Developmental changes in the hypoxic response of the hypoglossus respiratory motor output in vitro

The transverse brain stem slice of mice containing the pre-B?tzinger complex (PBC), a region essential for respiratory rhythm generation in vitro, was used to study developmental changes of the response of the in vitro respiratory network to severe hypoxia (anoxia). This preparation generates, at different postnatal stages [postnatal day (P)0-22], spontaneous rhythmic activity in hypoglossal (XII) rootlets that are known to occur in synchrony with periodic bursts of neurons in the PBC. It is assumed that this rhythmic activity reflects respiratory rhythmic activity. At all examined stages anoxia led to a biphasic response: the frequency of rhythmic XII activity initially increased ("primary augmentation") and then decreased ("secondary depression"). In neonates (P0-7), anoxia did not significantly affect the amplitude of integrated XII bursts. Secondary depression never led to a cessation of rhythmic activity. In mice older than P7, augmentation was accompanied by a significant increase in the amplitude of XII bursts. A significant decrease of the amplitude of XII bursts occurred during secondary depression. This depression led always to cessation of rhythmic activity in XII rootlets. The anoxia-induced response of the respiratory rhythmic XII motor output is biphasic and changes during development in a similar way to the in vivo respiratory network. Whether this biphasic response is due to a biphasic response of the respiratory rhythm generator and/or to a biphasic modulation of the XII motor nucleus remains unresolved and needs further cellular analysis. We propose that the transverse slice is a useful model system for examination of the mechanisms underlying the hypoxic response.     

Measuring memory for source: some theoretical assumptions and technical limitations

In a search for pathogenetic mechanisms underlying retention hyperkeratosis, we examined the pH gradient over the stratum corneum in 13 male patients suffering from either x-linked recessive (XRI) or autosomal dominant ichthyosis vulgaris. For recording pH values, a flat glass electrode was repeatedly applied to the skin during tape stripping of mildly involved forearm skin. Before stripping, surface pH was higher in ichthyosis vulgaris (5.3 +/- 0.7; n = 7) than in XRI (4.6 +/- 0.4; n = 6; p < 0.05) and healthy control men (4.5 +/- 0.2; n = 7; p < 0.01). Removal of stratum corneum, which required 100-240 strippings in ichthyotic skin and 80-120 strippings in healthy control skin, disclosed markedly different pH variations in the two types of ichthyosis. The major abnormality in ichthyosis vulgaris skin was that a neutral pH was attained already halfway through the horny layer, possibly reflecting a congenital lack of acidic breakdown products from keratohyaline. By contrast, stripping of XRI skin revealed a shallow pH gradient that plateaued at 6.2-6.6, instead of about 7 as in normal and ichthyosis vulgaris skin. A likely explanation is the XRI-associated accumulation of cholesterol sulfate in lower stratum corneum. Our results suggest that the "acid mantle" of normal skin, which penetrates deep into the stratum corneum, is the combined result of cornification-associated organic acids and back-diffusion of acid material from the surface. Because corneocyte desquamation involves many pH-dependent enzymes, abnormalities in the transcorneal pH gradient might play a role in the pathogenesis of ichthyosis.     

Probing heterotypic cell interactions: hepatocyte function in microfabricated co-cultures

Replacement of liver function using extracorporeal bioartificial systems has been attempted with limited success. The instability of the hepatocyte phenotype in vitro has restricted the useful lifetime of these devices. Co-cultivation of hepatocytes with mesenchymal cells is one method that has been widely utilized to stabilize the liver-specific function of isolated cells; however, co-culture has yet to be successfully incorporated in a bioreactor setting. In this study, we probed heterotypic cell interactions in co-cultures of hepatocytes and 3T3 in order to better understand the cellular microenvironment necessary to induce and stabilize liver-specific functions. Using microfabrication and conventional techniques to control the heterotypic interface, the effects of varying degrees of heterotypic interaction on tissue function (albumin and urea synthesis) were examined. Our data indicated maximal induction of liver-specific functions in cultures with maximal initial heterotypic interaction, and that induction of hepatic functions in hepatocytes was increased in the vicinity of fibroblasts as compared to hepatocytes far from the heterotypic interface. Furthermore, our data suggested that heterotypic cell contact is necessary for induction of these functions. These studies will aid in the formation of design criteria for a co-culture based bioartificial liver, as well as provide a useful tool to study the role of heterotypic and homotypic interactions in liver physiology and pathophysiology.     

Technical bases and acquisition conditions of electron-beam computed tomography

PURPOSE: In this review the technical principle and scanner characteristics of electron beam computer tomography (EBCT) are discussed. METHODS: In contrast to conventional CT, image acquisition in EBCT is achieved without mechanically moving parts. This construction allows for short acquisition times in investigating given anatomical regions (100 ms per slice) or up to 8 levels without table movement and short interscan delays (50 ms per slice). RESULTS: Depending on the nature of the investigation, the scanner can be used in the single slice, continuous volume scanning and multi slice mode. The single slice mode is used for detection and quantification of coronary calcifications and for CT angiography of the coronary vessels. Equivalent to the spiral mode in conventional CT, continuous volume scanning may be used for routine investigation of the chest and abdomen. Functional investigations of the heart and perfusion measurement of different organs can be performed in multi slice mode. Because of the geometry of the electron beam scanner, radiation exposure for certain investigations is above the exposure with conventional CT. CONCLUSION: Future developments will focus on dose efficient radiation collimation, high resolution detector systems and artefact reducing reconstruction kernels.     

Solid-phase extraction on C18 silica as a purification strategy in the solution synthesis of a 1-thio-beta-D-galactopyranoside library

A novel strategy for the purification of carbohydrate-based chemical libraries synthesized in solution was developed. Purification of reaction products was accomplished by means of solid-phase extraction enabled by protecting the 2-, 3-, 4-, and 6-hydroxyl groups of a galactose derivative as their hydrophobic O-laurates. The presence of multiple O-laurates allowed adsorption of reaction products onto C18 silica while reagents and by-products were washed away with MeOH. Products were quantitatively eluted with pentane. Purification of products using solid-phase extraction offers the combined advantages of solution synthesis (normal solution reactivity and ease of reaction monitoring) with those of solid-phase synthesis (facile product isolation permitting the use of large excesses of reagents). To demonstrate the utility of the hydrophobic recovery-procedure, tetra-O-lauroyl-beta-D-galactopyranose-1-thiol was subjected to high-yielding reactions with a panel of Michael-acceptors and an alpha-chloro ketone. The resulting ketone adducts were then either reduced to the alcohols or reductively aminated with a selection of amino acids to give 30 different 1-thio-beta-D-galactosides as mixtures of four diastereomers after removal of protecting groups. At each step, the product was separated from the reagents and their by-products by simple adsorption onto C18 silica, washing with MeOH and elution of product with pentane. After completion of the combinatorial chemistry sequence, the O-laurates were cleaved by methanolysis and the product methyl laurate in turn removed from the desired water-soluble products by C18 adsorption. Individual library members were thus conveniently produced on 10-30 mg scales at purity levels of > 90%. One of the 1-thio-beta-D-galactosides thus produced was found to be a competitive inhibitor of the beta-galactosidase from E. coli with Ki value of 1.7 microM.     

Modified intra-arterial calcium stimulation with venous sampling test for preoperative localization of insulinomas

BACKGROUND: Both genetic and environmental factors are thought to contribute to specific IgE responses, however, the relative contribution of each in the responses to individual ryegrass pollen allergens is largely unknown even though some responses to allergens have been linked to certain HLA complexes. OBJECTIVE: Using a large group of monozygotic and dizygotic twins, this study was designed to investigate the IgE binding profiles of individual ryegrass pollen (Lolium perenne) components to assess the relative contribution of genetic and environmental factors in determining IgE responses to specific allergens. METHODS: Ryegrass pollen proteins were separated by electrophoresis and immunoblotted with sera from 191 pairs of twins where at least one sibling had a SPT > 2 mm to perennial ryegrass. Concordance levels for individual ryegrass pollen components were compared between monozygotic and dizygotic twins in a subset group where both twins had SPT > 3 mm to perennial ryegrass. RESULTS: Immunoblotting revealed 23 individual IgE-binding components from ryegrass pollen. Although there was a significantly greater proportion of monozygotic twins with SPT wheals greater than 3 mm when compared with the dizygotic twins, the mean case-wise concordance for the immunoblot components was similar for both groups of twins. The mean case-wise concordance when at least four pairs of sera were involved was 44% for the MZ twins (n=11 components) and 45% for the DZ twins (n=12 components). We found no significant differences in concordance levels for any of the 23 individual components including allergens previously associated with HLA. CONCLUSION: Evidence for genetic control of allergen-specific IgE responses in a large population sample of twins to individual ryegrass allergens is limited, indicating that the IgE responses to specific ryegrass pollen allergens are determined largely by environmental factors.     

Identification of a hypoperfused segment in bull's-eye myocardial perfusion images using a feed forward neural network

Artificial neural networks are computer systems which can be trained to recognize similarities in patterns and which learn by example; one of the more straightforward types being the feed forward neural network (FFNN). We previously reported the use of FFNNs for classification of hypoperfusion patterns in bull's-eye representation of 201Tl single photon emission tomography myocardial perfusion studies and showed that, when such an image was divided into 24 segments, FFNNs could detect perfusion defects without direct comparison to a normal data base. This has been extended in this investigation to assess the ability of an FFNN, trained on data in which only a single segment was hypoperfused, to detect this abnormal segment when the hypoperfusion pattern of the other segments in the image varied. The results indicated that the network could reliably determine whether a segment was normally or under perfused, with accuracies of 99% and 100%, respectively, if all other segments were normally perfused. It could also reliably detect a normally perfused segment, even if other segments were hypoperfused, with accuracies of 95% and 98%. The network was less reliable, however, in detecting a hypoperfused segment when other segments were also hypoperfused, showing accuracies of only 74% and 88%.     

The possible significance of parallel changes in plasma lutein and retinol in Pakistani infants during the summer season

We established new cell lines from head and neck cancer patients for studies of adhesion molecules and cellular behavior in nine patients with primary or metastatic cancer treated at the Asan Medical Center. Explant cultures of fresh tumor tissue were used to develop new permanent tumor cell lines. Lines were tested for tumor formation and histology in nude mice. Flow cytometry and indirect immunofluorescence were used to assess DNA content and expression of the alpha 6, beta 4, and beta 1 integrin subunits and the intercellular adhesion molecule 1 (ICAM-1). In vitro growth patterns and adhesion to plastic were assessed using phase contrast microscopy. AMC-HN-1 to -8 were derived from patients with squamous cell carcinoma. AMC-HN-9 was from an undifferentiated carcinoma of the parotid gland. The 8 lines we tested produced nude mouse tumors that are identical to the histology of the original tumors. AMC-HN-1, -2, -5, and -9 have epithelioid or spindle cell morphology with poor cell-to-cell and cell-to-substrate adhesiveness. AMC-HN-3, -4, -7, and -8 grow as adherent epithelioid monolayers. AMC-HN-6 exhibits multilayer stratification. Four lines are near diploid, 4 are hyperdiploid and 1 is hypodiploid. Only three express ICAM-1. All lines express the alpha 6, beta 4, and beta 1 integrin subunits but to different extent. Four, AMC-HN-1, -2, -5, and -6, express the beta 4 integrin at low levels, AMC-HN-3, -4, -7, and -9, have intermediate beta 4 expression, and AMC-HN-8 has extremely high beta 4 expression. The AMC-HN cell lines are representative in vitro models for the study of head and neck cancer biology. Our preliminary results indicate a close relationship between integrin expression and cell adhesion in vitro.     

Scintigraphic evaluation of experimental colitis in rabbits

Scintigraphic techniques are frequently used for evaluation of inflammatory bowel disease. The radiopharmaceutical of choice is labeled leukocytes. In this study, two new agents, 111In-labeled polyethylene glycol-coated liposomes and 111In-labeled human nonspecific gamma globulin (immunoglobulin G; IgG), were compared with 111In-leukocytes in a rabbit model of colitis. METHODS: In rabbits, acute colitis was induced by colonic instillation of trinitrobenzene sulfonic acid at 25 cm from the anal sphincter. After 24 hr, 15 MBq of the radiopharmaceuticals was injected intravenously in groups of four rabbits. Twenty-four hours after injection, the animals were killed and macroscopic abnormalities were scored in seven consecutive affected colonic segments of 5 cm each (0 = normal, 1 = inflammation, 2 = ulcers). The ex vivo uptake was measured in the normal ascending colon and the affected colonic segments. The colitis index (CI, affected-to-normal colon-uptake ratio) was calculated. RESULTS: Histologically, an acute, patchy, transmural colitis was observed at the site of instillation and the distal colon. The CI of all agents in colitis lesions correlated with the severity of the abnormalities. With increasing severity, the CI for liposomes was 1.86 +/- 0.24, 4.88 +/- 0.42 and 7.42 +/- 0.54 (r2 = 0.68, p < 0.001); for leukocytes 1.77 +/- 0.32, 3.10 +/- 0.58 and 5.54 +/- 0.83 (r2 = 0.31, p < 0.01); for IgG 1.60 +/- 0.29, 2.81 +/- 0.21 and 2.65 +/- 0.21 (r2 = 0.29, p < 0.02). CONCLUSION: Indium-111-labeled-leukocytes, -IgG and -liposomes all show increased uptake in inflamed colonic tissue. Indium-111-liposomes showed the highest CI, which correlates best with the morphological abnormalities. Indium-111-leukocytes and 111In-liposomes are superior to 111In-IgG for this indication.