Risk factors for invasive aspergillosis in ICU patients with COVID-19: current insights and new key elements.

Invasive pulmonary aspergillosis (IPA) has always been a challenging diagnosis and risk factors an important guide to investigate specific population, especially in Intensive Care Unit. Traditionally recognized risk factors for IPA have been haematological diseases or condition associated with severe immunosuppression, lately completed by chronic conditions (such as obstructive pulmonary disease, liver cirrhosis, chronic kidney disease and diabetes), influenza infection and Intensive Care Unit (ICU) admission. Recently, a new association with SARS-CoV2 infection, named COVID-19-associated pulmonary aspergillosis (CAPA), has been reported worldwide, even if its basic epidemiological characteristics have not been completely established yet. In this narrative review, we aimed to explore the potential risk factors for the development of CAPA and to evaluate whether previous host factors or therapeutic approaches used in the treatment of COVID-19 critically ill patients (such as mechanical ventilation, intensive care management, corticosteroids, broad-spectrum antibiotics, immunomodulatory agents) may impact this new diagnostic category. Reviewing all English-language articles published from December 2019 to December 2020, we identified 21 papers describing risk factors, concerning host comorbidities, ICU management, and COVID-19 therapies. Although limited by the quality of the available literature, data seem to confirm the role of previous host risk factors, especially respiratory diseases. However, the attention is shifting from patients' related risk factors to factors characterizing the hospital and intensive care course, deeply influenced by specific features of COVID treatment itself. Prolonged invasive or non-invasive respiratory support, as well as the impact of corticosteroids and/or immunobiological therapies seem to play a pivotal role. ICU setting related factors, such as environmental factors, isolation conditions, ventilation systems, building renovation works, and temporal spread with respect to pandemic waves, need to be considered. Large, prospective studies based on new risk factors specific for CAPA are warranted to guide surveillance and decision of when and how to treat this particular population.

Carbapenem-resistant Klebsiella pneumoniae in ICU-admitted COVID-19 patients: Keep an eye on the ball.

Here we report on seven intensive care unit (ICU) patients with coronavirus disease 2019 (COVID-19)-related acute respiratory distress syndrome (ARDS) who developed positive rectal swabs and invasive infections due to carbapenemase-producing Klebsiella pneumoniae (CP-Kp). Notwithstanding the infection prevention measures introduced during the COVID-19 pandemic and changes in the hospitalised population, attention to CP-Kp infections must remain high, especially in the critically ill setting.

Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation.

BACKGROUND: Thrombopoietin (TPO) is a humoral growth factor that does not induce platelet aggregation per se, but enhances platelet activation in response to several agonists. Circulating levels of TPO are increased in patients with sepsis and are mainly related to sepsis severity. OBJECTIVES: To investigate the potential contribution of elevated TPO levels in platelet activation during burn injury complicated or not by sepsis. METHODS: We studied 22 burned patients, 10 without and 12 with sepsis, and 10 healthy subjects. We measured plasma levels of TPO, as well as leukocyte-platelet binding and P-selectin expression. The priming activity of plasma from burned patients or healthy subjects on platelet aggregation and leukocyte-platelet binding, and the role of TPO in these effects were also studied in vitro. RESULTS: Burned patients without and with sepsis showed higher circulating TPO levels and increased monocyte-platelet binding compared with healthy subjects. Moreover, TPO levels, monocyte-platelet binding and P-selectin expression were significantly higher in burned patients with sepsis than in burned patients without sepsis. In vitro, plasma from burned patients without and with sepsis, but not from healthy subjects, primed platelet aggregation, monocyte-platelet binding and platelet P-selectin expression. The effect of plasma from burned patients with sepsis was significantly higher than that of plasma from burned patients without sepsis. An inhibitor of TPO prevented the priming effect of plasma from burned patients. CONCLUSIONS: Increased TPO levels may enhance platelet activation during burn injury and sepsis, potentially participating in the pathogenesis of multi-organ failure in these diseases.

Clinical usefulness of ELISPOT assay on pericardial fluid in a case of suspected tuberculous pericarditis.

Rapid and accurate diagnosis of tuberculous pericarditis is often difficult, considering the low specificity of both clinical picture and laboratory tests on pericardial fluid, as well as the low sensitivity of microbiological tests. This report documents the feasibility and clinical usefulness of an Interferon (IFN) - gamma ELISpot - TB assay on pericardial fluid cells in a case of suspected tuberculous pericarditis presenting with tamponade. As large pericardial effusions requiring pericardiocentesis are relatively frequent in tuberculous pericarditis, the physician may consider this particular application of ELISpot-TB as a rapid decision aid for starting the treatment.

Phosphoinositide 3-kinase gamma: kinase-dependent and -independent activities in cardiovascular function and disease.

Cardiac function is controlled by GPCRs (G-protein-coupled receptors) which exert their function by triggering numerous signalling pathways, including the activation of PI3K (phosphoinositide 3-kinase). The GPCR-activated PI3Kgamma is weakly expressed in the heart, but the deletion of its expression in mice causes remarkable phenotypes. Indeed, the lack of PI3Kgamma does not modify heart rate and blood pressure, but does increase contractility, particularly in response to stimuli that enhance cardiac contractile force, such as catecholamines. Consistently, treatment of mutant cardiomyocytes with beta-adrenergic agonists causes an abnormal increase in the elevation of cAMP production. On the other hand, PI3Kgamma appears to play a role in mediating the contractile depression exerted by other GPCR agonists, such as PAF (platelet-activating factor), that are released in pathological conditions, such as after an ischaemic insult. The receptor for PAF coupled to G(i) activates PI3Kgamma, which, in turn, is essential to promote Akt phosphorylation, NOSIII (nitric oxide synthase isoform III) activation and the production of nitric oxide, a well characterized cardiodepressing agent. As a whole, PI3Kgamma appears to negatively control cardiac contractility through different signalling mechanisms, thus becoming a possible drug target for the treatment of critical human cardiac pathologies, such as infarction or heart failure.

Resistance to thromboembolism in PI3Kgamma-deficient mice.

Platelet aggregation and subsequent thrombosis are the major cause of ischemic diseases such as heart attack and stroke. ADP, acting via G protein-coupled receptors (GPCRs), is an important signal in thrombus formation and involves activation of phosphoinositide 3-kinases (PI3K). When platelets from mice lacking the G protein-activated PI3Kgamma isoform were stimulated with ADP, aggregation was impaired. Collagen or thrombin, however, evoked a normal response. ADP stimulation of PI3Kgamma-deficient platelets resulted in decreased PKB/Akt phosphorylation and alpha(IIb)beta(3) fibrinogen receptor activation. These effects did not influence bleeding time but protected PI3Kgamma-null mice from death caused by ADP-induced platelet-dependent thromboembolic vascular occlusion. This result demonstrates an unsuspected, well-defined role for PI3Kgamma downstream of ADP and suggests that pharmacological targeting of PI3Kgamma has a potential use as antithrombotic therapy.

Detection of platelet-activating factor in gingival tissue surrounding failed dental implants.

BACKGROUND: Dental implant therapy has entered routine clinical practice. However, the failure rate of implants at 5 years, due to biological factors, is still around 7%. The pathogenesis of implant loss involves a complex network of cells and inflammatory mediators. This study evaluated platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, in soft tissue surrounding failed dental implants versus healthy implants. METHODS: PAF was estimated on extracted lipids by bioassay on washed rabbit platelets; inflammatory cell populations were assessed semiquantitatively after staining, and microvessel density was evaluated after immunohistochemical staining. RESULTS: Biologically active PAF was detected in the lipid extracts of samples excised from gingival tissue of patients with failed implants, but not in samples from patients with osseointegrated implants or from healthy edentulous subjects. The amount of PAF detected in failed implants was significantly higher than in healthy implants, suggesting a local production of this mediator. CONCLUSIONS: The presence of PAF was associated with histopathological findings of local inflammation and increased blood vessel density.

PAF produced by human breast cancer cells promotes migration and proliferation of tumor cells and neo-angiogenesis.

Platelet-activating factor (PAF), a phospholipid mediator of inflammation, is present in breast cancer tissue and correlates with microvessel density. In the present study, we investigated the biological significance of PAF synthesized within breast cancer. In vitro, we observed the production of PAF by two estrogen-dependent (MCF7 and T-47D) and an estrogen-independent (MDA-MB231) breast cancer cell lines after stimulation with vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor, thrombin but not with estrogen, progesterone, and oxytocin. The sensitivity to agonist stimulation and the amount of PAF synthesized as cell-associated or released varied in different cell lines, being higher in MDA-MB231 cells, which are known to be highly invasive. We further demonstrate, by reverse transcriptase-polymerase chain reaction and cytofluorimetry, that all of the breast cancer cells express the PAF receptor and respond to PAF stimulation in terms of proliferation. Moreover, in MDA-MB231 cells PAF elicited cell motility. In vivo, two structurally different PAF receptor antagonists WEB 2170 and CV 3988 significantly reduced the formation of new vessels in a tumor induced by subcutaneous implantation of MDA-MB231 cells into SCID mice. In conclusion, these results suggest that PAF, produced and released by breast cancer cells, can contribute to tumor development by enhancing cell motility and proliferation and by stimulating the angiogenic response.

Role of platelet-activating factor in cardiovascular pathophysiology.

Platelet-activating factor (PAF) is a phospholipid mediator that belongs to a family of biologically active, structurally related alkyl phosphoglycerides. PAF acts via a specific receptor that is coupled with a G protein, which activates a phosphatidylinositol-specific phospholipase C. In this review we focus on the aspects that are more relevant for the cell biology of the cardiovascular system. The in vitro studies provided evidence for a role of PAF both as intercellular and intracellular messenger involved in cell-to-cell communication. In the cardiovascular system, PAF may have a role in embryogenesis because it stimulates endothelial cell migration and angiogenesis and may affect cardiac function because it exhibits mechanical and electrophysiological actions on cardiomyocytes. Moreover, PAF may contribute to modulation of blood pressure mainly by affecting the renal vascular circulation. In pathological conditions, PAF has been involved in the hypotension and cardiac dysfunctions occurring in various cardiovascular stress situations such as cardiac anaphylaxis and hemorrhagic, traumatic, and septic shock syndromes. In addition, experimental studies indicate that PAF has a critical role in the development of myocardial ischemia-reperfusion injury. Indeed, PAF cooperates in the recruitment of leukocytes in inflamed tissue by promoting adhesion to the endothelium and extravascular transmigration of leukocytes. The finding that human heart can produce PAF, expresses PAF receptor, and is sensitive to the negative inotropic action of PAF suggests that this mediator may have a role also in human cardiovascular pathophysiology.

Increased blood levels of platelet-activating factor in insulin-dependent diabetic patients with microalbuminuria.

BACKGROUND: Platelet-activating factor (PAF), a phospholipid mediator of inflammation, may induce an enhanced size- and charge-dependent glomerular permeability in experimental animals. Studies on the role of PAF in enhanced glomerular permeability in the early phase of diabetic nephropathy are still lacking. METHODS: We evaluated the intravascular levels of PAF and its main catabolic enzyme, the PAF-specific plasma acetyl-hydrolase (PAF-AH), in basal conditions and after exercise, in normo- or micro-albuminuric insulin-dependent diabetic (IDD) patients and in normal subjects. RESULTS: The results obtained indicate that the concentration of PAF in whole blood was significantly enhanced in basal conditions, during and after exercise in all microalbuminuric IDD patients, but not in normoalbuminuric IDD or in control subjects. The increased concentration of PAF did not correlate with changes in the activity of PAF-AH, suggesting an enhanced production rather than a decreased catabolism of PAF. CONCLUSIONS: These results indicate an association between increased production of PAF and enhanced glomerular permeability in microalbuminuric IDD patients.

Platelet-activating factor enhances vascular endothelial growth factor-induced endothelial cell motility and neoangiogenesis in a murine matrigel model.

We previously reported that platelet-activating factor (PAF) enhances the angiogenic activity of certain polypeptide mediators such as tumor necrosis factor and hepatocyte growth factor by promoting endothelial cell motility. The purpose of the present study was to evaluate whether the synthesis of PAF induced by vascular endothelial growth factor (VEGF) might affect endothelial cell motility, microvascular permeability, and angiogenesis. The neoangiogenesis and synthesis of PAF induced by VEGF were studied in vivo in a murine Matrigel model. Dermal permeability was studied in mice by injection of (125)I-albumin. The synthesis of PAF, cell motility, and the increased (125)I-albumin transfer across endothelial monolayers were studied in vitro by using cultures of human umbilical cord vein-derived endothelial cells (HUVECs). The results obtained demonstrate that the neoangiogenesis induced by VEGF in vivo was associated with a local synthesis of PAF and was inhibited by WEB2170 and CV3988, 2 chemically unrelated, specific PAF-receptor antagonists. In contrast, WEB2170 did not inhibit VEGF-enhanced dermal permeability, suggesting that the latter was independent of the synthesis of PAF. In vitro, it was found that VEGF induced the synthesis of PAF by HUVECs in a dose- and time-dependent manner. The cell motility induced by VEGF was inhibited by PAF-receptor antagonists. In contrast, VEGF-induced proliferation of HUVECs and albumin transfer through HUVEC monolayer were unaffected by PAF-receptor antagonists. These results suggest that the synthesis of PAF induced by VEGF enhances endothelial cell migration and contributes to the angiogenic effect of VEGF in the in vivo Matrigel model.

Role of nitric oxide and platelet-activating factor in cardiac alterations induced by tumor necrosis factor-alpha in the guinea-pig papillary muscle.

OBJECTIVE: Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine with negative inotropic properties, is implicated in several pathophysiological events. To clarify the mechanism of action of TNF-alpha on myocardium, we investigated the possible role of platelet-activating factor (PAF) and nitric oxide (NO) as secondary mediators of the depressant effect of this cytokine. METHODS: Isometric twitches and intracellular action potentials were recorded from guinea pig papillary muscles. The effects of TNF-alpha (1-10 ng/ml) were studied in controlled conditions and after treatment with 0.5% Triton X-100, to destroy the endocardial endothelium NG-nitro-L-arginine methyl ester (L-NAME), D-NAME (1 mM) and the two different PAF-receptor antagonists WEB 2170 (3 microM) and CV 3988 (5 microM) were used to study the role of NO and PAF in cardiac depression induced by TNF-alpha. To study the role of NO in cardiac alterations induced by PAF, papillary muscles were pretreated with L-NAME or D-NAME and then challenged with PAF (0.1-1 microM). Nitrite production by papillary muscles challenged with TNF-alpha alone. TNF-alpha in the presence of WEB 2170 or CV 3988, or PAF was studied with the Greiss reagent method. PAF production by papillary muscles stimulated by TNF-alpha was studied by a bioassay method. RESULTS: TNF-alpha induced an initial, transient positive inotropic effect, then reduced the contractility and the action potential duration in a concentration-dependent manner. Treatment of papillary muscle with Triton X-100 did not modify the response to TNF-alpha, suggesting that the effect of TNF-alpha is not mediated by endocardial endothelial cells. Pretreatment with indomethacin reduced the negative effect of TNF-alpha, while propranolol abolished the initial increase of contractility. The role of PAF and NO as mediators of TNF-alpha was suggested by: (1) the protective effect of L-NAME, but not of D-NAME, on electrical and mechanical alterations; (2) the stimulatory effect of TNF-alpha on nitrite production; (3) the inhibitory effect of WEB 2170 and CV 3988, on both the electromechanical alterations and the nitrite production; (4) the synthesis of PAF induced by TNF-alpha. L-NAME blocked the negative effect of PAF and PAF enhanced nitrite production by papillary muscle. CONCLUSIONS: The present results suggest that in cardiac muscle: (1) the release of PAF triggered by TNF-alpha may account for the stimulation of NO production; (2) both PAF and NO contribute to the development of the electrical and mechanical alterations induced by TNF-alpha; (3) NO production was down-stream to the synthesis of PAF.

Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism.

In this study, we demonstrate that human umbilical cord vein-derived endothelial cells (HUVECs) expressed c-Mpl, the thrombopoietin (TPO) receptor, and that TPO activates HUVECs in vitro, as indicated by directional migration, synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF) and interleukin-8 (IL-8), and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT5B. The observation that WEB 2170 and CV3988, 2 structurally unrelated PAF receptor antagonists, prevented the motility of HUVECs induced by TPO suggests a role of PAF as secondary mediator. Moreover, kinetic analysis of TPO-induced tyrosine phosphorylation of STAT demonstrated that STAT5B activation temporally correlated with the synthesis of PAF. PAF, in turn, induced a rapid tyrosine phosphorylation of STAT5B and PAF receptor blockade, by WEB 2170, preventing both TPO- and PAF-mediated STAT5B activation. The in vivo angiogenic effect of TPO, studied in a mouse model of Matrigel implantation, demonstrated that TPO induced a dose-dependent angiogenic response that required the presence of heparin. Moreover, the in vivo angiogenic effect of TPO was inhibited by the PAF receptor antagonist WEB 2170 but not by the anti-basic fibroblast growth factor neutralizing antibody. These results indicate that the effects of TPO are not restricted to cells of hematopoietic lineages, because TPO is able to activate endothelial cells and to induce an angiogenic response in which the recruitment of endothelial cells is mediated by the synthesis of PAF. Moreover, biochemical analysis supports the hypothesis that STAT5B may be involved in the signaling pathway leading to PAF-dependent angiogenesis.

Potential angiogenic role of platelet-activating factor in human breast cancer.

This study investigated the presence of platelet-activating factor (PAF) in the lipid extracts of 18 primary breast carcinomas and 20 control breast tissues. The amount of PAF detected in breast carcinomas was significantly higher than in controls. The mass spectrometric analysis of PAF-bioactive lipid extract from breast carcinomas showed the presence of several molecular species of PAF, including C16-alkylPAF, C18-lysophosphatidylcholine (LPC), C16-LPC, lyso-PAF, and C16-acylPAF. The amount of bioactive PAF extracted from breast specimens significantly correlated with tumor vascularization revealed by the number of CD34-and CD31-positive cells. As C16-alkylPAF was previously shown to induce angiogenesis in vivo, we evaluated whether the thin layer chromatography-purified lipid extracts of breast specimens elicited neoangiogenesis in a murine model of subcutaneous Matrigel injection. The lipid extracts from specimens of breast carcinoma containing high levels of PAF bioactivity, but not from breast carcinomas containing low levels of PAF bioactivity or from normal breast tissue, induced a significant angiogenic response. This angiogenic response was significantly inhibited by the PAF receptor antagonist WEB 2170. T47D and MCF7 breast cancer cell lines, but not an immortalized nontumor breast cell line (MCF10), released PAF in the culture medium. A significant in vivo neoangiogenic response, inhibited by WEB 2170, was elicited by T47D and MCF7 but not by MCF10 culture medium. These results indicate that an increased concentration of PAF is present in tumors with high microvessel density and that PAF may account for the neoangiogenic activity induced in mice by the lipid extracts obtained from breast cancer. A contribution of PAF in the neovascularization of human breast cancer is suggested.

Nitric oxide mediates angiogenesis induced in vivo by platelet-activating factor and tumor necrosis factor-alpha.

We evaluated the role of an endogenous production of nitric oxide (NO) in the in vitro migration of endothelial cells and in the in vivo angiogenic response elicited by platelet-activating factor (PAF), tumor necrosis factor-alpha (TNF), and basic fibroblast growth factor (bFGF). The NO synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME), but not its enantiomer D-NAME, prevented chemotaxis of endothelial cells induced in vitro by PAF and by TNF. The motogenic activity of TNF was also inhibited by WEB 2170, a specific PAF-receptor antagonist. In contrast, chemotaxis induced by bFGF was not prevented by L-NAME or by WEB 2170. Angiogenesis was studied in vivo in a murine model in which Matrigel was used as a vehicle for the delivery of mediators. In this model, the angiogenesis induced by PAF and TNF was inhibited by WEB 2170 and L-NAME but not by D-NAME. In contrast, angiogenesis induced by bFGF was not affected by L-NAME or by WEB 2170. TNF, but not bFGF, induced PAF synthesis within Matrigel. These results suggest that NO mediates the angiogenesis induced by PAF as well as that induced by TNF, which is dependent on the production of PAF. In contrast, the angiogenic effect of bFGF appears to be both PAF and NO independent.

Regulation of polymorphonuclear cell activation by thrombopoietin.

Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation.

Modulatory effect of interleukin-10 on the production of platelet-activating factor and superoxide anions by human leucocytes.

We observed that human monocytes (MO) and polymorphonuclear neutrophils (PMN) stimulated by lipopolysaccharide (LPS) produce platelet-activating factor (PAF) in a pattern characterized by an early and a delayed peak of synthesis. The early peak of PAF synthesis was due to a direct stimulation of these cells through mCD14 receptor as it was inhibited by anti-CD14 monoclonal antibody. The delayed and sustained peak of PAF synthesis was dependent on protein synthesis and cytokine production as shown by the inhibitory effect of cycloheximide on both MO and PMN, and of anti-tumour necrosis factor-alpha (anti-TNF-alpha) and of anti-interleukin-8 (anti-IL-8) neutralizing antibodies on MO and PMN respectively. IL-10 completely prevented this second, cytokine-dependent peak of PAF synthesis. In contrast, IL-10 markedly enhanced the first peak of PAF synthesis both in MO and PMN. Moreover, IL-10 was shown to modulate the production of superoxide anions (O2-) on both MO and PMN. As suggested by previous studies, IL-10 inhibited the delayed production of O2-. In the present study, we observed that IL-10 directly stimulated an early production of O2-. In addition, IL-10 enhanced the synthesis of O2- by MO and PMN challenged with LPS. The IL-10-induced O2- production was dependent, at least in part, from its effect on PAF synthesis, as it was inhibited by the PAF receptor antagonist WEB 2170. These results suggest that IL-10 may upregulate the early synthesis of PAF and O2- triggered by direct LPS stimulation, whereas it may downregulate the delayed production of these mediators.