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Biomedical subjects

P Monaghan

Publications and source records attributed to P Monaghan.

At least 19 recordsLinked to original sources
PubMed1992

Characterisation of chondroitin/dermatan sulphate proteoglycans synthesised by rat mammary myoepithelial and fibroblastic cell lines.

Chondroitin sulphate proteoglycans were isolated from the culture medium of rat mammary gland fibroblast (Rama 27) and myoepithelial (Rama 401) cell lines which had been labelled with [35S]sulphate. Chromatography on Sepharose CL-4B indicated that the Rama 401 proteoglycan was larger than the Rama 27 proteoglycan (Kav values 0.47 and 0.56, respectively). Treatment of the proteoglycans with alkaline NaBH4 yielded chondroitin sulphate chains with average M(r) values of 37,000 (Rama 401) and 21,000 (Rama 27). Structural analysis of the glycosaminoglycan chains indicated that both were co-polymers of chondroitin and dermatan sulphate although there were differences in the amounts and distribution of the disaccharide repeating units. The M(r) values of the core proteins, determined by immunoblotting, were about 43,000 and 46,000 (Rama 27) and 44,500 (Rama 401). Using an antibody to chondroitin sulphate proteoglycan in immunofluorescence experiments, the proteoglycan was demonstrated on the surface of both cell lines. Rama 27 cells additionally possessed an extensive fibrous extracellular matrix which also stained with the antibody. Staining of sections of lactating mammary gland suggested that the proteoglycan was present in the basement membrane as well as the stromal connective tissue. The presence of chondroitin sulphate proteoglycan in the basement membrane was confirmed by ultrastructural immunolocalisation.

Animals↗
PubMed1992

Dipeptidyl peptidase IV expression identifies a functional sub-population of breast fibroblasts.

The immunocytochemical distribution of the cell-surface enzyme dipeptidyl peptidase IV (DPP IV) has been studied in the human breast at the light and ultrastructural level. The presence of the enzyme was demonstrated on the cell membranes of interlobular fibroblasts, whilst intralobular fibroblasts were DPP-IV-negative. A fluorograph, after immunoprecipitation of 35S-methionine-labelled proteins of fibroblasts from primary breast cultures with an anti-serum to DPP IV, demonstrated a band at 135 kDa consistent with the presence of the enzyme. The clear delineation of 2 functionally distinct subpopulations of breast fibroblasts was maintained in benign fibro-adenomas and cystosarcoma phyllodes, both tumour types having growth characteristics of intralobular stroma. This observation has important implications for both normal breast biology and for breast carcinogenesis.

Adenofibroma↗
PubMed1992

Effects of growth factors on proliferation on basal and luminal cells in human breast epithelial explants in serum-free culture.

A method of culturing human breast epithelium is described in which viable explants can be maintained in protein-free medium while retaining the capacity of responding to added hormones and growth factors for at least 7 days. Culture parameters were chosen to provide maximum sensitivity of detection of proliferative responses by autoradiography. Under basal conditions, the mean thymidine labeling index of the explants was 0.08%. After stimulation with insulin, hydrocortisone, and cholera toxin (I,H,CT), a combination known to stimulate proliferation in human breast epithelium in vitro, the mean labeling index was 15.7%. Stimulation of explants with epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha resulted in mean labeling indices of 6.6 and 10.8%, respectively. Autoradiography at the ultrastructural level demonstrated that in I,H,CT-stimulated explants the majority of the labeled cells were luminal, with only 1.5% being basal cells. In contrast, after EGF and TGF-alpha basal cells accounted for 11.5 and 18.5% of the labeled population. These results indicate that this system provides an in vitro assay of proliferative activity in the normal human breast that enables comparisons to be made between both the luminal and the basal cells in the explants and their counterparts in monolayer culture prepared from flow sorted cells. Thus, growth responses dependent on cell-to-cell interactions or stromal modulation can be identified.

Breast↗
PubMed1992

Effects of home bleaching preparations on composite resin color.

The lightening of teeth is done professionally by exposure to warm hydrogen peroxide solutions or at home with an active bleaching gel of perhydrol urea or carbamide peroxide. This study compared the effects of two commercial bleaching gels on the color of composite resin with those in a previous study using the dental office technique. Commercial composite resins test specimens were exposed to the bleaching gels or water (control) for 312 hours at 37 degrees C at 100% relative humidity. Color change value was calculated with before-and-after solution exposure L*a*b* color values. ANOVA (alpha = 0.01) revealed no differences in the color of the specimens after use of the test solutions. All color change values were less than 2 or the normal limit of visual acuity. No visual color change was evident for these composite resins. The at-home method recorded no shade-altering effects compared with the professional technique, which created a noticeable color change.

Analysis of Variance↗
PubMed1992

Composite resin color change after vital tooth bleaching.

Color change of composite resin was determined with the Minolta Chroma meter (CR-100) after four sessions of vital bleaching. Specimens of selected composite resin materials were subjected to vital bleaching (37% H3PO4/1 minute, then 30% H2O2/infrared light/30 minutes.) Specimens were stored in water between bleaching. Control specimens were used to determine the effects of water storage alone. Initial L*a*b* color readings were made on 24-hour hydrated specimens. Final L*a*b* readings were made on 24-hour hydrated specimens. Calculations were made for the delta E values for each specimen. Mean delta E values and standard deviations were obtained for each material. Two-way ANOVA and Newman-Keuls analyses showed significant (alpha = 0.05) color change for most bleached materials. Additionally, some materials had delta E values greater than 3. These were visibly lighter compared to their controls. Vital bleaching produced color change in most composites as measured with the Chroma meter. This technique may be used to lighten dark-colored or stained composite resin restorations.

Acrylic Resins↗
PubMed1992

An appraisal of low-temperature embedding by progressive lowering of temperature into Lowicryl HM20 for immunocytochemical studies.

The progressive lowering of temperature (PLT) method of embedding for electron microscope immunolabelling has been examined with the objective of formulating a standardized protocol which can be applied to a wide variety of samples. The methods described cover fixation, processing of samples by the PLT method, embedding in Lowicryl HM20 and subsequent immunolabelling. Each of the steps in the fixation and embedding protocol has been assessed for its potential to retain both morphology and antigenicity. Comparison of samples embedded in Lowicryl K4M and HM20 at -25 degrees C indicate an increased membrane contrast in HM20 sections, and a further improvement in morphology when samples were embedded in HM20 at -50 degrees C rather than at -25 degrees C. The results of applying the methods described are demonstrated in a range of samples of both mammalian and botanical origin, which includes solid tissues, cells in suspension, and cells grown in vitro as a monolayer culture and embedded in situ. Samples processed by this method have been immunolabelled using a wide range of antibodies recognizing nuclear, cytoplasmic, cell membrane and extracellular matrix antigens.

Acrylic Resins↗
PubMed1992

Ultrastructural localization of bcl-2 protein.

Previous cell subfractionation studies have indicated that bcl-2 is an inner mitochondrial membrane protein. We have sought to determine the ultrastructural localization of bcl-2 protein in lymphoma and breast carcinoma cell lines and biopsy material known to overexpress bcl-2 using immunoelectron microscopy. To avoid the possibility of processing artifacts, samples were prepared by three different methods: progressive lowering of temperature, cryosectioning, and freeze-substitution. In all instances the labeling of bcl-2 protein was relatively weak but the distribution the same. In both lymphoma and breast carcinoma tissues, bcl-2 protein was detected on the periphery of mitochondria: little labeling of either the mitochondrial matrix or cristae could be detected. Labeling was also detected on the perinuclear membrane and throughout the cytoplasm, as also indicated by confocal microscopy. These data therefore indicate that bcl-2 protein can be detected at several intracellular sites and that at the likely functional destination, the mitochondria, there appears to be, contrary to expectations, a preferential association with the outer membrane.

Breast Neoplasms↗
PubMed1992

Immunocytochemical localization of the ectoenzyme aminopeptidase N in the human breast.

The ectoenzyme aminopeptidase N (APN) was localized in the normal human breast at both the light microscopic and the ultrastructural level. APN was expressed on intralobular and interlobular fibroblasts and on the apical surface of some luminal epithelial cells. This enzyme was not detected on either myoepithelial cells and their associated basement membrane or capillary endothelium. Furthermore, the staining pattern was maintained in benign and malignant breast disease. APN belongs to a family of enzymes that hydrolyze peptides in the extracellular space. As with other ectoenzymes present in the breast, APN expression is restricted to specific cell types. This pattern of expression may indicate a role for these enzymes in the biology of the normal breast.

Adolescent↗
PubMed1991

Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region.

Based on the map location of the aniridia (AN) locus in human chromosomal band 11p13, we have cloned a candidate AN cDNA (D11S812E) that is completely or partially deleted in two patients with AN. The less than 70 kb smallest region of overlap between the two deletions encompasses the 3' coding region of the cDNA. This cDNA, which spans over 50 kb of genomic DNA, detects a 2.7 kb message specifically within all tissues affected in AN. The predicted polypeptide product possesses a paired domain, a homeodomain, and a serine/threonine-rich carboxy-terminal domain, structural motifs characteristic of certain transcription factors. The concordance between expression and pathology, map location, structure, and predicted function argues that the cDNA corresponds to the AN gene.

Amino Acid Sequence↗
PubMed1991

Growth and development of the human infant breast.

Seventy-two samples of infant breasts, aged from newborn to 2 years, were collected at necropsy. Whole-mount preparations and histological sections were made. A system of classification was devised to study the extent of the structural development of the ductal system (morphological types I, II, and III) and the functional differentiation of the lining epithelium (functional stages I to V). There was no correlation between the age of the infant and the type of development of the ductal system. In contrast, the epithelial differentiation followed a chronological pattern, starting with secretory changes and apparently going through a period characterized by apocrine metaplasia before post-secretory involution. These epithelial changes were not associated with the morphological type of the ductal system. There were no distinguishing features between the breasts from the two sexes. Immunoperoxidase staining for actin and kappa-casein was carried out to study the myoepithelial cells and secretory cells, respectively. Myoepithelial cells were present at all stages and prominent staining for casein was observed up to 2 months of age. Embryonic-type adipose tissue was seen in 7 cases, in one of which it was associated closely with the developing ductal system. Extramedullary hematopoiesis was observed in the periductal connective tissue until 4 months of age. This paper describes the most extensive anatomical and histological study of the human infant breast to date and lays the foundation for a detailed study of the epithelial and stromal changes that take place during human breast development.

Actins↗
PubMed1991

Optimising technetium 99m diethylene triamine penta-acetate lung clearance in patients with the acquired immunodeficiency syndrome.

Pneumocystis carinii pneumonia (PCP) has become a major cause of morbidity and mortality due to infectious diseases, largely as a result of the acquired immunodeficiency syndrome (AIDS) epidemic. Since the mortality from recurrent infection is between 40% and 60%, early diagnosis and therapy are the keys to survival. The role of technetium 99m diethylene triamine penta-acetate (DTPA) aerosol pulmonary clearance was studied in 81 patients with AIDS. The mathematical technique of curve stripping was found to be the optimal method of analysis and to provide an overall sensitivity of 94% for the detection of interstitial pneumonitis. The procedure was superior to standard pathology parameters and radiography and more convenient than gallium 67 scintigraphy.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Growth factor stimulation of proliferating cell nuclear antigen (PCNA) in human breast epithelium in organ culture.

Normal human breast explants were maintained in serum-free culture for 7 days in the presence of either insulin hydrocortisone and cholera toxin (I/H/CT), epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha). Explants were labelled with [3H] thymidine, fixed in methacarn and processed for autoradiography. Parallel sections were immunolabelled with anti-PCNA antibody and analysed with a CAS 200 image analyser. Thymidine labelling index (TLI) and PCNA expression produced similar results with both indices increased in response to I/H/CT, EGF and TGF-alpha. In sections double labelled for PCNA and autoradiography the majority of labelled cells were positive for both markers.

Autoantigens↗
PubMed1991

Characterization in vitro of luminal and myoepithelial cells isolated from the human mammary gland by cell sorting.

Luminal and myoepithelial cells have been separated from normal adult human breast epithelium using fluorescence activated cell sorting. Their isolation was based on the exclusive expression of two surface antigens, epithelial membrane antigen (EMA) and the common acute lymphoblastic leukaemia antigen (CALLA/CD10/neutral endopeptidase 24.11). Sorted luminal and myoepithelial cells displayed distinctively different morphologies when maintained in monolayer culture, differences which were enhanced by the addition of hydrocortisone, insulin and cholera toxin to the culture medium. The EMA-positive cells formed an attenuated monolayer with indistinct cell boundaries while CALLA-positive cells, by contrast, formed tightly packed arrays of refractile cells. The distribution of the cell type-specific markers cytokeratin 18 (luminal cells) and smooth muscle alpha-actin (myoepithelial cells) indicated that the sorted populations were approximately 98% pure. However, a significant minority (approximately 15%) of sorted luminal cells consistently expressed the basal-cell marker cytokeratin 14 in culture. A marked difference was noted in the proliferative behaviour of the two types of sorted cells, with myoepithelial cells dividing rapidly in response to the humoural additives, in contrast to the luminal cells which proliferated slowly. Both types of sorted cells could be cloned in the presence of feeder layers of mouse fibroblasts. Clones of luminal and myoepithelial cells were also distinctive; all "spread" luminal clones were similar in appearance to each other, although some cellular heterogeneity, including squamous metaplasia, was observed in "compact" myoepithelial clones. Both types were shown to have retained their original surface markers and to exhibit different cytoskeletal antigenic phenotypes when they were re-analysed after a 3-week growth period. Both spread and compact phenotypes were obtained when separately isolated ducts and alveoli were cloned. This detailed characterization of cells isolated from the human breast epithelium by flow cytometry provides the basis for further studies of luminalmyoepithelial interactions and growth responses of purified cell types in vitro.

Antigens, Differentiation↗
PubMed1991

Recent advances in electron immunocytochemistry.

Considerable progress has been made in the development of methods for the localisation of antigens at the ultrastructural level using both transmission and scanning electron microscopy. There is currently no technique which is appropriate for all antigens, although, almost without exception, colloidal gold-linked reagents provide the electron-dense marker. For post-embedding techniques, several approaches are available all utilising low temperatures during the preparative stages. Cryosectioning and embedding by the PLT method both require the antigen to withstand some degree of fixation. For fixation-sensitive antigens, freeze substitution in solvents lacking fixative agents is an approach offering considerable potential.

Frozen Sections↗
PubMed1991

An improved radionuclide technique for the detection of altered pulmonary permeability.

Technegas, an ultra-fine dry aerosol with prolonged retention in the lungs, can be modified by altering the atmosphere in which the carbon particles are generated. The modified Technegas has much faster clearance from the lung. The half-time pulmonary clearances with modified Technegas were compared to those obtained with conventional 99mTc DTPA aerosol in 50 patients. Interstitial lung disease was suspected in 12 while 38 were infected with the human immunodeficiency virus and suspected of having opportunistic lung infection. In 22 nonsmokers in whom no evidence of active pulmonary pathology was demonstrable, the mean half-time with DTPA was 52.5 min whereas the mean half-time with modified aerosol was 10.1 min. The mean half-time in 14 smokers in whom there was also no evidence of active pulmonary disease was 28.3 min with DTPA and 7.0 min with the modified method. In the 14 patients in whom altered pulmonary permeability was demonstrated by a short DTPA half-time (mean 4.8 min) there was also an accelerated half-time with modified Technegas (mean 2.5 min). It is concluded that the modified Technegas procedure offers a simple but accurate method of identifying individuals having opportunistic infection or other diffuse lung pathology.

Acquired Immunodeficiency Syndrome↗