Characterization of a standardized glucagon challenge test as a pharmacodynamic tool in pharmacological research.

The aim of this study was to characterize a glucagon challenge test as a tool in diabetes research by assessing the inter- and intra-individual variability, and investigating the activity of the autonomic nervous system (ANS) during the challenge, as this might have an indirect impact on glucose homeostasis. The study was performed in 24 healthy volunteers separated in 2 groups. The first group of 12 volunteers underwent a 5-h glucagon challenge during a pancreatic clamp procedure with infusion of [6,6-2H2]-glucose infusion in combination with heart rate variability measurements. In the second group, 12 other healthy volunteers underwent two 6-h glucagon challenges separated by 6 weeks, and fat biopsies were taken for analysis of glucagon receptor expression. Serum glucose rose rapidly after glucagon infusion, and reached a plateau at 90 min. The time profiles suggested rapid development of tolerance for glucagon-induced hyperglycemia. During the glucagon challenge intra- and inter-individual variabilities for hepatic glucose production, the rate of disappearance of glucose, and plasma glucose were approximately 10-15% for all variables. Hyperglucagonemia did not affect heart rate variability. Human adipose tissue had a low, but variable, expression of glucagon receptor mRNA. This standardized glucagon challenge test has a good reproducibility with only limited variability over 6 weeks. It is a robust tool to explore in detail the contribution of glucagon in normal and altered glucose homeostasis and can also be used to evaluate the effects of drugs antagonizing glucagon action in humans without confounding changes in ANS tone.

Eosinophil lipid bodies: specific, inducible intracellular sites for enhanced eicosanoid formation.

The specific intracellular sites at which enzymes act to generate arachidonate-derived eicosanoid mediators of inflammation are uncertain. We evaluated the formation and function of cytoplasmic lipid bodies. Lipid body formation in eosinophils was a rapidly (<1 h) inducible response which was platelet-activating factor (PAF) receptor-mediated, involved signaling through protein kinase C, and required new protein synthesis. In intact and enucleated eosinophils, the PAF-induced increases in lipid body numbers correlated with enhanced production of both lipoxygenase- and cyclooxygenase-derived eicosanoids. All principal eosinophil eicosanoid-forming enzymes, 5-lipoxygenase, leukotriene C4 synthase, and cyclooxygenase, were immunolocalized to native as well as newly induced lipid bodies in intact and enucleated eosinophils. Thus, lipid bodies are structurally distinct, inducible, nonnuclear sites for enhanced synthesis of paracrine eicosanoid mediators of inflammation.

Pathogenesis of ascites tumor growth: angiogenesis, vascular remodeling, and stroma formation in the peritoneal lining.

In the accompanying papers, we demonstrated that two murine ascites tumors (MOT and TA3/St) induced peritoneal lining blood vessels to become hyperpermeable to plasma proteins, leading to extravasation of fibrinogen and its clotting to cross-linked fibrin in peritoneal lining tissues (peritoneal wall, mesentery, and diaphragm). In solid tumors, vascular hyperpermeability and fibrin deposition lead to the generation of vascularized connective tissue. In order to determine whether fibrin had similar consequences in ascites tumors, the vasculature and stroma of peritoneal lining tissues were analyzed at successive intervals after i.p. tumor cell injection. In both MOT and TA3/St ascites tumors, the size and number of peritoneal lining microvessels increased significantly by 5-8 days. Subsequently, peritoneal lining vessels increased in cross-sectional area by as much as 15-fold and peritoneal vascular frequency increased by up to 11-fold. Incorporation of [3H]thymidine by mesenteric blood vessels was negligible in control animals but came to involve 20 and 40% of endothelial cells lining mesenteric vessels in MOT and TA3/St ascites tumor-bearing mice, respectively. After an early dramatic increase in cross-sectional area, peritoneal lining microvessels subsequently underwent a novel form of remodeling to smaller average size as the result of transvascular bridging by endothelial cell cytoplasmic processes. Thus, both of the ascites tumors studied here induced angiogenesis and stroma similar to that elicited when these same tumors were grown in solid form. However, stroma developed more slowly in ascites than in solid tumors and was entirely confined to a compartment (peritoneal lining tissues) that was distinct from that (peritoneal cavity) containing the majority of tumor cells and ascites fluid. These findings are consistent with the hypothesis that vascular hyperpermeability, induced in both solid and ascites tumors by tumor cell-secreted vascular permeability factor, is a common early step in tumor angiogenesis, resulting in fibrinogen extravasation, fibrin deposition, and likely other alterations of the extracellular matrix that together stimulate new vessel and fibroblast ingrowth.

The vesiculo-vacuolar organelle (VVO): a distinct endothelial cell structure that provides a transcellular pathway for macromolecular extravasation.

The vesiculo-vacuolar organelle (VVO) is a recently described organelle found in the cytoplasm of endothelial cells that line tumor microvessels and normal venules. VVOs are grape-like clusters of interconnecting uncoated vesicles and vacuoles, bounded by trilaminar unit membranes, that span the entire thickness of vascular endothelium, thereby providing a potential trans-endothelial connection between the vascular lumen and the extravascular space. Macromolecular tracers preferentially cross hyperpermeable tumor microvessels through VVOs. The present investigation was undertaken to elucidate further the ultrastructure and function of VVOs in a murine ovarian carcinoma (MOT) and in normal venules. Morphometry revealed that VVOs were enormous cytoplasmic structures (median area, 0.12-0.14 microns2 in single electron micrographs). Moreover, the individual vesicles and vacuoles that comprised VVOs were on average substantially larger than capillary caveolae and followed a non-normal distribution that was skewed to the right. Specimen tilting provided conclusive evidence that individual VVO vesicles and vacuoles communicated with each other and with the endothelial cells' plasma membranes by stomata, some of which were closed by diaphragms composed of a single membrane. Studies with two tracers, ferritin (FE, diameter approximately 11 nm) and horseradish peroxidase (HRP, diameter approximately 5 nm), revealed that passage of macromolecules through VVOs was regulated at the level of stomatal diaphragms, thereby demonstrating a mechanism for controlling the passage of macromolecules across endothelial cells. Thus, compared with tumor microvessels, little circulating FE and HRP entered the VVOs of normal venular endothelium because stomata joining vesicles and vacuoles to each other and to the lumen and ablumen were closed. VVOs and their component vesicles/vacuoles were readily distinguished from endosomal organelles such as coated vesicles and multivesicular bodies, which also accumulated FE and HRP. Our findings indicate that VVOs provide a major pathway for the extravasation of circulating macromolecules across endothelia taller than capillary endothelium and suggest that upregulated VVO function accounts for the well-known hyperpermeability of tumor blood vessels.

Diamine oxidase-gold ultrastructural localization of histamine in isolated human lung mast cells stimulated to undergo anaphylactic degranulation and recovery in vitro.

A new enzyme-affinity-gold ultrastructural method makes use of the affinity of the enzyme, diamine oxidase coupled to gold, for its substrate, histamine, for localization of histamine in isolated human lung mast cells (HLMCs). The method works with routinely prepared ultrastructural samples, thereby allowing precise identification of ultrastructural structures that contain histamine. We used this method to identify the release of histamine from granule stores in anti-immunoglobulin-E (IgE)-stimulated HLMCs and the replacement of histamine in secretory granules of HLMCs during recovery from anaphylactic degranulation in vitro. The findings show that electron-dense granules in unstimulated HLMCs, maintained up to 24 h in culture, and in responding anti-IgE-stimulated HLMCs, over the same time period in vitro, contained histamine. Alteration of granules, resulting in decreased electron density of their contents, was associated with decreased label for histamine. Electron-lucent intracytoplasmic degranulation chambers were devoid of histamine. Recovering HLMCs developed new granule stores of histamine by a mixture of conservation, synthetic, and endocytotic mechanisms.

Ribonuclease-gold labels heparin in human mast cell granules. New use for an ultrastructural enzyme affinity technique.

We evaluated an enzyme affinity-gold ultrastructural technique designed to identify RNA-rich structures, based on an RNase-gold (R-G) probe in human mast cells (HMCs). As expected, the R-G technique labeled RNA-containing ribosomes and nucleoli in HMCs. The heparin-rich secretory granules in HMCs were also labeled. Extensive studies revealed that HMCs isolated from lung or skin and sustained in short-term cultures, derived de novo in growth factor-supplemented cord blood cell cultures, or present in vivo in multiple sites all shared this property. We performed a large number of controls designed to examine the HMC granule binding characteristics of gold alone, of irrelevant protein- or enzyme-gold reagents, of the role of charge and enzyme activity after various enzyme digestions, after blocking with macromolecules, after exposure to inhibitors of RNase, of heparin, or to irrelevant enzyme inhibitors, including staining of macromolecule-containing test agar blocks and a variety of combined absorption and digestion experiments of the binding of R-G to HMC granules. These studies established that the R-G method detected heparin in this site in conventionally prepared, well-preserved electron microscopic samples. These findings demonstrate a new use for this enzyme affinity-gold technique in mast cell biology, based on the known property of heparin as an inhibitor of RNase.

RNA is closely associated with human mast cell secretory granules, suggesting a role(s) for granules in synthetic processes.

The distribution of ribosomes in mature human mast cells, a major granulated secretory cell, does not resemble that in other secretory cells, such as pancreatic acinar cells and plasma cells. By routine ultrastructural analysis, ribosomes in human mast cells are often close to, attached to, or even appear to be within secretory granules. To document better these relationships, we used multiple electron microscopic imaging methods, based on different principles, to define RNA, ribosome, and granule relationships in mature human mast cells. These methods included EDTA regressive staining, RNase digestion, immunogold labeling of ribonucleoproteins or uridine, direct binding or binding after ultrastructural in situ hybridization of various polyuridine probes to polyadenine in mRNA, and ultrastructural autoradiographic localization of [3H]-uridine incorporated into cultured human mast cells. These different labeling methods demonstrated ribosomes, RNA, U1SnRNP (a small nuclear RNP specific for alternative splicing of mRNA), mRNA, and uridine to be associated with secretory granules in human mast cells, implicating granules in a larger synthetic role in mast cell biology.

Diamine oxidase-gold labels histamine in human mast-cell granules: a new enzyme-affinity ultrastructural method.

We developed a post-embedding ultrastructural enzyme affinity-gold technique to label histamine. Diamine oxidase (DAO)-gold complex was prepared and tested for specificity with routinely processed, Epon-embedded, histamine-rich human mast-cell granules and histamine-agar test blocks, both of which were labeled. Specificity controls included removal of staining by DAO digestion of Epon sections containing mast cells or by filtering DAO-gold over histamine-agarose beads, and a wide variety of controls (for these specificity controls) that did not abrogate mast-cell granule or histamine-agar block labeling with DAO-gold. Internal negative controls in each sample included the failure of DAO-gold to stain Type II alveolar pneumocyte lamellar bodies or altered, swollen mast-cell granules from which histamine has been released. The new post-embedding enzyme affinity-gold technique is effective in optimally prepared ultrastructural samples that allow clear identification of key ultrastructural morphology.

Ribonuclease-gold labels proteoglycan-containing cytoplasmic granules and ribonucleic acid-containing organelles--a survey.

An enzyme-affinity-gold method to detect RNA in routinely prepared ultrastructural samples is based on the affinity of the gold-coupled enzyme, ribonuclease, for its substrate, RNA. High concentrations of a known inhibitor of RNase, heparin, are uniquely located in human mast cell granules. Specific labeling for the presence of heparin in these structures was determined using the RNase-gold (R-G) reagent based on the RNase inhibitor property of heparin. This property was used to probe for the presence of proteoglycans (PG) known to be present in a wide variety of ultrastructural samples, none of which contain heparin. In addition to known subcellular sites of RNA, the R-G reagent was shown to bind to PG-rich cytoplasmic granules in a wide variety of leukocytes and secretory cells of epithelial, endocrine, and neuroendocrine origin. This newly recognized property was used to image the changing distribution of labeled PGs during cellular maturation, secretion, and recovery from secretion of secretory cells in vivo, ex vivo, in vitro and in isolated, biochemically defined guinea pig basophil granule preparations.

RNA is closely associated with human mast cell lipid bodies.

Both novel and multiple ultrastructural studies based on different principles show relationships of cytoplasmic lipid bodies and ribonucleic acid (RNA) of potential importance to RNA metabolism in human mast cells. The methods include general ultrastructural morphological observations, imaging of RNA with an EDTA regressive stain, imaging of the incorporation of radio labeled uridine by ultrastructural autoradiography, postembedding immunogold labeling of uridine, ribosomes and small nuclear ribonuclear proteins and ultrastructural in situ hybridization detection of poly(A)-positive messenger RNA. Altogether these studies implicate human mast cell lipid bodies in RNA metabolism and are analogous to earlier similar studies which showed that human mast cell granules also curtain RNA.

Maternal obesity and venous thromboembolism.

The incidence of obesity in pregnancy has increased rapidly in the last decade. Obesity is a risk factor for venous thromboembolism outside of pregnancy and previous studies of maternal death in the UK have identified obesity as a risk factor in pregnancy. As a result the Royal College of Obstetricians and Gynaecologists have targeted obesity as a risk factor in evaluation of the need for thromboprophylaxis in pregnancy. This article highlights the evidence that obesity increases the risk of venous thromboembolism in pregnancy and the puerperium, discusses thromboprophylaxis and appropriate dosing in obese parturients and details the anaesthetic implications of the 2009 Royal College of Obstetricians and Gynaecologists' guidelines. More clinical studies are required to clarify the appropriate dose of low-molecular-weight heparin in an obese parturient.

Ultrastructural cytochemical, immunocytochemical and in situ hybridization methods with polyuridine probes detect mRNA in human mast cell granules.

Mature human mast cells are classical secretory cells that are filled with secretory-storage granules but are poorly endowed with visible free or membrane-bound cytoplasmic ribosomes. We recently reported close associations of ribosomes and various components essential to RNA metabolism in and close to human mast cell granules using multiple ultrastructural imaging methods. In view of these findings and an increased awareness of RNA sorting and localization to specific subcellular sites and organelles, we used human mast cells purified from non-tumour portions of lung samples resected at surgery for carcinoma and ultrastructural methods to investigate this further. Poly(U) probes were used to detect direct en grid binding, and radiolabelled as well as non-radiolabelled poly(U) probes were used in in situ hybridization protocols to detect poly(A)-positive pre-mRNA and mRNA in nuclear, cytoplasmic and granular compartments of mature human mast cells. Negative controls verified specificity of label; expected nuclear and cytoplasmic locations of poly(A)-positive RNA served as positive controls for each sample. These findings lend support to the hypothesis that site-specific synthesis in secretory-storage granules may occur in secretory cells.

Ultrastructural immunogold cytochemistry with autoimmune human sera and an antibody to uridine implicate human mast cell granules in RNA biology.

Human mast cells are professional secretory cells that store synthetic products in large granules filling their cytoplasm. Unlike many secretory cells, the principal synthetic organelle, ribosome-rich endoplasmic reticulum, is a minor component of their cytoplasm. Sightings of nonmembrane-bound ribosomes in and near their secretory granules stimulated detailed ultrastructural studies of various RNA species to implicate secretory-storage granules in RNA biology. In the work reported here, postembedding immunogold ultrastructural cytochemistry indicates that human mast cells contain uridine, an integral ingredient of RNA, and ribonucleoproteins, known to associate with small nuclear RNAs important for splicing RNA precursors, several ribonucleoproteins with possible functions in other aspects of RNA biology and ribonucleoproteins known to associate with ribosomes. These findings should catalyse future work toward establishing the full functional repertoire of secretory-storage granules.

Ultrastructural detection of histamine in human mast cells developing from cord blood cells cultured with human or murine recombinant c-kit ligands.

Immature human mast cells, arising from cord blood mononuclear cells cultured in the presence of the c-kit ligand of human or murine origin, contain mixtures of morphologically immature and mature secretory granules. Use of a new cytochemical technique to localize histamine in ultrastructural samples (based on the affinity of the enzyme, histaminase, for its substrate, histamine) localized this amine to mature mast cell granules of all substructural patterns present, as well as to condensation foci appearing in immature cytoplasmic granules. This cytochemical evidence of histamine bound to condensation foci during granule building in developing mast cells is analogous to evidence obtained during granule recovery of degranulated human basophils and mast cells.

Diamine oxidase-gold enzyme-affinity ultrastructural demonstration that human gut mucosal mast cells secrete histamine by piecemeal degranulation in vivo.

Biopsy specimens of human ilea were prepared for the ultrastructural detection of histamine in vivo with a new enzyme-affinity method (Dvorak et al., J Histochem Cytochem 1993; 41:787-800). Human gut mucosal mast cells contained histamine in cytoplasmic secretory granules that were electron dense, regardless of underlying substructural patterns. Partially and completely empty cytoplasmic granules, characteristic for piecemeal degranulation (Dvorak et al., Int Arch Allergy Immunol 1992;99:74-83) had diminished and absent histamine stores, whereas electron-dense granules in the same cells retained their histamine. Interstitial collagen (but not basal lamina of other cells) near secretory mast cells avidly bound histamine, thereby providing a potential source for re-release of extracellular histamine stores. These studies provide the first documentation of histamine secretion in vivo during piecemeal degranulation of human mast cells.

Ribonuclease-gold labels chondroitin sulphate in guinea pig basophil granules.

Basophilic leucocytes are metachromatic granule-containing secretory granulocytes that contain a mixture of granular proteoglycans devoid of heparin. In guinea pigs, isolated basophilic leucocyte granules primarily contain chondroitin sulphate. We have recently demonstrated that an enzyme-affinity-gold technique to image RNA, using the reagent RNase gold, also binds specifically to heparin in human mast cell granules. Such binding is based on the known property of heparin as a competitive inhibitor of RNase. Using similar methods, we show here that RNase-gold binds to the chondroitin sulphate in the secretory granules of guinea pig basophils, thus broadening the applicability of this post-embedding affinity-gold method to studies that require imaging of chondroitin sulphate in routinely prepared electron microscopical samples.

Ribosomes and secretory granules in human mast cells: close associations demonstrated by staining with a chelating agent.

The distribution of ribosomes was investigated in mature human mast cells with a chelation-based staining protocol known to bleach DNA-rich structures, leaving RNA-rich structures unbleached. With this method, electron-dense ribosomes were adjacent to, attached to, and within secretory granules, which were also bleached with the chelation method that we used. The finding of these ribosome-secretory granule relationships suggests that secretory granules in mature human mast cells may participate in RNA metabolism.

Ribonuclease-gold ultrastructural localization of heparin in isolated human lung mast cells stimulated to undergo anaphylactic degranulation and recovery in vitro.

BACKGROUND: Human mast cells are a rich and unique source of heparin, which is stored in cytoplasmic secretory granules and accounts for metachromasia, a staining property used to identify mast cells by light microscopy. OBJECTIVE: The sub-cellular locations of heparin in secretory human mast cells and human mast cells recovering from secretion are not known. Acquisition of this knowledge requires ultrastructural imaging of well-preserved cells with a visible probe which binds to heparin. We sought to develop this knowledge regarding human mast cell secretion by using a labelling method for heparin that depends on the well-known property of ribonuclease inhibition by heparin. METHODS: Human lung mast cells were isolated, partially purified, either stimulated or not stimulated to secrete with anti-IgE, and recovered 20 min or 6 h later for routine electron microscopy. Histamine secretion was also determined. A previously developed post-embedding, enzyme-affinity-gold electron microscopic technique to image ribonucleic acid (RNA) with ribonuclease-gold (R-G), which also binds to the enzyme inhibitor, heparin, was employed to determine the sub-cellular locations of heparin in non-secretory and secretory mast cells as well as in mast cells recovered from short-term cultures after secretion. Specificity controls for the novel use of this method and quantification of granule labelling in these controls were performed. RESULTS: Heparin was labelled by R-G in electron-dense granules within non-secretory human lung mast cells (HLMCs), in electron-dense granules that persisted in secretory HLMCs at the maximum histamine secretion time (20 min), and in electron-dense granules within recovering HLMCs. Specificity controls showed that gold alone did not label HLMCs and that absorption with heparin significantly reduced or abrogated HLMC granule staining with R-G, but that RNA absorption did not. Heparin stores were absent in newly formed, electron-lucent intracytoplasmic degranulation channels in secretory HLMCs. Electron-dense granule matrices in the process of extrusion to the cell exterior still retained heparin at the instant of cellular secretion. Non-granule heparin stores bound R-G in recovering HLMCs. These locations included resolving degranulation channels, as newly emergent granules partitioned and condensed within them, and electron-dense content-containing vesicles and progranules within synthetic mast cells. Ultimately, all known ultrastructural patterns of HLMC granules developed in recovering cells, and each of them contained heparin. CONCLUSION: Heparin was secreted from HLMCs which were stimulated by anti-IgE, and heparin was recovered by a combination of conservative and synthetic mechanisms in HLMCs after a secretory event.

Ultrastructural immunolocalization of basic fibroblast growth factor to lipid bodies and secretory granules in human mast cells.

Isolated human lung mast cells were used to identify subcellular sites of basic fibroblast growth factor using a postembedding immunogold method. The factor was present in quantity in secretory granules and cytoplasmic lipid bodies. Cisterns of smooth endoplasmic reticulum and ribosome clusters, closely associated with lipid bodies, contained the factor as did the nuclear matrix. Factor-positive lipid bodies were adjacent to nuclear pores and often indented perinuclear cisternae. Altered secretory granules with reduced density, characteristic of secretion by piecemeal degranulation in mast cells, showed reduced gold label for basic fibroblast growth factor; small, electron-lucent (80-100 nm) transport vesicles near altered granules were labelled for the factor. Since these mature mast cells do not display extensive arrays of classical secretory organelles, such as rough endoplasmic reticulum and Golgi structures, these new subcellular localizations for basic fibroblast growth factor suggest several possible alternative release routes for a cytokine devoid of a signal sequence characteristic of regulated secretory proteins.