Fatality rate and predictors of mortality in an Italian cohort of hospitalized COVID-19 patients

Clinical features and natural history of coronavirus disease 2019 (COVID-19) differ widely among different countries and during different phases of the pandemia. Here, we aimed to evaluate the case fatality rate (CFR) and to identify predictors of mortality in a cohort of COVID-19 patients admitted to three hospitals of Northern Italy between March 1 and April 28, 2020. All these patients had a confirmed diagnosis of SARS-CoV-2 infection by molecular methods. During the study period 504/1697 patients died; thus, overall CFR was 29.7%. We looked for predictors of mortality in a subgroup of 486 patients (239 males, 59%; median age 71 years) for whom sufficient clinical data were available at data cut-off. Among the demographic and clinical variables considered, age, a diagnosis of cancer, obesity and current smoking independently predicted mortality. When laboratory data were added to the model in a further subgroup of patients, age, the diagnosis of cancer, and the baseline PaO2/FiO2 ratio were identified as independent predictors of mortality. In conclusion, the CFR of hospitalized patients in Northern Italy during the ascending phase of the COVID-19 pandemic approached 30%. The identification of mortality predictors might contribute to better stratification of individual patient risk

Diversity and ethics in trauma and acute care surgery teams: results from an international survey

Background Investigating the context of trauma and acute care surgery, the article aims at understanding the factors that can enhance some ethical aspects, namely the importance of patient consent, the perceptiveness of the ethical role of the trauma leader, and the perceived importance of ethics as an educational subject. Methods The article employs an international questionnaire promoted by the World Society of Emergency Surgery. Results Through the analysis of 402 fully filled questionnaires by surgeons from 72 different countries, the three main ethical topics are investigated through the lens of gender, membership of an academic or non-academic institution, an official trauma team, and a diverse group. In general terms, results highlight greater attention paid by surgeons belonging to academic institutions, official trauma teams, and diverse groups. Conclusions Our results underline that some organizational factors (e.g., the fact that the team belongs to a university context or is more diverse) might lead to the development of a higher sensibility on ethical matters. Embracing cultural diversity forces trauma teams to deal with different mindsets. Organizations should, therefore, consider those elements in defining their organizational procedures. Level of evidence Trauma and acute care teams work under tremendous pressure and complex circumstances, with their members needing to make ethical decisions quickly. The international survey allowed to shed light on how team assembly decisions might represent an opportunity to coordinate team member actions and increase performance

Peripheral-type benzodiazepines inhibit calcium channels and aldosterone production in adrenal glomerulosa cells

Angiotensin-II (Ang-II), K+, and ACTH are important stimulators of aldosterone secretion that require Ca2+ influx to be active. However, Ang-II and K+ are linked to the Ca2+ messenger system, while ACTH is coupled to the cAMP pathway. Peripheral-type binding sites for benzodiazepines are particularly abundant in steroidogenic tissues and have been proposed to be involved in the steroidogenic action of ACTH in Y-1 adrenocortical cells. We report here that in adrenal glomerulosa cells, peripheral-type [4'-chlor-diazepam (CDZ), 1-(2-chlorophenyl)N-methyl-N-(1-methylpropyl)3-isoquinolinecarboxamid e (RP 52028), and flunitrazepam], but not a central-type (flumazenil) benzodiazepine reversibly abolished the stimulation of aldosterone output induced by Ang-II or K+, while they had no significant effect on basal aldosterone secretion. This inhibitory effect depended upon drug concentration (IC50 30 microM for CDZ) and affected the potencies of both stimulators, without altering their respective EC50 values. Similar results were obtained when aldosterone production was stimulated with ACTH, forskolin, or (Bu)2cAMP. Aldosterone production from exogenous 25-hydroxycholesterol or progesterone was partially inhibited by CDZ. In glomerulosa cells loaded with a fluorescent Ca2+ probe, benzodiazepines blocked Ca2+ influx triggered by K+ or Ang-II without affecting the release of Ca2+ from intracellular stores induced by Ang-II. T- and L-type Ca2+ channel activities, monitored with the patch-clamp technique, were both inhibited within the same range of concentrations as aldosterone synthesis and Ca2+ influx. These results indicate that in adrenal zona glomerulosa cells, peripheral-type benzodiazepines block Ca2+ influx through voltage-activated channels. The combined action of peripheral-type benzodiazepines on calcium influx and precursor conversion may be responsible for the observed inhibition of Ang-II-, K(+)-, or ACTH-induced aldosterone secretion

Role of the capacitative calcium influx in the activation of steroidogenesis by angiotensin-II in adrenal glomerulosa cells

Angiotensin-II (AngII)-induced Ca2+ influx in adrenal glomerulosa cells, a signal necessary for the stimulation of steroidogenesis by the hormone, is believed to involve two distinct mechanisms: 1) opening of voltage-operated Ca2+ channels, and 2) activation of a capacitative Ca2+ entry pathway that is dependent on calcium release from intracellular stores. Nicardipine, a dihydropyridine calcium antagonist, has been used to investigate the role of these Ca2+ entry mechanisms in the steroidogenic response to AngII. As demonstrated with the patch-clamp technique, micromolar concentrations of nicardipine completely blocked voltage-operated Ca2+ channel activity of both T- and L-types. This agent similarly inhibited the rise of cytosolic free calcium concentration induced by potassium, but did not significantly affect the response to thapsigargin, an activator of the capacitative pathway. Nicardipine reduced by only 22% the calcium influx stimulated by AngII, and the nicardipine-insensitive part of this response was abolished after exhausting the intracellular Ca2+ stores with thapsigargin. Similarly, aldosterone secretion induced by AngII was only partially inhibited (40%) by nicardipine at concentrations that completely abolished the steroidogenic response to potassium. Thapsigargin by itself was able to stimulate aldosterone production, an action highly potentiated by physiological concentrations of extracellular potassium. These data strongly suggest that the major part of the calcium influx response to AngII, leading to aldosterone formation, involves a capacitative calcium entry pathway activated by the release of calcium from intracellular stores. This mechanism of calcium influx could be responsible for some features of aldosterone response to the hormone, such as its poor sensitivity to dihydropyridines or its potentiation by potassium

Angiotensin II negatively modulates L-type calcium channels through a pertussis toxin-sensitive G protein in adrenal glomerulosa cells

In bovine adrenal glomerulosa cells, angiotensin II and extracellular K+ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K+ is inhibited by angiotensin II. Because K+-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT1 receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K+ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K+ concentrations. Finally, the expression of both Go and Gi proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II

Blocking T-type calcium channels with tetrandrine inhibits steroidogenesis in bovine adrenal glomerulosa cells

Tetrandrine, an alkaloid extracted from a Chinese medicinal herb traditionally used in hypertension treatment, inhibited aldosterone production induced in bovine adrenal glomerulosa cells by either potassium ion, angiotensin II, or ACTH in a concentration-dependent manner (IC50 = 10 microM). The inhibition of the response to potassium by tetrandrine had a pattern very similar to that of nickel, a blocker of T-type calcium channels. In addition, tetrandrine prevented calcium influx induced by potassium or angiotensin II without affecting the calcium release phase stimulated by the hormone. The effect of tetrandrine on voltage-activated barium currents was investigated using the whole cell configuration of the patch clamp technique. T-type currents were isolated by recording the slowly deactivating currents elicited during repolarization of the cell to -65 mV after various depolarizing pulses. These currents were blocked by micromolar concentrations of the drug. The voltage sensitivity of channel activation was not affected by tetrandrine; nevertheless, the drug significantly slowed the deactivation of the current. The action of tetrandrine did not require the activation of the channel. Tetrandrine also affected L-type currents, as assessed after inactivating T channels for 100 msec, but at higher concentrations of the drug. Thus, tetrandrine affects with a similar potency aldosterone production, calcium influx, and T-type calcium channel activity. This finding strongly suggests a role for these channels in calcium signaling and control of steroidogenesis in adrenal glomerulosa cells

Control of cytosolic free calcium by intracellular organelles in bovine adrenal glomerulosa cells. Effects of sodium and inositol 1,4,5-trisphosphate

The regulation of cytosolic free Ca2+ concentration ([Ca2+]c) by intracellular organelles was studied in permeabilized bovine adrenal glomerulosa cells. Two compartments, with distinct characteristics, were able to pump Ca2+. A first pool, sensitive to ruthenium red and presumably mitochondrial, required respiratory chain substrates to maintain [Ca2+]c around 700 nM. Ca2+ efflux from this compartment was activated by Na+ (ED50 = 5 mM). Inositol 1,4,5-trisphosphate (IP3) had no effect on this pool. A second nonmitochondrial pool required ATP to lower [Ca2+]c to about 200 nM and released Ca2+ transiently upon addition of IP3. When the two systems were allowed to work simultaneously, the nonmitochondrial pool regulated [Ca2+]c and IP3 released Ca2+ in a concentration-dependent manner (EC50 = 0.6 microM). Under these conditions the mitochondria seemed Ca2+ depleted. Upon repeated stimulations with IP3, a marked attenuation of the response was observed. This phenomenon was due to Ca2+ sequestration by a nonmitochondrial IP3-insensitive pool. Neither dantrolene (200 microM) nor 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (10 microM) were able to abolish IP3-induced Ca2+ release, though both compounds efficiently inhibited aldosterone production in intact cells stimulated with angiotensin II (10 nM) or K+ (12 mM). These results suggest that in permeabilized adrenal glomerulosa cells: the nonmitochondrial pool is responsible for buffering [Ca2+]c and for releasing Ca2+ in response to IP3; at resting [Ca2+]c levels, the mitochondria appear Ca2+ depleted; when [Ca2+]c rises above their set point, the mitochondria accumulate Ca2+ as a function of [Na+]c; 4) the mitochondria are not involved in the desensitization mechanism of the response to IP3

1,25-dihydroxyvitamin D3 induces responsiveness to the chemotactic peptide f-Met-Leu-Phe in the human monocytic line U937: dissociation between calcium and oxidative metabolic responses

In the human premonocytic line U937, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) induces a functional NADPH oxidase, that is responsive to both phorbol esters and opsonized zymosan. The chemotactic peptide f-Met-Leu-Phe (fMLP) did not, however, induce superoxide generation by these cells. This was not due to the absence of receptors for fMLP. Although there was no significant binding of [3H]-fMLP to undifferentiated U937 cells, preincubation with 1,25-(OH)2D3 induced expression of specific and saturable binding sites. Moreover, fMLP induced a rapid and reversible rise in cytosolic free Ca2+ concentration ([Ca2+]i) in 1,25-(OH)2D3-treated U937 cells, but not in control or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3)-treated cells. This [Ca2+]i response was dependent on concentrations of both fMLP and 1,25-(OH)2D3 and was observed at physiologic concentrations of the hormone (approximately 25 pM). The rise in [Ca2+]i induced by fMLP in 1,25-(OH)2D3-treated U937 cells was blocked by pertussis toxin and presumably mediated by inositol (1,4,5)-trisphosphate generation. These results indicate that in U937 cells differentiated with 1,25-(OH)2D3, inositol phosphate-mediated [Ca2+]i responses to fMLP are uncoupled from NADPH oxidase activation

Angiotensin II promotes selective uptake of high density lipoprotein cholesterol esters in bovine adrenal glomerulosa and human adrenocortical carcinoma cells through induction of scavenger receptor class B type I.

Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. The hormone stimulates intracellular cholesterol mobilization to the mitochondrion for steroid biosynthesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is exerted on the selective transport of high density lipoprotein-derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine adrenal glomerulosa and human NCI H295R adrenocortical carcinoma cells, high density lipoprotein stimulated steroid production. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of reconstituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accumulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Fluorescence incorporation into cells was prevented by a monoclonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175 +/- 15% of controls after 2 h and to 260 +/- 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 +/- 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Similar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II regulates the scavenger receptor class B type I-mediated selective transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorticoid biosynthesis in both human and bovine adrenal cells

Insulin-secreting beta-cell dysfunction induced by human lipoproteins

Diabetes is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent low density lipoprotein (LDL) and high density lipoprotein (HDL) particles in insulin-secreting beta-cells. Purified human very low density lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels and beta-cell proliferation and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of beta-cells involved caspase-3 cleavage and reduction in the levels of the c-Jun N-terminal kinase-interacting protein-1. In contrast, the proapoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of c-Jun N-terminal kinase. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of Akt/protein kinase B. In conclusion, human lipoproteins are critical regulators of beta-cell survival and may therefore contribute to the beta-cell dysfunction observed during the development of type 2 diabetes