Proteomics of serum-derived extracellular vesicles are associated with the severity and different clinical profiles of patients with COVID-19: An exploratory secondary analysis.

BACKGROUND AIMS: Coronavirus disease 2019 (COVID-19) is characterized by a broad spectrum of clinical manifestations with the potential to progress to multiple organ dysfunction in severe cases. Extracellular vesicles (EVs) carry a range of biological cargoes, which may be used as biomarkers of disease state. METHODS: An exploratory secondary analysis of the SARITA-2 and SARITA-1 datasets (randomized clinical trials on patients with mild and moderate/severe COVID-19) was performed. Serum-derived EVs were used for proteomic analysis to identify enriched biological processes and key proteins, thus providing insights into differences in disease severity. Serum-derived EVs were separated from patients with COVID-19 by size exclusion chromatography and nanoparticle tracking analysis was used to determine particle concentration and diameter. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to identify and quantify protein signatures. Bioinformatics and multivariate statistical analysis were applied to distinguish candidate proteins associated with disease severity (mild versus moderate/severe COVID-19). RESULTS: No differences were observed in terms of the concentration and diameter of enriched EVs between mild (n = 14) and moderate/severe (n = 30) COVID-19. A total of 414 proteins were found to be present in EVs, of which 360 were shared while 48 were uniquely present in severe/moderate compared to mild COVID-19. The main biological signatures in moderate/severe COVID-19 were associated with platelet degranulation, exocytosis, complement activation, immune effector activation, and humoral immune response. Von Willebrand factor, serum amyloid A-2 protein, histone H4 and H2A type 2-C, and fibrinogen ß-chain were the most differentially expressed proteins between severity groups. CONCLUSION: Exploratory proteomic analysis of serum-derived EVs from patients with COVID-19 detected key proteins related to immune response and activation of coagulation and complement pathways, which are associated with disease severity. Our data suggest that EV proteins may be relevant biomarkers of disease state and prognosis.

Beneficial effects of empagliflozin and liraglutide on the cerebral microcirculation of diabetic rats.

OBJECTIVES: This study aimed to evaluate the effects of the antidiabetics liraglutide, a GLP-1 analog, and empagliflozin, an SGLT-2 inhibitor, on the brain microcirculation of diabetic rats. METHODS: Type 2 diabetes mellitus (DM) was experimentally induced in male Wistar rats by combining a high-fat diet and a low dose of streptozotocin (35 mg/kg). Liraglutide (100 µg/kg s.c.) and empagliflozin (10 mg/kg, oral) were administered for 5 weeks. Body weight was monitored periodically. Oral glucose tolerance, fasting glycemia, and blood triglycerides were evaluated after the treatments. Endothelial-leukocyte interactions in the brain microcirculation and structural capillary density were assessed. RESULTS: DM rats presented metabolic and cerebrovascular alterations. Liraglutide treatment decreased body weight and blood triglycerides of DM rats. Empagliflozin treatment improved glucose tolerance but only the combination therapy significantly reduced fasting blood glucose. Both treatments and their combination reduced leukocyte adhesion into the endothelium of brain venules. However, empagliflozin was more effective in preventing DM-induced microvascular rarefaction. CONCLUSION: These findings suggest that chronic treatment with SGLT2 inhibitors and GLP-1 receptor agonists may serve as potential therapeutic approaches to prevent microvascular complications associated with diabetes.

Neurological impairment caused by Schistosoma mansoni systemic infection exhibits early features of idiopathic neurodegenerative disease.

Schistosomiasis, a neglected tropical disease caused by trematodes of the Schistosoma genus, affects over 250 million people around the world. This disease has been associated with learning and memory deficits in children, whereas reduced attention levels, impaired work capacity, and cognitive deficits have been observed in adults. Strongly correlated with poverty and lack of basic sanitary conditions, this chronic endemic infection is common in Africa, South America, and parts of Asia and contributes to inhibition of social development and low quality of life in affected areas. Nonetheless, studies on the mechanisms involved in the neurological impairment caused by schistosomiasis are scarce. Here, we used a murine model of infection with Schistosoma mansoni in which parasites do not invade the central nervous system to evaluate the consequences of systemic infection on neurologic function. We observed that systemic infection with S. mansoni led to astrocyte and microglia activation, expression of oxidative stress-induced transcription factor Nrf2, oxidative damage, Tau phosphorylation, and amyloid-ß peptide accumulation in the prefrontal cortex of infected animals. We also found impairment in spatial learning and memory as evaluated by the Morris water maze task. Administration of anthelmintic (praziquantel) and antioxidant (N-acetylcysteine plus deferoxamine) treatments was effective in inhibiting most of these phenotypes, and the combination of both treatments had a synergistic effect to prevent such changes. These data demonstrate new perspectives toward the understanding of the pathology and possible therapeutic approaches to counteract long-term effects of systemic schistosomiasis on brain function.

6-Shogaol Exerts a Neuroprotective Factor in Offspring after Maternal Immune Activation in Rats.

6-Shogaol is one of the main active phenolic components of ginger and has neuroprotective effects by protecting brain against the oxidative stress and regulate the levels of neurotrophic factors. The objective of the present study was to verify the effect of 6-shogaol on neurochemical parameters in offspring after maternal immune activation by lipopolysaccharide (LPS) in rats. Twelve pregnant Wistar rats received 100 µg/kg of LPS or saline solution on the gestational day 9.5. Male offspring participated in the study and from the postnatal days (PND) 30 and 55, respectively, they were supplemented with 6-shogaol or saline solution, by gavage at a dose of 10 mg/kg/day, orally for 5 days. In PND 37 and 62, analysis of kinase signaling regulated by extracellular signal 1/2 (ERK 1/2), levels of neurotrophic factor derived from the brain (BDNF), and neuron-specific enolase (NSE), lipid and protein oxidative damage was evaluated by 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine (3-NT), respectively, and myeloperoxidase (MPO) activity was performed in the hippocampus. Prenatal exposure to LPS significantly decreased ERK and BDNF levels in PND 37 and 62, increased NSE levels and lipid damage in rats in PND 37, and increased 3-NT level in rats in PND 62. With treatment using 6-shogaol, an increase in ERK and BDNF levels was identified in PND 37 and 62 and a reduction in HNE and MPO activity in rats in PND 37 and 62, respectively. 6-Shogaol positively increased markers of neuronal growth, plasticity and synaptic activity and reduced oxidative damage in the hippocampus in an animal model of autism by maternal immune activation.

TLR4 mutation protects neurovascular function and cognitive decline in high-fat diet-fed mice.

BACKGROUND: Metabolic syndrome (MS) is defined as a low-grade proinflammatory state in which abnormal metabolic and cardiovascular factors increase the risk of developing cardiovascular disease and neuroinflammation. Events, such as the accumulation of visceral adipose tissue, increased plasma concentrations of free fatty acids, tissue hypoxia, and sympathetic hyperactivity in MS may contribute to the direct or indirect activation of Toll-like receptors (TLRs), specifically TLR4, which is thought to be a major component of this syndrome. Activation of the innate immune response via TLR4 may contribute to this state of chronic inflammation and may be related to the neuroinflammation and neurodegeneration observed in MS. In this study, we investigated the role of TLR4 in the brain microcirculation and in the cognitive performance of high-fat diet (HFD)-induced MS mice. METHODS: Wild-type (C3H/He) and TLR4 mutant (C3H/HeJ) mice were maintained under a normal diet (ND) or a HFD for 24 weeks. Intravital video-microscopy was used to investigate the functional capillary density, endothelial function, and endothelial-leukocyte interactions in the brain microcirculation. Plasma concentrations of monocyte chemoattractant protein-1 (MCP-1), adipokines and metabolic hormones were measured with a multiplex immunoassay. Brain postsynaptic density protein-95 and synaptophysin were evaluated by western blotting; astrocytic coverage of the vessels, microglial activation and structural capillary density were evaluated by immunohistochemistry. RESULTS: The HFD-induced MS model leads to metabolic, hemodynamic, and microcirculatory alterations, as evidenced by capillary rarefaction, increased rolling and leukocyte adhesion in postcapillary venules, endothelial dysfunction, and less coverage of astrocytes in the vessels, which are directly related to cognitive decline and neuroinflammation. The same model of MS reproduced in mice deficient for TLR4 because of a genetic mutation does not generate such changes. Furthermore, the comparison of wild-type mice fed a HFD and a normolipid diet revealed differences in inflammation in the cerebral microcirculation, possibly related to lower TLR4 activation. CONCLUSIONS: Our results demonstrate that TLR4 is involved in the microvascular dysfunction and neuroinflammation associated with HFD-induced MS and possibly has a causal role in the development of cognitive decline.

Fluorine Atoms on C6H5-Corrole Affect the Interaction with Mpro and PLpro Proteases of SARS-CoV-2: Molecular Docking and 2D-QSAR Approaches.

The chymotrypsin-like cysteine protease (3CLpro, also known as main protease-Mpro) and papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been used as the main targets for screening potential synthetic inhibitors for posterior in vitro evaluation of the most promising compounds. In this sense, the present work reports for the first time the evaluation of the interaction between Mpro/PLpro with a series of 17 porphyrin analogues-corrole (C1), meso-aryl-corrole (C2), and 15 fluorinated-meso-aryl-corrole derivatives (C3-C17) via molecular docking calculations. The impact of fluorine atoms on meso-aryl-corrole structure was also evaluated in terms of binding affinity and physical-chemical properties by two-dimensional quantitative structure-activity relationship (2D-QSAR). The presence of phenyl moieties increased the binding capacity of corrole for both proteases and depending on the position of fluorine atoms might impact positively or negatively the binding capacity. For Mpro the para-fluorine atoms might decrease drastically the binding capacity, while for PLpro there was a certain increase in the binding affinity of fluorinated-corroles with the increase of fluorine atoms into meso-aryl-corrole structure mainly from tri-fluorinated insertions. The 2D-QSAR models indicated two separated regions of higher and lower affinity for Mpro:C1-C17 based on dual electronic parameters (σI and σR), as well as one model was obtained with a correlation between the docking score value of Mpro:C2-C17 and the corresponding 13C nuclear magnetic resonance (NMR) chemical shifts of the sp2 carbon atoms (δC-1 and δC-2) of C2-C17. Overall, the fluorinated-meso-aryl-corrole derivatives showed favorable in silico parameters as potential synthetic compounds for future in vitro assays on the inhibition of SARS-CoV-2 replication.

Inflammatory, synaptic, motor, and behavioral alterations induced by gestational sepsis on the offspring at different stages of life.

BACKGROUND: The term sepsis is used to designate a systemic condition of infection and inflammation associated with hemodynamic changes that result in organic dysfunction. Gestational sepsis can impair the development of the central nervous system and may promote permanent behavior alterations in the offspring. The aim of our work was to evaluate the effects of maternal sepsis on inflammatory cytokine levels and synaptic proteins in the hippocampus, neocortex, frontal cortex, and cerebellum of neonatal, young, and adult mice. Additionally, we analyzed the motor development, behavioral features, and cognitive impairments in neonatal, young and adult offspring. METHODS: Pregnant mice at the 14th embryonic day (E14) were intratracheally instilled with saline 0.9% solution (control group) or Klebsiella spp. (3 × 108 CFU) (sepsis group) and started on meropenem after 5 h. The offspring was sacrificed at postnatal day (P) 2, P8, P30, and P60 and samples of liver, lung, and brain were collected for TNF-α, IL-1ß, and IL-6 measurements by ELISA. Synaptophysin, PSD95, and ß-tubulin levels were analyzed by Western blot. Motor tests were performed at all analyzed ages and behavioral assessments were performed in offspring at P30 and P60. RESULTS: Gestational sepsis induces a systemic pro-inflammatory response in neonates at P2 and P8 characterized by an increase in cytokine levels. Maternal sepsis induced systemic downregulation of pro-inflammatory cytokines, while in the hippocampus, neocortex, frontal cortex, and cerebellum an inflammatory response was detected. These changes in the brain immunity were accompanied by a reduction of synaptophysin and PSD95 levels in the hippocampus, neocortex, frontal cortex, and cerebellum, in all ages. Behavioral tests demonstrated motor impairment in neonates, and depressive-like behavior, fear-conditioned memory, and learning impairments in animals at P30 and P60, while spatial memory abilities were affected only at P60, indicating that gestational sepsis not only induces an inflammatory response in neonatal mouse brains, but also affects neurodevelopment, and leads to a plethora of behavioral alterations and cognitive impairments in the offspring. CONCLUSION: These data suggest that maternal sepsis may be causatively related to the development of depression, learning, and memory impairments in the litter.

Systemic microvascular endothelial dysfunction and disease severity in COVID-19 patients: Evaluation by laser Doppler perfusion monitoring and cytokine/chemokine analysis.

BACKGROUND: Microvascular dysfunction, serum cytokines and chemokines may play important roles in pathophysiology of coronavirus disease 2019 (COVID-19), especially in severe cases. METHODS: Patients with COVID-19 underwent non-invasive evaluation of systemic endothelium-dependent microvascular reactivity - using laser Doppler perfusion monitoring in the skin of the forearm - coupled to local thermal hyperemia. Maximal microvascular vasodilatation (44 °C thermal plateau phase) was used as endpoint. A multiplex biometric immunoassay was used to assess a panel of 48 serum cytokines and chemokines. Severe COVID-19 (S-COVID) was defined according to WHO criteria, while all other cases of COVID-19 were considered mild to moderate (M-COVID). A group of healthy individuals who tested negative for SARS-CoV-2 served as a control group and was also evaluated with LDPM. RESULTS: Thirty-two patients with COVID-19 (25% S-COVID) and 14 controls were included. Basal microvascular flow was similar between M-COVID and controls (P = 0.69) but was higher in S-COVID than in controls (P = 0.005) and M-COVID patients (P = 0.01). The peak microvascular vasodilator response was markedly decreased in both patient groups (M-COVID, P = 0.001; S-COVID, P < 0.0001) compared to the healthy group. The percent increases in microvascular flow were markedly reduced in both patient groups (M-COVID, P < 0.0001; S-COVID, P < 0.0001) compared to controls. Patients with S-COVID had markedly higher concentrations of dissimilar proinflammatory cytokines and chemokines, compared to patients with M-COVID. CONCLUSIONS: In patients with COVID-19, especially with S-COVID, endothelium-dependent microvascular vasodilator responses are reduced, while serum cytokines and chemokines involved in the regulation of vascular function and inflammation are increased.

SARS-CoV-2 Infection in Pregnant Women: Neuroimmune-Endocrine Changes at the Maternal-Fetal Interface.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has devastating effects on the population worldwide. Given this scenario, the extent of the impact of the disease on more vulnerable individuals, such as pregnant women, is of great concern. Although pregnancy may be a risk factor in respiratory virus infections, there are no considerable differences regarding COVID-19 severity observed between pregnant and nonpregnant women. In these circumstances, an emergent concern is the possibility of neurodevelopmental and neuropsychiatric harm for the offspring of infected mothers. Currently, there is no stronger evidence indicating vertical transmission of SARS-CoV-2; however, the exacerbated inflammatory response observed in the disease could lead to several impairments in the offspring's brain. Furthermore, in the face of historical knowledge on possible long-term consequences for the progeny's brain after infection by viruses, we must consider that this might be another deleterious facet of COVID-19. In light of neuroimmune interactions at the maternal-fetal interface, we review here the possible harmful outcomes to the offspring brains of mothers infected by SARS-CoV-2.

Neurovascular Interactions in Malaria.

Malaria is caused by Plasmodium infection and remains a serious public health problem worldwide, despite control efforts. Malaria can progress to severe forms, affecting multiple organs, including the brain causing cerebral malaria (CM). CM is the most severe neurological complication of malaria, and cognitive and behavior deficits are commonly reported in surviving patients. The number of deaths from malaria has been reducing in recent years, and as a consequence, neurological sequelae have been more evident. Neurological damage in malaria might be related to the neuroinflammation, characterized by glia cell activation, neuronal apoptosis and changes in the blood-brain barrier (BBB) integrity. The neurovascular unit (NVU) is responsible for maintaining the homeostasis of the BBB. Endothelial and pericytes cells in the cerebral microvasculature and neural cells, as astrocytes, neurons, and microglia, compose the NVU. The NVU can be disturbed by parasite metabolic products, such as heme and hemozoin, or cytokines that can promote activation of endothelial and glial cells and lead to increased BBB permeability and subsequently neurodegeneration. In this review, we will approach the main changes that happen in the cells of the NVU due to neuroinflammation caused by malaria infection, and elucidate how the systemic pathophysiology is involved in the onset and progression of CM.

Should COVID-19 be branded to viral thrombotic fever?

Coronaviruses can cause a diverse array of clinical manifestations, from fever with symptoms of the common cold to highly lethal severe acute respiratory syndrome (SARS) and middle east respiratory syndrome (MERS). SARS-CoV-2, the coronavirus discovered in Hubei province, China, at the end of 2019, became known worldwide for causing coronavirus disease 2019 (COVID-19). Over one year's time period, the scientific community has produced a large bulk of knowledge about this disease and countless reports about its immune-pathological aspects. This knowledge, including data obtained in postmortem studies, points unequivocally to a hypercoagulability state. However, the name COVID-19 tells us very little about the true meaning of the disease. Our proposal is more comprehensive; it intends to frame COVID-19 in more clinical terminology, making an analogy to viral haemorrhagic fever (VHF). Thus, we found irrefutable evidence in the current literature that COVID-19 is the first viral disease that can be branded as a viral thrombotic fever. This manuscript points out that SARS-CoV-2 goes far beyond pneumonia or SARS. COVID-19 infections promote remarkable interactions among the endothelium, coagulation, and immune response, building up a background capable of promoting a "thrombotic storm," much more than a "cytokine storm." The importance of a viral protease called main protease (Mpro) is highlighted as a critical component for its replication in the host cell. A deeper analysis of this protease and its importance on the coagulation system is also discussed for the first time, mainly because of its similarity with the thrombin and factor Xa molecules, as recently pointed out by structural comparison crystallographic structures.

Physical exercise promotes astrocyte coverage of microvessels in a model of chronic cerebral hypoperfusion.

BACKGROUND: Brain circulation disorders such as chronic cerebral hypoperfusion have been associated with a decline in cognitive function during the development of dementia. Astrocytes together with microglia participate in the immune response in the CNS and make them potential sentinels in the brain parenchyma. In addition, astrocytes coverage integrity has been related to brain homeostasis. Currently, physical exercise has been proposed as an effective intervention to promote brain function improvement. However, the neuroprotective effects of early physical exercise on the astrocyte communication with the microcirculation and the microglial activation in a chronic cerebral hypoperfusion model are still unclear. The aim of this study was to investigate the impact of early intervention with physical exercise on cognition, brain microcirculatory, and inflammatory parameters in an experimental model of chronic cerebral hypoperfusion induced by permanent bilateral occlusion of the common carotid arteries (2VO). METHODS: Wistar rats aged 12 weeks were randomly divided into four groups: Sham-sedentary group (Sham-Sed), Sham-exercised group (Sham-Ex), 2VO-sedentary group (2VO-Sed), and 2VO-exercised group (2VO-Ex). The early intervention with physical exercise started 3 days after 2VO or Sham surgery during 12 weeks. Then, the brain functional capillary density and endothelial-leukocyte interactions were evaluated by intravital microscopy; cognitive function was evaluated by open-field test; hippocampus postsynaptic density protein 95 and synaptophysin were evaluated by western blotting; astrocytic coverage of the capillaries, microglial activation, and structural capillary density were evaluated by immunohistochemistry. RESULTS: Early moderate physical exercise was able to normalize functional capillary density and reduce leukocyte rolling in the brain of animals with chronic cerebral hypoperfusion. These effects were accompanied by restore synaptic protein and the improvement of cognitive function. In addition, early moderate exercise improves astrocytes coverage in blood vessels of the cerebral cortex and hippocampus, decreases microglial activation in the hippocampus, and improves structural capillaries in the hippocampus. CONCLUSIONS: Microcirculatory and inflammatory changes in the brain appear to be involved in triggering a cognitive decline in animals with chronic cerebral ischemia. Therefore, early intervention with physical exercise may represent a preventive approach to neurodegeneration caused by chronic cerebral hypoperfusion.

Mesenchymal Stromal Cells Protect the Blood-Brain Barrier, Reduce Astrogliosis, and Prevent Cognitive and Behavioral Alterations in Surviving Septic Mice.

OBJECTIVES: Survivors of sepsis are frequently left with significant cognitive and behavioral impairments. These complications derive from nonresolving inflammation that persists following hospital discharge. To date, no study has investigated the effects of mesenchymal stromal cell therapy on the blood-brain barrier, astrocyte activation, neuroinflammation, and cognitive and behavioral alterations in experimental sepsis. DESIGN: Prospective, randomized, controlled experimental study. SETTING: Government-affiliated research laboratory. SUBJECTS: Male Swiss Webster mice (n = 309). INTERVENTIONS: Sepsis was induced by cecal ligation and puncture; sham-operated animals were used as control. All animals received volume resuscitation (1 mL saline/mouse subcutaneously) and antibiotics (meropenem 10 mg/kg intraperitoneally at 6, 24, and 48 hours). Six hours after surgery, mice were treated with mesenchymal stromal cells IV (1 × 10 cells in 0.05 mL of saline/mouse) or saline (0.05 mL IV). MEASUREMENTS AND MAIN RESULTS: At day 1, clinical score and plasma levels of inflammatory mediators were increased in cecal ligation and puncture mice. Mesenchymal stromal cells did not alter clinical score or survival rate, but reduced levels of systemic interleukin-1ß, interleukin-6, and monocyte chemoattractant protein-1. At day 15, survivor mice completed a battery of cognitive and behavioral tasks. Cecal ligation and puncture mice exhibited spatial and aversive memory deficits and anxiety-like behavior. These effects may be related to increased blood-brain barrier permeability, with altered tight-junction messenger RNA expression, increased brain levels of inflammatory mediators, and astrogliosis (induced at day 3). Mesenchymal stromal cells mitigated these cognitive and behavioral alterations, as well as reduced blood-brain barrier dysfunction, astrocyte activation, and interleukin-1ß, interleukin-6, tumor necrosis factor-α, and interleukin-10 levels in vivo. In cultured primary astrocytes stimulated with lipopolysaccharide, conditioned media from mesenchymal stromal cells reduced astrogliosis, interleukin-1ß, and monocyte chemoattractant protein-1, suggesting a paracrine mechanism of action. CONCLUSIONS: In mice who survived experimental sepsis, mesenchymal stromal cell therapy protected blood-brain barrier integrity, reduced astrogliosis and neuroinflammation, as well as improved cognition and behavior.

Haem oxygenase protects against thrombocytopaenia and malaria-associated lung injury.

BACKGROUND: Malaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host's response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65. METHODS: C57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer. RESULTS: Plasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia. CONCLUSION: Data here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.

Simvastatin Posttreatment Controls Inflammation and Improves Bacterial Clearance in Experimental Sepsis.

Sepsis is characterized by a life-threatening organ dysfunction caused by an unbalanced host response to microbe infection that can lead to death. Besides being currently the leading cause of death in intensive care units worldwide, sepsis can also induce long-term consequences among survivors, such as cognitive impairment. Statins (lipid-lowering drugs widely used to treat dyslipidemia) have been shown to possess pleiotropic anti-inflammatory and antimicrobial effects. These drugs act inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, an enzyme that catalyzes the conversion of HMG-CoA to mevalonate, the limiting step in cholesterol biosynthesis. In this work, we evaluated the therapeutic effects of simvastatin in an animal model of sepsis. In previous study from our group, statin pretreatment avoided cognitive damage and neuroinflammation in sepsis survivors. Herein, we focused on acute inflammation where sepsis was induced by cecal ligation and puncture (CLP), and the animals were treated with simvastatin (2 mg/kg) 6 h after surgery. We measured plasma biochemical markers of organ dysfunction, cell migration, cell activation, bacterial elimination, production of nitric oxide 24 h after CLP, survival rate for 7 days, and cognitive impairment 15 days after CLP. One single administration of simvastatin 6 h after CLP was able to prevent both liver and kidney dysfunction. In addition, this drug decreased cell accumulation in the peritoneum as well as the levels of TNF-α, MIF, IL-6, and IL-1ß. Simvastatin diminished the number of bacterial colony forming units (CFU) and increased the production of nitric oxide production in the peritoneum. Simvastatin treatment increased survival for the first 24 h, but it did not alter survival rate at the end of 7 days. Our results showed that posttreatment with simvastatin hampered organ dysfunction, increased local production of nitric oxide, improved bacterial clearance, and modulated inflammation in a relevant model of sepsis.

Beyond Members of the Flaviviridae Family, Sofosbuvir Also Inhibits Chikungunya Virus Replication.

Chikungunya virus (CHIKV) causes a febrile disease associated with chronic arthralgia, which may progress to neurological impairment. Chikungunya fever (CF) is an ongoing public health problem in tropical and subtropical regions of the world, where control of the CHIKV vector, Aedes mosquitos, has failed. As there is no vaccine or specific treatment for CHIKV, patients receive only palliative care to alleviate pain and arthralgia. Thus, drug repurposing is necessary to identify antivirals against CHIKV. CHIKV RNA polymerase is similar to the orthologue enzyme of other positive-sense RNA viruses, such as members of the Flaviviridae family. Among the Flaviviridae, not only is hepatitis C virus RNA polymerase susceptible to sofosbuvir, a clinically approved nucleotide analogue, but so is dengue, Zika, and yellow fever virus replication. Here, we found that sofosbuvir was three times more selective in inhibiting CHIKV production in human hepatoma cells than ribavirin, a pan-antiviral drug. Although CHIKV replication in human induced pluripotent stem cell-derived astrocytes was less susceptible to sofosbuvir than were hepatoma cells, sofosbuvir nevertheless impaired virus production and cell death in a multiplicity of infection-dependent manner. Sofosbuvir also exhibited antiviral activity in vivo by preventing CHIKV-induced paw edema in adult mice at a dose of 20 mg/kg of body weight/day and prevented mortality in a neonate mouse model at 40- and 80-mg/kg/day doses. Our data demonstrate that a prototypic alphavirus, CHIKV, is also susceptible to sofosbuvir. As sofosbuvir is a clinically approved drug, our findings could pave the way to it becoming a therapeutic option against CF.

IGF-1 and IGF-1R modulate the effects of IL-4 on retinal ganglion cells survival: The involvement of M1 muscarinic receptor.

Trophic factors are involved in different cellular responses. Previously we demonstrated that IL-4 treatment induces an increase in retinal ganglion cell survival (RGCS) and regulates cholinergic differentiation of retinal cells in vitro. Data from literature show that IGF-1 also promotes RGCS, an effect mediated by PI-3K/AKT pathway. The aim of this study was to investigate the role of IGF-1 and IGF-1R on RGCS mediated by IL-4 treatment and the role of M1 acetylcholine receptors in this effect. Here we show that the effect of IL-4 on RGCS depends on IGF-1 and IGF-1R activation, the PI-3K/AKT and NFkB intracellular pathways and depends on M1 mAChRs activation. IGF-1 increases the levels of M1 mAChRs in 15min, 45min, 24 h and 48 h in mixed retinal cells culture, modulates the levels of IL-4, pIGF-1R, IGF-1R. IL-4 modulates IGF-1, pIGF-1R and IGF-1R levels in different time intervals. These results put in evidence a crosstalk between IL-4 and IGF-1 and a role of M1 mAChRs, IGF-1 and IGF-1R in RGCS mediated by IL-4.

Age-related cognitive impairment is associated with long-term neuroinflammation and oxidative stress in a mouse model of episodic systemic inflammation.

BACKGROUND: Microglia function is essential to maintain the brain homeostasis. Evidence shows that aged microglia are primed and show exaggerated response to acute inflammatory challenge. Systemic inflammation signals to the brain inducing changes that impact cognitive function. However, the mechanisms involved in age-related cognitive decline associated to episodic systemic inflammation are not completely understood. The aim of this study was to identify neuropathological features associated to age-related cognitive decline in a mouse model of episodic systemic inflammation. METHODS: Young and aged Swiss mice were injected with low doses of LPS once a week for 6 weeks to induce episodic systemic inflammation. Sickness behavior, inflammatory markers, and neuroinflammation were assessed in different phases of systemic inflammation in young and aged mice. Behavior was evaluated long term after episodic systemic inflammation by open field, forced swimming, object recognition, and water maze tests. RESULTS: Episodic systemic inflammation induced systemic inflammation and sickness behavior mainly in aged mice. Systemic inflammation induced depressive-like behavior in both young and aged mice. Memory and learning were significantly affected in aged mice that presented lower exploratory activity and deficits in episodic and spatial memories, compared to aged controls and to young after episodic systemic inflammation. Systemic inflammation induced acute microglia activation in young mice that returned to base levels long term after episodic systemic inflammation. Aged mice presented dystrophic microglia in the hippocampus and entorhinal cortex at basal level and did not change morphology in the acute response to SI. Regardless of their dystrophic microglia, aged mice produced higher levels of pro-inflammatory (IL-1ß and IL-6) as well as pro-resolution (IL-10 and IL-4) cytokines in the brain. Also, higher levels of Nox2 expression, oxidized proteins and lower antioxidant defenses were found in the aged brains compared to the young after episodic systemic inflammation. CONCLUSIONS: Our data show that aged mice have increased susceptibility to episodic systemic inflammation. Aged mice that showed cognitive impairments also presented higher oxidative stress and abnormal production of cytokines in their brains. These results indicate that a neuroinflammation and oxidative stress are pathophysiological mechanisms of age-related cognitive impairments.

In vivo and in vitro antimalarial effect and toxicological evaluation of the chloroquine analogue PQUI08001/06.

Antimalarial interventions mostly rely upon drugs, as chloroquine. However, plasmodial strains resistant to many drugs are constantly reported, leading to an expansion of malaria cases. Novel approaches are required to circumvent the drug resistance issue. Here, we describe the antimalarial potential of the chloroquine analogue 2-[[2-[(7-chloro-4-quinolinyl)amino]ethyl]amino] ethanol (PQUI08001/06). We observed that PQUI08001/06 treatment reduces parasitemia of both chloroquine-resistant and -sensitive strains of Plasmodium falciparum in vitro and P. berghei in vivo. Our data suggests that PQUI08001/06 is a potential antimalarial therapeutic alternative approach that could also target chloroquine-resistant plasmodial strains.