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Biomedical subjects

C A Poole

Publications and source records attributed to C A Poole.

At least 19 recordsLinked to original sources
PubMed1999

Influence of a family history of cancer within and across multiple sites on patterns of cancer mortality risk for women.

A case-control study nested within a large cohort, the American Cancer Society Cancer Prevention Study-1, was conducted to test associations between a family history of cancer and cancer mortality in women. By using logistic regression, the authors analyzed family history, as reported by 429,483 women enrolled in 1959, relative to subsequent mortality through 1972 from cancer within and across multiple sites. The associations between family history and cancer mortality were generally stronger within cancer sites than across cancer sites. Within-site associations were found for breast cancer (odds ratio (OR) = 1.9), colorectal cancer (OR = 1.6), stomach cancer (OR = 1.9), and lung cancer (OR = 1.7). Across-site associations were observed for a family history of 1) breast cancer as a risk factor for ovarian cancer mortality (OR = 1.6), 2) stomach cancer as a risk factor for ovarian cancer mortality (OR = 1.5), and 3) uterine cancer as a risk factor for pancreatic cancer mortality (OR = 1.6). A general pattern of positive associations was observed between a family history of cancer at several sites and subsequent death from pancreatic cancer. These findings support the growing body of evidence from cancer genetics suggesting that inherited cancer-susceptibility genes increase the risk for cancer at many sites and are not specific to cancer risk within a single site.

Adult↗
PubMed1998

Loss of glucose transporter-2 precedes insulin loss in the nonobese diabetic and the low-dose streptozotocin mouse models: a comparative immunohistochemical study by light and confocal microscopy.

Glucose transporter-2 (glut2) is underexpressed in beta cells of several rodent models of non-insulin-dependent diabetes mellitus (NIDDM). This may also be true for rodent models of insulin-dependent diabetes mellitus (IDDM). The present study examines two murine models of autoimmune IDDM, the nonobese diabetic (NOD) and the low-dose streptozotocin (stz) murine models for changes in the expression of glut2 by double-label light and confocal microscopy during various stages of the disease. The spatial distribution of glut2 cells was also examined in relation to insulin immunoreactive cells and the islet inflammatory cells during these stages. In both the female NOD mouse and the female Swiss mouse without stz treatment, glut2 colocalized with insulin in virtually all the beta cells. In the NOD mouse, islets with moderate to advanced insulitis showed either an absence or considerably reduce expression of glut2 in insulin-containing beta cells. Cells with reduced glut2 expression were usually located adjacent to the region of insulitis. At onset of diabetes, glut2 immunolabeling was reduced despite the preservation of weak insulin immunoreactivity. In Swiss mice treated repeatedly with stz, glut2 labeling began to decline in select Beta cells after the fourth injection in approximately 50% of the islets, despite the lack of insulitis. At this stage expression of glut2 fell in a small number of islets with evidence of early macrophage infiltration. Loss of glut2 became more pronounced in nondiabetic Swiss mice after the fifth injection. At this stage glut2 labeling in the plasma membrane appeared diffuse and variable. At onset of stz-induced diabetes, glut2 expression significantly fell, despite weak immunoreactivity for insulin. This loss was associated with an enhanced influx of both macrophages and T lymphocytes within the islets of diabetes mice. In both the NOD and the low-dose stz mouse models, loss of glut2 thus occurs from an early stage and precedes hyperglycaemia. This loss may be mediated by immune and nonimmune mechanisms.

Animals↗
PubMed1998

Identification of distinct metabolic pools of aggrecan and their relationship to type VI collagen in the chondrons of mature bovine articular cartilage explants.

The metabolism and distribution of newly synthesized aggrecan present in the extracellular matrix of intact explant cultures of mature articular cartilage was investigated with respect to type VI collagen-stained chondrons. Using biochemical, autoradiographical and novel confocal immunohistochemical techniques it was shown that aggrecan exists as a number of distinct pools that are located within the extracellular matrix of the tissue. The first was identified as a pool of high specific radioactivity, much of which appeared in the medium one day after incubation with radiolabeled sulfate. Of the radiolabeled aggrecan remaining within the extracellular matrix, three pools were differentiated on the basis of time and location within the extracellular matrix. One pool was resident within the pericellular microenvironment associated with the chondron, one migrated into the territorial matrix adjacent to the chondron and one was sequestered long term in the interterritorial matrix. Analysis of the kinetics of loss of radiolabeled aggrecan macromolecules present in the region of matrix defined by the chondron suggests that this pool rapidly turns over and is a precursor to the pools of aggrecan present in the territorial and interterritorial matrix. There were marked differences in the distribution of newly synthesized aggrecan present in these regions of the extracellular matrix in explant cultures maintained with or without fetal calf serum. In the absence of serum, more of the newly synthesized aggrecan moved into the territorial and interterritorial matrix indicating that the presence of serum in the culture medium influenced the tissue distribution of aggrecan. This work indicates that the pericellular microenvironment of the chondron plays an important role in the retention and maturation of aggrecan prior to the sequestration of aggrecan complexes into the functional load bearing matrices of adult articular cartilage.

Aggrecans↗
PubMed1997

Confocal analysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and aortic smooth muscle cells.

Detyrosinated and acetylated alpha-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium-centrosome and centrosome-Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

Acetylation↗
PubMed1997

Double-label immunofluorescence study of glutamic acid decarboxylase in the fetal and adult ovine pancreas by light and confocal microscopy: evidence for predominant beta-cell coexpression.

Glutamic acid decarboxylase (GAD) is present in the central nervous system and in several nonneuronal tissues including the pancreatic islets. There are two isoforms with molecular weights of 65 kDa (GAD65) and 67 kDa (GAD67). The cellular specificity of the two molecular forms of GAD and their levels within the mammalian islets may be species-dependent, being coexpressed in both beta and in non-beta cells. We have examined the ovine pancreas, from the adult and fetal stages of late gestation, for the expression of GAD65 within the islet cells by double-label immunofluorescence light and confocal microscopy. In the adult tissue, GAD65 was colocalized in a majority of the beta cells (> 95%), with only a few glucagon and somatostatin cells (< 5%) showing immunolocalization. During the fetal stages GAD65 also showed a similar predominant beta-cell coexpression. The enzyme was also detected in a few fetal glucagon (< 5%) but not somatostatin cells. In the degenerating large fetal islets, GAD65 was also observed in the majority of the residual beta cells. These results demonstrate that in the ovine pancreas GAD65 is expressed during fetal development and is predominantly beta-cell-restricted. This pattern of expression is maintained during adult life. However, the physiological role of pancreatic GAD and/or its biosynthetic product, gamma-aminobutyric acid, in islet function in the sheep and in other ruminants remains unclear.

Aging↗
PubMed1997

Immunolocalization of type IX collagen in normal and spontaneously osteoarthritic canine tibial cartilage and isolated chondrons.

OBJECTIVE: The pericellular localization of type IX collagen in avian and mammalian hyaline cartilages remains controversial, while its distribution during osteoarthritic degeneration is poorly understood. This study aimed to compare and contrast the immunohistochemical distribution of type IX collagen in normal mature and spontaneously osteoarthritic canine tibial cartilage. DESIGN: Thick vibratome sectioning techniques were evaluated and compared with isolated chondrons using a range of streptavidin-linked probes in combination with light, confocal and transmission electron microscopy. RESULTS: In normal intact samples, type IX collagen was concentrated in the pericellular microenvironment, while a weaker extracellular reaction around each chondron separated the territorial matrix from the unstained interterritorial matrix. Further differentiation was evident in isolated chondrons where the fibrous pericellular capsule stained more intensely than the tail and interconnecting segments between columnated chondrons. Two regions of type IX reactivity were identified in osteoarthritic tissue: an intensely stained superficial reactive region below the eroding margins, and normal deep layer cartilage where pericellular staining persists. The superficial reactive region was characterized by chondron swelling and chondrocyte cluster formation, a loss of pericellular type IX staining, and a significant increase in matrix staining between clusters. Disintegration and loss of fibrillar collagens was evident in both the swollen microenvironment and adjacent territorial matrices. CONCLUSIONS: The results suggest that changes in type IX distribution, expansion of the pericellular microenvironment and chondrocyte proliferation represent key elements in the chondron remodeling and chondrocyte cluster formation associated with osteoarthritic degeneration.

Animals↗
PubMed1997

Isolated chondrons: a viable alternative for studies of chondrocyte metabolism in vitro.

OBJECTIVE: To develop and test a simple enzymatic procedure for isolating chondrons, which consist of the chondrocytes and their surrounding pericellular microenvironment. DESIGN: Chondrons were obtained by digesting adult human articular cartilage with a mixture of dispase and collagenase. Chondrons and chondrocytes were cultured in alginate beads, immunofluorescence labeled and examined by confocal microscopy. RESULTS: Comparison of freshly isolated chondrons with cryostat sections of cartilage revealed that type VI collagen, type II collagen and aggrecan were retained, but fibronectin and a unique chondroitin sulfate epitope recognized by the antibody, 7D4, were lost. Comparison of enzymatic and mechanical homogenization methods revealed subtle changes in chondron morphology and retention of fibronectin in mechanically isolated chondrons. Average yield of enzyme-isolated chondrons was slightly lower than that of chondrocytes isolated by pronase and collagenase digestion, but was much greater than that reported for mechanically isolated chondrons. Enzyme-isolated chondron viability was greater than 80% 1 day after isolation, and continued to be above 80% through 7 weeks of alginate bead culture. Viability of isolated chondrocytes was initially greater than 80% but fell to 60-80% with time in culture. Chondrons and isolated chondrocytes had a similar division rate except osteoarthritic chondrons were significantly slower after 2 weeks in culture. Cell division was more rapid for nonosteoarthritic chondrons than for osteoarthritic ones. CONCLUSIONS: Enzymatic isolation of chondrons is relatively simple, gives better yield and viability than mechanical isolation, but comparable yield and viability of traditional chondrocyte isolation. Enzymatic chondron isolation allows the effect of the in vivo-formed pericellular matrix on chondrocyte metabolism to be studied in vitro.

Adolescent↗
PubMed1997

Inducible nitric oxide synthase in pancreatic islets of the non-obese diabetic mouse: a light and confocal microscopical study of its ontogeny, co-localization and up-regulation following cytokine administration.

Nitric oxide has been shown to mediate beta-cell destruction in rodent islets exposed to interleukin 1 beta in culture. The inhibitory effect is potentiated by tumour necrosis factor-alpha and interferon-gamma. Cytokine stimulation leads to gene transcription and translation of inducible nitric oxide synthase, the biosynthetic enzyme of nitric oxide. In the non-obese diabetic mouse, progressive invasion of pancreatic islets by immune cells may lead to local production of inflammatory cytokines, resulting in inducible nitric oxide synthase expression within the islets. In this study, the ontogeny of this enzyme and its cellular expression were examined in pancreatic sections of female non-obese diabetic mice by double-label immunofluorescence. Light and confocal microscopy were employed to study the up-regulation, co-localization and immunocytoplasmic distribution of the enzyme in female non-obese, diabetic and Swiss mice following cytokine treatment. From day 40 to day 220 a small number of beta-cells and a proportion of macrophages, usually in peri-islet and exocrine areas, expressed the enzyme. At onset of diabetes, an increasing number of macrophages within and surrounding the islets were positive for the enzyme. Treatment of day 60 female non-obese diabetic mice with interleukin 1 beta alone and in combination with tumour necrosis factor-alpha and/or interferon-gamma resulted in a significant influx of macrophages into the pancreas, while this was lower in female Swiss mice treated similarly. Cytokine administration led to intense but sometimes eccentric immunocytoplasmic labelling for the enzyme in a considerable proportion of macrophages and beta-cells. Macrophages positive for inducible nitric oxide synthase were located in peri- and intra-islet areas, being distal and adjacent to enzyme-positive and negative beta-cells. Treatment with tumour necrosis factor-alpha and/or interferon-gamma did not lead to enzyme up-regulation. These results show that in the non-obese diabetic mouse there is low and sustained expression of islet inducible nitric oxide synthase in the prediabetic period, which is followed by an increase around onset. However, treatment of female non-obese diabetic and Swiss mice with interleukin-1 beta, alone or together with tumour necrosis factor-alpha and/or interferon-gamma leads to a marked expression of this enzyme within macrophages and beta-cells.

Animals↗
PubMed1997

Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel.

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1-3 microns in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4-6 weeks in culture and progressively developed a broad territorial region up to 12 microns wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly.

Agar↗
PubMed1997

Structural colocalisation of type VI collagen and fibronectin in agarose cultured chondrocytes and isolated chondrons extracted from adult canine tibial cartilage.

Cell-matrix and matrix-matrix interactions are of critical importance in regulating the development, maintenance and repair of articular cartilage. In this study, we examined the structural colocalisation of type VI collagen and fibronectin in isolated chondrons and long-term agarose cultured chondrocytes extracted from normal adult canine articular cartilage. Using double labelling immunohistochemistry in conjunction with dual channel confocal microscopy and digital image processing we demonstrate that type VI collagen and fibronectin are distributed in a similar staining pattern and are colocalised at the surface of cultured chondrocytes and isolated chondrons. The results suggest that type VI collagen and fibronectin may play a role in both cell-matrix adhesion and matrix-matrix cohesion in the pericellular microenvironment surrounding articular cartilage chondrocytes.

Animals↗
PubMed1997

Articular cartilage chondrons: form, function and failure.

The chondrocyte and its pericellular microenvironment together represent the chondron, historically considered the primary structural, functional and metabolic unit of articular and other hyaline cartilages. This review summarises research over the last 10 years to establish the molecular anatomy, functional properties and metabolic contribution of the chondron in articular cartilage homeostasis, and its failure during the initiation and progression of degenerative osteoarthritis.

Animals↗
PubMed1996

Glutamic acid decarboxylase 65 and 67 isoforms in fetal, neonatal and adult porcine islets: predominant beta cell co-localization by light and confocal microscopy.

Glutamic acid decarboxylase (GAD) has been shown to exist as two isoforms with molecular weights of 65 kD (GAD65) and 67 kD (GAD67) in the central nervous system as well as in several non-neuronal tissues, including the pancreatic islets. Recently, this enzyme has been proposed as a key beta-cell autoantigen in insulin-dependent diabetes mellitus (IDDM). In the present study, we used double label light and confocal microscopy to examine the expression of the two GAD isoforms in islet cells of fetal, neonatal and adult porcine pancreas. We also aimed to identify the islet cell-type(s) which co-express GAD. In the adult pig, GAD65 was localized exclusively in most of the beta cells, whereas GAD67, in addition to being present in a majority of the beta cells, was also seen in a proportion of glucagon and somatostatin labelled cells. In the 90-day fetus and the 7-day neonate, while GAD65 was also observed in a majority of beta cells, a proportion of glucagon cells also co-expressed this isoform. The cellular expression of GAD67 in the fetal and neonatal stages was similar to that in the adult. Detailed confocal analysis of GAD65 immunoreactive cells showed a granular cytoplasmic staining, with labelled granules often concentrated in specific perinuclear regions, possibly the Golgi apparatus. In contrast, GAD67 positive cells showed more diffuse cytoplasmic staining. The predominant expression of both the isoforms in porcine beta cells suggests that islet cells from this species may act as a suitable cellular model for study of GAD autoreactivity during the early stages of IDDM.

Animals↗
PubMed1996

Confocal imaging of the keratocyte network in porcine cornea using the fixable vital dye 5-chloromethylfluorescein diacetate.

This study reports on the combined use of an aldehyde fixable, cell viability fluoroprobe, 5-chloromethylfluorescein diacetate (CMFDA), confocal laser scanning microscopy and digital image reconstruction, to produce high resolution images of corneal keratocyte preparations in situ. The central region of freshly enucleated porcine corneae were removed and stained overnight at 4 degrees C with CMFDA. The tissue was washed, fixed, and frozen for cryosectioning in either a horizontal or antero-posterior orientation. Sections from anterior, central and posterior stroma were examined with a confocal microscope, and the digital images rendered as three-dimensional stereo reconstructions. Fluorescent CMFDA which completely permeated the cell bodies and extremes of the finest ramifying cell processes of all keratocytes provided exceptional high resolution images of the three morphologically distinct cell subpopulations at different levels of the stroma, and enabled improved characterisation of each cell type. Anteriorly was a thin, dense, non-lamellar network of keratocytes subjacent Bowman's membrane. In the central stroma, keratocytes were arranged in layers, the cell bodies had a flattened pyramidal or stellate shape, and the fine cell processes formed extensive distal ramifications. Immediately anterior to Descemet's membrane a small subpopulation of keratocytes with large cell bodies and short branched processes was identified. Extensive and diverse cell-to-cell contacts were orientated in all stromal planes, including ramping cell bridges between keratocyte lamellae in the central stroma. The use of the cell viability dye CMFDA is feasible and valuable for enhancing the visibility of entire keratocyte population in the intact cornea. Diverse multi-directional cell processes and intercellular contacts throughout the keratocyte network suggest a strong capacity for direct communication and cohesion in the maintenance and repair of the stromal matrix. Keratocytes closely related to the epithelium and endothelium have unique morphologies which may relate to specialised functions of these interface cells.

Animals↗
PubMed1996

Detection of viable and non-viable cells in connective tissue explants using the fixable fluoroprobes 5-chloromethylfluorescein diacetate and ethidium homodimer-1.

The efficacy of connective tissue explants is difficult to determine, particularly where autopsy material is required for research or clinical applications. We report here an optimised protocol using 5-chloromethylfluorescein diacetate (CMFDA) and ethidium homodimer-1 to distinguish viable and non-viable cells in a range of connective tissue explants. Biopsies and explants of corneae, arteries, cartilage and skin were loaded with fluoroprobes for extended periods (< or = 24h) at low temperatures (4 degrees C), fixed in paraformaldehyde, and processed using a variety of embedding, sectioning, autoradiographic, and immunohistochemical procedures. Detection of fluorescent green CMFDA and red ethidium homodimer was achieved using epi-illuminated light or dual channel confocal microscopy, and clearly differentiated live from dead cells throughout the explants. Furthermore, the intracellular distribution of CMFDA provided superior images of cell shape and morphology not previously available using conventional histochemical techniques. Adaptations of this protocol could prove valuable in a variety of research and clinical applications.

Adolescent↗
PubMed1995

Operative gastric perforation: a rare complication of ventriculoperitoneal shunt.

Complications of ventriculoperitoneal shunts are reported in 24-47 % of cases. These include mechanical malfunction and infection, cerebrospinal fluid collection, shunt migration, and visceral perforation. Intestinal perforation, most commonly colonic, may be associated with meningitis or cerebral abscess. Gastric migration has been previously described as a late complication related to the use of stiff Raimondi coil-spring peritoneal catheters in children with malnutrition and abdominal adhesions. We report a case of operative gastric perforation, a rare complication which was detected prior to development of symptoms.

Dandy-Walker Syndrome↗
PubMed1993

Keratocyte networks visualised in the living cornea using vital dyes.

Fluorescent viability probes have been used to visualise and investigate the viability, morphology and organisation of the keratocyte within the stroma of the intact living cornea. The live cell probe, calcien-AM, in combination with a dead cell probe, ethidium homodimer (Live/Dead Assay, Molecular Probes, U.S.A.) proved superior to earlier generation vital dyes such as fluorescein diacetate or 5,6-carboxyfluorescein diacetate, initially used in combination with ethidium bromide. The ubiquitous distribution of esterase enzymes that cleave calcien-AM within the keratocyte cytoplasm produced a high concentration of fluorescently active calcein throughout the cell, including fine cell processes. Epi-illuminated fluorescence microscopy on transparent corneal dissections subsequently revealed details of keratocyte microanatomy and three-dimensional network organisation in situ. Three morphologically discrete subpopulations of keratocytes were identified: two formed relatively small bands of cells, immediately subjacent to either Bowman's or Descemet's membranes, the third subpopulation constituting the majority of keratocytes typically located within the corneal stroma. The results indicate that calcein-AM is able to penetrate intact living cornea revealing cell viability, and it also has the capacity to 'trace' cellular elements and reveal fine structure within a dense connective tissue matrix.

Animals↗
PubMed1992

Microbiological assay of avoparcin in animal feeds and pre-mixes--co-operative study.

A microbiological method for the determination of the antibiotic growth promoter avoparcin in animal feeds and pre-mixes was first published in 1979. The existing method has been applied to new matrices as commercial use of avoparcin has spread. Problems with the existing analytical procedure have been reported, particularly in Germany. This paper identifies the causes of the problems and includes revisions to the existing analytical procedure. The testing of these revisions in co-operative studies with up to five German official laboratories is also reported.

Animal Feed↗