Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca 2+ , ATP, and proteins and allows micromolar Ca 2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 Μ M ) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [ 3 H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [ 3 H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [ 3 H]norepinephrine uptake was inhibited by 1 Μ M reserpine, 30 m M NH 4 + , or 1 Μ M carbonyl cyanide p -trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [ 3 H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H + electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules within digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 Μ M or greater. Catecholamine released into the medium by micromolar Ca 2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-Β-hydroxylase. The studies demonstrate that 20 Μ M of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca 2+ , the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65144/1/j.1471-4159.1985.tb07226.x.pd

Effects of Osmolality and Ionic Strength on Secretion from Adrenal Chromaffin Cells Permeabilized with Digitonin

Hyperosmotic solutions inhibit exocytosis of catecholamine from adrenal chromaffin cells at a step after Ca 2+ entry into the cells. The possibility that the inhibition resulted from an inability of shrunken secretory granules to undergo exocytosis was investigated in cells with plasma membranes permeabilized by digitonin. The osmoticants and salts used in this study rapidly equilibrated across the plasma membrane and bathed the intracellular organelles. When sucrose was the osmoticant, secretion was not significantly inhibited unless the osmolality was raised above 1,000 mOs. When the osmolality was raised with the tetrasaccharide stachyose or a low-molecular-weight maltodextrin fraction (average size a tetrasaccharide), one-half maximal inhibition occurred at 900–1,000 mOs. Prior treatment of permeabilized cells with Ca 2+ in hyperosmotic solution did not result in enhanced secretion when cells were restored to normal osmolality. Increased concentrations of potassium glutamate or sodium isethionate were more potent than carbohydrate in inhibiting secretion. Half-maximal inhibition occurred at 600–700 mOs or when the ionic strength was approximately doubled. The inhibition by elevated potasium glutamate also occurred when the osmolality was kept constant with sucrose. Increasing the ionic strength did not alter the Ca 2+ sensitivity of the secretory response. Reducing the ionic strength by substituting sucrose for salt reduced the Ca 2+ concentration required for half-maximal stimulated secretion from approximately 1.2 Μ M . Chromaffin granules, the secretory granules, are known to shrink in hyperosmotic solution. The experiments indicate that shrunken chromaffin granules can undergo exocytosis and suggest that in intact cells elevated ionic strength rather than chromaffin granule shrinkage contributes to the inhibition of secretion by hyperosmotic solutions. The experiments place limits on the possible osmotic mechanisms that could be involved in exocytosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66041/1/j.1471-4159.1986.tb08502.x.pd

Relationship Between Ca 2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Carbachol or elevated K + stimulated 45 Ca 2+ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca 2+ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca 2+ uptake occurred within 15 s of stimulation by carbachol or elevated K + at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K + was similar to that of Ca 2+ uptake. There was a close correlation between Ca 2+ uptake and catecholamine secretion at various concentrations of Ca 2+ . The concentration dependencies for inhibition of both processes by Mg 2+ or Cd 2+ were similar. Ca 2+ uptake saturated with increasing Ca 2+ concentrations, with an apparent K m for both carbachol-induced and elevated K + -induced Ca 2+ uptake of approximately 2 m M . The Ca 2+ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca 2+ entry and a presumed increase in cytosolic Ca 2+ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca 2+ - dependent processes associated with catecholamine secretion. Ca 2+ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca 2+ channels.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66166/1/j.1471-4159.1982.tb07940.x.pd

Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Improved Outcomes Associated with the use of Shock Protocols: Updates from the National Cardiogenic Shock Initiative

BACKGROUND: The National Cardiogenic Shock Initiative is a single-arm, prospective, multicenter study to assess outcomes associated with early mechanical circulatory support (MCS) in patients presenting with acute myocardial infarction and cardiogenic shock (AMICS) treated with percutaneous coronary intervention (PCI). METHODS: Between July 2016 and February 2019, 35 sites participated and enrolled into the study. All centers agreed to treat patients with AMICS using a standard protocol emphasizing invasive hemodynamic monitoring and rapid initiation of MCS. Inclusion and exclusion criteria mimicked those of the SHOCK trial with an additional exclusion criteria of intra-aortic balloon pump counter-pulsation prior to MCS. RESULTS: A total of 171 consecutive patients were enrolled. Patients had an average age of 63 years, 77% were male, and 68% were admitted with AMICS. About 83% of patients were on vasopressors or inotropes, 20% had a witnessed out of hospital cardiac arrest, 29% had in-hospital cardiac arrest, and 10% were under active cardiopulmonary resuscitation during MCS implantation. In accordance with the protocol, 74% of patients had MCS implanted prior to PCI. Right heart catheterization was performed in 92%. About 78% of patients presented with ST-elevation myocardial infarction with average door to support times of 85 ± 63 min and door to balloon times of 87 ± 58 min. Survival to discharge was 72%. Creatinine ≥2, lactate \u3e4, cardiac power output (CPO) CONCLUSION: In contemporary practice, use of a shock protocol emphasizing best practices is associated with improved outcomes

Improved Outcomes Associated with the use of Shock Protocols: Updates from the National Cardiogenic Shock Initiative.

BACKGROUND: The National Cardiogenic Shock Initiative is a single-arm, prospective, multicenter study to assess outcomes associated with early mechanical circulatory support (MCS) in patients presenting with acute myocardial infarction and cardiogenic shock (AMICS) treated with percutaneous coronary intervention (PCI). METHODS: Between July 2016 and February 2019, 35 sites participated and enrolled into the study. All centers agreed to treat patients with AMICS using a standard protocol emphasizing invasive hemodynamic monitoring and rapid initiation of MCS. Inclusion and exclusion criteria mimicked those of the SHOCK trial with an additional exclusion criteria of intra-aortic balloon pump counter-pulsation prior to MCS. RESULTS: A total of 171 consecutive patients were enrolled. Patients had an average age of 63 years, 77% were male, and 68% were admitted with AMICS. About 83% of patients were on vasopressors or inotropes, 20% had a witnessed out of hospital cardiac arrest, 29% had in-hospital cardiac arrest, and 10% were under active cardiopulmonary resuscitation during MCS implantation. In accordance with the protocol, 74% of patients had MCS implanted prior to PCI. Right heart catheterization was performed in 92%. About 78% of patients presented with ST-elevation myocardial infarction with average door to support times of 85 ± 63 min and door to balloon times of 87 ± 58 min. Survival to discharge was 72%. Creatinine ≥2, lactate \u3e4, cardiac power output (CPO) CONCLUSION: In contemporary practice, use of a shock protocol emphasizing best practices is associated with improved outcomes