Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction.

The effect of glutathione (GSH) depletion by L-buthionine-[S,R]-sulphoximine (BSO) on tumor necrosis factor-alpha (TNF-alpha)-induced adhesion molecule expression and mononuclear leukocyte adhesion to human umbilical vein endothelial cells (HUVECs) was investigated. Cells with marked depletion of cytoplasmic GSH, but with an intact pool of mitochondrial GSH, only slightly enhanced TNF-alpha-induced E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression, compared with the control. However, TNF-a-induced expression of both molecules was markedly enhanced when the mitochondrial GSH pool was diminished to <15% of the control. In contrast, TNF-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression was not affected by the depletion of either cytoplasmic or mitochondrial GSH. Marked enhancement of TNF-alpha-induced adhesion molecule expression by the depletion of mitochondrial GSH resulted in increased in mononuclear leukocyte adhesion to treated HUVECs, compared with the control. These effects parallel reactive oxygen species (ROS) formation by the depletion of mitochondrial but not cytoplasmic GSH. Our findings demonstrate that depletion of mitochondrial GSH renders more ROS generation in HUVECs, and mitochondrial GSH modulates TNF-alpha-induced adhesion molecule expression and mononuclear leukocyte adhesion in HUVECs.

Laser cytophotometric detection of variations in spatial distribution of concanavalin-A cell surface receptors following viral infection.

Human cells in culture (H.Ep. 2) were infected with herpes simplex virus type 2 at a multiplicity of infection (m.o.i.) of 3 for 3, 18 and 36 hr. Spatial redistribution of concanavalin (Con-A) cell surface receptor sites was detected by an indirect double-antibody (peroxidase-conjugated goat anti-rabbit to rabbit anti-Con-A) immunoenzymatic staining reaction which rendered the Con-A sites visible to light microscopy and to flow laser cytophotometry. Significant spatial redistribution of cell surface Con-A receptor sites occurred within the first 3 hr after infection with herpes simplex virus type 2. The infected cells must be in the presence of Con-A for the redistribution to occur, and significant redistribution occurs only at 37 degrees C. Fixation of infected cells prior to application of Con-A prevented this spatial redistribution of Con-A receptor sites.

Lasar flow cytophotometric immunoperoxidase detection of herpes simplex virus type 2 antigens in infected cultured human cells.

Human cells in culture (HEp-2) were infected with herpes simplex virus type 2 (HSV-2) at multiplicities of infection varying from 0.2 to 10, and fixed 6, 12, 18 and 24 hr after infection. Infection-related antigens were detected by an indirect double antibody (peroxidase conjugated goat anti-rabbit to rabbit anti-herpes simplex virus type 2) immunoenzymatic staining reaction that rendered infection-related antigens visible by light microscopy. A corresponding series of laser flow cytophotometric experiments yielded reproducible large-angle (1-19 degrees) laser-light scattering distributions that depended upon multiplicities of infection and the location of the infection-related antigens in the infected cells.

Laser flow cytometric light scatter and fluorescence pulse width and pulse rise-time sizing of mammalian cells.

In laser flow cytometry, an increasingly popular technique of analytical cytology, quantitative measurements of interest include cell and nuclear diameters. Electronic circuitry for a new cell sizing technique has been developed which measured the time that signal pulses from either fluorescence or light scatter sensors exceed a preset constant fraction of the peak signal amplitude (pulse width) or the time that it takes a signal to rise between constant fractions of the peak signal amplitude on the rising side of the pulse (pulse rise-time). These pulse width or pulse rise-time measurements were related to cell or nuclear diameters and were used in combination to determine nuclear size to cell size ratios. This method of sizing was found to be independent of fluorescent or light-absorbing stain intensity, linearly related to cell or nuclear diameter, and capable of resolving small diameter differences.

Induction of the fms proto-oncogene product in HL-60 cells by vitamin D: a flow cytometric analysis.

Agents which induce monocytic characteristics in HL-60 human acute promyelocytic leukemia cells induce mRNA for the fms proto-oncogene, which encodes the receptor for M-CSF. Previous studies of fms expression in HL-60 cells have characterized chiefly induction by phorbol esters of fms mRNA. Our studies of fms expression in HI-60 cells have characterized induction by vitamin D3 of the fms protein. We have used flow cytometry to correlate fms antigen with a monocyte-specific differentiation antigen recognized by antibody MO2 (CD14), with DNA content, and with the nuclear antigen Ki-67, a marker of cell cycling. HL-60 cells were cultured with or without 1 microM vitamin D for 7 days. fms antigen was found on 42 +/- 5.8% of the cells cultured without vitamin D, but on 63 +/- 4.3% of the cells cultured with vitamin D. MO2 binding was detected on only 2 +/- 0.5% of the cells without vitamin D, but on 59 +/- 9% with vitamin D. Cells cultured with vitamin D that were fms-positive were also predominantly (83%) MO2-positive. Analysis of DNA content, measured by propidium iodide staining, showed that 57 +/- 1.5% of cells cultured without vitamin D, but 93 +/- 0.5% of cells cultured with vitamin D, were in the G0/G1 cell cycle phase. Analysis of nuclear antigen Ki-67 revealed that, of the vitamin D-treated cells that were fms-positive, a significant proportion (37%) were still cycling. We conclude that (1) fms is demonstrable on some uninduced HL-60 cells, (2) when HL-60 cells are induced to develop monocytic characteristics by vitamin D, fms induction is part of the program for monocytic differentiation that includes MO2 expression, yet (3) some induced cells expressing fms are still cycling.

Multipotent human hematopoietic cell line K562: lineage-specific constitutive and inducible antigens.

K562 cells have been reported to display a variety of non-erythroid properties. Using 28 lineage-specific monoclonal antibodies, we analysed which antigens are present spontaneously and which are inducible by a variety of agents. The data suggest that (1) antigens of a given lineage are preferentially responsive to certain inducers, e.g. megakaryocytic antigens to phorbol ester, and (2) a given inducer may influence antigens of different lineages in opposite directions, e.g. phorbol dibutyrate, not only induces megakaryocytic antigens, but also decreases granulocyte and erythroid antigens. We conclude that the K562 cell, despite its malignant origin, retains some capacity for expression of alternative programs of differentiation, a characteristic of the normal multipotent hematopoietic stem cell.

Alveolar epithelial changes in rabbits after a 21-day exposure to 60% O2.

This study characterizes the biochemical and physiological effects of prolonged exposure of rabbits to sublethal (60%) O2 concentrations. After 3 wk in 60% O2, rabbits had arterial PO2 values of 69 +/- 2 vs. 79 +/- 3 Torr for control animals (means +/- SE; P less than 0.05) and a small but significant rise in pulmonary wet weight-to-dry weight ratios (5.6 +/- 0.3 vs. 4.1 +/- 0.3; P less than 0.05). Alveolar permeability to solute, lung compliance, total lung capacity, and alveolar protein levels were unchanged from control, but the amount of lavagable alveolar phospholipid was 90% higher in the O2-exposed rabbits. The lipid biosynthetic ability of isolated alveolar type II pneumocytes, measured by radiolabeled precursor [3H]choline incorporation, indicated that type II cells isolated from hyperoxic animals synthesized phosphatidylcholine at a rate 110% higher than those from control animals. Laser flow cytometric analyses of isolated type II cells showed a significant increase in type II cell diameter, based on time-of-flight measurements, and an average 60% increase in lipid content per cell, based on phosphine-3R fluorescence intensity. These findings indicate that exposure to 60% O2 for 21 days results in a decrease in arterial PO2 and induces several important biochemical and morphological changes in alveolar type II pneumocytes.

Obliteration of the gallbladder without formal cholecystectomy. A feasibility study.

Obliteration of the gallbladder without formal cholecystectomy was attempted in 36 rabbits, using an open technique for cannulation of the gallbladder during laparotomy. The gallbladder was cannulated, the proximal cystic duct was occluded with cyanoacrylate-nitrocellulose 2% wt/wt, and the gallbladder mucosa was exposed to various sclerosing agents to induce fibrosis. Heated 60% diatrizoate meglumine and absolute alcohol induced a controllable chronic cholecystitis with transmural fibrosis within two weeks, a result not seen with morrhuate sodium or in the control groups. All animals survived without complications, and no instances of common bile duct damage or occlusion was noted. The results reported are promising and indicate the need for further studies of this technique for its efficacy and safety.

Tetanus toxin binding to neuroblastoma cells differentiated by antimitotic agents.

We have examined the binding of tetanus toxin (TT) to surface receptors of neuroblastoma cells by flow cytometry following chemically induced differentiation. Cells were treated with mitomycin C, bromodeoxyuridine, prostaglandin E1 or cyclic adenosine monophosphate at different doses, alone or in combination for 4 days. Cells extended long neurites within 24 h in the presence of prostaglandin/cyclic AMP or mitomycin/bromodeoxyuridine treatment while single-drug treatment was less efficient in morphological differentiation of these cells. Cells exposed to the drug combinations stopped growing after 3 days while flow-cytometric analysis of DNA levels of each cell stained with propidium iodide indicated that at least 60% of these cells were arrested in phase G0/G1 of the cell cycle. Drug-treated cultures were stained for TT binding by immunofluorescence of cells in suspension and analyzed by flow cytometry. Chemically differentiated N2AB-1 cells were shown to bind significantly more TT than control cultures. Receptors for TT could be saturated by increasing doses of TT and differentiated cells bound twice as much toxin at saturation as did control cells. Immunofluorescence of TT binding to monolayers revealed staining in a stippled fashion along all neurites and cell bodies. These data support the concept that drugs which stimulate differentiation of neuroblastoma cells as determined by morphological and cell-cycle criteria also increase the presence of ganglioside receptors on the cell surface available for toxin binding.

Selective flow cytometric sorting of viable dopamine neurons.

Flow cytometry has been revealed as a powerful technique for studying cell populations. The availability of the method to identify, analyze and isolate specific populations of central nervous system cells will be of great aid for studying the in vivo (i.e. neural transplants) and in vitro (i.e. cell cultures) behavior of these cells. The present report describes the analysis and cell sorting of a population of retrogradely fluorescence-labeled dopamine-containing neurons from ventral mesencephalon. Dopamine neurons were identified by immunohistochemical localization of neuron-specific enolase and tyrosine hydroxylase. After being maintained in culture, this relatively pure population differentiates toward a mature phenotype bearing a prominent neuropil.

Recovery of central respiratory function following anticholinesterase intoxication.

Spontaneous recovery of central respiratory function was studied in anesthetized guinea pigs intoxicated with pinacolyl methylphosphonofluoridate (Soman) or isopropyl methylphosphonofluoridate (Sarin). I.v adiministration of either agent produced an immediate disruption phrenic nerve activiity and resulting ventilatory failure. Animals were maintained on artifical respiration until spontaneous functional recovery was complete, as evidenced by the re-establishment of synchronized burst activity on the phrenic nerve and return of tracheal airflow. This usually occurred within 1 h. Animals were sacrificed at predetermined intervals after intoxication, and the brainstem homogenates were analyzed for AChE activity. Results showed no significant return of AChE activity after 1 h, although functional recovery of respiration was complete within this time. Additional doses of the agents were administered at various times after recovery from the respiratory blockade. Following spontaneous restoration of ventilatory function, subsequent injections of the organophosphorus compounds failed to reinstate central respiratory paralysis, although they further depressed brainstem AChE levels. These data suggest that spontaneous recovery of central respiratory function after intoxication with Soman or Sarin may not be related to the return of AChE activity.

Flow cytometric detection of lymphocyte alterations in Huntington's disease.

Several recent reports indicate that patients with Huntington's Disease (HD) may manifest membrane abnormalities in a wide variety of cells including peripheral blood lymphocytes. In this study, flow cytometry is used in conjunction with the fluorescent membrane probe, 8-anilino-1-naphthalene sulfonate (ANS), to examine peripheral blood lymphocytes from 16 HD patients and 14 age- and diet-matched control subjects. Increased ANS fluorescence intensity of lymphocytes (p less than 0.02) was found in HD patients as compared to control subjects. These differences are masked when the mean fluorescence of the total leukocyte population is measured, possibly explaining conflicting data of other investigators. These observed differences in ANS fluorescence intensity between HD patients and control subjects support the concept of a gene defect which may be expressed as membrane alterations in non-neural as well as neural cells. The selective alterations of lymphocytes may also reflect altered immunological activity reported in HD.

Hyperplasia of pancreatic duct epithelium produced by exposure to sodium deoxycholate.

Severe epithelial hyperplasia was produced in a canine model by the perfusion of the main pancreatic duct with 15 mM of deoxycholate at rates as low as 1.5 ml/day in 6 to 14 days. At higher rates (5 ml/day) deoxycholate caused complete epithelial cell lysis in the duct closest to the tip of the cannula with hyperplastic changes downstream from this section. Perfusion with a buffer solution alone and cannulation alone produced none of these changes in similar duct segments. No hyperplasia was seen in the upstream cannula obstructed duct, even in the presence of severe atrophy. Long-term (81 days) perfusion with 3 mM of deoxycholate at 3 ml/day resulted in more severe hyperplasia that still appeared benign. When glycine-conjugated deoxycholate was perfused through the duct, hyperplasia but no cell lysis was seen. In vitro, deoxycholate caused epithelial cell lysis in pancreatic duct fragments at concentrations of 0.5 mM and above. The results of this study suggest that secondary bile salts or other similar surface-active cytotoxic agents present in the biliary tree or duodenum may play a more important role in the pathogenesis of pancreatic ductal epithelial hyperplasia associated with pancreatic cancer than ductal obstruction.

Induction of megakaryocytic characteristics in human leukemic cell line K562: polyploidy, inducers, and secretion of mitogenic activity.

Because of the rarity of megakaryocytes in the bone marrow, a cell line inducible for megakaryocytic characteristics provides a valuable model for study. When cultured with phorbol esters, the human multipotent hematopoietic leukemic cell line K562 can be induced to develop many megakaryocytic characteristics, viz. increased cell size, reduced growth rate, megakaryocytic antigens, and expression of the sis proto-oncogene, the structural gene for the B-chain of platelet-derived growth factor. Further aspects of this process are here presented. First, it induces the release of mitogenic activity into the medium. Second, phorbol dibutyrate induces polyploidy, a feature of normal megakaryocyte development. Third, mezerein and teleocidin, nonphorbol ester tumor promotors, also induce development of multinuclearity and polyploidy.

Comparison of time-tradeoff utilities and rating scale values of cancer patients and their relatives: evidence for a possible plateau relationship.

Because they are easy to administer, rating scales are often used as proxies for utility measures. The authors investigated the relationship between time-tradeoff utilities and rating scale values in two populations: 124 cancer patients asked to evaluate their current states of health and 102 relatives and close friends of cancer patients asked to evaluate health-state scenarios. None of the models tested effectively described the relationship between individual patients' rating scale values and time-tradeoff utilities for their current states of health. In contrast, both a plateau and a power-function model explained the variability in the responses of the relatives reasonably well (R2 = 0.56 and R2 = 0.58, respectively). Given that many respondents who were unwilling to trade off any time assigned rating scale values of well below 100, a plateau model may represent the best approach to adjusting rating scale values for health-state scenarios when it is not feasible to elicit time-tradeoff utilities.

Effects of insecticide, diazinon, on pancreas of dog, cat and guinea pig.

The organophosphate insecticide Diazinon has been reported to cause acute pancreatitis in dogs. Based on histochemical examination of the acinar tissue, it was suggested that pancreatic tissue-fixed butyrylcholinesterase (BuChE) is the target enzyme of organophosphate toxicity. To further evaluate this theory, we exposed dogs, cats, and guinea pigs to a single sublethal dose of the organophosphate insecticide Diazinon (75 mg/kg). In cats, which lack pancreatic BuChE, no pathological changes occurred after two, three, and six hours, whereas in the guinea pigs as in dogs, both having abundant pancreatic BuChE, vacuolization of the acinar cells, interstitial edema and vasculitis indicate acute edematous pancreatitis as early as two hours. Atropine pretreatment (0.2 mg/kg) gave complete protection against pancreatitis. It was concluded that inhibition of pancreatic BuChe leads to cholinergic hyperstimulation of the acinar cell, which results in acute pancreatitis, and that pancreatic BuChE is essential for dogs and guinea pigs to downregulate cholinergic excitation. The insecticide pancreatitis model is considered a simple, non-invasive, reproducible, and cheap and useful method to evaluate early changes and methods of treatment in acute pancreatitis. Pancreatitis in humans has also been reported after accidental insecticide exposure.

Comparison of quantitative acid-elution technique and flow cytometry for detecting fetomaternal hemorrhage.

A study of the Kleihauer-Betke acid-elution technique for quantitating fetomaternal hemorrhage was performed to assess intra- and inter-technologist accuracy and precision as well as to delineate the statistically valid domain of the test as usually performed. The results were then compared to a parallel study quantitating fetomaternal hemorrhage by flow cytometry. Additionally, a statistical model for estimating efficacy of treatment with Rh immune globulin in the prevention of pregnancy-associated Rh(D) isoimmunization was developed. The results indicate that the acid-elution technique can be performed in a reproducible manner with acceptable accuracy and precision with whole blood fetomaternal hemorrhages 25 ml and higher if a background correction for false positive identification of fetal cells is included. Flow cytometric determination reveals significantly increased accuracy in comparison to the corresponding Kleihauer-Betke results.

Spontaneous rupture of the oesophagus.

Two cases of spontaneous rupture of the oesophagus are described, one of which was a rare middle third tear.The possible aetiological factors are reviewed and the problems of diagnosis and treatment are discussed.