Immunohistochemical localization of epinephrine, norepinephrine, catecholamine-synthesizing enzymes, and chromogranin in neuroendocrine cells and tumors.

The immunohistochemical localization of epinephrine (E), norepinephrine (NE), and chromogranin was analyzed in normal and neoplastic neuroendocrine cells. The immunohistochemical detection of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) was used to distinguish between uptake and biosynthesis of catecholamines. E, NE, chromogranin, TH, DBH, and PNMT were found in the normal human adrenal medulla and in pheochromocytomas. Although many neuroendocrine tissues outside of the adrenal gland contained immunoreactive NE, only a small percentage of these tissues contained DBH. E was found in a few neuroendocrine tissues outside of the adrenal, including cardiac paragangliomas, and the enzyme PNMT was localized in some of these neoplasms. There was very close agreement between the localization of chromogranin and of catecholamines in normal and neoplastic neuroendocrine tissues. These results indicate that the presence of catecholamines and chromogranin in neuroendocrine cells and tumors within the adrenal medulla and in many other sites may be closely related.     

Giardia lamblia produces alanine anaerobically but not in the presence of oxygen

Proton nuclear magnetic resonance was used to follow glucose metabolism in Giardia lamblia. Under strictly anaerobic conditions this organism produces equimolar ethanol and alanine as well as CO2 and some acetate. Aerobically the production of both alanine and ethanol are inhibited and more acetate and CO2 are formed. These changes in the balance of products are reversible over the range 0-46 microM O2. In the presence of 46 microM O2, alanine was not detectable. The O2-sensitivity of alanine production may highlight the necessity for redox-balancing reactions in an organism exposed in situ to fluctuating concentrations of O2.     

Monocyte-to-macrophage transition in vitro. A systematic study using human cells isolated by fractionation on Percoll

Improved density-gradient methods, using Percoll or Nycodenz, have recently been introduced for the isolation of human monocytes, but the capacity of cells thus isolated to differentiate into macrophages has not been systematically studied. We have compared Percoll and Nycodenz methods for the isolation of monocytes from human blood. The Nycodenz method yielded a monocyte population of high purity, but the yield was low. The Percoll method gave almost quantitative yield of monocytes, and the contaminating cells, mostly lymphocytes, were readily washed away after allowing the monocytes to adhere to a plastic surface. The Percoll method was then successfully scaled up, providing a simple method to obtain the monocytes from 180 ml blood. These monocytes were maintained in culture and their capacity to mature into macrophages was studied, using the following criteria: increase in cell size and protein content, increase in specific activity of hexosaminidase, differential hexosaminidase release on exposure to opsonized zymosan and unopsonized polystyrene beads, loss of peroxidase activity, and development of fluoride-insensitivity by the cells' cytochemically demonstrable esterase. The cells also displayed morphological changes typical of the monocyte-to-macrophage transition. The procedures reported constitute a simple and reliable method for the production of human macrophages in increased yield.     

Microvascular structural entropy: A novel approach to assess angiogenesis in pituitary tumors

Entropy, a measure of the degree of disorder in a system, has recently been used in different morphologic studies to quantify regularity. Our aims were (a) to study the structural organization of the microvascular bed in prolactin (PRL)-producing adenomas and carcinomas, the most vascularized of pituitary tumors, by assessing microvascular structural entropy (MSE), and (b) to determine whether the degree of disorder of the capillary bed correlates with tumor cell proliferation as estimated by MIB-1 labeling, microvessel density (MVD), the most widely used method of quantifying blood vessel formation, and various clinicopathologic parameters (gender, age, tumor size and invasiveness). The morphometric study demonstrated statistically significant differences in MIB-1 labeling, MVD, and MSE between PRL-producing adenomas and carcinomas. Unlike MIB-1 labeling index (PRL-producing adenomas 1.5±0.27; carcinomas 15.0±4.04) and MVD (PRL-producing adenomas 2.7±0.34; carcinomas 4.2±0.72), the MSE values were significantly higher in adenomas (171.5±25.37) than in carcinomas (67.9±17.45). These results indicate that PRL-producing carcinomas have a less chaotic distribution of vessels than benign adenomas. In contrast to a lack of correlation between, microvessel density and other morphometric parameters, a strong negative correlation was found between MSE and MIB-1 labeling index (r=0.511, p=0.003). It thus appears that regular, less chaotic microvascular geometry contributes to increased proliferative activity in PRL cell tumors. Analysis of MSE may provide an independent parameter of tumor behavior, and contributes to a better understanding of the role of microvasculature in pituitary tumor progression.     

Distinction between papillary thyroid hyperplasia and papillary thyroid carcinoma by immunohistochemical staining for cytokeratin 19, galectin-3, and HBME-1

The histopathology of papillary thyroid hyperplasia and papillary thyroid carcinoma is similar enough to cause a diagnostic dilemma in a few cases. Both lesions may have papillary fronds with fibrovascular cores, nuclear crowding, and nuclear anisocytosis. Formalin-fixed paraffin-embedded tissues from 30 randomly selected patients with papillary thyroid hyperplasia and an equal number from patients with papillary thyroid carcinoma were analyzed for expression of cytokeratin 19 (CK19), galectin-3, and HBME-1. Cases of papillary thyroid carcinoma had moderate to strong CK19, galectin-3, and HBME-1 reactivity although both CK19 and galectin-3 showed positive staining in a significant number of nonneoplastic thyroid cases. HBME-1 was uncommon in the nonneoplastic cases. These results indicate that HBME-1 may be useful in helping to distinguish papillary thyroid carcinoma from hyperplasia in diagnostically difficult cases.     

Intrinsic and extrinsic modulation of a single central pattern generating circuit

Intrinsic and extrinsic neuromodulation are both thought to be responsible for the flexibility of the neural circuits (central pattern generators) that control rhythmic behaviors. Because the two forms of modulation have been studied in different circuits, it has been difficult to compare them directly. We find that the central pattern generator for biting in Aplysia is modulated both extrinsically and intrinsically. Both forms of modulation increase the frequency of motor programs and shorten the duration of the protraction phase. Extrinsic modulation is mediated by the serotonergic metacerebral cell (MCC) neurons and is mimicked by application of serotonin. Intrinsic modulation is mediated by the cerebral peptide-2 (CP-2) containing CBI-2 interneurons and is mimicked by application of CP-2. Since the effects of CBI-2 and CP-2 occlude each other, the modulatory actions of CBI-2 may be mediated by CP-2 release. Although the effects of intrinsic and extrinsic modulation are similar, the neurons that mediate them are active predominantly at different times, suggesting a specialized role for each system. Metacerebral cell (MCC) activity predominates in the preparatory (appetitive) phase and thus precedes the activation of CBI-2 and biting motor programs. Once the CBI-2s are activated and the biting motor program is initiated, MCC activity declines precipitously. Hence extrinsic modulation prefacilitates biting, whereas intrinsic modulation occurs during biting. Since biting inhibits appetitive behavior, intrinsic modulation cannot be used to prefacilitate biting in the appetitive phase. Thus the sequential use of extrinsic and intrinsic modulation may provide a means for premodulation of biting without the concomitant disruption of appetitive behaviors.     

Clinical importance of Campylobacter pyloridis and associated serum IgG and IgA antibody responses in patients undergoing upper gastrointestinal endoscopy.

Campylobacter pyloridis was isolated from 77% of 220 (35%) unselected adults undergoing gastroscopy. Isolation was significantly associated with histological gastritis (p less than 0.0001), duodenal ulcer (p less than 0.0001), and to a much lesser extent, with gastric ulcer (p less than 0.05). The relation between the isolation of C pyloridis and peptic ulcer seemed to be independent of coexisting gastritis. In those with no endoscopic or histological evidence of disease there was no relation between isolation and increasing age. Antibody responses to a whole cell sonicate of a strain of C pyloridis were measured by means of an enzyme linked immunosorbent assay (ELISA). Increased IgA (p less than 0.0001) and IgG (p less than 0.0001) antibody titres were found in patients with C pyloridis. Peptic ulceration or gastritis were present in 78% and 100% of patients with a high concentration of IgG and IgA, respectively, but in only 9% and 18% of those with low titres. These results provide further evidence for a possible pathogenic role of these organisms in gastric disease and suggest that immunological markers of their presence might be useful non-invasive indicators of disease.     

Site-specific mutagenicity of stereochemically defined 1,N2-deoxyguanosine adducts of trans-4-hydroxynonenal in mammalian cells

Trans-4-hydroxynonenal (HNE) is a toxic compound produced endogenously during lipid peroxidation. HNE is a potent electrophile that is reactive with both proteins and nucleic acids. HNE preferentially reacts with deoxyguanosine to form four stereoisomeric HNE-deoxyguanosine (HNE-dG) adducts: (6R, 8S, 11R), (6S, 8R, 11S), (6R, 8S, 11S), and (6S, 8R, 11R). These adducts were synthesized into 12-mer oligodeoxynucleotides, inserted into a DNA shuttle vector and evaluated for the ability of each stereoisomer to induce mutagenesis when replicated through mammalian cells. The resultant mutagenicity of these adducts was related to their stereochemistry, in that two of the HNE-dG adducts, (6R, 8S, 11R) and (6S, 8R, 11S), were significantly more mutagenic than the (6R, 8S, 11S) and (6S, 8R, 11R) HNE-dG adducts. These data conclusively demonstrate that HNE-derived DNA adducts can be mutagenic in mammalian cells and their ability to cause mutations is dictated by their stereochemistry.     

Characterization of damage modes in dental ceramic bilayer structures

Results of contact tests using spherical indenters on flat ceramic coating layers bonded to compliant substrates are reported for selected dental ceramics. Critical loads to produce various damage modes, cone cracking, and quasiplasticity at the top surfaces and radial cracking at the lower (inner) surfaces are measured as a function of ceramic-layer thickness. It is proposed that these damage modes, especially radial cracking, are directly relevant to the failure of all-ceramic dental crowns. The critical load data are analyzed with the use of explicit fracture-mechanics relations, expressible in terms of routinely measurable material parameters (elastic modulus, strength, toughness, hardness) and essential geometrical variables (layer thickness, contact radius). The utility of such analyses in the design of ceramic/substrate bilayer systems for optimal resistance to lifetime-threatening damage is discussed.     

Suppression of acute proinflammatory cytokine and chemokine upregulation by post-injury administration of a novel small molecule improves long-term neurologic outcome in a mouse model of traumatic brain injury

High levels of serum cholesterol and disruptions of the blood brain barrier (BBB) have all been implicated as underlying mechanisms in the pathogenesis of Alzheimer's disease. Results from studies conducted in animals and humans suggest that caffeine might be protective against Alzheimer's disease but by poorly understood mechanisms. Using rabbits fed a cholesterol-enriched diet, we tested our hypothesis that chronic ingestion of caffeine protects against high cholesterol diet-induced disruptions of the BBB. New Zealand rabbits were fed a 2% cholesterol-enriched diet, and 3 mg caffeine was administered daily in drinking water for 12 weeks. Total cholesterol and caffeine concentrations from blood were measured. Olfactory bulbs (and for some studies hippocampus and cerebral cortex as well) were evaluated for BBB leakage, BBB tight junction protein expression levels, activation of astrocytes, and microglia density using histological, immunostaining and immunoblotting techniques. We found that caffeine blocked high cholesterol diet-induced increases in extravasation of IgG and fibrinogen, increases in leakage of Evan's blue dye, decreases in levels of the tight junction proteins occludin and ZO-1, increases in astrocytes activation and microglia density where IgG extravasation was present. Chronic ingestion of caffeine protects against high cholesterol diet-induced increases in disruptions of the BBB, and caffeine and drugs similar to caffeine might be useful in the treatment of Alzheimer's disease.