of endometriosis-associated infertility.

Calcinosis↗
PubMed2002

Primary mediastinal seminomas: evidence of single and multiple KIT mutations.

Primary mediastinal seminomas (MS) are rare tumors that are histologically similar to their testicular counterparts. Reports document KIT mutations in gastrointestinal stromal tumors, mastocytosis, and germ cell tumors. Although rare exon 17 mutations have been reported in gonadal seminomas, their mediastinal counterparts have not been studied. To determine whether primary MS harbor KIT mutations, eight formalin-fixed, paraffin-embedded primary MS were microdissected; KIT exons 11 and 17 were sequenced. Four (50%) of the eight cases demonstrated KIT exon 17 mutations. Two of the cases showed single monoallelic base pair alterations; one of these mutations were silent. The other two cases each demonstrated two monoallelic point mutations. In each case, one of these mutations results in protein sequence alteration, and the other is silent. Sequencing of cloned PCR products showed both the silent and amino acid-altering mutations to be found on the same allele (cis). The codon 816 mutation previously identified in mastocytosis and gonadal germ cell tumors was not observed. Non-neoplastic tissues from these patients did not demonstrate KIT mutations; exon 11 mutations were not seen in either tumors or normal tissues. Only the three cases in which amino acid-altering mutations were observed showed a predominantly cytoplasmic CD177 KIT immunohistochemical staining, whereas the one non-amino acid mutating and all wild-type cases were immunonegative. Our findings demonstrate a unique KIT sequence and expression pattern among MS. KIT sequencing may assist in differentiating primary from metastatic MS.

Adolescent↗
PubMed2002

Diagnosis of gastrointestinal stromal tumors: a consensus approach.

As a result of major recent advances in understanding the biology of gastrointestinal stromal tumors (GIST), specifically recognition of the central role of activating KIT mutations and associated KIT protein expression in these lesions, and the development of novel and effective therapy for GISTs using the receptor tyrosine kinase inhibitor STI-571, these tumors have become the focus of considerable attention among pathologists, clinicians, and patients. Stromal/mesenchymal tumors of the gastrointestinal tract have long been a source of confusion and controversy with regard to classification, line(s) of differentiation, and prognostication. Characterization of the KIT pathway and its phenotypic implications has helped to resolve some but not all of these issues. Given the now critical role of accurate and reproducible pathologic diagnosis in ensuring appropriate treatment for patients with GIST, the National Institutes of Health (NIH) convened a GIST workshop in April 2001 with the goal of developing a consensus approach to diagnosis and morphologic prognostication. Key elements of the consensus, as described herein, are the defining role of KIT immunopositivity in diagnosis and a proposed scheme for estimating metastatic risk in these lesions, based on tumor size and mitotic count, recognizing that it is probably unwise to use the definitive term benign for any GIST, at least at the present time.

Antineoplastic Agents↗
PubMedDate not supplied

Detection of hTERT mRNA in colonic brush specimens as an adjunct to cytopathologic diagnosis of colonic adenocarcinoma.

OBJECTIVE: To investigate the role of reverse transcriptase polymerase chain reaction (RT-PCR) in determining telomerase catalytic subunit (hTERT) mRNA to assist with the diagnosis of colonic adenocarcinoma (CCA) in colonic brush cytology specimens. STUDY DESIGN: Twenty-seven colonic brushes of CCA were obtained. Initial cytologic diagnoses included CCA, suspicious for CCA (SFC), atypical (ATY) and unsatisfactory. The cytologic specimens were reviewed. A homogeneous RT-PCR assay for hTERT mRNA was performed on 27 colonic brushes of CCA and 24 controls (10 negative brushes, 6 resected CCA and 8 benign colonic mucosa from resected specimens). A RT-PCR assay for beta 2-microglobulin was used as an internal control for mRNA quality. RESULTS: On review, the initial cytopathologic diagnosis of CCA was confirmed in all 7 cases. In addition, 7 of 19 initially interpreted as SFC and ATY seemed to demonstrate unequivocal cytomorphologic features of malignancy. Telomerase mRNA was more often expressed in CCA than in negative controls in both brush (30%) and resected specimens (57%) (P < .05). Seven cases with hTERT mRNA expression were initially diagnosed as CCA (three), SFC (three) and ATY (one). CONCLUSION: CCA may be underdiagnosed in brush cytopathology. The expression of hTERT mRNA may be determined by RT-PCR in brush specimens and may eventually prove to be a useful diagnostic adjunct in the interpretation of inconclusive cases of CCA. Sparse cellularity in brush specimens may result in a relatively low rate of hTERT detection in colonic brushes of CCA; inflammatory changes may contribute to a higher-than-expected rate of hTERT expression in benign colonic epithelium.

Adenocarcinoma↗