Biomedical subjects
Publications and source records attributed to D Hopwood.
Microsomes isolated from adult human gall-bladders have for the first time been shown to contain specific glucose-6-phosphatase activity. The gall-bladder glucose-6-phosphatase enzyme has the same molecular weight (36,500 daltons) and similar immunological properties and kinetic characteristics to the hepatic microsomal glucose-6-phosphatase enzyme.
Human gall-bladder epithelium obtained straight from the operating theatre was incubated in an Ussing chamber with the fluid phase marker, horseradish peroxidase (HRP), for up to 60 min. When the marker was presented on the apical surface, within 30 min it had moved readily across the apical cytoplasm in transport vesicles to receptosomes and into the lateral intercellular space, extending across the basement membrane into the lamina propria. When HRP was presented at the basal aspect, within 30 min it had moved through the lamina propria, across the basement membrane and into the lateral intercellular space. By 60 min, only small amounts had been taken up by the epithelial cells and transported to receptosomes. These data indicate a rapid transmucosal endocytotic pathway for blood- or bile-borne macromolecules.
The addition of 2% phenol had a marked accelerating effect on neutral buffered 4% formaldehyde as a fixative. Histopathological material fixed in buffered phenol-formaldehyde (pH 7.0) and rapidly advanced to paraffin in an enclosed tissue-processor showed improved nuclear and cytoplasmic detail, reduced shrinkage and distortion, and an absence of formalin pigment. Good results were obtained in less time when sequential fixation in phenol-formaldehyde buffered to pH 7.0 and pH 5.5 was carried out at an elevated temperature (40 degrees C) in the enclosed tissue-processor. Standard histological stains and immunoperoxidase methods worked well. In resin-embedded tissue, buffered phenol-formaldehyde (pH 7.0) gave satisfactory ultrastructural results. The penetration rate of buffered phenol-formaldehyde (pH 7.0) in gelatin models did not differ from that of neutral buffered 4% formaldehyde. Polyacrylamide gel electrophoresis showed enhanced protein polymer formation with buffered phenol-formaldehyde (pH 7.0) as compared with neutral buffered 4% formaldehyde. Protein polymer formation increased in response to increased time and temperature. Cells fixed in suspension in buffered phenol-formaldehyde (pH 7.0) and neutral buffered 4% formaldehyde showed similar volume changes.
Sodium ion (Na+) transport, a principal function of the gallbladder epithelium, was studied by measuring the flux of 22Na across isolated human gallbladder mucosa maintained in a modified 'Ussing' flux chamber. Tissue was obtained from cholecystectomy specimens in symptomatic patients with cholelithiasis. Out of 26 gallbladders studied, 13 had a net Na+ flux from mucosa to serosa which indicated active Na+ absorption. The hormone secretin, when added to the serosal fluid, reversed the direction of net flux in these gallbladders and caused a secretion of Na+ from serosa to mucosa. These results suggest that secretin may be involved in the physiological regulation of fluid transport in the human gallbladder, and also suggest a possible role for this hormone in gallbladder emptying.
The addition of 2% phenol has a marked adjuvant effect on 4% formaldehyde as a fixative. Sequential use of phenol formaldehyde buffered to pH 7.0 and pH 5.5 results in improved fixation in material rapidly advanced to paraffin in an enclosed tissue processor. Sections stained with alum haematoxylin and eosin show improved nuclear and cytoplasmic detail, reduced tissue shrinkage and an absence of formalin pigment. Excellent results are obtained with conventional special stains and immunoperoxidase methods.
The 3-fucosyl N-acetyllactosamine residue is the antigen recognized by the monoclonal antibody MC2. Using MC2, we demonstrated the distribution of this antigen in a variety of squamous epithelia. The antigen is expressed to a variable degree on supra-basal cells in most normal non-keratinizing squamous mucosae, with a similar distribution in metaplastic squamous epithelia; antibody-labelled latex microspheres and immunogold electron microscopy show the antigen to form part of the glycocalyx. In dysplastic and neoplastic squamous lesions, expression is reduced or absent except in cells around areas of differentiation. Prior neuraminidase treatment of sections had little effect on the amount or distribution of demonstrable antigen. Expression of this antigen by cells in non-keratinizing squamous epithelia gives an indication of cell maturity and may provide a histological marker for the grading of dysplastic and malignant squamous mucosal lesions. A possible role for these carbohydrate residues in squamous mucosal defence is discussed.
Biopsies from the common bile ducts from seven patients undergoing surgery for biliary obstruction due to stones or malignancy were studied histochemically and electron microscopically. The surface of the bile duct is lined by a tall epithelium which extends into diverticula. Apically, they contain some neutral and sialated mucosubstances. Fucosyl residues were found in the Golgi apparatus and along the apical cell membrane. The latter is lined by microvilli. There was a well-developed rough endoplasmic reticulum and Golgi apparatus and a small number of apical secretory droplets. Large numbers of lipid droplets were present basally in some cells. Lipid-containing macrophages were also seen intra-epithelially and in the lamina propria. This suggests a possible pathway for lipid transport. The glands were lined by cuboidal cells, some containing much mucus--sulphated, sialated, and neutral with a basal nucleus. A well-developed endoplasmic reticulum and Golgi apparatus were found with abundant secretory droplets. The glandular epithelium contained lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin. These may play a protective role. The lamina propria contained scattered smooth muscle cells amongst the fibroblasts and inflammatory cells.
The CD 15 antigen (3-fucosyl N-acetyllactosamine), present on the outer cell membrane of cervical squamous epithelial cells, is recognized by the monoclonal antibody MC2, which is similar to several commercially available antibodies. Staining sections of cervical biopsies with MC2 clearly demonstrates the zone of supra-basal differentiated cells in the normal squamous epithelium. Staining with MC2 also demonstrates the diminished proportion of this zone occurring with grades of CIN, reflecting progressive de-differentiation of the epithelium. In immature squamous metaplastic epithelium, absence of cytoplasmic differentiation is reflected by lack of staining. As expression of the CD 15 antigen by cervical squamous cells mirrors cytoplasmic maturity and is a marker of cellular differentiation, staining colposcopic biopsies with MC2 may aid the routine histopathological grading of CIN. A comparison is made between the staining pattern observed using MC2 with that of two commercially available antibodies (Leu M1 and Dako M1), and a possible role for the CD 15 antigen in cellular adhesion in squamous mucosae is discussed.
Using cuprolinic blue as a stain along with enzymic digestion, heparan sulphate has been identified as the main glycosaminoglycan in the basement membrane of human gallbladder epithelium. The amount of glycosaminoglycans was quantified by counting the number of molecular profiles cm-2 in electron micrographs of mildly, moderately and severely inflamed gallbladders. There is a significant increase (P = 0.009) in the amount of glycosaminoglycans in the basement membranes of severely inflamed gallbladders compared with cases of mild chronic cholecystitis. Differences, although present, are less significant when mild and moderate or moderate and severe cholecystitis are compared. The findings suggest that there is a continuous accumulation of heparan sulphate in the basement membrane in chronic cholecystitis which increases in amount with the severity of inflammation.
1. Sodium ion (Na+) transport, a principal function of the gallbladder epithelium, was studied by measuring the flux of 22Na across isolated, inflamed human gallbladder mucosa maintained in a modified 'Ussing' flux chamber. Tissue was obtained from cholecystectomy specimens in symptomatic patients with cholelithiasis. 2. In 30 gallbladders studied, 57% had a net Na+ flux from mucosa to serosa (Na+ absorption), while 23% had a net Na+ flux from serosa to mucosa (Na+ secretion). The remaining 20% showed no overall net Na+ flux. 3. Indomethacin added to the serosal fluid reversed the direction of net Na+ flux in secreting gallbladders and caused an absorption of Na+. In Na+-absorbing gallbladders, indomethacin caused a slight reduction in Na+ absorption. No change in Na+ flux was induced in gallbladders with no initial net Na+ flux. 4. These results demonstrate that instead of absorbing Na+, some inflamed human gallbladders may secrete Na+. As this secretion can be reversed to the more usual absorption by indomethacin, it is likely that this secretion is mediated by prostaglandins.
Oral cholecystography is a well established method for studying the human gallbladder and radiological non-visualization of the gallbladder has been shown to correlate highly with the presence of disease. The exact mechanism by which diseased gallbladders fail to visualize is unclear, but may be due to a failure of the gallbladder to concentrate the luminal contents. Concentration of gallbladder contents is achieved by the reabsorption of water, the driving force for which is active sodium (Na+) absorption. Therefore Na+ transport was studied by measuring the flux of Na22 across isolated human gallbladder mucosa (obtained at cholecystectomy) and compared with the results of oral cholecystography and histological grading. In 27 gallbladders studied, 59% absorbed Na+, whilst the remainder secreted Na+. Comparison with histological grading showed that as gallbladders became more diseased they absorbed less Na+ and were more likely to secrete Na+. In addition, gallbladders that absorbed Na+ were significantly more likely to visualize on cholecystography than those that secreted Na+. These results indicate that some diseased human gallbladders secrete, rather than absorb, Na+ and suggest that the mechanism for radiological non-visualization is failure of fluid absorption and the development of active fluid secretion.
The determination of uptake rates for lipids across absorptive epithelia, using radiolabelled compounds has normally used non-absorbable volume markers to correct gross tissue counts for contamination by adherent mucosal fluid. There is evidence however to indicate that the macromolecules traditionally used as non-absorbable markers are in fact absorbed. This may result in an overestimate of the apparent adherent mucosal fluid with consequent errors in the calculation of lipid uptake. An in-vitro method was used to assess cholesterol absorption by human gallbladder from artificial bile, containing 3H-dextran 70,000 as a 'non-absorbable' volume marker. Both cholesterol and dextran were transported through the mucosa and appeared on the serosal side of the gallbladder. Confirmation of dextran absorption was obtained by electron microscopic examination of the gallbladder epithelium. By using the serosal appearance of dextran as a guide to the amount of absorbed (intracellular) dextran in the tissue samples, we have derived a formula to calculate tissue dextran absorption and, from the revised adherent mucosal volume, have consequently obtained more realistic estimates of tissue lipid uptake. In this study, comparison made between the previous estimate of lipid uptake compared to this new calculation, showed that the previous technique significantly underestimated true lipid uptake.
Horseradish peroxidase (HRP) was administered intravenously to mice by bolus injection. The subsequent uptake and fate of the HRP by the lateral and basal cell surfaces of resting and stimulated gallbladder epithelial cells was followed by light and electron microscopy. At 10 min after injection, HRP was visible in the lamina propria of the gallbladder and within 20 min of injection, HRP had permeated the basement membrane and had entered the lateral intercellular space, extending as far as the apical tight junction. Over the following 30 min, there was evidence of vesicular epithelial HRP uptake and 1 h after injection, HRP was visible in epithelial secretory granules within the lumen of the gallbladder and apical transport vesicles. These data provide evidence of a blood-to-bile transport pathway which could represent an important route of entry to bile by various blood-borne macromolecules.
Endoscopic gastric biopsy specimens taken in 1976 from 174 patients were reviewed. Biopsy specimens from 44 patients showed intestinal metaplasia, and subtyping by mucin histochemistry showed that 16 were of type I, 14 of type II, and 14 of type III. Only two of these 174 patients developed gastric adenocarcinoma over the next 10 to 11 years: one with type II and one with type III intestinal metaplasia. Case notes of a separate group of 68 patients with gastric adenocarcinoma diagnosed in 1985 were reviewed for evidence of intestinal metaplasia in a previous gastric biopsy. Only two patients had previously been biopsied; one of these biopsy specimens showed type II intestinal metaplasia and the other showed no intestinal metaplasia. These findings suggest that subtyping of intestinal metaplasia in endoscopic gastric biopsy specimens is of only limited value in identifying patients at risk of gastric adenocarcinoma who require long term follow up.
The characteristics of cholesterol uptake by 83 human gall bladders (obtained at cholecystectomy) were studied with a modified Ussing technique. Real and artificial biles labelled with 14C-cholesterol and 3H-dextran (the latter to correct for adherent mucosal bile) were used; all gall bladders absorbed cholesterol (average 3.5 nmol/cm2/minute). Recovery of the absorbed cholesterol from the tissue showed that about 4% was esterified over 60 minutes. In artificial bile the rate of absorption of cholesterol increased as the bile saturation index rose, but became constant once supersaturation was achieved. In contrast, supersaturated real bile permitted greater absorption of cholesterol, possibly due to enhanced cholesterol solubilisation. Preincubation of gall bladder tissue in sodium cyanide (5 mM) caused a 30% reduction in cholesterol uptake indicating that, although absorption is predominantly a "passive" process, there is a partial "active" component. There were no pronounced differences in the rate of cholesterol absorption as gall bladders became more diseased, but there was a reduction in the amount of cholesterol ester formed.
Veterinarians tend to look upon themselves as professionals first, and as businessmen either second or not at all. This was the main conclusion of a study of 10 livestock practices mainly in the Lakelands area. The study attempts to show that by ignoring standard marketing and accounting procedures, they are wasting an opportunity to improve their financial efficiency and the quality of service they provide. Both these qualities will become more and more important in an increasingly competitive world, it says.
Human oesophageal submucosal glands may be regularly demonstrated by first exposing the oesophageal lumen to toluidine blue which reveals the duct ostia. Four types of cell were identified in the glands - mucous, subsidiary or serous, myoepithelial and oncocytes. The mucous cell contained neutral, sialated and sulphated mucins. The subsidiary cells held smaller amounts of neutral and sialated mucin, plus fucosyl residues. No lipids were detectable histochemically. ATP-ase and alkaline phosphatase were shown in the capillary endothelium. The duct epithelium showed some nonspecific esterase activity not sensitive to E 600. By immunoperoxidase techniques, the duct epithelium was shown to be rich in cytokeratin. The subsidiary cells contained lysozyme, CEA and pepsinogen. B lymphocytes composed most of the periductular lymphoid aggregates, although some T cells were found there and also intraepithelial and subepithelial in relation to the stratified squamous epithelium lining the oesophagus. Langerhans' cells were also demonstrated as intraepithelial by several techniques.
Human gallbladder and gastric epithelial cells are normally covered with a layer of mucus. When specimens were exposed to cationized ferritin (CF) in vitro, they did not regularly bind nor internalise it. If the tissues were first exposed to the mucolytic agents cysteamine or pepsin, then the gallbladder epithelium readily bound CF and the gastric epithelium irregularly. The in vivo binding of CF by guinea pig gallbladder could be abolished by the induction of mucous hypersecretion by the antibiotic lincomycin. The removal of the mucus by mucolytic agents restored the binding of CF. The irregular binding of CF by gastric mucosa after the use of mucolytic agents suggests other factors may be at play.