Secondary infections in mechanically ventilated patients with COVID-19: An overlooked matter?

OBJECTIVE: The susceptibility to infection probably increases in COVID-19 patients due to a combination of virusand drug-induced immunosuppression. The reported rate of secondary infections was quite low in previous studies. The objectives of our study were to investigate the rate of secondary infections, risk factors for secondary infections and risk factors for mortality in COVID-19 critically ill patients. METHODS: We performed a single-center retrospective study in mechanically ventilated critically ill COVID-19 patients admitted to our Critical Care Unit (CCU). We recorded the patients' demographic data; clinical data; microbiology data and incidence of secondary infection during CCU stay, including ventilator-associated pneumonia (VAP) and nosocomial bacteremia (primary and secondary). RESULTS: A total of 107 patients with a mean age 62.2 ± 10.6 years were included. Incidence of secondary infection during CCU stay was 43.0% (46 patients), including nosocomial bacteremia (34 patients) and VAP (35 patients). Age was related to development of secondary infection (65.2 ± 7.3 vs. 59.9 ± 12.2 years, p=0.007). Age ≥ 65 years and secondary infection were independent predictors of mortality (OR=2.692, 95% CI 1.068-6.782, p<0.036; and OR=3.658, 95% CI 1.385- 9.660, p=0.009, respectively). The hazard ratio for death within 90 days in the ≥ 65 years group and in patients infected by antimicrobial resistant pathogens was 1.901 (95% CI 1.198- 3.018; p= 0.005 by log-rank test) and 1.787 (95% CI 1.023-3.122; p= 0.036 by log-rank test), respectively. CONCLUSIONS: Our data suggest that the incidence of secondary infection and infection by antimicrobial resistant pathogens is very high in critically ill patients with COVID-19 with a significant impact on prognosis.

A novel interplay between membrane and nuclear melatonin receptors in human lymphocytes: significance in IL-2 production.

Human lymphocyte melatonin, through membrane and nuclear receptors binding, acts as an activator in IL-2 production. Antagonism of membrane melatonin receptors using luzindole exacerbates the drop of the IL-2 production induced by PGE(2) in peripheral blood mononuclear and Jurkat cells. This paper studies the melatonin membrane and nuclear receptors interplay in PGE(2)-diminished IL-2 production. The decrease in IL-2 production after PGE(2) and/or luzindole administration correlated with downregulation in the nuclear receptor RORalpha. We also highlighted a role of cAMP in the pathway, because forskolin mimicked the effects of luzindole and/or PGE(2) in the RORalpha expression. Finally, a significant RORalpha downregulation was observed in T cells permanently transfected with inducible MT(1) antisense. In conclusion, we show a novel connection between melatonin membrane receptor signalling and RORalpha expression, opening a new way to understand melatonin regulation in lymphocyte physiology.

Melatonin biosynthesis in the thymus of humans and rats.

Melatonin is an indoleamine widely distributed in the evolution that shows a great functional versatility, playing an important role as a transmitter of photoperiodic information and exhibiting antioxidant, oncostatic, anti-aging and immunomodulatory properties. In vertebrates, this molecule is produced by the pineal gland and other extrapineal sites. The present study was carried out to investigate the presence of melatonin in thymus and the possibility of an endogenous melatonin synthesis in this organ, in which T cells are matured. In this work, we demonstrate in humans and rats that thymus contains melatonin, expresses the mRNAs encoding N-acetyltransferase and hydroxyindol-O-methyltransferase, the two key enzymes of the melatonin synthesis, and has this biosynthetic machinery activated. In addition, rat thymocytes cultured for 24 h exhibited high levels of melatonin. The results presented here suggest that human and rat thymuses are able to synthesize melatonin, which could have intracrine, autocrine and paracrine functions.

Nuclear receptors are involved in the enhanced IL-6 production by melatonin in U937 cells.

This report shows that melatonin enhances IL-6 production by U937 cells via a nuclear receptor-mediated mechanism. Resting U937 cells only express membrane (mt1) melatonin receptors. In these cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus lipopolysaccharide, a treatment that downregulates the expression of mt1 receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the ROR alpha 1 and ROR alpha 2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor but not membrane receptors is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.

Melatonin is responsible for the nocturnal increase observed in serum and thymus of thymosin alpha1 and thymulin concentrations: observations in rats and humans.

This paper shows that melatonin regulates both thymosin alpha1 and thymulin production as well as the expression of the prothymosin alpha gene. The results revealed the following facts: (a) The concentrations of thymosin alpha1 in both serum and thymus of rat showed a nyctohemeral profile with peak values late at night and basal values during the day. The concentrations of thymulin in rat serum also showed a 24-h rhythm with an increase in their values at night. This rhythmical character for thymosin alpha1, and thymulin was also found in the human serum. (b) Rats injected with melatonin during the day exhibited a significant increase in the concentrations of both peptides. Moreover, continuous light exposure on the animals at daytime and pinealectomy cause a decrease in thymosin a1 and thymulin concentrations with regards to those found in control rats. (c) Melatonin regulates the expression of the prothymosin alpha gene, analyzed by Northern blot. These results suggest that melatonin may be involved in the regulation of immune functions by increasing the thymic peptides production.

Involvement of nuclear receptors in the enhanced IL-2 production by melatonin in Jurkat cells.

This report shows that melatonin enhances IL-2 production by Jurkat cells via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZR alpha, ROR alpha 1, and ROR alpha 2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mt1 receptor. In the absence of the membrane receptor, melatonin still activates the IL-2 production. These results show that the expression of the nuclear melatonin receptor is sufficient for melatonin to activate IL-2 production by Jurkat cells.

Circadian variations in the rat serum total antioxidant status: correlation with melatonin levels.

In this paper, we show for the first time, a nyctohemeral rhythm in serum total antioxidant status (TAS) in rats which parallels the 24-H melatonin cycle. Both TAS and melatonin in rat serum exhibited 24 hr variations with nocturnal peak values at 05.00 hr and low basal values during the day. When rats were maintained under light exposure (>500 lux) from 20.00 h to 05.00 hr, serum TAS was significantly reduced when compared with control rat killed in darkness. Moreover, when animals were maintained under continuous light exposure for 5 days and killed at 05.00 hr, serum TAS exhibited an additional decrease when compared with control rats. Since administering exogenous melatonin also increased TAS in the rat serum, results suggest that melatonin may be relevant in terms of participating in the antioxidative capacity of the rat serum.

Nocturnal increases in the triiodothyronine/thyroxine ratio in the rat thymus and pineal gland follow increases of type II 5'-deiodinase activity.

The type II 5'-deiodinase (5'D-II) is regulated by the light-dark cycle in some tissues in which the enzyme is present. This prompted us to investigate putative influences of light-dark cycle on thyroid hormone concentrations in these tissues. The results revealed the following facts: (a) Deiodinase activity in the rat thymus exhibits a nyctohemeral profile with peak values late at night and basal values during the day. The thyroid hormone concentrations in the thymus also show a 24 h rhythm with an increase in the triiodothyronine/thyroxine (TT3/TT4) ratio at night. (b) The content of thyroid hormones in the pineal gland exhibits, like in the thymus, nyctohemeral variations with increase values in the TT3/TT4 ratio during the dark period coinciding with the maximal enzyme activity. (c) Other tissue, like the anterior pituitary, in which 5'D-II, activity does not exhibit a diurnal variation, the concentration of thyroid hormones does not show modifications. In conclusion, the nocturnal increase of 5'D-II activity produces an increase of T3 concentration and a decrease of T4 concentration in both thymus and pineal gland. Therefore, these diurnal changes in 5'D-II activity is a mean by which the cell can regulate the intracellular availability of the most active thyroid hormone T3.

Continuous light exposure modifies the nocturnal increase in rat thymus type II thyroxine 5'-deiodinase.

In the present study we show that thymus type II thyroxine deiodinase activity exhibits a nyctohemeral profile, with basal values during the day and high values at night. This rhythmic character is dependent on neuroadrenergic input since exposure to continuous light at night completely abolished the nocturnal rise of the enzyme activity. However, treatment with isoproterenol under light exposure at night restored it.

Different experimental conditions which regulate type II 5'-deiodinase mRNA in rat Harderian gland.

In the present study, we describe the modifications in the expression of type II 5'deiodinase activity (5'D) in Xenopus laevis oocytes by injection of polyadenylated (poly A) mRNA from hypothyroid rat Harderian gland. The time-course study showed that the expression of the enzyme was dependent on time. Thus, enzyme activity was observed in oocytes 6 and 12 hours after the injection with poly A mRNA, reaching a maximal value at 24 hours. The activity was partially inhibited by 6-n-propyl-thiouracil, completely inhibited by iopanoic acid and exhibited a higher affinity for the T4 (Km=1.5 nM) than rT3 (Km=20 nM). The expression of the enzyme was modified in different experimental conditions: (a) exhibited diurnal variations with maximal peak values at night, (b) was inhibited by light at night and, (c) was activated by isoproterenol. On the other hand, we have also identified, for the first time, the size of mRNA capable of inducing 5'D in rats.

Characterization of binding sites for beta-adrenergic agonists and vasoactive intestinal peptide in the rat harderian gland.

Vasoactive intestinal peptide (VIP) receptors and beta-adrenergic receptors were investigated in rat Harderian gland membranes using 125I-VIP and 125I-cyanopindolol (125I-CYP), respectively, as ligands. The receptor bindings were rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The stoichiometric data suggested the presence of two classes of VIP receptors with Kd values of 0.36 and 65.37 nM and binding capacities of 323 and 39,537 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP as follows: VIP > helodermin > rGRF > PHI > > secretin. Glucagon, somatostatin, insulin, and pancreastatin were ineffective at concentrations up to 1 microM. However, the stoichiometric data suggest the presence of one class of binding sites for 125I-CYP. The Kd for the single site was 290 pM with a binding capacity of 32 pmol/L. The pharmacological characterization of 125I-CYP binding to membranes showed that only isoproterenol, a beta-adrenergic agonist, and norepinephrine, an alpha beta-adrenergic agonist, was as effective as propranolol in inhibiting 125I-CYP binding to Harderian gland membranes. However, alpha 1- and alpha 2-adrenergic agonists and blockers such as methoxamine, prazosin, clonidine, and yohimbine were shown to be ineffective. These results demonstrate the presence of specific VIP and beta-adrenergic receptors in the Harderian gland and suggest a role for VIP and beta-adrenergic agonists in the physiology of this gland.

Beta- and alpha-adrenergic mechanisms are involved in regulating type II thyroxine 5'-deiodinase in rat thymus.

The role of adrenergic receptors in regulation of rat thymus type II thyroxine 5'-deiodinase (5'-D) activity was investigated. Our results show that norepinephrine, an alpha- and beta-adrenergic agonist elicited an increase in thymus 5'-D activity. Isoproterenol, beta-adrenergic agonist, also increased the enzyme activity, being less effective than norepinephrine. Moreover, alpha-adrenergic agonists, i.e., methoxamine, an alpha1-agonist, and clonidine, an alpha2-agonist, did not increase 5'-D activity. The effect of isoproterenol was potentiated by methoxamine, but the potentiating effect was observed only at doses of isoproterenol which induce submaximal activation of the enzyme. Administration of propranolol, beta-adrenergic blocker, and prazosin, an alpha1-adrenergic blocker, inhibited significantly the activation of the enzyme induced by norepinephrine. However, yohimbine, an alpha2-adrenergic blocker, had small effect. These results show, in hypothyroid rats, a clear regulation by adrenergic mechanisms of 5'-D activity in the thymus, where alpha- and beta-adrenergic receptors might be involved.

Type II thyroxine 5'-deiodinase in the rat thymus.

In the present study we have shown type II thyroxine 5'-deiodination (5'D) in the rat thymus. The enzyme activity was identified in crude extract homogenates by measuring the 125I released from [3',5'-125I]thyroxine which is used as a substrate of the reaction. The release of 125I is dependent on protein tissue concentration, time, temperature and pH, and is saturable by increasing the substrate concentration, indicating its enzymatic nature. Characteristics of the enzyme activity also include a low Km (9.1 nM), its dependence on dithiothreitol, and its inhibition by iopanoic acid, but not by propylthiouracil. Experiments to investigate the cellular location of the enzyme in the thymic gland showed that the enzyme is present in both stromal cells and thymocytes. At the subcellular level, 5'D activity was associated with cellular membranes. Thyroid status appears to regulate 5'D activity in rat thymus. Hypothyroidism caused an increase in thymus 5'D activity. The Km value remained unchanged (9.1 vs 10.5 nM) during hypothyroidism, but Vmax increased significantly from 17.7 fmol/mg protein per h in euthyroid rats to 53.5 fmol/mg protein per h in hypothyroid rats. 5'D activity was also modulated by catecholamines through beta-adrenergic receptors because isoproterenol, but not methoxamine or clonidine, could activate the enzyme. Because these characteristics define the type II iodothyronine-deiodinating pathway in other tissues, we suggest that the rat thymus also shares this pathway.

Expression of type II thyroxine 5'-deiodinase from rat harderian gland in Xenopus laevis oocytes.

The presence of isoenzymes mediating the conversion of thyroxine to 3,5,3'-triiodothyronine has been studied according to characteristic kinetics and physiological regulation. In this paper, we report the expression of type II 5'-deiodinase (5'D) activity in oocytes of Xenopus laevis. Oocytes injected with total RNA extracted from rat Harderian gland, and then incubated up to five days demonstrated a progressive increase in 5'D activity, reaching a maximal value at 24 h; then, 5'D activity remained almost stable for an additional period of four days. Characteristics of the enzyme activity expressed by oocytes included its inhibition by iopanoic acid, but not by propylthiouracil, and its increase during beta-adrenergic agonist treatment and hypothyroidism. The expressed activity manifests characteristics typical of the type II isoenzyme. Deiodinating activity in oocytes also exhibited diurnal variations. In this study, 5'D activity expressed in oocytes exhibited low values when animals were killed during the day, and high values when animals were killed at night. Maximal values were reached 3-4 h before the nocturnal peak of 5'D activity in Harderian gland crude homogenates. Results suggest that the in vivo activation of 5'D by isoproterenol, hypothyroidism, or dark exposure may be caused by an increase in the synthesis and/or maturation of the RNA expressing the enzyme.

Thyroxine type II 5'-deiodinase activity in pineal and Harderian gland is enhanced by hypothyroidism but is independent of serum thyroxine concentrations during hyperthyroidism.

1. This paper studies the effect of thyroid status on 5'-D activity in pineal gland, Harderian gland, brown adipose tissue (BAT), pituitary gland, brain frontal cortex (BFC), and cerebellum. 2. Hypothyroidism clearly increased diurnal 5'-D activity in Harderian gland, BAT, pituitary gland, BFC, and cerebellum. In pineal gland, diurnal values of 5'-D activity were not affected by hypothyroidism. 3. Hypothyroidism in adult rats clearly enhanced nocturnal increase of 5'-D activity in pineal and Harderian gland. Congenital hypothyroidism also enhanced the nocturnal increase of 5'-D activity in pineal gland. 4. Hyperthyroidism inhibited 5'-D activity in pituitary gland, BFC, and cerebellum. A small inhibition, although significant, was found in BAT. 5. In pineal and Harderian gland, hyperthyroidism did not inhibit either the basal diurnal values of the enzyme or the nocturnal increase of its activity. 6. Results suggest that, in tissues where 5'D-activity is regulated by adrenergic mechanisms, mostly pineal gland and Harderian gland, the enzyme activity is independent of serum T4 concentrations during hyperthyroidism.

Vasoactive intestinal peptide stimulates type II thyroxine 5'-deiodinase and N-acetyltransferase activities in dispersed pineal cells of euthyroid and hypothyroid rats.

The effect of vasoactive intestinal peptide (VIP) on thyroxine type II 5'-deiodinase (5'-D) and N-acetyltransferase (NAT) activities were studied using pineal cells of euthyroid and hypothyroid rats. VIP activated 5'-D activity in a dose-dependent manner in both euthyroid and hypothyroid animals. However, basal and VIP stimulated activity was higher in pinealocytes from hypothyroid than in cells from euthyroid rats. VIP was also able to stimulate NAT activity but hypothyroidism did not induce modifications in its activity. Both 5'-D and NAT activities were stimulated not only by VIP, but also by isoproterenol, a beta-adrenergic receptor agonist, and forskolin, a potent activator of adenylate cyclase activity. The results suggest that VIP may be involved in the physiological regulation of pineal 5'-D activity.

Chronic ethanol intake inhibits both the vasoactive intestinal peptide binding and the associated cyclic AMP production in rat enterocytes.

1. Chronic ethanol intake during 6, 8, 10 or 12 weeks resulted in a decrease of 125I-vasoactive intestinal peptide (VIP) binding to rat enterocytes. 2. Native peptide displaced 125I-VIP binding to enterocytes, exhibiting a IC50 at about 4 nM native VIP in control and ethanol-treated animals. 3. The number of binding sites in ethanol-treated animals were significantly diminished when compared to control animals. This reduction is observed in both the high-affinity and the low-affinity binding sites. 4. Increasing concentrations of native VIP produced a similar cyclic AMP rise in enterocytes from control or ethanol-treated rats during 6 weeks. However, after 8 weeks of ethanol treatment, a significant decrease in cyclic AMP production stimulated by VIP was observed.

In vivo activation of pineal N-acetyltransferase but not type II thyroxine 5'-deiodinase by phenylephrine in young rats.

The regulation by alpha- and beta-adrenergic agonists of pineal N-acetyltransferase (NAT) and type II thyroxine 5'-deiodinase (5'-D) in rats at either 2 or 6 weeks of age was studied. The pattern of stimulation was different because NAT activity could be clearly activated by an alpha-adrenergic agonist, phenylephrine, only in 2-week-old rats. However, isoproterenol, a beta-adrenergic agonist, was able to stimulate NAT activity in rats at both 2 and 6 weeks of life. On the other hand, phenylephrine was always ineffective in stimulating 5'-D activity, while isoproterenol clearly activated it at both ages. These results strongly suggest a role for alpha-adrenergic receptors, in addition to beta-adrenergic receptors, in regulating rat pineal NAT activity during development.

Decreased binding of vasoactive intestinal peptide to intestinal epithelial cells from hypothyroid rats.

The binding of vasoactive intestinal peptide (VIP) and stimulation of adenylate cyclase by VIP were studied in intestinal epithelial cells during hypothyroidism. Experimental hypothyroidism was induced in rats by the administration of KC10(4). The binding capacity, but not the affinity, of VIP receptors decreased in the hypothyroid rats. Besides, the stimulation of cyclic AMP production by VIP was also diminished in cells from hypothyroid rats. These observations indicate a decrease of the responsiveness of intestinal epithelial cells to VIP in the hypothyroid status, suggesting a role of the peptide in the pathophysiologic mechanism of intestinal manifestations during hypothyroidism.

Evaluation of maternal serum alpha-foetoprotein assay using dry blood spot samples.

The quantification of alpha-foetoprotein in dry blood spots from pregnant women was evaluated, using a conventional radioimmunoassay (RIA) with a monospecific antibody. The stability of alpha-foetoprotein in dry blood spots on filter paper was evaluated with respect to mailing, distances travelled, and the existence of high summer temperatures in our region. The results obtained show that the blood alpha-foetoprotein is stable on dry filter spots sent by mail and is stable for up to four weeks at 4, 25 and 37 degrees C. The analytical method used has a minimal detectable concentration of 10 +/- 1.9 international kilo-units/l. Both inter- and intra-assay variabilities are smaller than 10% and this method can provide results comparable with those of conventional serum assays. Results from dry blood spots and serum samples (the latter analysed by both RIA and two-site enzyme immunoassay) exhibited a good correlation (r = 0.98 and r = 0.97, p less than 0.001). The design of the assay and the nature of the samples make this method suitable for a screening programmes for the antenatal detection of open neural tube defects.