Follow-up after emergency laparotomy suggests high one- and five-year mortality with risk stratified by ASA.

INTRODUCTION: We investigated all-cause mortality following emergency laparotomy at 1 and 5 years. We aimed to establish a basis from which to advise patients and relatives on long-term mortality. METHODS: Local data from a historical audit of emergency laparotomies from 2010 to 2012 were combined with National Emergency Laparotomy Audit (NELA) data from 2017 to 2020. Covariates collected included deprivation status, preoperative blood work, baseline renal function, age, American Society of Anesthesiologists (ASA) grade, operative time, anaesthetic time and gender. Associations between covariates and survival were determined using multivariate logistic regression and Kaplan-Meier analysis. We used patients undergoing laparoscopic cholecystectomy between 2015 and 2020 as controls. RESULTS: ASA grade was the best discriminator of long-term outcome following laparotomy (n=894) but was not a predictor of survival following cholecystectomy (n=1,834), with mortality being significantly greater in the laparotomy group. Following cholecystectomy, 95% confidence intervals for survival at 5 years were 98-99%. Following laparotomy these intervals were: ASA grade 1, 79-96%; ASA grade 2, 69-82%; ASA grade 3, 44-58%; ASA grade 4, 33-48%; and ASA grade 5, 4-51%. The majority of deaths occurred after 30 days. CONCLUSIONS: Emergency laparotomy is associated with a significantly increased risk of death in the following 5 years. The risk is strongly correlated to ASA grade. Thirty-day mortality estimation is not a good basis on which to advise patients and carers on long-term outcomes. ASA grade can be used to predict long-term outcomes and to guide patient counsel.

The immunofluorescent era of membrane traffic.

In recent years immunofluorescence microscopy has been increasingly used to study membrane traffic. In this article seven electron microscopists, all with considerable experience in using light microscopy, take a critical look at the immunofluorescence approach and argue that results obtained with this method are often overinterpreted.

Formation of coated vesicles from coated pits in broken A431 cells.

Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti-transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.

Mitotic Golgi fragments in HeLa cells and their role in the reassembly pathway.

Immunoelectron microscopy and stereology were used to identify and quantitate Golgi fragments in metaphase HeLa cells and to study Golgi reassembly during telophase. On ultrathin frozen sections of metaphase cells, labeling for the Golgi marker protein, galactosyltransferase, was found over multivesicular Golgi clusters and free vesicles that were found mainly in the mitotic spindle region. The density of Golgi cluster membrane varied from cell to cell and was inversely related to the density of free vesicles in the spindle. There were thousands of free Golgi vesicles and they comprised a significant proportion of the total Golgi membrane. During telophase, the distribution of galactosyltransferase labeling shifted from free Golgi vesicles towards Golgi clusters and the population of free vesicles was depleted. The number of clusters was no more than in metaphase cells so the observed fourfold increase in membrane surface meant that individual clusters had increased in size. More than half of these had cisterna(e) and were located next to "buds" on the endoplasmic reticulum. Early in G1 the number of clusters dropped as they congregated in the juxtanuclear region and fused. These results show that fragmentation of the Golgi apparatus yields Golgi clusters and free vesicles and reassembly from these fragments is at least a two-step process: (a) growth of a limited number of dispersed clusters by accretion and fusion of vesicles to form cisternal clusters next to membranous "buds" on the endoplasmic reticulum; (b) congregation and fusion to form the interphase Golgi stack in the juxtanuclear region.

Immunolocalization of the oligosaccharide trimming enzyme glucosidase II.

We used immunoelectron microscopy to localize glucosidase II in pig hepatocytes. The enzyme trims the two inner alpha 1,3-linked glucoses from N-linked oligosaccharide precursor chains of glycoproteins. Immunoreactive enzyme was concentrated in rough (RER) and smooth (SER) endoplasmic reticulum but not detectable in Golgi apparatus cisternae. Transitional elements of RER and smooth membraned structures close to Golgi apparatus cisternae contained labeling for glucosidase II. Specific labeling was also found in autophagosomes. These results indicate strongly that glucosidase II acts on glycoproteins before their transport to, and processing in Golgi apparatus cisternae, and suggest that an important transitional region for glucosidase II exists between RER and Golgi apparatus cisternae. Degradation in autophagolysosomes could form a normal catabolic pathway for glucosidase II.

A mitotic form of the Golgi apparatus in HeLa cells.

Galactosyltransferase, a marker for trans-Golgi cisternae in interphase cells, was localized in mitotic HeLa cells embedded in Lowicryl K4M by immunoelectron microscopy. Specific labeling was found only over multivesicular structures that we term Golgi clusters. Unlike Golgi stacks in interphase cells, these clusters lacked elongated cisternae and ordered stacking of their components but did comprise two distinct regions, one containing electron-lucent vesicles and the other, smaller, vesiculo-tubular structures. Labeling for galactosyltransferase was found predominantly over the latter region. Both structures were embedded in a dense matrix that excluded ribosomes and the cluster was often bounded by cisternae of the rough endoplasmic reticulum, sometimes on all sides. Clusters were present at all stages of mitosis examined, which included prometaphase, metaphase, and telophase. They were also identified in conventionally processed mitotic cells and shown to contain another trans-Golgi marker, thiamine pyrophosphatase. Serial sectioning showed that clusters were discrete and globular and multiple copies appeared to be dispersed in the cytoplasm. Their possible role in the division of the Golgi apparatus is discussed.

Association of a phosphatidylinositol-specific 3-kinase with a human trans-Golgi network resident protein.

The eukaryotic trans-Golgi network (TGN) is a key site for the formation of transport vesicles destined for different intracellular compartments [1]. A key marker for the mammalian TGN is TGN38/46 [2]. This integral membrane glycoprotein cycles between the TGN and the cell surface and is implicated in recruitment of cytosolic factors and regulation of at least one type of vesicle formation at the mammalian TGN [2] [3]. In this study, we have identified a phosphatidylinositol (PtdIns)-specific 3-kinase activity associated with the human orthologue (TGN46), which is sensitive to lipid kinase inhibitors. Treatment of HeLa cells with low levels of these inhibitors reveals subtle morphological changes in TGN46-positive compartments. Our findings suggest a role for PtdIns 3-kinases and presumably for the product, PtdIns 3-phosphate (PtdIns3P), in the formation of secretory transport vesicles by mechanisms conserved in yeast and mammals.

Okadaic acid induces selective arrest of protein transport in the rough endoplasmic reticulum and prevents export into COPII-coated structures.

Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15 degrees C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the juxtanuclear region and peripheral cytoplasm. During OA treatment, p53/p58 accumulated in the RER but was excluded from sec 13p-containing membrane structures. Taken together our data indicate that OA induces an early defect in RER export which acts to prevent entry into COPII-coated structures of the IC region.

ERK2 signalling from internalised epidermal growth factor receptor in broken A431 cells.

Activation of extracellular signal-regulated kinase 2 (ERK2) signalling from epidermal growth factor receptors (EGFRs) is widely assumed to originate in the plasma membrane. Using an in vitro assay, we investigated whether EGF/EGFR complexes internalised by endocytosis in A431 cells can initiate signalling in the ERK2 pathway. At 0 degrees C, binding of EGF induced tyrosine phosphorlyation of EGFR and, when the cells were subsequently broken by scraping and warmed in the presence of exogenous cytosol, activation of ERK2 occurred. At 0 degrees C, washes with pH 4.5 media reversed EGF binding, tyrosine phosphorylation and ERK-2 activation in exogenous cytosol, providing conditions in which signalling from the cell surface and internalised EGFRs could be distinguished. When cells containing internalised EGF/EGFR complexes were first washed in low pH media at 0 degrees C and then broken and incubated in exogenous cytosol, substantial activation of ERK2 occurred. This activation reached a maximum after a 5-min internalisation and was almost completely prevented by incubation in 0.45 M sucrose, a known inhibitor of receptor-mediated endocytosis. These data are consistent with activation of the ERK2 signalling pathway by internalised EGRFs situated in endosomal compartments. Our observation that EGFR tyrosine dephosphorylation is incomplete above pH 5.5 suggests that signalling is initiated in early endosomes.

Evidence for fusion between multilamellar endosomes and autophagosomes in HeLa cells.

The organelles of the endocytic and autophagic pathways were studied in HeLa cells using immunoelectron microscopy and stereological techniques. In the absence of autophagic stimulation, characteristic particulate structures containing closely packed layers of membrane received the fluid-phase marker horseradish peroxidase after 25 min uptake. These multilamellar endosomes contained the majority of the cellular immunogold labeling obtained by using a monoclonal antibody (1B5) directed against a lysosomal glycoprotein. Lysosomes with homogeneous dense content were only poorly labeled. After stimulation of autophagy, two classes of autophagosome profile appeared. One had a double limiting membrane and content that resembled the surrounding cytoplasm. The other most abundant type possessed a single limiting membrane and contained multilamellar structures which were strikingly similar to multilamellar endosomes, not only in form, but also in volume and membrane packing density. Immunogold labeling showed that now the majority of 1B5 labeling was located in the class of autophagosomes which contained multilamellar structures. Stereological methods showed that, after autophagic stimulation, multilamellar endosomes had become depleted, while multilamellar structures had appeared within the autophagosomes. Taken together, these data provide evidence that autophagosomes of HeLa cells fuse with preexisting multilamellar endosomes.

Quantitative immunoelectron microscopy reveals alpha2,6 sialyltransferase is concentrated in the central cisternae of rat hepatocyte Golgi apparatus.

The Golgi apparatus is a membrane bound organelle involved in synthesis of N-linked oligosaccharides which are trimmed and then lengthened by a series of sugar transferases adding N-acetylglucosamine, galactose and sialic acid in sequence. We previously published qualitative work which localized Galbeta1,4GlcNAc alpha2,6 sialyltransferase of rat hepatocytes to the trans cisternae and the trans Golgi network. We now report the use of combined stereological and immunoelectron microscopical techniques for mapping the Golgi stack composition and distribution of sialyltransferase protein in rat hepatocytes. The Golgi stack showed substantial variation in composition consisting of 1, 2, 3, 4, or 5 cisternae with an average of 2.5 cisternae. Sialyltransferase labeling was mainly located in the central cisternae of the Golgi stacks irrespective of whether the stacks were oriented in a cis/trans direction using morphological criteria. Only 20% of the total sialyltransferase labeling was present in the transmost cisterna and 2% in the trans Golgi Network. The low labeling in the transmost cisterna was essentially due to the presence of a sialyltransferase negative cisterna. These data emphasize the importance of quantitation in obtaining a representative picture of Golgi enzyme distribution in three dimensions. They indicate that central cisternae, rather than the transmost cisterna and TGN, function in sialylation along the secretory pathway of rat hepatocytes.

Preparation of protein colloidal gold complexes in the presence of commonly used buffers.

In the presence of various commonly used buffers, phosphate-buffered saline (PBS), tris-buffered saline (TBS), Na-cacodylate, bovine serum albumin and a wide range of cytochemically active proteins (monoclonal and polyclonal IgG, concanavalin A, Ricinus communis lectin I, Helix pomatia lectin, protein A) were complexed to colloidal gold of different particle sizes (6 nm, 9 nm, 22 nm). The resulting complexes were active in cytochemical labelling. Complex-formation in the presence of electrolyte opens the possibilities of: maintenance of ionic environment during complexing of proteins sensitive to low ionic strength, pH control by addition of buffers to the protein solution or to the gold sol, direct coupling of protein supplied in PBS or saline avoiding dialysis against low ionic strength buffers. Using the electron microscope to estimate the protein amounts needed for stabilization provided a sensitive and economical method to obtain aggregate-free protein-gold complexes.

Coated pits in interphase and mitotic A431 cells.

Endocytosis is inhibited during mitosis in A431 cells (Warren et al., 1984) but the site of inhibition is unknown. A quantitative method measuring the extent of budding was used to compare coated pits in interphase and mitotic cells. Every stage of budding found in interphase cells was also found in cells at every stage of mitosis. Flatter coated pits appeared more frequent in mitotic cells but this can be partly, if not entirely, explained by their greater size. We conclude that, if budding is inhibited, inhibition must occur at all stages of the budding process.

Distinct compartmentalization of TGN46 and beta 1,4-galactosyltransferase in HeLa cells.

The Golgi proteins, TGN46 and GalT, were characterized in human HeLa cells using specific polyclonal and monoclonal antibodies. A bacterially expressed soluble recombinant TGN46 protein was used to raise rabbit polyclonal antibodies and used to probe HeLa cell extracts. Human TGN46 had an apparent molecular mass of 100 to 120 kDa which reflects extensive glycosylation. Epifluorescence light microscopy indicated substantial colocalization of TGN46 and GalT. However, confocal laser microscopy and three-dimensional reconstruction of double-labeled HeLa cells revealed large areas of colocalization but also specific differences in the distribution of these two proteins within the Golgi apparatus. Importantly, quantitative immunoelectron microscopy showed that there was little overlap between the distribution of GalT and TGN46. Approximately 75% of GalT was in the Golgi stack, whereas 80% of TGN46 was detected in tubules. Distinct GalT-positive regions within the Golgi cisternal stack were not labeled for TGN46.

Immunolocalisation of phospholipase D1 on tubular vesicular membranes of endocytic and secretory origin.

We have examined the localisation of overexpressed phospholipase D1 (PLD1) using antibodies against its amino- and carboxyl-terminal domains. PLD1 overexpressed in COS-7 cells showed variable distribution by immunofluorescence but was mainly in punctate structures in the perinuclear region and at the plasma membrane. Downregulation by an anti-sense plasmid resulted in almost exclusively perinuclear distribution in punctate structures that contained immunoreactivity for the endogenous KDEL receptor and the early endosomal antigen EEA1 protein. Influenza haemagglutinin (HA) and HA-derived mutants designed to locate primarily to secretory or endocytic membranes were present in PLD1-positive membranes. Immunofluorescence analysis in permanent CHO cell lines that express PLD1 inducibly confirmed the presence of PLD1 on both endocytic and secretory membranes. Analysis of PLD1 distribution by immunocytochemistry and electron microscopy of intact CHO cells and of isolated membranes revealed that PLD1 was present in tubulovesicular elements and multivesicular bodies. Some of these were close to the Golgi region whereas others stained positive for endocytic cargo proteins. Morphometric analysis assigned the majority of PLD1 immunoreactivity on endosomal membranes and a smaller amount on membranes of secretory origin. PLD1, via signals that are currently not understood, is capable of localising in tubulovesicular membranes of both endocytic and secretory origin.

Ultrastructural localization of keratin proteins in human skin using low-temperature embedding and the protein A-gold technique.

Human skin was embedded in Lowicryl K4M and keratin proteins were localized by incubation with antikeratin antisera, followed by protein A-gold. The antikeratin antisera labeled all intermediate filament (tonofilament) structures in all layers of the epidermis. The association of keratin filaments with hemidesmosomes, desmosomes, and keratohyaline granules was clearly visualized. Desmosomes and keratohyaline granules were not labeled by the antikeratin antisera. No nonfilamentous structures were labeled. The technique described is suitable for studying the distribution of keratin filaments in normal and diseased tissue.

Ultrastructural localization of hyaluronan in myelin sheaths of the rat central and rat and human peripheral nervous systems using hyaluronan-binding protein-gold and link protein-gold.

Neural tissue of central (rat spinal cord) and peripheral origin (rat sciatic nerve, nerve fascicles of rat skin and iris and of human conjunctiva) was processed by osmium tetroxide/microwave fixation and embedded in epoxy resin. Hyaluronan-binding proteins and link proteins coupled to 15-20-nm gold particles were used as markers in a one-step post-embedding procedure for identifying hyaluronan (hyaluronic acid) at the ultrastructural level. All myelin sheaths in both rat and human material were found to be intensely labelled. The specificity of the hyaluronan-binding probes was demonstrated by the total loss of labelling following treatment of sections with hyaluronidase or by preincubating either the probes with hyaluronan oligosaccharides or the sections with unlabelled hyaluronan-binding protein. The identified hyaluronan appears to be located extracellularly, but is precise role here remains to be elucidated.

Quantitation of gold labeling and estimation of labeling efficiency with a stereological counting method.

The amount of immunolabeling over a cell compartment of an average cell was estimated by use of an adaptation of the double disector method introduced by Gundersen. The first and last sections of a stack of ultra-thin sections formed a disector in which cell number could be estimated and related to a defined reference volume to give the cell density. Another stack section, selected at random, was immunolabeled and the number of gold particles associated with unit volume reference space (gold "density") estimated in quadrats placed systematically across the section. The ratio of gold density to cell density was used to estimate the number of gold particles lying over a chosen compartment of an average cell, N(gold)/N(cell). Such estimates required neither cell volume nor section thickness measurement and were reproducible. By combining the number of gold particles per cell with estimates of the number of protein antigens per cell, the number of gold particles associated with each antigen could be found (labeling efficiency).

Applications of immunocolloids in light microscopy. III. Demonstration of antigenic and lectin-binding sites in semithin resin sections.

Previous studies have demonstrated that antigens or lectin-binding sites can be localized in sections from paraffin-embedded tissues with protein A or lectins bound to colloidal gold or colloidal silver (Roth J: J Histochem Cytochem 30:691, 1982 and 31:547, 1983). In the present study the protein A-gold technique and lectin-gold complexes have been applied to semithin sections (0.5-1.5 micron) of Epon- or low temperature Lowicryl K4M-embedded rat pancreas, kidney and submandibular gland. The results show that an increase in resolution and, therefore, in amount of information can be obtained. The optimal mode of imaging was determined on sections without counterstaining. Bright-field illumination gives the maximum information about the staining signal, while phase-contrast and Nomarski differential interference contrast give predominantly structural and, to a lesser extent, staining information. Polarization epi- and transillumination microscopy is inferior in all aspects. The application of a battery of lectin-gold complexes to rat submandibular gland revealed a specific staining pattern for each lectin in acinar and excretory duct cells.

Detection of terminal N-linked N-acetylglucosamine residues in the Golgi apparatus using galactosyltransferase and endoglucosaminidase F/peptide N-glycosidase F: adaptation of a biochemical approach to electron microscopy.

Purified human milk beta-N-acetylglucosaminide beta 1, 4 galactosyltransferase (EC 2.4.1.38) was used to galactosylate N-acetylglucosamine (GlcNAc) residues present in ultra-thin sections of Lowicryl K4M-embedded rat and pig liver. Both endogenous galactose and galactosylated transferase products could be revealed by Ricinus communis lectin I-gold complexes (RcL I-g15). Without galactosyltransferase (GT) treatment, labeling for galactose (gal) was limited to the trans region of rat and pig hepatocyte Golgi apparatus. After exposure to GT, additional labeling was found over cis Golgi apparatus cisternae. RcL I-g15 labeling was sensitive to a purified preparation of endoglucosaminidase F/peptide N-glycosidase F (at pH 9). This indicates that endogenous gal and gal transferred by GT to terminal GlcNAc residues are present N-linked oligosaccharides. The RcL I-g15 labeling produced by GT was insensitive to extensive washing with solutions containing either EDTA and urea or SDS and 2-mercaptoethanol or 0.1 M GlcNAc. Substrate inhibition studies showed that 50 mM GlcNAc specifically inhibited the additional RcL I-g15 labeling produced by GT. The use of purified glycosyltransferases therefore appears to allow specific detection of oligosaccharide substrates and their high resolution localization in thin sections by electron microscopy.