Biomarkers of cell damage, neutrophil and macrophage activation associated with in-hospital mortality in geriatric COVID-19 patients.

BACKGROUND: The risk for symptomatic COVID-19 requiring hospitalization is higher in the older population. The course of the disease in hospitalised older patients may show significant variation, from mild to severe illness, ultimately leading to death in the most critical cases. The analysis of circulating biomolecules involved in mechanisms of inflammation, cell damage and innate immunity could lead to identify new biomarkers of COVID-19 severity, aimed to improve the clinical management of subjects at higher risk of severe outcomes. In a cohort of COVID-19 geriatric patients (n= 156) who required hospitalization we analysed, on-admission, a series of circulating biomarkers related to neutrophil activation (neutrophil elastase, LL-37), macrophage activation (sCD163) and cell damage (nuclear cfDNA, mithocondrial cfDNA and nuclear cfDNA integrity). The above reported biomarkers were tested for their association with in-hospital mortality and with clinical, inflammatory and routine hematological parameters. Aim of the study was to unravel prognostic parameters for risk stratification of COVID-19 patients. RESULTS: Lower n-cfDNA integrity, higher neutrophil elastase and higher sCD163 levels were significantly associated with an increased risk of in-hospital decease. Median (IQR) values observed in discharged vs. deceased patients were: 0.50 (0.30-0.72) vs. 0.33 (0.22-0.62) for n-cfDNA integrity; 94.0 (47.7-154.0) ng/ml vs. 115.7 (84.2-212.7) ng/ml for neutrophil elastase; 614.0 (370.0-821.0) ng/ml vs. 787.0 (560.0-1304.0) ng/ml for sCD163. The analysis of survival curves in patients stratified for tertiles of each biomarker showed that patients with n-cfDNA integrity < 0.32 or sCD163 in the range 492-811 ng/ml had higher risk of in-hospital decease than, respectively, patients with higher n-cfDNA integrity or lower sCD163. These associations were further confirmed in multivariate models adjusted for age, sex and outcome-related clinical variables. In these models also high levels of neutrophil elastase (>150 ng/ml) appeared to be independent predictor of in-hospital death. An additional analysis of neutrophil elastase in patients stratified for n-cfDNA integrity levels was conducted to better describe the association of the studied parameters with the outcome. CONCLUSIONS: On the whole, biomarkers of cell-free DNA integrity, neutrophil and macrophage activation might provide a valuable contribution to identify geriatric patients with high risk of COVID-19 in-hospital mortality.

Telomere/telomerase system impairment in circulating angiogenic cells of geriatric patients with heart failure.

BACKGROUND: The functional characteristics of circulating angiogenic cells (CACs) are impaired in congestive heart failure (CHF) patients, suggesting that CAC dysfunction could contribute to CHF pathogenesis. However, the underlying mechanisms are only partly unraveled. No data are currently available regarding telomere/telomerase system in CACs of CHF patients. METHODS: CACs were obtained from 80 subjects: 40 healthy control subjects (CTR) [median age (IQR), 80 (76-85 yrs)] and 40 patients affected by post-ischemic cardiomyopathy CHF [median age (IQR), 82 (77-89)]. CAC and leukocyte telomere length, assessed as T/S ratio, and telomerase (TERT) activity were determined in all the enrolled subjects. Specificity and sensitivity of CAC and leukocyte T/S in discriminating between CHF and CTR were evaluated using Receiver Operator Characteristic (ROC) curve analysis and reported as AUC values. CD34+/VEGFR2+ number and pro-inflammatory cytokines plasma levels, such as IL-6 and TNF-α, were also measured. RESULTS: CAC T/S and TERT activity were significantly reduced in CHF patients compared to CTR subjects. In leukocytes, only a significant T/S reduction was observed. AUC values were higher for CAC T/S with respect to leukocyte T/S (AUC=0.89, and AUC=0.73, P<0.01, respectively). In multivariate analysis, leukocyte T/S, CAC T/S, CAC TERT activity and NT-proBNP levels were confirmed as parameters significantly associated with CHF. CD34+/VEGFR2+ number, IL-6 and TNF-α plasma levels were significantly increased in CHF patients. CONCLUSIONS: CACs from CHF patients are characterized by telomere/telomerase system impairment, providing new insight into the clinical relevance of CACs in CHF pathogenesis.

Crystalline silica induces apoptosis in human endothelial cells in vitro.

We investigated whether incubation of cultured human aortic endothelial cells (HAEC) with crystalline silica at the concentration 1 cm2/ml (chosen on the basis of a pilot experiment) leads to alterations typical of apoptosis. The binding of annexin V as early, and DNA fragmentation as late events of apoptosis were measured besides the number of cells with depolarized mitochondria. The generation of reactive oxygen species (ROS) by HAEC in presence of silica was determined as well as silica ability to in vitro generate hydroxyl radicals was investigated. After 18 h of silica incubation, about 30% of viable cells bound annexin V. After 24 h of silica treatment, the percentage of cells with fragmented DNA (Tunel positive) was 27% and it increased up to 50% after 48 h, whereas in untreated cells this percentage was 7% and 11% after 24 and 48 h, respectively. The presence of fragmented DNA in cells treated with silica was confirmed by agarose gel electrophoresis. In agreement with these results showing an induction of HAEC apoptosis by silica incubation, the number of cells with depolarized mitochondria was significantly higher after silica treatment as compared to the control. Apoptosis was also obtained with silica added to aliquots of anti-C5a-absorbed-medium. In the cells exposed to silica there was a significant increasing of ROS generation in comparison to the untreated cells. Apoptosis might be due to peroxidative stress since silica can generate hydroxyl radicals.

Melatonin increases the intensity of respiratory burst and prevents L-selectin shedding in human neutrophils in vitro.

Effects of melatonin priming of neutrophils and subsequent increase of phorbol 12-miristate 13-acetate stimulated respiratory burst were investigated on the modulation of L-selectin shedding and MAC-1 upregulation. Respiratory burst related H2O2 production and adhesion molecule expression were quantified by flow cytometry. Phorbol 12-miristate 13-acetate dose dependence of intracellular oxidation and adhesion molecule expression showed no relationship between respiratory burst intensity and MAC-1 expression or L-selectin shedding. Treatment of cells with 12.5 nM phorbol 12-miristate 13-acetate resulted in less than 20% of the respiratory burst response, however it induced 91.7% of total MAC-1 expression and 62.8% of L-selectin shedding. Melatonin priming experiments showed also no connection between the extent of respiratory burst and MAC-1 expression, however melatonin priming almost completely prevented L-selectin down-regulation elicited by phorbol 12-miristate 13-acetate, without affecting MAC-1 expression. It is suggested that melatonin may inhibit metalloproteases responsible for L-selectin cleavage.

Melatonin regulates the respiratory burst of human neutrophils and their depolarization.

The effect of different doses of melatonin on the respiratory burst as well as on the membrane potential changes of human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) was investigated. The intracellular production of reactive oxygen species (ROS) in stimulated neutrophils was quantified in individual cells by flow cytometry, measuring the oxidation of nonfluorescent dihydrorhodamine 123 to the green fluorescent rhodamine 123. The transmembrane potential change was measured using the fluorescent probe oxonol. Preincubating the cells with micromolar concentrations of the indole resulted in an increase of the response to PMA. In two of six subjects investigated, the respiratory burst was also increased by a 10 nM concentration of the indole, but when the melatonin concentration was increased to 2 mM the respiratory burst was inhibited. The change in the transmembrane potential of neutrophils paralleled the respiratory burst. Indeed, the treatment of the cells with doses of melatonin up to 0.5 mM increased the depolarization occurring subsequent to PMA stimulation, whereas 2 mM melatonin concentration decreased the extent of depolarization. To investigate whether melatonin could directly affect the transmembrane potential changes of neutrophils, the extent of depolarization, induced by increasing the extracellular potassium concentration, was measured in cells preincubated with 2 mM melatonin. This treatment resulted in a decrease of the extent of depolarization, which suggests that melatonin can directly alter membrane ion conductance in human neutrophils.

Effect of acetylcholine on the electrophysiology and proliferative response of human lymphocytes.

Using the patch-clamp technique, we determined that 1-15 mM extracellular acetylcholine reduced whole-cell n-type K+ currents in human peripheral blood lymphocytes and accelerated their inactivation. The percentage increase in K+ channel inactivation rate and the degree of drug induced block were independent of membrane potential. In flow cytometric membrane potential measurements with the oxonol dye similar doses of acetylcholine depolarized the lymphocyte population. Both acetylcholine induced K+ channel block and depolarization fully developed within 2 minutes. The depolarizing and K+ channel blocking effects of acetylcholine are in concert. [3H]thymidine incorporation experiments proved that the proliferative response of PHA stimulated peripheral blood lymphocytes was decreased by increasing concentrations of acetylcholine in the 1-50 mM range.

The impairment of mitochondrial membrane potential and mass in proliferating lymphocytes from vitamin E deficient animals is recovered by glutathione.

The time-dependent changes of mitochondrial membrane potential and mass have been investigated on splenocytes from control and vit. E deficient rats, stimulated to proliferate with Concanavalin A, in the presence and absence of reduced glutathione (GSH, 5 mM). Rhodamine-123 (Rh-123) and nonyl acridine orange (NAO) were used as specific probes to monitor the membrane potential and mass of mitochondria, respectively, by means of flow cytometry. Rh-123 uptake was high in an increasing number of cells from normally fed animals during the three-day culture period. On the contrary, splenocytes from vitamin E deficient rats showed a biphasic pattern. The number of cells showing a high uptake of Rh-123 increased after 24 hrs. from mitogenic stimulation, then it decreased at the other two time points considered. In parallel, a continuous increase of the number of cells with depolarized organelles (up to 60% by 72 hrs.) has been observed in vit. E deficiency. This impairment was fully prevented by GSH supplementation to the culture medium. In the presence of the thiol, about 80-85% of cells showed activated mitochondria, whereas the number of splenocytes with depolarized organelles did not exceed 17%, irrespective of the diet applied to the animals. The same pattern was observed considering the changes of mitochondrial mass, measured using NAO as a probe. Present results support that GSH may substitute vitamin E in protecting mitochondria from peroxidative damage.

The modulation of intracellular glutathione level modulates the mitochondrial response in proliferating rat splenocytes.

We have investigated the effect of intracellular reduced glutathione (GSH) levels on the mitochondrial parameters of proliferating rat splenocytes. The intracellular GSH content of the cells was altered by continuous culture with buthionine-S, R-sulfoximine (BSO), a specific inhibitor of GSH synthesis. BSO decreased the GSH levels, inhibited DNA synthesis and caused depolarization of mitochondria in 52% of cells stimulated to proliferate for 72 h. These data support the proposition that GSH influences the lymphocyte proliferation at more than one site, one of which is the energy producing machinery.

Melatonin is an efficient antioxidant.

We have compared the peroxyl radical scavenger ability of melatonin with that of vitamin E, ascorbic acid (As.A.), reduced glutathione (GSH) and mannitol. All the antioxidants, except mannitol, prevented the lysis of human erythrocytes exposed to an azo-initiator of peroxyl radicals (2,2'-azo-bis(2-amidinopropane)dihydrochloride) at 37 degrees C. The percentage of this inhibition of erythrocyte lysis varied with the concentration of antioxidants, but the efficiency was melatonin > vitamin E > As.A. > GSH. Based on the assumption that each molecule of vitamin E scavenges two peroxyl radicals, the scavenging capacity of melatonin was four peroxyl radicals/molecule.

Effect of reduced glutathione on mitochondrial parameters of proliferating splenocytes from young and old rats.

The time-dependent changes of mitochondrial membrane potential and mass have been investigated on splenocytes from young, adult and old rats stimulated with Con A in the presence and absence of reduced glutathione (GSH). In addition, the basal level as well as the level of GSH during a 3-day culture period has been determined. No age-dependent changes of cellular GSH content were observed in freshly prepared splenocytes; however, in proliferating cells from old animals the expected increase in GSH levels was delayed. As regards the mitochondrial parameters, their membrane potential and mass were measured by means of the fluorescent probes rhodamine-123 (Rh-123) and nonyl acridine orange (NAO), respectively, and flow cytometry. During aging and with time of culture, an increased number of cells showed depolarization and loss of mitochondrial mass. This age-dependent impairment was completely prevented by addition of GSH to the culture medium, which resulted in a sharp increase in intracellular GSH. The present findings support the view that an impairment of the antioxidant defense system may be responsible for the damage observed in the mitochondria of proliferating splenocytes from old animals.

Food restriction in female Wistar rats. VII. Mitochondrial parameters in resting and proliferating splenic lymphocytes.

The effect of food restriction on the mitochondria of resting and proliferating rat splenocytes was examined, measuring the membrane potential and mass of these organelles, by means of the specific fluorescent probes Rhodamine-123 and Nonyl Acridine Orange, respectively. Food restriction was applied on an every-other-day schedule (EOD) starting at the age of 3.5 months. The ad libitum fed (AL) animals were killed when they were 4, 11 and 24 months old, whereas the EOD rats were killed at 11 and 26 months. Resting lymphocytes from AL rats showed an age-dependent increase of both membrane potential and mass of their mitochondria. However, the mitochondrial mass increased to a larger extent when compared with the membrane potential resulting in a decrease of the respiratory quotient (RQ), i.e. of the respiratory activity per unit of mitochondrial mass. In EOD animals, the mitochondrial membrane potential was lower and the mitochondrial mass was higher in the corresponding age-matched controls, resulting in a further decrease of RQ. Following mitogenic stimulation, most of the cells from young and adult AL rat showed an increase of membrane potential and mass of their mitochondria. In contrast about 50% of cells from old AL rats had depolarized organelles after 72 h from the stimulation. Food restriction was able to prevent these alterations allowing the majority of cells, including those from old animals, to maintain the hyperpolarization of their mitochondria during the 3-day culture. In light of the well known sensitivity of mitochondrial membrane potential to peroxidative stress, present data suggest that the increase of respiration occurring during mitogenesis may increase free radical production, which is better tolerated by cells from EOD animals than by those from AL animals.

Melatonin: a peroxyl radical scavenger more effective than vitamin E.

We have compared the peroxyl radical scavenger ability to melatonin with that of vitamin E, vitamin C and reduced glutathione (GSH). In the assay system, beta-phycoerythrin (beta-PE) was used as fluorescent indicator protein, 2-2'-azo-bis(2-amidinopropane)dihydrochloride as a peroxyl radical generator and the water soluble vitamin E analogue. Trolox, as reference standard. Results are expressed as oxygen radical absorbing capacity (ORAC(perox)) units, where 1 ORAC unit equals the net protection produced by 1 microM Trolox. A linear correlation of ORAC values with concentration (0.5-4 microM) of all the substances tested has been observed. However, on molar basis, the relative ORAC(perox) of Trolox, vitamin C, GSH and melatonin was 1:1.12:0:68:2.04, respectively. Thus, melatonin, which is a lipid-soluble compound, was twice more active than vitamin E, believed to be the most effective lipophilic antioxidant.

The response of human lymphocytes to phytohemagglutinin is impaired at different levels during aging.

Several parameters generally believed to be necessary for the activation and progression of proliferation of human lymphocytes have been investigated and compared with special reference to aging. The responding capacity of plasma membrane potential to depolarizing and also repolarizing conditions induced by exposure to mitogens like PHA was lower in lymphocytes from old donors as compared to those of young ones. This indicates a significant age-dependent difference in the readiness to respond to channel-activating perturbations. As an early signal of activation, after one hour PHA stimulation the merocyanine 540 uptake by the lipid regions was chosen, based on the property of this fluorescent probe to bind to loosely packed lipids of the plasma membrane. The proteins encoded by the c-myc and c-myb genes were chosen as markers of the G0/G1 and G1/S phased transition, respectively. The mean number of cells that increased the uptake of MC 540 following mitogenic stimulation did not differ in young vs. old individuals. However, 4 samples out of 10 from the old population showed lower MC 540 fluorescence than the lowest signal from the young population. The number of responding cells was decreased during aging when the presence of the c-myc protein was taken as its measure; and this decrease was further accentuated, determining the expression of the c-myb protein. This frequently encountered age-dependent pattern, however, was not followed by the lymphocytes of all old donors. One example is reported in which the MC 540 uptake, the c-myc and c-myb expression in the cells from one old subject fell in the range of the young subjects. However, even in this case, the response of the lymphocytes as measured by 3H-thymidine incorporation was only 64% of that of young subjects. For this sample, we found an impairment of the response at the mitochondrial level. In addition to these parameters, the amount of 3H-thymidine incorporated by the cells expressing the c-myb protein was calculated. The values in old individuals were lower than those in the young, suggesting that not all the cells expressing the c-myb protein were able to synthesize DNA in lymphocyte populations from the elderly. Our data support the view that the age-dependent decline of lymphocyte responsiveness to mitogens can be accounted for by impairments at different levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipid peroxidation causes an increase of lipid order and a decrease of 5'-nucleotidase activity in the liver plasma membrane.

The effect of peroxidation on 5'-nucleotidase activity as well as on membrane microviscosity has been investigated in liver plasma membranes from Wistar rats. The peroxidation was performed with 100 microM H2O2 and 200 microM FeSO4 and/or with 5 mM t-butylhydroperoxide. Treatment of the membranes with these oxidizing agents resulted in an elevation of the transition temperatures of the polarization of the lipid fluorescent probes 1,6 diphenyl-1,3,5 hexatriene (DPH), 3-p-(6-phenyl) 1,3,5 hexatriene phenylpropionic acid (PA-DPH) as well as of the fluorescent thiol reagent N-(1-pyrene) maleimide (1-PM). The peroxidation resulted in a decrease of the activity of 5'nucleotidase. Our data support that the increase of membrane microviscosity of the lipid domain regulates the activity of 5'-nucleotidase.

Phytohemagglutinin induced changes of membrane lipid packing, c-myc and c-myb encoded protein expression in human lymphocytes during aging.

Three parameters which signal different stages of cell activation were analyzed in lymphocytes from young and old subjects. Merocyanine 540 (MC-540) incorporation into the membrane lipid phase was used as a very early marker of activation and was measured after 1 h of phytohemagglutinin (PHA) stimulation. The proteins coded by c-myc and c-myb protooncogenes were determined by appropriate antibodies and were taken as markers of the G0/G1 and G1/S phase transition, respectively. The number of cells which increased the uptake of MC-540 following PHA stimulation did not differ when comparing young and old individuals. Both the number of the responding cells and the size of the response were decreased during aging when the presence of the c-myc protein was taken into account. A consistent decrease of the percentage of lymphocytes able to express the c-myb protein was observed in the cells from old donors as compared to those from the young ones, but the amount of detectable protein per cell remained unchanged. Our data suggest that the deficiency of responsiveness which accompanies aging is due to impairments at different points of the cell cycle. The very low number of cells expressing the c-myb protein is likely the result of step by step elimination of those cells not able to fulfill the requirements to progress along the cell cycle.

A sodium channel opener inhibits stimulation of human peripheral blood mononuclear cells.

The role of membrane potential changes in T cell activation was studied on human peripheral blood lymphocytes stimulated with phytohemagglutinin. Addition of bretylium tosylate, a sodium channels opener, to PHA treated lymphocytes modified the membrane potential and consequently blocked cell activation in a dose-dependent fashion. BT was non-toxic even in long-term (72 hr) incubations. It was reversibly removable, and the removal restored the stimulatory effect of PHA. 3H-thymidine incorporation was blocked if BT was present during the first 20-24 hr of the mitogenic activation. The later BT was added after PHA, the less inhibition of proliferation was observed. BT hyperpolarized the lymphocytes also in the presence of PHA. BT hindered the depolarizing effect of high extracellular potassium concns. The sustained polarized state of the lymphocytes did not influence the intracellular calcium increase upon PHA treatment. IL-2 and transferrin receptor expression was not hindered by BT during PHA stimulation of lymphocytes. Addition of rIL-2 did not abolish the inhibitory effect of BT. According to cell-cycle analysis BT arrested the majority of the cells in G1 phase. It is suggested that cell activation demands the flexible maintenance of a relatively narrow membrane potential "window". Any sustained and significant hyper-, or depolarization, may dramatically decrease the effectivity of transmembrane signalling.

Cholesterol-rich rabbit serum modulates beta-adrenergic receptor density of human lymphocytes. A possible role of LDL-cholesterol.

The effect of in vitro treatment of human lymphocytes with rabbit cholesterol-rich serum (RCS) on the membrane microviscosity as well as on the beta-adrenergic receptor density has been investigated. RCS treatment of cells resulted in a 30% decrease of receptor density without any effect on membrane microviscosity. A complete recovery was observed incubating the RCS cells either with the "Active Lipids" (AL) or with heparin. The AL are a mixture of neutral lipids, phosphatidylcholine and phosphatidylethanolamine from hen egg yolk known to fluidify the cell membrane. The AL modified membrane microviscosity of control lymphocytes without altering their beta-receptor number. These observations support the proposition that beta-receptor density of human lymphocytes is not regulated by membrane microviscosity and suggest that probably low density lipoprotein-cholesterol complex is involved in such a regulation.

Aging impairs membrane potential responsiveness as well as opening of voltage and ligand gated Na+ channels in human lymphocytes.

Depolarizing effects of increasing concentrations of extracellular K(+), as well as the repolarizing effect of bretylium tosylate (BT) were evaluated in human lymphocytes from young and elderly volunteers. Cells from elderly volunteers were less responsive to depolarization induced by increased extracellular potassium concentrations than those from young volunteers. Upon a near complete depolarization induced by 140 mM K(+) in the extracellular space, a significant amount of non-responding cells were found in samples from elderly volunteers. BT, which opens the otherwise silent Na(+) channels of partially depolarized cells, with subsequent activation of the Na(+)-K(+) pump (Pieri et al., 1989). repolarized both young and old lymphocytes. However, the degree of the repolarization was only 40% in the case of lymphocytes from elderly volunteers than from that of the young. It is suggested that an increase of membrane microviscosity, characteristic of old cells, may be at least partially responsible for the decreased responsiveness of plasma membrane functions which were observed.