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Biomedical subjects

R O'Grady

Publications and source records attributed to R O'Grady.

At least 19 recordsLinked to original sources
PubMed2001

Histopathological features of ocular toxoplasmosis in the fetus and infant.

BACKGROUND: Ocular disease is a frequent manifestation of congenital Toxoplasma gondii infection. There are only limited data available in the literature concerning early stages of this disease in fetuses and infants. The purpose of our study was to characterize histopathological features in the eyes of 10 fetuses and 2 infants with congenital toxoplasmosis. METHODS: Fifteen eyes from 10 fetuses, 3 eyes from 2 premature infants, and both eyes from a 2-year-old child with congenital toxoplasmosis were examined by light microscopy. Immunohistochemical analysis to identify inflammatory cells and T gondii antigens was performed. The findings in infected eyes were compared with those of age-matched control eyes. RESULTS: Retinitis (10/18 eyes), retinal necrosis (4/18 eyes), disruption of the retinal pigment epithelium (12/18 eyes), and choroidal inflammation and congestion (15/18 eyes) were characteristic findings. Optic neuritis was present in 5 of 8 fetal eyes with associated optic nerve available for evaluation. An eye obtained from a 32-week-old fetus showed retinal rosettes at the edge of a scar. T cells predominated in retinal lesions and choroid. Parasites were identified by immunohistochemical analysis in 10 of 18 eyes. CONCLUSIONS: Ocular toxoplasmosis causes irreversible damage to the retina in utero. The fetus and infant mount inflammatory responses that may contribute to ocular damage. These findings have important implications for serological screening programs and in utero therapy.

Antigens, CD↗
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↗
PubMed2000

Detection of rare mutant alleles by restriction endonuclease-mediated selective-PCR: assay design and optimization.

BACKGROUND: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site. METHODS: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR. RESULTS: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status. CONCLUSION: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.

Alleles↗
PubMed1999

Pigmented adenoma mimicking a juxtapapillary melanoma. A 20-year follow-up.

A 52-year-old white woman was first diagnosed with a tumor of the right optic nerve in 1972. She remained asymptomatic until 1992, when she had a seizure on the left side of her body from a frontoparietal glioblastoma multiforme. Ophthalmic examination revealed enlargement of the eye tumor. This case provides clinical documentation spanning 20 years of a growing, pigmented tumor of the optic nerve head shown histopathologically to be a retinal pigment epithelial adenoma.

Adenoma↗
PubMed1998

Methicillin-resistant Staphylococcus aureus in a high school wrestling team and the surrounding community.

OBJECTIVES: To describe a community outbreak of methicillin-resistant Staphylococcus aureus (MRSA) and to investigate risk factors for MRSA transmission and infection in a wrestling team. DESIGN: Case series and retrospective cohort study. SETTING: A high school wrestling team and the surrounding community in southern Vermont, 1993 to 1994. PATIENTS OR OTHER PARTICIPANTS: The case series included persons whose MRSA-positive infections were identified at a hospital laboratory from January 1, 1993, through February 28, 1994, and a health maintenance organization laboratory from July 1, 1993, through February 28, 1994. A wrestling team case-patient was a 1993-1994 team member with an MRSA-positive culture during the period from January 1, 1993, through February 28, 1994. INTERVENTIONS: Visual inspection of wrestlers before matches was instituted. Affected wrestlers were excluded from wrestling and advised to seek appropriate medical care. Heightened attention was given to personal and environmental hygiene. MAIN OUTCOME MEASURES: Colonization or infection with MRSA. RESULTS: Seven of 32 team members were MRSA positive (6 infected, 1 colonized). All lesion-positive wrestlers were tested by pulsed field gel electrophoresis and found to be infected with the same MRSA strain, as were 6 nonwrestlers. No risk factors for MRSA infection were identified. CONCLUSIONS: The MRSA was transmitted among members of a wrestling team. Infection with MRSA should be suspected in outbreaks of boils that are nonresponsive to standard antibiotic therapy among healthy participants of contact sports and their close contacts.

Adolescent↗
PubMed1998

Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.

The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.

Adenocarcinoma↗
PubMed1996

Epidermal growth factor-like molecular species in normal bronchoalveolar lavage fluid.

Normal bronchoalveolar lavage fluid (BALF) contains mitogenic activity for fibroblasts and type 2 pneumocytes. A number of growth factors that might contribute to this activity have been identified in BALF. We found that a molecule or molecules able to bind to epidermal growth factor (EGF) receptors on mouse lung fibroblasts were present in normal mouse BALF and could be blocked by an antiserum to mouse EGF. Receptor binding was partially blocked by preincubation with heparin, indicating a relationship to the heparin-binding subgroup of EGF-like growth factors. Heparin markedly enhanced the mitogenic activity of BALF for fibroblasts, but we were unable to establish whether the EGF-like molecule contributed to this activity. Immunoblotting using the anti-EGF serum identified a protein of M(r) 88,000 in concentrated BALF. The cellular source and physiological role of this growth factor merit further investigation.

Animals↗
PubMed1996

Epithelial cell-derived transforming growth factor-beta in bleomycin-induced pulmonary injury.

We have investigated whether enhanced secretion of transforming growth factor-beta (TGF-beta) by distal respiratory epithelial cells was associated with the development of bleomycin-induced pulmonary fibrosis. Type 2 pneumocyte-enriched preparations of bronchioloalveolar epithelial cells from normal mouse lung tissue released latent TGF-beta when cultured in serum-free medium. TGF-beta in culture supernatants could be detected using a sensitive enzyme immunoassay which employed enzyme complex amplification as a reporter system, as well as by a radiolabelled receptor competition assay. Exposure to bleomycin and other potentially fibrogenic stimuli in vitro did not stimulate production of TGF-beta by the epithelial cells but release was enhanced by treatment of the cells with interferon-gamma. Type 2 pneumocyte-enriched cell preparations obtained following induction of a pulmonary inflammatory response by administration of intratracheal bleomycin to susceptible C57BL/6 mice did not demonstrate increased release of TGF-beta in culture. However, the concentration of TGF-beta in bronchoalveolar lavage (BAL) fluids was significantly elevated compared to controls at 1 and 2 weeks after bleomycin-induced injury in these mice. No such increase was detected in BAL fluids from BALB/c mice, which are resistant to the effects of bleomycin. These results provide no support for a pathogenetic role of alveolar epithelial cell-derived TGF-beta in bleomycin-induced pulmonary fibrosis. Nevertheless, elevated levels of TGF-beta in BAL fluids may provide a marker of the progression of pulmonary injury to fibrosis.

Animals↗
PubMed1994

Public health nursing services for drug-exposed infants and mothers: a pilot study.

The purpose of this exploratory study was to identify the pattern of public health nursing service provided to substance-using mothers and their infants. To determine the effectiveness of public health nursing in a later randomized clinical trial (subsequent study), the first essential step was to identify clearly the nursing services that would constitute the intervention. For consistency, a clinical-practice checklist was developed by the public health nurses (PHNs) with the assistance of the investigators. Over a 6-month period, six PHNs made home, telephone, and office or clinic visits to mothers and their infants who were referred for follow-up due to a positive toxicology screen test at the time of delivery. For this client group (N = 28), the PHNs made one or two home visits to 17 mothers (61%), one or two telephone visits to 12 mothers (43%), and one or two office or clinic visits to 5 mothers (2%). Nearly one third of the time (29%), home-visit attempts were unsuccessful, and the client attrition rate was 50%. Polydrug use was common in this group, with 71% using two or more substances at the time of delivery. Referrals to substance-abuse treatment programs were usually not accepted by the clients, and many referrals were made for medical care and infants supplies. The most frequently mentioned area for advocacy was housing. Overall, public health nursing services were provided in a very limited time span of client availability.

Adolescent↗
PubMed1994

Epidermal growth factor-like activity in bronchoalveolar lavage fluid in experimental silicosis.

We investigated whether development of pulmonary fibrosis following inhalational exposure of mice to silica (quartz) dust was accompanied by enhanced secretion of activity resembling epidermal growth factor (EGF). Mitogenic activity for pulmonary fibroblasts was assessed in bronchoalveolar lavage fluid (BALF) using a serum-free bioassay. Activity in BALF from mice exposed to nonfibrogenic titanium dioxide dust was comparable to that in BALF from normal animals. In contrast, mitogenic activity was significantly increased at 6 and 12 weeks after inhalation of silica particles, coinciding with the appearance of collagenised lesions in the lung. BALF from mice exposed to silica 6 weeks previously had significantly higher concentrations of growth factor(s) able to bind to EGF receptors on pulmonary fibroblasts. In parallel, macrophages within inflammatory lesions in the airspaces acquired immunoreactivity for EGF. The presence of an increased concentration of EGF-like growth factor(s) in BALF might constitute a marker of particle-induced pulmonary fibrosis.

Animals↗
PubMed1994

Immunoenzymatic detection of collagen production by pulmonary fibroblasts in serum-free culture.

We have developed an improved assay for the production of collagen by fibroblasts. Early passage adult mouse lung fibroblasts, established and maintained in serum-free culture, were employed as the target cells. An enzyme immunoassay was used for detection of type I collagen deposited on the substratum, permitting adaptation of the technique to cultures in 96-well microplates. Approximately two-fold enhancement of collagen deposition was induced by exposure to a concentration of 3 ng/ml of transforming growth factor-beta 1 or of 100 ng/ml of insulin-like growth factor-1 for 48 hr.

Animals↗
PubMed1993

Secretion of epidermal growth factor-like molecular species by lung parenchymal macrophages: induction by interferon-gamma.

A population of cells enriched for pulmonary interstitial macrophages was obtained by differential adherence of lung parenchymal cells released by dissociation with trypsin. These cells secreted a molecule or molecules that bound to epidermal growth factor (EGF) receptors expressed on pulmonary fibroblasts. Secretion was reproducibly stimulated by exposure of the macrophages to interferon-gamma. Binding to EGF receptors could be blocked by a polyclonal antibody to EGF. It could also be partially blocked by incubation with heparin, suggesting that at least a component of the activity might be due to a member of the heparin-binding subgroup of the EGF family of growth factors. Because pulmonary fibrosis is consistently associated with inflammatory accumulation of activated T-lymphocytes, induction by interferon-gamma of growth factor secretion by macrophages could have pathogenetic importance. We speculate that similar cellular interactions may play a role in the progression of other chronic inflammatory lesions to fibrosis.

Animals↗
PubMed1992

Primary malignant melanoma of the optic nerve simulating a melanocytoma.

Primary malignant melanoma of the optic nerve head is an extremely rare tumor. We describe a patient with a pigmented optic nerve lesion that simulated a growing melanocytoma. The pigmented tumor slowly infiltrated the optic nerve causing disc edema, retinal venous congestion, progressive visual field loss, and eventually, loss of central acuity. Following enucleation of the eye, and later resections of the optic nerve to the chiasm, histopathologic examination of the optic nerve, disc, and peripapillary region demonstrated a primary malignant melanoma of the optic nerve without choroidal involvement. Involvement of the optic nerve extended 26 mm beyond the lamina cribrosa. Malignant melanoma of the optic nerve is a real entity and may clinically simulate a melanocytoma.

Cranial Nerve Neoplasms↗
PubMed1992

Mitogenic activity for fibroblasts induced by silica and titanium dioxide particles in vitro and in vivo.

Experimental studies on particle-induced pulmonary fibrosis have not provided consistent evidence for the specific induction of fibroblast-regulating cytokines by pulmonary macrophages in response to fibrogenic as compared to non-fibrogenic particles. Using an optimized, wholly serum-free bioassay, we assessed mitogenic activity for pulmonary fibroblasts in supernatants of short-term cultures of alveolar macrophages exposed to either fibrogenic silica or non-fibrogenic titanium dioxide ducts. The responses to these supernatants were influenced by the replicative status of the target cells, in that samples which stimulated non-cycling fibroblasts caused inhibition of DNA synthesis by cycling cells when tested at the same concentration. However, both silica and titanium dioxide elicited comparable secretion of growth factor activity by macrophages, following either in-vitro or in-vivo administration of particles. In contrast, bronchoalveolar lavage fluids from animals that received intratracheal injections of silica, but not from those that received titanium dioxide, exhibited a sustained reduction in fibroblast-stimulating activity. We conclude that secretion of growth factor activity by alveolar macrophages in culture is induced by particles in a non-specific manner. However, alterations in mitogenic activity in bronchoalveolar lavage fluid may constitute a biological marker of the pattern of pulmonary injury which progresses to fibrosis.

Animals↗
PubMed1992

Role of lymphocytes in silicosis: regulation of secretion of macrophage-derived mitogenic activity for fibroblasts.

We investigated the role of pulmonary lymphocytes in regulating the secretion by alveolar macrophages (AM) of mitogenic activity for lung fibroblasts, in an experimental model of the initial stages of silicotic inflammation and fibrosis. Following intratracheal instillation of silica, pulmonary parenchymal lymphocytes produced a lymphokine(s) that caused modest stimulation of the secretion of mitogenic activity by normal AM. Co-culture of small numbers of lymphocytes from silica-injected animals with AM induced enhanced secretion of fibroblast growth factor activity which was comparable to the maximal response elicited by recombinant interferon-gamma. Lymphocytes from animals given non-fibrogenic titanium dioxide exhibited no such effects. The stimulatory effect of lymphocytes from silica-treated animals in co-culture with macrophages was abrogated when the cells were separated by a microporous membrane. Our findings demonstrate that lymphocytes participating in the response to pulmonary deposition of silica are able to induce the secretion of a growth factor(s) for fibroblasts by pulmonary macrophages, possibly via lymphokines expressed on the cell surface or secreted at sites of cell-to-cell contact.

Animals↗
PubMed1991

Primary culture of adult mouse lung fibroblasts in serum-free medium: responses to growth factors.

We report a completely serum-free system for primary culture of fibroblasts from explants of adult mouse lung tissue which permits bioassays for cytokine activity to be performed using unselected populations of cells at low passage number, without interference by serum binding proteins or interacting growth factors. Cultures were established on collagen-coated surfaces in medium MCDB 201 containing albumin, transferrin, epidermal growth factor, lipids, prostaglandin E1, vitamin E, and reducing agents. The cells were morphologically and ultrastructurally typical of fibroblasts in culture and demonstrated expression of vimentin and induction of expression of desmin in culture. Proliferation of the cells was reproducible between different primary cultures and was growth factor dependent. Both cycling and growth-arrested cells exhibited increased DNA synthesis when stimulated with epidermal growth factor, platelet-derived growth factor, or basic fibroblast growth factor, which functioned as complete mitogens, but did not respond to insulin, tumor necrosis factor or interleukin-1 beta. Maximal induction of DNA synthesis by epidermal growth factor required the continued presence of the mitogen in the culture medium. These results cannot be satisfactorily explained by the competence-progression model of responses to mitogenic stimuli but support and extend the findings of other studies using diploid fibroblasts.

Animals↗