Induction of surface tumor necrosis factor (TNF) expression and possible facilitation of surface TNF release from human monocytic cells by granulocyte-macrophage colony-stimulating factor or gamma interferon in combination with 1,25-dihydroxyvitamin D3

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) can regulate monocyte maturation and activation. Using the human monocytoid cell line U937, we have shown that these agents increase surface tumor necrosis factor (TNF) expression without directly affecting TNF release. GM-CSF and IFN-gamma combined with 1,25(OH)2D3 increased cellular TNF secretion to levels not seen with these agents alone. Ability to express and secrete TNF in part depended on degree of monocytic maturation. The combination of 1,25(OH)2D3 and GM-CSF, however, facilitated lipopolysaccharide (LPS)-mediated release of surface TNF from U937 cells, an effect that was temporally independent of maximal maturation. 1,25(OH)2D3 plus IFN-gamma was less effective than 1,25(OH)2D3 plus GM-CSF at facilitating TNF secretion. We postulate that 1,25(OH)2D3 and GM-CSF are required together to prime a specific mechanism, probably a protease, which cleaves TNF from the surface of monocytic cells. This protease, once primed, can be activated by a secondary stimulus such as LPS.     

Stimulation of leptin release by actinomycin D in rat adipocytes

A greater understanding of the factors causing the enhanced release of leptin by adipocytes in obesity is needed. Experiments were designed to determine the effects of actinomycin D on leptin release by isolated rat adipocytes during primary culture for 24 hr. In adipocytes from fed hypothyroid rats, the initial rate of leptin release over the first 6 hr was not maintained over the next 18 hr. The decline in leptin release by adipocytes in primary culture between 6 and 24 hr was reduced markedly by either dexamethasone or actinomycin D. Both actinomycin D and dexamethasone also reduced the loss of leptin mRNA seen over the 24-hr incubation. Maximal effects on leptin release and leptin mRNA accumulation required only 0.1 microM of actinomycin D, a concentration that had no significant effect on the 18S RNA content of adipocytes at the end of a 24-hr incubation. In contrast to the reduced loss of leptin mRNA seen at 24 hr, the loss of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid (GAPDH mRNA) was enhanced in the presence of 0.1 microM of actinomycin D. The effects of dexamethasone could be differentiated from those of actinomycin D by the finding that cycloheximide blocked the reduced loss of leptin mRNA due to dexamethasone while having no effect on that due to actinomycin D. These results point to a unique regulation of leptin release and leptin mRNA levels by actinomycin D.     

Evaluation of Unsteady Flow Resistances by Quasi-2D or 1D Models

This note considers the evaluation of resistances in unsteady flow by means of a 1D unsteady friction model. The model is applied for the case of a water hammer, both in a single gravity pipeline and in a single pumped pipeline. Comparisons with experimental results show that the model does not predict the exact shape of the oscillation, but it gives, with high precision, the maximums and minimums of the pressure head oscillation if correct values of the parameter of the model are used. Because experimental evaluations can be made only for a few cases and can give rise to very different values depending on experimental conditions, the sensitivity of the model to physical and numerical parameters that influence the phenomenon is examined by comparison with the results of a quasi-2D model previously proposed.     

Measurement of histamine release from human lung tissue ex vivo by microdialysis technique

OBJECTIVE AND DESIGN: Currently no method is available for measurement of mediator release from intact human lung. In this study, a microdialysis technique was used to measure histamine release from mast cells in human lung tissue ex vivo. MATERIAL: Microdialysis fibers of 216 microm were inserted into lung tissue and perfused with Krebs Ringer buffer at a rate of 3 microl/min. After a 15 min period of steady-state perfusion, anti-IgE and vehicle were injected into the lung tissue above individual fibers. Samples from each fibre were collected for 20 min at 2 min intervals. Histamine was assayed fluorometrically. RESULTS: Anti-IgE concentrations of 40-40,000 U/ml dose-dependently released histamine, significant histamine release being demonstrated with anti-IgE concentrations of 400 U/ml and greater. The kinetics of histamine release showed peak values 2-8 min after the injection. Great individual responses were observed but data could be reproduced within individual donors. Monocyte chemoattractant protein-1, a potent basophil secretagogue, did not induce histamine release in lung tissue which indicated mast cells to be the histamine source. Substance P did not release histamine in the lung tissue. CONCLUSIONS: The microdialysis technique allowed measurements of histamine release from mast cells in intact lung ex vivo. The method may prove useful since a number of experiments can be performed in a few hours in intact lung tissue without any dispersion or enzymatic treatment.     

Ease of insertion of the laryngeal mask airway by inexperienced personnel when using an introducer

The Portex introducer for the laryngeal mask airway was designed as an aid to successful insertion, acting as an idealised 'artificial hard palate' to guide the tip of the laryngeal mask into the correct position. A number of authors have investigated laryngeal mask insertion by unskilled personnel in certain situations, one example being nurses during in-hospital cardiopulmonary resuscitation. We investigated whether the introducer had any effect on the incidence of first-time successful LMA placement by unskilled personnel. These were nonanaesthetist doctors, randomly assigned to have one attempt at LMA insertion in an anaesthetised patient, with and without the introducer. In 44 patients with the LMA being inserted according to the manufacturer's instructions, there was a 68% success rate (14 failures). In 45 patients with the LMA being inserted with the aid of a Portex introducer, there was a 96% success rate (two failures). This was a highly significant improvement (p < 0.001).     

Cell-permeable ceramides prevent the activation of phospholipase D by ADP-ribosylation factor and RhoA

The mechanism of inhibition of phospholipase D (PLD) by ceramides was determined using granulocytes differentiated from human promyelocytic leukemic (HL-60) cells. In a cell-free system, hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon phosphatidylinositol 4,5-bisphosphate, guanosine 5'-3-O-(thio)triphosphate) (GTPgammaS), and cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C2-(N-acetyl-), C8- (N-octanoyl-), and long-chain ceramides, but not dihydro-C2-ceramide, inhibited PLD activity. Apyrase or okadaic acid did not modify the inhibition of PLD by ceramides, indicating that the effect in the cell-free system was unlikely to be dependent upon a ceramide-stimulated kinase or phosphoprotein phosphatases. C2- and C8-ceramides prevented the GTPgammaS-induced translocation of ARF1 and RhoA from the cytosol to the membrane fraction. In whole cells, C2-ceramide, but not dihydro-C2-ceramide, inhibited the stimulation of PLD by N-formylmethionylleucylphenylalanine and decreased the amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-alpha and -beta1 that were associated with the membrane fraction, but did not alter the distribution of protein kinase C-epsilon and -zeta. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibition of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity.     

Granulocyte-macrophage colony stimulating factor suppresses LHRH release by inhibition of nitric oxide synthase and stimulation of gamma-aminobutyric acid release

Several cytokines produced by immune cells act within the hypothalamus and/or on the pituitary to produce the pattern of pituitary hormone secretion that characterizes infection. Granulocyte-macrophage colony stimulating factor (GMCSF) was first described as a hematopoietic cytokine; however, its synthesis is also stimulated during infection, and it has been found in glia in the brain. Previous research indicates that interleukin-1 inhibits release of luteinizing hormone-releasing hormone (LHRH) both in vivo and in vitro. In the present study, we determined that GMCSF inhibited the release of LHRH in vitro and evaluated the mechanisms involved. After a 1-hour preincubation in Krebs-Ringer bicarbonate glucose buffer (KRB), medial basal hypothalamic explants were incubated in KRB together with recombinant murine GMCSF for 0.5 h in a Dubnoff metabolic shaker (50 cycles/min) in an atmosphere of 95% O2/5% CO2. LHRH release into the media was determined by radioimmunoassay. At concentrations of 10(-12) and 10(-11) M, GMCSF significantly inhibited LHRH release. There was a U-shaped dose-response curve and LHRH release was not inhibited at lower or higher cytokine concentrations. The inhibition was specific since it was completely blocked by GMCSF antiserum. Since sodium nitroprusside (NP; 300 microM), a releaser of nitric oxide (NO), stimulates LHRH, presumably by acting within the LHRH neurons, we examined the effect of GMCSF (10(-11) M) on NP-induced LHRH release. It completely suppressed NP-induced release of LHRH. Bicuculline (10(-5) M), a gamma-aminobutyric acid (GABA) receptor antagonist, partially reversed the inhibitory effects of GMCSF on LHRH release. This dose completely reversed the suppression of LHRH release induced by GABA. The present results indicate that the inhibitory effects of GMCSF on LHRH release are partially caused by blockade of NO-induced LHRH release by its activation of GMCSF receptors on GABAergic neurons. The stimulated release of GABA acts on the GABA-a receptors on the LHRH terminals to inhibit their response to NO. At the end of the experiment, NO synthase (NOS) activity was measured in the tissue homogenate by the citrulline method. NOS activity was highly significantly reduced by GMCSF (10(-11) M) indicating that part of its suppressive action on LHRH release is mediated by reduction in NOS activity in the medial basal hypothalamus.     

Measurements of colour constancy by using a forced-choice matching technique

In order to compare the reactivity of glutamic acid decarboxylase (GAD) antibodies recognizing linear and conformational epitopes as islet cell cytoplasmic antibodies (ICA), monoclonal antibodies were generated. An ELISA displacement test using two biotinylated monoclonals recognizing a linear (M61/7E11) or a conformational GAD65 epitope (M65/6B12) was performed to identify epitope regions recognized by monoclonal GAD antibodies. The GAD binding by monoclonal GAD antibodies was tested by immunofluorescence on fixed and unfixed pancreatic sections of human, rat, and mouse, and by Dot-blot experiments. 16/23 (69.6%) of the monoclonals were specifically reactive with GAD65 and 7/23 (30.4%) were reactive with both GAD isoforms. 8/16 (50%) of monoclonal GAD65 antibodies recognized a linear GAD epitope located at the N-terminus (pattern 1). 5/16 (31.3%) displaced M65/6B12, indicating the recognition of a conformational GAD epitope (pattern 2). Monoclonals belonging to patterns 1 and 2 showed strong ICA binding. 3/16 (18.8%) of monoclonals specific for GAD65 with weak or no immunostaining of pancreatic islets (pattern 3) did not inhibit the binding of both biotinylated antibodies in the displacement test, indicating other epitope specificities. In conclusion, GAD antibodies recognizing both conformational and linear epitopes of the GAD65 molecule are involved in ICA binding with strong reactivity. Furthermore, results obtained with monoclonals of pattern 3 suggest the occurrence of GAD65 epitopes partly inaccessible on cryosections, which may result in an ICA-negative test of GAD65 autoantibody positive sera.     

Enhancement of neurotransmitter release induced by brain-derived neurotrophic factor in cultured hippocampal neurons

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, recent work has shown that BDNF also can induce rapid changes in synaptic efficacy. We have investigated the mechanism(s) of these synaptic effects on cultured embryonic hippocampal neurons. In the presence of the GABAA receptor antagonist, picrotoxin, the application of BDNF (100 ng/ml) for 1-5 min increased the amplitude of evoked synaptic currents by 48 +/- 9% in 10 of 15 pairs of neurons and increased the frequency of EPSC bursts to 205 +/- 20% of the control levels. There was no detectable effect of BDNF on various measures of electrical excitability, including the resting membrane potential, input resistance, action potential threshold, and action potential amplitude. In addition, BDNF did not change the postsynaptic currents induced by the exogenous application of glutamate. BDNF did increase the frequency of miniature EPSCs (mEPSCs) (268.0 +/- 46.8% of control frequency), however, without affecting the mEPSC amplitude. The effect of BDNF on mEPSC frequency was blocked by the tyrosine kinase inhibitor K252a and also by the removal of extracellular calcium ([Ca2+]o). Fura-2 recordings showed that BDNF elicited an increase in intracellular calcium concentration ([Ca2+]c). This effect was dependent on [Ca2+]o; it was blocked by K252a and by thapsigargin, but not by caffeine. The results demonstrate that BDNF enhances glutamatergic synaptic transmission at a presynaptic locus and that this effect is accompanied by a rise in [Ca2+]c that requires the release of Ca2+ from IP3-gated stores.     

Fiber fracture during processing of continuous fiber,metal-matrix composites using the foil/fiber/foil technique

The fracture of continuous fibers during processing of foil/fiber/foil (F/F/F) metal-matrix composites (MMCs) has been investigated both experimentally and theoretically. Experimental observations revealed that fiber fracture occurs during the heat-up portion of the consolidation cycle primarily in a bending mode in regions where cross-weave wires are present. Based on these observations, a general model that describes fiber fracture as a function of the processing stress and fiber mat geometry was developed. Model results showed that fiber stresses and, hence, the propensity for fracture are very sensitive to the distance between cross-weave wires in adjacent fiber mats; analytical expressions that allow the definition of a critical distance between such cross-weave wires were derived. The model relations demonstrated that fiber fracture is more likely in areas of a composite in which the fibers are arranged in a rectangular, rather than a triangular, pattern. The experimental and theoretical results were used to develop guide-lines for the design of F/F/F layups to avoid fiber fracture during processing. Formerly Graduate Student, Department of Materials Science and Engineering, Carnegie Mellon University     

Leak measurements in spontaneously breathing premature newborns by using the flow-through technique

A new method for measuring and correcting air leaks during lung-function testing in infants has been validated in vitro and in vivo by using a flow-through system that measured the inflow and outflow of a face mask. An adjustable leak was quantified by using suction flow to validate the accuracy of leak measurements. To validate the leak correction, the volume of a pump was measured with different air leaks (0-30%). The method developed was tested in 67 infants breathing spontaneously. There was good agreement between measured and simulated leaks (r = 0.998, P < 0.001; 95% limits of agreement were -0.3 and 0.1%, respectively). The volume was generally underestimated because of leaks, and the volume error was up to 94% compared with the maximum error of 5% after leak correction. With continuous leak measurements in vivo, there were <4% actual leaks (median 2.6%), and we did not observe any leaks in >7% of cases. The leak correction improved the accuracy of ventilatory measurements. The monitoring of leaks is helpful for airtight placement of the face mask and for prevention of serious measurement errors caused by leaks.