Materials characterization and histological analysis of explanted polypropylene, PTFE, and PET hernia meshes from an individual patient.

During its tenure in vivo, synthetic mesh materials are exposed to foreign body responses, which can alter physicochemical properties of the material. Three different synthetic meshes comprised of polypropylene, expanded polytetrafluoroethylene (ePTFE), and polyethylene terephthalate (PET) materials were explanted from a single patient providing an opportunity to compare physicochemical changes between three different mesh materials in the same host. Results from infrared spectroscopy demonstrated significant oxidation in polypropylene mesh while ePTFE and PET showed slight chemical changes that may be caused by adherent scar tissue. Differential scanning calorimetry results showed a significant decrease in the heat of enthalpy and melt temperature in the polypropylene mesh while the ePTFE and PET showed little change. The presence of giant cells and plasma cells surrounding the ePTFE and PET were indicative of an active foreign body response. Scanning electron micrographs and photo micrographs displayed tissue entrapment and distortion of all three mesh materials.

Conjugation of gold nanoparticles to polypropylene mesh for enhanced biocompatibility.

Polypropylene mesh materials have been utilized in hernia surgery for over 40 years. However, they are prone to degradation due to the body's aggressive foreign body reaction, which may cause pain or complications, forcing mesh removal from the patient. To mitigate these complications, gold nanomaterials were attached to polypropylene mesh in order to improve cellular response. Pristine samples of polypropylene mesh were exposed to hydrogen peroxide/cobalt chloride solutions to induce formation of surface carboxyl functional groups. Gold nanoparticles were covalently linked to the mesh. Scanning electron microscopy confirmed the presence of gold nanoparticles. Differential scanning calorimetry and mechanical testing confirmed that the polypropylene did not undergo any significantly detrimental changes in physicochemical properties. A WST-1 cell culture study showed an increase in cellularity on the gold nanoparticle-polypropylene mesh as compared to pristine mesh. This study showed that biocompatibility of polypropylene mesh may be improved via the conjugation of gold nanoparticles.

Pain and discomfort levels in patients during root surface debridement with sonic metal or plastic inserts.

The study was designed to evaluate whether root surface debridement with a sonic scaler plastic insert would cause less pain and discomfort to patients than an ordinary, probe-shaped metal sonic insert. One quadrant in each of 23 patients was debrided with each insert. Blood pressure, mean arterial pressure, and heart rate were monitored before, during, and immediately after each treatment. Pain was also evaluated on a visual analogue scale (VAS) after each treatment, as well as 2 weeks later following pain-provoking stimuli. Blood pressure, mean arterial pressure, and heart rate did not reveal any differences between quadrants treated with plastic or metal inserts. Heart rate had a weak, positive association with treatment time. The VAS gave a higher pain score for the plastic (30.8) than for the metal insert (24.4), but this difference was not statistically significant (P = 0.055). Following pain-provoking stimuli at the 2-week follow-up visit, quadrants debrided with the metal insert (31.3) scored significantly lower (less pain) (P < 0.01) on the VAS than quadrants treated with the plastic insert (30.7). It is concluded that the sonic metal insert caused less pain and discomfort to the patients due to its superior accessibility and water spray cooling. The significant difference between metal and plastic tip debrided quadrants at the follow-up visit was probably caused by the smearing effect of the metal insert with partial closure of the dentin tubule orifices.

Differential screening of a human pancreatic adenocarcinoma lambda gt11 expression library has identified increased transcription of elongation factor EF-1 alpha in tumour cells.

A human pancreatic adenocarcinoma lambda gt11 expression library was differentially screened with mRNA derived from normal and cancerous pancreatic tissues. Five clones preferentially hybridized with pancreatic adenocarcinoma mRNA. cDNA inserts from 4 of these clones were amplified by PCR, labelled with alpha 32P and used in Northern blot analysis against mRNA prepared from a variety of tumour and normal tissues. lambda GER-4 identified a pancreas-associated mRNA (greater than 10 kb) with no homology with known sequences at either the nucleic or amino-acid level. lambda GER-2 identified a 1.7-kb mRNA transcript that was over-expressed in mRNA prepared from pancreas, colon, breast, lung and gastric tumours relative to normal tissues. Sequence analysis and restriction-enzyme mapping showed that this clone was completely homologous with the active form of human elongation factor EF-1 alpha. This high level of EF-1 alpha-mRNA expression in tumour tissues lends support to the increasing evidence that EF-1 alpha is an important regulator of the cell cycle.

The identification of a novel NCA-related pancreatic tumour-associated antigen, DD9-antigen: a comparison with the expression of other tumour antigens by the pancreatic tumour cell line GER.

Pancreatic tumour-associated monoclonal antibody DD9E7, raised against the GER pancreatic adenocarcinoma cell line, recognises a protein epitope on a novel family of membrane-bound cell surface glycoproteins (Mr 80-115,000). Western blot analysis of SDS/PAGE gels of tumour biopsies and of normal adult pancreas has shown that these glycoproteins are highly expressed in most pancreatic tumours but cannot be detected in normal adult pancreas. Using monoclonal antibodies directed against other antigens that have been associated with pancreatic adenocarcinoma (Du-Pan-2, Ca 19-9, CEA, NCA-95/55, EMA, and FAP), we have been able to show that although some of the antigens are also expressed by the GER pancreatic tumour cell line, the glycoproteins identified by monoclonal antibody DD9E7 are distinct from those other antigens in both molecular weight and antibody binding characteristics. Neuraminidase, periodic acid, and tunicamycin treatment of cultured cells has shown that monoclonal antibody DD9E7 recognises an epitope on the protein core of the antigen. This epitope is also present in NCA-1, but not in CEA, which suggest that there may be an association between DD9-antigen and other members of the NCA/CEA supergene family.

Fe3+(2)-transferrin and Fe3+(2)-asialotransferrin deliver iron to hepatocytes by an identical mechanism.

We have been unable to demonstrate the unequivocal presence of transferrin receptors on rat hepatocytes. The binding and presumed internalisation of 125I-Fe3+(2)-transferrin by freshly isolated hepatocytes was only partially inhibited by up to a 10(4)-fold molar excess of unlabelled ligand and was virtually insensitive to chloroquine. There would appear to be only a weak association between this ligand and some component of the hepatocyte cell surface. These results were not compatible with the commitment of Fe3+(2)-transferrin to either a receptor-mediated endocytotic pathway nor to a rapid recycling pathway through sorting endosomes. Desialylation of the biantennary oligosaccharide side chains of Fe3+(2)-transferrin engendered a low affinity (Kd greater than or equal to 0.25 microM) for the asialoglycoprotein receptor. 125I-Fe3+(2)-asialotransferrin was only superficially internalised by isolated hepatocytes but this was characteristic of ligands for the asialoglycoprotein receptor which have only biantennary (or single triantennary) side chains. This was also incompatible with the delivery of the ligand to sorting endosomes where the release of iron has been presumed to occur. Despite the different properties of the two ligands, the rates of iron uptake from 59Fe3+(2)-transferrin and 59Fe3+(2)-asialotransferrin were identical, suggesting a common mechanism for the translocation of iron across the plasma (or possibly endosomal) membrane such as a transmembrane oxidoreductase. Competition studies with unlabelled ligand or impermeable ferric ammonium citrate gave an IC50 of 1-15 micrograms Fe3+/ml for this process. The absence of transferrin receptor from the surface of the terminally differentiated, quiescent hepatocyte would be compatible with the dual roles suggested for transferrin as an iron transport protein and as a growth factor. The release of iron at the hepatocyte cell surface would effectively uncouple the two functions and render the hepatocyte unresponsive to growth stimulation by transferrin.

Receptor-mediated endocytosis of asialoglycoproteins and diferric transferrin is independent of second messengers.

The intracellular concentrations of cyclic AMP, polyphosphoinosides and free Ca2+ were unaffected during receptor-mediated endocytosis of the neoglycoprotein beta-D-galactosyl-bovine serum albumin (D-Gal-BSA) by isolated hepatocytes. Elevation of either intracellular cyclic AMP by glucagon or inositol phosphates and Ca2+ by vasopressin were without effect on the binding and internalization of D-Gal-BSA. The normal response of this cell to glucagon- and vasopressin-mediated mobilization of these second messengers was not modified in the presence of saturating concentrations of D-Gal-BSA. Receptor-mediated endocytosis of diferric transferrin (Fe3+-TRF) by both hepatocytes and HL60 cells was also shown to be independent of second messengers, although the unequivocal expression of the transferrin receptor by hepatocytes could not be satisfactorily demonstrated. The results of the present study are at variance with a suggested regulatory role for second messengers in receptor-mediated endocytosis of serum-derived ligands such as asialoglycoproteins and Fe3+-TRF. Receptor phosphorylation by protein kinase C in particular has been proposed to regulate the distribution and recycling of these receptors in actively endocytosing cells. We would suggest that if receptor phosphorylation has a regulatory role during endocytosis, it is likely to be mediated by a second-messenger-independent protein kinase analogous to casein kinase II. An alternative interpretation is that phosphorylation has no physiological significance and receptor-mediated endocytosis is a constitutive event coupled to membrane turnover.

Inhibition of breathing associated with gallbladder stimulation in dogs.

The effect of mechanical stimulation of the gallbladder on breathing was studied in anesthetized spontaneously breathing dogs. Measurements of tidal volume, breathing frequency, rib cage and abdominal diameter, transdiaphragmatic pressure, and electrical activity of the diaphragm were made while traction or compression was applied to the gallbladder for periods of 30 s. Both forms of mechanical stimulation produced similar changes, including large decreases in tidal volume, respiratory rate, electrical activity of the diaphragm, and transdiaphragmatic pressure swings. Inspiratory rib cage expansion was little affected, but abdominal expansion was greatly reduced, and swings in gastric pressure were reduced more than swings in pleural pressure, indicating a selective decrease in diaphragmatic activity. Recovery of all measured parameters returned toward control values, despite continued traction or compression. Some inhibition persisted after the stimulus was withdrawn. The very brief interval between stimulus and response suggested that the mechanism was a neural reflex. The afferents involved are unknown but are not purely vagal in nature, since qualitatively similar results were seen in animals after vagotomy. The alteration in breathing frequency indicates that at least part of the reflex is supraspinally mediated. The change in pattern of breathing closely resembles that seen in subjects after abdominal surgery and supports the theory that reflex inhibition of breathing contributes to postoperative pulmonary complications seen in those subjects.

Intraduct enterokinase is lethal in rats with experimental bile-salt pancreatitis.

Controlled intraduct infusion and peri-acinar dispersal of 100 microliter buffer containing sodium glycodeoxycholate (GDOC) at concentrations of 8.5, 17 and 34 mmol/l in rats caused a progressively severe acute pancreatitis from which none of the animals died over the experimental period. Infusion of affinity-purified active human enterokinase in buffer did not cause pancreatitis, presumably because of the inability of the macromolecule to gain access to its specific intracellular substrate trypsinogens. The addition of enterokinase 200 ng to GDOC 34 mmol/l in the infusate resulted in a severe systemic disturbance and a form of acute necrotizing pancreatitis which was uniformly and rapidly lethal. This effect was not seen when equimolar trypsin was substituted for enterokinase. These findings show that enterokinase specifically increases the lethality of experimental bile salt pancreatitis and suggest that this bile-borne enzyme may in some cases pose a significant clinical threat.

A differential effect between the acute and chronic administration of ethanol on the endocytotic rate constant, ke, for the internalisation of asialoglycoproteins by hepatocytes.

The endocytotic rate constant, ke, originally described for the quantification of epidermal growth factor by fibroblasts (Wiley, H.S. and Cunningham, D.D. (1982) J. Biol. Chem. 257, 4222-4229) has been adapted to measure receptor-mediated endocytosis of asialoglycoproteins by hepatocytes. A ke value of 0.21 min-1 was obtained for the internalisation of beta-D-galactosyl bovine serum albumin by freshly isolated hepatocytes. The addition of ethanol to the incubation medium had a biphasic effect on ke. The value of ke was increased by up to 30% by low concentrations of ethanol, whereas higher concentrations progressively decreased ke and in 500 mM ethanol the ke value was 0.1 min-1. The amount of ligand bound to the cell surface was independent of the extracellular concentration of ethanol and the changes in ke were exclusively due to changes in the amount of internalised ligand. There was a progressive decrease in the value of ke in hepatocytes prepared from rats that were maintained on an ethanol-impregnated liquid diet for up to 20 days. The decrease was already apparent by day 2 when blood alcohol levels were only 50 mg%, indicating that the effect of chronic alcoholism on endocytosis are manifested at an early stage.

Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.

We report a novel assay method for enterokinase capable of detecting approx. 1 fmol of enzyme. The method depends on quantification of the release of specifically radiolabelled activation peptides from bovine trypsinogen and is unaffected by trypsin inhibitors. The assay is applicable to biological fluids such as serum. The substrate was produced by selective epsilon-amidination of bovine trypsinogen followed by acetylation with [3H]acetic anhydride and deprotection. The assay has been used to study the effects of pH, Ca2+, ionic strength abd glycodeoxycholate on enterokinase activity.

Immunofluorescent localisation of enterokinase in human small intestine.

The distribution of enterokinase in human intestine was studied in operative mucosal biopsies using specific antiserum to human enterokinase, previously purified to apparent homogeneity by affinity chromatography and immunoabsorption. Fluorescence was observed in the brush-border and glycocalyx of the duodenum and proximal 15 cm of jejunum distal to the D/J flexure. Distal jejunum and ileum as well as stomach and colon were consistently negative. Brunner's glands and goblet cells were never stained by specific antibody. Preliminary evidence was obtained that the human enterokinase molecule contains a specific antigenic determinant in its polypeptide component and a second determinant in the oligosaccharide moiety which cross-reacts with blood group A. Preliminary evidence was also obtained that mucosal synthesis of enterokinase may be impaired in jaundice due to carcinoma of the pancreas and induced in the small intestine distal to the normal limit of synthesis after pancreatico-duodenectomy.