PubMed Health⌕ Search

Biomedical subjects

E Gibney

Publications and source records attributed to E Gibney.

18 recordsLinked to original sources
PubMed2001

Progesterone receptor immunohistochemical quantitation compared with cytosolic assay: correlation with prognosis in breast cancer.

Quantitation of estrogen and progesterone receptors (PR) represents the standard of care in the treatment of patients with breast cancer. Historically this was performed by cytosolic assay; current methods utilize immunohistochemical staining, which may be quantitated visually or by image cytometry. Formalin-fixed paraffin embedded sections from 95 breast carcinomas were immunostained with an avidin-biotin complex technique. steam antigen retrieval, and a monoclonal PR antibody (1/40 Biogenex). Nuclear immunostain was quantitated visually as the percentage of immunopositive nuclei, scored as 0 to 4. By image cytometry, the percentage of positively staining nuclear area (PPNA) was determined in 15 hpf using the CAS 200 Image Analyzer. Dextran-coated charcoal (DCC) ligand binding assay data were divided into negative (<10 fmol), low positive (10-50), or positive (>50). A statistically significant correlation was found between stage (P = 0.0001), the presence of nodal metastases (P = 0.0001), cytosolic assay (P = 0.036), image cytometry (P = 0.01), and disease-free survival. Only stage (P = 0.0001) and PR quantitation per cytosolic assay (P = 0.0001) correlated with overall survival. The method of choice for the assessment of PR hormone status in breast carcinomas is the DCC ligand binding assay. This method correlates with both survival and disease-free survival. Image cytometric quantitation of PR immunohistochemical staining correlates only with disease-free survival. The commonly used method of visual quantitation of PR immunostaining fails to relate either to survival or disease-free survival.

Breast Neoplasms↗
PubMed1997

Total energy expenditure in patients with small-cell lung cancer: results of a validated study using the bicarbonate-urea method.

The bicarbonate-urea method for measuring CO2 production was applied to eight free-living patients (mean age, 68 +/- 10 years; mean weight, 69 +/- 10 kg; mean height, 1.65 +/- 0.10 m) with unresectable small-cell lung cancer for a period of 1 day (n = 5) or 2 days (n = 3). The basal metabolic rate (BMR) was measured in all subjects. The technique was first validated against whole-body indirect calorimetry over an additional 24-hour period in five of these subjects. The bicarbonate-urea method predicted net CO2 production to be 102.1% +/- 3.4% of that measured by whole-body indirect calorimetry, and energy expenditure, 101.5% +/- 3.8% of the measured calorimeter value (8.1 +/- 1.6 MJ/d). The 24-hour recovery of label in CO2 excreted by the body was 95.6% +/- 0.5%. In free-living conditions, the bicarbonate-urea method predicted energy expenditure to be 9.0 +/- 2.6 MJ/d. BMR was elevated by a mean of 6% (P < .05) compared with the Schofield standards. The physical activity level ([PAL] the ratio of total energy expenditure [TEE] to BMR) was variable (1.15 to 1.87), but the mean value was only 1.36 +/- 0.22, considerably less than that of moderately active healthy subjects with estimated PAL values of 1.55 (P < .05) to 1.65 (P < .01) and the mean results obtained by doubly labeled water (previous studies) in healthy age- and sex-matched subjects. This is the first time a tracer method for measuring CO2 production and energy expenditure has been validated against whole-body 24-hour indirect calorimetry in patients with lung cancer or a systemic inflammatory reaction. The agreement between the two methods is similar to that observed in normal subjects. This is also the first time a tracer method has been used to measure energy expenditure in free-living patients with lung cancer. The results suggest that TEE and the energy requirements necessary to maintain energy balance were not increased despite basal hypermetabolism, because of the associated decrease in physical activity.

Adult↗
PubMed1996

Positive regulation of corneal type V collagen mRNA: analysis by chicken-human heterokaryon formation.

Our previous studies have suggested that type V collagen is at least one factor responsible for the characteristically small, uniform diameter of striated collagen fibrils of the corneal stroma. These fibrils, which are heterotypic combinations of collagen types I and V, contain four- to fivefold more type V collagen than those of tendon and sclera. The latter are much larger and more heterodisperse. This high content of type V collagen in cornea is reflected by an equally elevated content of alpha1(V) chain mRNA in corneal fibroblasts. Thus, the increased production of the molecule in cornea appears to be regulated at the level of transcription and/or mRNA stability. One possible explanation for this is that corneal fibroblasts contain positive regulatory factors that specifically upregulate transcription of the type V collagen genes and/or increase their mRNA stability. To test this possibility, we have produced transient heterokaryons by fusing chicken corneal fibroblasts with two human noncorneal cell lines selected as containing little if any alpha1(V) mRNA. If the chicken corneal cells contain positive regulators that can act across species, these regulators should result in increased levels of the human alpha1(V) transcript. The results were evaluated by reverse transcript-polymerase chain reaction employing a primer pair selected for its ability specifically to amplify part of the human alpha1(V) mRNA. In fusions between chicken corneal fibroblasts and the human cell lines, after a lag of 10-14 h the heterokaryon-containing cultures showed de novo appearance or upregulation of human alpha1(V) chain mRNA, compared with that of the parental cell lines. Cultures of the mixed cell types that had not been fused showed no such upregulation, so the effect was not mediated by diffusible substances acting between the cells. Chicken tendon fibroblasts, a low producer of type V collagen, when tested in the same assay, evoked no detectible increase in the human transcript. Thus, corneal cells do contain positive regulators for alpha1(V) chain mRNA, and this effect is at least somewhat cell specific.

Animals↗
PubMed1996

Characterization and developmental regulation of avian corneal beta-1,4-galactosyltransferase mRNA.

Corneal development involves the synthesis and assembly of a number of specialized extracellular matrices. These matrices have distinctive properties derived from a unique assembly of collagens, proteoglycans and glycoproteins. The synthesis of each of these requires a number of enzymes. By probing a corneal cDNA library for genes that appeared to be up-regulated in cornea we have isolated a cDNA that represents an mRNA encoding the enzyme beta-1,4-galactosyltransferase. In cornea, a major function for this enzyme is likely to be in the synthesis of the keratan sulfate proteoglycan, lumican. Employing quantitative reverse transcript-polymerase chain reaction, we have observed that the steady-state level of mRNA for the molecule is elevated during certain stages of corneal development. It is also elevated in corneal fibroblasts in culture that have a greatly decreased synthesis of the mature lumican molecule. These data are consistent with, and complement, studies by others that show a corresponding regulation of the lumican core protein during development and in corneal fibroblast cultures.

Amino Acid Sequence↗
PubMed1993

Type II collagen during cartilage and corneal development: immunohistochemical analysis with an anti-telopeptide antibody.

We have examined the pattern of immunoreactivity of a monoclonal antibody, II-5B2, with specificity for an epitope which resides within the NH2-terminal extension peptide (telopeptide) of the avian type II collagen molecule. This epitope is available in regions of matrix where de novo synthesis of the molecule is ongoing, but not where synthesis has ceased and maturation and crosslink formation have occurred. Within the cartilaginous growth plate, the epitope disappears from the matrix soon after the chondrocytes become hypertrophic; within the cornea, the epitope disappears subjacent to the epithelium. The II-5B2 epitope is not made available by a variety of procedures shown to remove potentially masking substances and to disrupt fibrillar organization. It is rendered available, however, when covalent crosslink formation between collagen molecules is blocked through administration of beta-aminopropionitrile or penicillamine. In contrast, the epitope of another monoclonal antibody against type II collagen, II-II6B3, which resides in the triple-helical domain of the molecule, in cartilage is present throughout the growth plate including the hypertrophic zone, and in cornea extends for a considerable distance into the stroma. Thus, it is available for antibody binding regardless of fibril maturation and crosslinking. These data suggest that the II-5B2 epitope becomes unavailable when the telopeptide becomes crosslinked. By using these two monoclonal antibodies in serial sections, one can establish the crosslinking pattern of type II collagen in the tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminopropionitrile↗
PubMed1993

Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis.

Previous work from our laboratories has demonstrated that: (a) the striated collagen fibrils of the corneal stroma are heterotypic structures composed of type V collagen molecules coassembled along with those of type I collagen, (b) the high content of type V collagen within the corneal collagen fibrils is one factor responsible for the small, uniform fibrillar diameter (25 nm) characteristic of this tissue, (c) the completely processed form of type V collagen found within tissues retains a large noncollagenous region, termed the NH2-terminal domain, at the amino end of its alpha 1 chain, and (d) the NH2-terminal domain may contain at least some of the information for the observed regulation of fibril diameters. In the present investigation we have employed polyclonal antibodies against the retained NH2-terminal domain of the alpha 1(V) chain for immunohistochemical studies of embryonic avian corneas and for immunoscreening a chicken cDNA library. When combined with cDNA sequencing and molecular rotary shadowing, these approaches provide information on the molecular structure of the retained NH2-terminal domain as well as how this domain might function in the regulation of fibrillar structure. In immunofluorescence and immunoelectron microscopy analyses, the antibodies against the NH2-terminal domain react with type V molecules present within mature heterotypic fibrils of the corneal stroma. Thus, epitopes within at least a portion of this domain are exposed on the fibril surface. This is in marked contrast to mAbs which we have previously characterized as being directed against epitopes located in the major triple helical domain of the type V molecule. The helical epitopes recognized by these antibodies are antigenically masked on type V molecules that have been assembled into fibrils. Sequencing of the isolated cDNA clones has provided the conceptual amino acid sequence of the entire amino end of the alpha 1(V) procollagen chain. The sequence shows the location of what appear to be potential propeptidase cleavage sites. One of these, if preferentially used during processing of the type V procollagen molecule, can provide an explanation for the retention of the NH2-terminal domain in the completely processed molecule. The sequencing data also suggest that the NH2-terminal domain consists of several regions, providing a structure which fits well with that of the completely processed type V molecule as visualized by rotary shadowing.

Amino Acid Sequence↗
PubMed1991

Collagen types IX and X in the developing chick tibiotarsus: analyses of mRNAs and proteins.

To examine the regulation of collagen types IX and X during the hypertrophic phase of endochondral cartilage development, we have employed in situ hybridization and immunofluorescence histochemistry on selected stages of embryonic chick tibiotarsi. The data show that mRNA for type X collagen appears at or about the time that we detect the first appearance of the protein. This result is incompatible with translational regulation, which would require accumulation of the mRNA to occur at an appreciably earlier time. Data on later-stage embryos demonstrate that once hypertrophic chondrocytes initiate synthesis of type X collagen, they sustain high levels of its mRNA during the remainder of the hypertrophic program. This suggests that these cells maintain their integrity until close to the time that they are removed at the advancing marrow cavity. Type X collagen protein in the hypertrophic matrix also extends to the marrow cavity. Type IX collagen is found throughout the hypertrophic matrix, as well as throughout the younger cartilaginous matrices. But the mRNA for this molecule is largely or completely absent from the oldest hypertrophic cells. These data are consistent with a model that we have previously proposed in which newly synthesized type X collagen within the hypertrophic zone can become associated with type II/IX collagen fibrils synthesized and deposited earlier in development (Schmid and Linsenmayer, 1990; Chen et al. 1990).

Animals↗
PubMed1990

Long-range movement and fibril association of type X collagen within embryonic cartilage matrix.

A recent immunoelectron microscopic study of type X collagen in developing cartilage gave results that could be explained by movement of the molecule from one region of the cartilage matrix to another, there becoming associated with preexisting collagen fibrils. In the present study, to test the feasibility of this model we incubated pieces of nonhypertrophic, embryonic chicken sternal cartilage (which has no endogenous type X collagen) in medium with type X collagen and then used immunofluorescence and immunoelectron microscopy to evaluate movement of the molecule through the matrix. The results show that type X collagen molecules can indeed pass through embryonic sternal cartilage matrix and subsequently become fibril-associated.

Animals↗
PubMed1990

Extracellular matrices of the developing chick retina and cornea. Localization of mRNAs for collagen types II and IX by in situ hybridization.

In the embryonic avian eye, the vitreous and the primary corneal stroma are composed in part of collagen types II and IX. Previous biochemical and immunohistochemical studies have suggested that the production of these molecules changes dramatically during development. In the current investigation, we employed in situ hybridization to determine the temporospatial distribution of the messenger ribonucleic acids (mRNAs) for these collagens in the eyes of selected stages of chick embryos. Our observations suggest that through much of development, the ciliary region is the major source for the production of the collagens found within the vitreous. For the cornea, they confirm that both collagen mRNAs are indeed present, and suggest that each is independently regulated.

Animals↗
PubMed1986

Segmental appearance of type X collagen in the developing avian notochord.

To determine whether short-chain cartilage collagen, collagen type X, is a component of notochordal matrix, we have performed immunohistochemistry with a monoclonal antibody (X-AC9) previously shown to be specific for this molecule (Schmid and Linsenmayer (1984). J. Cell Biol. 100, 598-605). We have also examined different stages of embryos to establish the temporo-spatial appearance of the molecule. The data show that type X collagen is indeed a component of notochordal matrix, but that its developmental appearance is quite late compared to that of type II collagen, another cartilage matrix molecule found in notochord. It is detected only in embryos older than 12 days. The appearance of type X collagen within the notochord is preceded by its appearance within the surrounding hypertrophic cartilage matrix of the vertebral bodies. Spatially, within the notochord the appearance of type X collagen is initially restricted to sites at which the surrounding vertebral cartilage matrix is also reactive for type X. With subsequent development, the notochordal reactivity extends, in a decreasing gradient, anteriorly and posteriorly toward the intervertebral zones. However, the brightest immunofluorescence of notochordal type X is maintained at the midvertebral sites. The ultimate fate of the notochordal tissue at such midvertebral sites is to be removed during endochondral bone formation within the vertebrae. We have observed that type II collagen is also found within notochordal tissue, but this molecule has a distribution which is the converse of that of the type X collagen. The type II collagen is preferentially deposited at intervertebral sites (i.e., the locations of future intervertebral discs). That the type X collagen within the notochord is preferentially deposited at sites destined to be replaced is consistent with one of the hypotheses we previously raised for the function of the type X collagen molecule--it may "target" skeletal tissues for eventual removal.

Animals↗
PubMed1986

Intracellular avian type X collagen in situ and determination of its thermal stability using a conformation-dependent monoclonal antibody.

The thermal stability of the helical domain of intracellular and matrix-associated type X collagen was examined in situ within the hypertrophic region of embryonic chick vertebral cartilages. For this we employed indirect immunofluorescence histochemistry of unfixed tissue sections reacted at progressively higher temperatures (Linsenmayer et al., J cell biol 99 (1984) 1405) with a conformation-dependent monoclonal antibody (X-AC9) (Schmid & Linsenmayer, J cell biol 100 (1985) 598). The hypertrophic chondrocytes which had most recently initiated synthesis of type X did not immediately secrete it, but instead retained it intracellularly within cytoplasmic organelles. This allowed for clear visualization of the intracellular type X. Within the pool of intracellular type X collagen, the epitope recognized by the antibody was stable up to 55 degrees C, but was destroyed at 60 degrees C. This is 5-10 degrees C higher than the thermal stability of the epitope when the molecule is in neutral solution (as determined by competition ELISA). The matrix-associated type X collagen is stable at least to 65-67.5 degrees C. We conclude that in situ the stability of the collagen helix in its normal intracellular environment is considerably greater than might be predicted from measurements made on molecules in solution.

Animals↗
PubMed1986

Embryonic avian cornea contains layers of collagen with greater than average stability.

A unique morphological feature of the embryonic avian cornea is the uniformity of its complement of striated collagen fibrils, each of which has a diameter of 25 nm. We have asked whether this apparent morphological uniformity also reflects an inherent uniformity of the structural and physical properties of these fibrils. For this we have examined the in situ thermal stability of the type I collagen within these fibrils. Corneal tissue sections were reacted at progressively higher temperatures with conformation-dependent monoclonal antibodies directed against the triple-helical domain of the type I collagen molecule. These studies show that the cornea contains layers of collagen fibrils with greater than average stability. The two most prominent of these extend uninterrupted across the entire width of the cornea, and then appear to insert into thick bundles of scleral collagen, which in turn appear to insert into the scleral ossicles, a ring of bony plates which circumscribe the sclera of the avian eye. Once formed, the bands may act to stabilize the shape of the cornea or, conversely, to alter it during accommodation.

Animals↗
PubMed1984

Thermal stability of the helical structure of type IV collagen within basement membranes in situ: determination with a conformation-dependent monoclonal antibody.

To examine the thermal stability of the helical structure of type IV collagen within basement membranes in situ, we have employed indirect immunofluorescence histochemistry performed at progressively higher temperatures using a conformation-dependent antibody, IV-IA8. We previously observed by competition enzyme-linked immunosorbent assay that, in neutral solution, the helical epitope to which this antibody binds undergoes thermal denaturation over the range of 37-40 degrees C. In the present study, we have reacted unfixed cryostat tissue sections with this antibody at successively higher temperatures. We have operationally defined denaturation as the point at which type IV-specific fluorescence is no longer detectable. Under these conditions, the in situ denaturation temperature of this epitope in most basement membranes is 50-55 degrees C. In capillaries and some other small blood vessels the fluorescent signal is still clearly detectable at 60 degrees C, the highest temperature at which we can confidently use this technique. We conclude that the stability of the helical structure of type IV collagen within a basement membrane is considerably greater than it is in solution, and that conformation-dependent monoclonal antibodies can be useful probes for investigations of molecular structure in situ.

Animals↗
PubMed1983

Monoclonal antibodies against chicken type V collagen: production, specificity, and use for immunocytochemical localization in embryonic cornea and other organs.

Two monoclonal antibodies have been produced against chick type V collagen and shown to be highly specific for separate, conformational dependent determinants within this molecule. When used for immunocytochemical tissue localization, these antibodies show that a major site for the in situ deposition of type V is within the extracellular matrices of many dense connective tissues. In these, however, it is largely in a form unavailable to the antibodies, thus requiring a specific "unmasking" treatment to obtain successful immunocytochemical staining. The specificity of these two IgG antibodies was determined by inhibition ELISA, in which only type V and no other known collagen shows inhibition. In ELISA, mixtures of the two antibodies give an additive binding reaction to the collagen, suggesting that each is against a different antigenic determinant. That both antigenic determinants are conformational dependent, being either in, or closely associated with, the collagen helix is demonstrated by the loss of antibody binding to molecules that have been thermally denatured. The temperature at which this occurs, as assayed by inhibition ELISA, is very similar to that at which the collagen helix melts, as determined by optical rotation. This gives strong additional evidence that the antibodies are directed against the collagen. The antibodies were used for indirect immunofluorescence analyses of cryostat sections of corneas and other organs from 17 to 18-day-old chick embryos. Of all tissues examined only Bowman's membrane gave a strong staining reaction with cryostat sections of unfixed material. Staining in other areas of the cornea and in other tissues was very light or nonexistent. When, however, sections were pretreated with pepsin dissolved in dilute HAc or, surprisingly, with the dilute HAc itself dramatic new staining by the antibodies was observed in most tissues examined. The staining, which was specific for the anti-type V collagen antibodies, was largely confined to extracellular matrices of dense connective tissues. Experiments using protease inhibitors suggested that the "unmasking" did not involve proteolysis. We do not yet know the mechanism of this unmasking; however, one possibility is that the dilute acid causes swelling or conformational changes in a type-V collagen-containing supramolecular structure. Further studies should allow us to determine whether this is the case.

Animals↗
PubMed1982

Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen.

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.

Animals↗
PubMedDate not supplied

A novel immunocytochemistry technique to measure hsp70 induction in L929 cells exposed to cadmium chloride.

The heat shock response has been suggested as a potential biomarker in toxicology. A vast amount of stimuli have been shown to induce heat shock proteins and new techniques to measure the response are constantly being assessed. In this study we use a novel immunocytochemistry technique to measure heat shock protein 70 (hsp70) induction in L929 cells exposed to cadmium chloride. Hsp70 induction was quantifiably measured using a soluble coloured substrate and qualitatively measured using a coloured substrate that precipitated at the location of hsp70. Using the insoluble coloured substrate hsp70 was identified predominantly within the cytoplasm of control cells. At intermediate cadmium concentrations hsp70 was observed to translocate to the nucleus. At these intermediate concentrations a heterogeneous heat shock response was observed. At lethal concentrations a strong heat shock response was observed with a widespread cellular response. This study demonstrates the potential of this immunocytochemistry technique to measure toxicological effects in cells by identifying the response quantitatively and qualitatively.

Animals↗