Changes in markers of inflammation and their correlation with death in patients with COVID-19 in the intensive care unit.

This study aimed to characterize the changes in serum inflammatory mediators in hospitalized patients with COVID-19 correlating with death. The study includes 58 participants: i) inpatients (n = 37): patients suffering from severe acute respiratory syndrome due to COVID-19, who were admitted at Intensive Care Unit (ICU) recovered and who died and ii) control group (n = 21): community volunteers. Inflammatory mediators evaluated interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17 (IL-17), interferon-gamma (IFN-γ) and interferon-gamma protein levels (IFN-γ), as well as, Urea, LDH, D-dimer, TAP/INR, AST, ALT and lymphocytes. Our results suggest that high levels of inflammatory markers, such as pro-inflammatory cytokines, and laboratory parameters, such as low levels of lymphocytes and high levels of IL-6, are associated with disease severity, especially in individuals who died. Constant measurement and monitoring of these inflammatory parameters is an effective tool in clinical practice, and it can help choosing appropriate therapies during the course of the disease.

Effects of calcium supplementation on changes in the IL2, IL4, IL6, IL10 axes and oxidative stress in pregnant women at risk for pre-eclampsia.

BACKGROUND: Pregnant women with hypertensive disorders are at increased risk for inflammatory diseases and oxidative stress. The dilemma raised by the best dosage of calcium supplementation on these factors is evident. The aim of the current study was to examine the effects of calcium on biomarkers of the purinergic system, inflammation and oxidative stress, which are factors contributing to vascular damage in pregnant women at high risk of pre-eclampsia. METHODS: A prospective, double-blind and placebo-controlled study conducted with 101 women at risk of pre-eclampsia were randomized to take 500 mg calcium/day or 1,500 mg calcium/day or placebo for 6 weeks from the 20th gestational week until delivery. Fasting blood samples were collected at the beginning of the study and 6 weeks after the intervention. RESULTS: Taking calcium supplements (500 mg calcium/day) led to a significant increase in ATP hydrolysis (p < 0.05), NTPDase activity with increased hydrolysis of ADP and AMP nucleotides in platelets and lymphocytes. In the intragroup analysis IL-2, IL-6, IL-4 and interferon-É£ presented lower values in the calcium 1,500 mg/day group (p < 0.005). Oxidative stress was assessed by TBARS pro-oxidant marker, with an increase for the calcium groups when compared to the placebo group. The Vitamin C antioxidant marker presented a significant increase (p < 0.005) for the group that received high calcium doses. CONCLUSIONS: Calcium administration for 6 weeks had antioxidant action and positively modulated the purinergic system and inflammatory markers in pregnant women at risk of pre-eclampsia.

Diosmetin attenuates fibromyalgia-like symptoms in a reserpine-induced model in mice.

Fibromyalgia is a potentially disabling idiopathic disease characterized by widespread chronic pain associated with comorbidities such as fatigue, anxiety, and depression. Current therapeutic approaches present adverse effects that limit adherence to therapy. Diosmetin, an aglycone of the flavonoid glycoside diosmin found in citrus fruits and the leaves of Olea europaea L., has antinociceptive, anti-inflammatory, and antioxidant properties. Here, we investigated the effect of diosmetin on nociceptive behaviors and comorbidities in an experimental fibromyalgia model induced by reserpine in mice. To induce the experimental fibromyalgia model, a protocol of subcutaneous injections of reserpine (1 mg/kg) was used once a day for three consecutive days in adult male Swiss mice. Mice received oral diosmetin on the fourth day after the first reserpine injection. Nociceptive (mechanical allodynia, muscle strength, and thermal hyperalgesia) and comorbid (depressive-like and anxiety behavior) parameters were evaluated. Potential adverse effects associated with diosmetin plus reserpine (locomotor alteration, cataleptic behavior, and body weight and temperature changes) were also evaluated. Oral diosmetin (0.015-1.5 mg/kg) reduced the mechanical allodynia, thermal hyperalgesia, and loss of muscle strength induced by reserpine. Diosmetin (0.15 mg/kg) also attenuated depressive-like and anxiety behaviors without causing locomotor alteration, cataleptic behavior, and alteration in weight and body temperature of mice. Overall, diosmetin can be an effective and safe therapeutic alternative to treat fibromyalgia symptoms, such as pain, depression and anxiety.

Curcumin modulates neurogliogenesis and purinergic receptor expression in neural precursor cells infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis, and its congenital transmission is of paramount concern. During embryonic development, infection with the parasite causes irreversible damage to the still-forming fetus's central nervous system (CNS). In the pathogenesis of neurotoxoplasmosis, purinergic receptors prejudice neuroprotection, neuroinflammation, and activation of microbicide mechanisms against the parasitic vacuole. This study used curcumin as a treatment for neural precursor cells (NPCs) infected with T. gondii. The congenital toxoplasmosis induction consisted of maternal infection with the VEG strain, and NPCs were obtained from the telencephalon of mouse embryos. Curcumin at increasing concentrations was administered in vitro to analyze NPC metabolic activity, cell number, and size, as well as neurogliogenesis, proving to be effective in recovering the size of infected NPCs. Curcumin partially re-established impaired neurogenesis. Purinergic A1, A2A, and P2X7 receptors may be related to neuroprotection, neuroinflammatory control, and activation of mechanisms for inducing the parasite's death. ERK 1/2 was highly expressed in infected cells, while its expression rates decreased after the addition of the treatment, highlighting the possible anti-inflammatory action of curcumin. These findings suggest that curcumin treats neurological perturbations induced by toxoplasmosis.

Istradefylline modulates purinergic enzymes and reduces malignancy-associated factors in B16F10 melanoma cells.

ATP and adenosine exert pivotal roles in the development, maintenance, and metastatic spreading of melanoma. The action of such key melanoma tumor microenvironment (TME) constituents might be complementary or opposed, and their effects are not exclusive to immune cells but also to other host cells and tumor cells. The effects of ATP are controlled by the axis CD39/73, resulting in adenosine, the main actor in the TME, and A2A is the crucial mediator of its effects. We evaluated ATP and adenosine signaling through A2A on B16F10 melanoma cells using istradefylline (IST) (antiparkinsonian A2A antagonist) and caffeine (CAF) treatments after exposure to ATP and adenosine. Adenosine increased melanoma cell viability and proliferation in a concentration-dependent manner. ATP increases viability only as a substrate by CD39 to produce adenosine. Both IST and CAF are toxic to B16F10 cells, but only IST potentialized paclitaxel-induced cytotoxic effects, even decreasing its IC50 value. IST positively modulated CD39 and CD73 expression. CD39 activity was increased, and E-ADA was reduced, indicating that the melanoma cells promoted compensatory feedback in the production and maintenance of adenosine levels. A2A antagonism by IST reduced the factors associated with malignancy, like migration, adhesion, colony formation, and the capacity to produce melanin. Moreover, IST significantly increases nitric oxide (NO) production, which correlates to a decline in melanoma cell viability by apoptotic events. Altogether, our results suggest that adenosine signaling through A2A is essential for B16F10 cells, and its inhibition by IST causes compensatory purinergic enzymatic modulations. Furthermore, IST is a promising therapy that provides new ways to improve current melanoma treatments.

Berberine increases the expression of cytokines and proteins linked to apoptosis in human melanoma cells.

BACKGROUND: Melanoma is the most lethal form of skin cancer, and its incidence has increased considerably in the last decades. Melanoma presents difficult treatment with strong resistance of tumor cells, due to its extremely invasive nature with high capacity to metastases. Berberine (BBR), an isoquinoline alkaloid, is a molecule found in several medicinal plants, and has been studied in several diseases, demonstrating antimicrobial, antidiabetic and anti-inflammatory properties and anti-tumorigenic effects. METHODS AND RESULTS: In SK-MEL-28 cells, 50 µM BBR treatment for 24 h decreased cell viability by 50 percent. This concentration generated cell death both by early apoptosis and necrosis, with an increase in the DNA damage index. BBR increased (*p < 0.05) the proportion of cells in G1/G0 phase and decreased (###p < 0.005) the percentage of cells in S phase. The alcaloid increased (****p < 0.001) ROS production compared to untreated controls with an increase in activated caspase 3 and phosphorylated p53 protein levels. In addition, BBR significantly enhanced ERK as well as both pro- and anti-inflammatory cytokine expression compared to untreated controls. CONCLUSIONS: BBR has important antiproliferative effects and may be alone or in adjunct therapy a promising candidate for melanoma treatment, a cancer with great incidence and high lethality.

Richardia brasiliensis Gomes: phytochemical characterization, antiproliferative capacity and in vitro and in vivo toxicity.

Richardia brasiliensis, known as poaia branca, is a medicinal species widely distributed throughout Brazil and used in folk medicine. However, studies on its toxicity are practically non-existent, and little is known about its biological activity. This study aimed to investigate its phytochemical compounds, assess its in vitro and in vivo toxicities, and determine its antiproliferative activity. UHPLC-ESI-HRFTMS performed the phytochemical characterization, and the antiproliferative activity was analyzed in different tumor cell lines. In vitro toxicity was evaluated in PBMC cells, and in vivo acute and repeated dose toxicity was evaluated according to OECD guidelines. It was identified alkaloids and terpenes as significant compounds. Regarding its antiproliferative activity, the human melanoma strain decreased its viability by about 95%. In vitro toxicity showed that the extracts maintained the viability of PBMCs; however, higher concentrations were able to increase the production of dsDNA quantity. In vivo tests showed no mortality nor signs of toxicity; the alterations found in hematological and biochemical parameters are within the standards for the species. The results indicate that R. brasiliensis has a good effect against the tumor cell line; still, more studies on its toxicity at higher concentrations are needed.

Mesenchymal stem cell-glioblastoma interactions mediated via kinin receptors unveiled by cytometry.

Glioblastoma (GBM) is one of the most malignant and devastating brain tumors. The presence of highly therapy-resistant GBM cell subpopulations within the tumor mass, rapid invasion into brain tissues and reciprocal interactions with stromal cells in the tumor microenvironment contributes to an inevitable fatal prognosis for the patients. We highlight the most recent evidence of GBM cell crosstalk with mesenchymal stem cells (MSCs), which occurs either by direct cell-cell interactions via gap junctions and microtubules or cell fusion. MSCs and GBM paracrine interactions are commonly observed and involve cytokine signaling, regulating MSC tropism toward GBM, their intra-tumoral distribution, and immune system responses. MSC-promoted effects depending on their cytokine and receptor expression patterns are considered critical for GBM progression. MSC origin, tumor heterogeneity and plasticity may also determine the outcome of such interactions. Kinins and kinin-B1 and -B2 receptors play important roles in information flow between MSCs and GBM cells. Kinin-B1 receptor activity favors tumor migration and fusion of MSCs and GBM cells. Flow and image (tissue) cytometry are powerful tools to investigate GBM cell and MSC crosstalk and are applied to analyze and characterize several other cancer types.

Insights in Chloroquine Action: Perspectives and Implications in Malaria and COVID-19.

Malaria is a threat to human mankind and kills about half a million people every year. On the other hand, COVID-19 resulted in several hundred thousand deaths since December 2019 and remains without an efficient and safe treatment. The antimalarials chloroquine (CQ) and its analog, hydroxychloroquine (HCQ), have been tested for COVID-19 treatment, and several conflicting evidence has been obtained. Therefore, the aim of this review was to summarize the evidence regarding action mechanisms of these compounds against Plasmodium and SARS-CoV-2 infection, together with cytometry applications. CQ and HCQ act on the renin angiotensin system, with possible implications on the cardiorespiratory system. In this context, flow and image cytometry emerge as powerful technologies to investigate the mechanism of therapeutic candidates, as well as for the identification of the immune response and prognostics of disease severity. Data from the large randomized trials support the conclusion that CQ and HCQ do not provide any clinical improvements in disease severity and progression of SARS-CoV-2 patients, as well as they do not present any solid evidence of increased serious side effects. These drugs are safe and effective antimalarials agents, but in SARS-CoV-2 patients, they need further studies in the context of clinical trials. © 2020 International Society for Advancement of Cytometry.

Ellagic Acid Inhibits Neuroinflammation and Cognitive Impairment Induced by Lipopolysaccharides.

Neuroinflammation is a predisposing factor for the development of cognitive impairment and dementia. Among the new molecules that are currently being studied, ellagic acid (EA) has stood out for its neuroprotective properties. The present study investigated the effects of ellagic acid in the object recognition test, oxidative stress, cholinergic neurotransmission, glial cell expression, and phosphorylated Tau protein expression. For this, 32 male Wistar rats received an intraperitoneal (IP) application of lipopolysaccharides (LPS) at a dose of 250 µg/kg or 0.9% saline solution (SAL) for 8 days. Two hours after the IP injections, the animals received 100 mg/kg of EA or SAL via intragastric gavage. Behavioral parameters (open field test and object recognition) were performed on days 5, 6, and 7 of the experimental periods. The results showed that the treatment with EA in the LPS group was able to inhibit cognitive impairment, modulate the immune system response by significantly reducing glial cell expression, attenuating phosphorylated Tau and oxidative damage with consequent improvement in the antioxidant system, as well as preventing the increase of acetylcholinesterase activity. Thus, the neuroprotective effects of EA and its therapeutic potential in cognitive disorders secondary to neuroinflammation were demonstrated.

Berberine induces apoptosis in glioblastoma multiforme U87MG cells via oxidative stress and independent of AMPK activity.

Glioblastoma multiforme (GM) is the most prevalent tumor among gliomas and presents the highest mortality rate among brain tumors. Berberine (BBR) is an alkaloid isoquinoline found in medicinal plants such as Coptis chinensis. Studies have been showed that BBR presents protective activity in mesenchymal cells and neurons, and antitumor properties in breast cancer and hepatocarcinoma. The aim of this study was to investigate the antitumor effects of BBR in GM U87MG cells, as well as to identify, whether such effects are mediated by oxidative stress and canonical apoptotic pathways. After treatment with several concentrations of BBR (10, 25, 100 and 250 µM) for 24, 48 and 72 h of exposure, BBR reduce cell viability of U87MG cells in a concentration- and time-dependent manner. Afterwards, it was observed that BBR, starting at a concentration of 25 µM of 24 h exposure, significantly suppressed proliferation and increased early apoptosis (53.5% ± 11.15 of annexin V+ propidium iodide- cells) compared to untreated cells (7.5% ± 4.6). BBR-induced apoptosis was independent from AMPK activity and did not change total caspase-3 and p-p53 levels. Moreover, BBR (25 µM/24 h) increased oxidative stress in U87MG cells, evidenced by high levels of reactive oxygen species, thiobarbituric acid reactive substance and protein carbonylation. Considering the antitumor effects of BBR in U87MG cells, this compound may be a potential candidate for adjuvant GM treatment.

Antiproliferative and apoptotic effects of caffeic acid on SK-Mel-28 human melanoma cancer cells.

Cutaneous melanoma (CM) is an extremely aggressive cancer presenting low survival and high mortality. The vast majority of patients affected by this disease does not respond or show resistance to the chemotherapeutic drugs, which makes the treatment ineffective. In this sense, the necessity for the development of new agents to assist in CM therapy is extremely important. One of the sources of great interest in this search are compounds of natural origin. Among these compounds, caffeic acid has demonstrated a broad spectrum of pharmacological activities as well as antitumor effects in some types of cancer. Therefore, the objective of this work was to investigate the possible antitumor effect of caffeic acid on the SK-Mel-28 cell line, human CM cells. Cells were cultured in flasks with culture medium containing fetal bovine serum, antibiotic, and antifungal, and maintained in ideal conditions. Cells were treated with 25 µM, 50 µM, 100 µM, 150 µM and 200 µM of caffeic acid and dacarbazine at 1 mg/mL. We verified the effect on cell viability and cell death, apoptosis, cell cycle, colony formation and gene expression of caspases. Results showed a decrease in cell viability, cell death induction by apoptosis, inhibition of colony formation, modulation of cell cycle and alterations in gene expression of caspases after caffeic acid treatment. These results suggest an antitumor effect of the compound on SK-Mel-28 cells. This study provides original information on mechanisms by which caffeic acid may play a key role in preventing tumor progression in human melanoma cells.

Surface immunoglobulins of erythrocytes and platelets in dogs naturally infected by Rangelia vitalii.

Rangelia vitalii is a protozoan of the Babesiidae family that parasitizes domestic and wild dogs in South American countries. The main laboratory findings in blood samples from animals infected by R. vitalii are anemia and thrombocytopenia. The aim of this study was to detect IgM and IgG immunoglobulins on the surface of red blood cells and platelets, as well as to determine the percentage of reticulated platelets and reticulocytes in dogs naturally infected by R. vitalii. Blood samples from twenty dogs seen at the Veterinary Hospital of the Federal University of Santa Maria (UFSM) were divided into two groups: the diseased group consisted of blood samples from 10 animals with the diagnosis of rangeliosis, and the healthy group (control) consisted of samples from 10 healthy animals. All diseased dogs showed normocytic normochromic anemia but showed no differences (p > 0.05) in reticulocyte counts compared to healthy dogs. Moreover, IgM and IgG immunoglobulins were detected on the surface of the plasma membrane of red blood cells from both groups, but the amounts did not differ between groups (p > 0.05). Thrombocytopenia in infected animals was classified as severe. The percentage of reticulated platelets was higher (p < 0.001) in diseased dogs than in healthy animals. Diseased animals showed more IgM immunoglobulins bound to the surface of platelets than did the healthy group (p < 0.001). However, the amount of IgG bound to the surface of platelets was not different between groups. In conclusion, we showed that R. vitalii caused immune-mediated thrombocytopenia since IgM immunoglobulins were found on the surface of platelets of diseased dogs. We suggest that the binding of immunoglobulins on platelet surfaces contributes to early destruction of these cells and, consequently, alterations in hemostasis. An increase in reticulated platelets was noted in response to thrombocytopenia, indicating active thrombopoiesis.

EP2 receptor agonist ONO-AE1-259-01 attenuates pentylenetetrazole- and pilocarpine-induced seizures but causes hippocampal neurotoxicity.

Epilepsy is a common and devastating neurological disease affecting more than 50 million people worldwide. Accumulating experimental and clinical evidence suggests that inflammatory pathways contribute to the development of seizures in various forms of epilepsy. In this context, while the activation of the PGE2 EP2 receptor causes early neuroprotective and late neurotoxic effects, the role of EP2 receptor in seizures remains unclear. We investigated whether the systemic administration of the highly selective EP2 agonist ONO-AE1-259-01 prevented acute pentylenetetrazole (PTZ)- and pilocarpine-induced seizures. The effect of ONO-AE1-259-01 on cell death in the hippocampal formation of adult male mice seven days after pilocarpine-induced status epilepticus (SE) was also evaluated. ONO-AE1-259-01 (10µg/kg, s.c.) attenuated PTZ- and pilocarpine-induced seizures, evidenced by the increased latency to seizures, decreased number and duration of seizures episodes and decreased mean amplitude of electrographic seizures. ONO-AE1-259-01 and pilocarpine alone significantly increased the number of pyknotic cells per se in all hippocampal subfields. The EP2 agonist also additively increased pilocarpine-induced pyknosis in the pyramidal cell layer of CA1 but reduced pilocarpine-induced pyknosis in the granule cell layer of the dentate gyrus (DG). Although the systemic administration of ONO-AE1-259-01 caused a significant anticonvulsant effect in our assays, this EP2 agonist caused extensive cell death. These findings limit the likelihood of EP2 receptor agonists being considered as novel potential anticonvulsant drugs.

Bradykinin promotes immune responses in differentiated embryonic neurospheres carrying APPswe and PS1dE9 mutations.

BACKGROUND: Neural progenitor cells (NPCs) can be cultivated from developing brains, reproducing many of the processes that occur during neural development. They can be isolated from a variety of animal models, such as transgenic mice carrying mutations in amyloid precursor protein (APP) and presenilin 1 and 2 (PSEN 1 and 2), characteristic of familial Alzheimer's disease (fAD). Modulating the development of these cells with inflammation-related peptides, such as bradykinin (BK) and its antagonist HOE-140, enables the understanding of the impact of such molecules in a relevant AD model. RESULTS: We performed a global gene expression analysis on transgenic neurospheres treated with BK and HOE-140. To validate the microarray data, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed on 8 important genes related to the immune response in AD such as CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF alpha and Iba-1. Furthermore, comparative analysis of the transcriptional profiles was performed between treatments, including gene ontology and reactome enrichment, construction and analysis of protein-protein interaction networks and, finally, comparison of our data with human dataset from AD patients. The treatments affected the expression levels of genes mainly related to microglia-mediated neuroinflammatory responses, with BK promoting an increase in the expression of genes that enrich processes, biological pathways, and cellular components related to immune dysfunction, neurodegeneration and cell cycle. B2 receptor inhibition by HOE-140 resulted in the reduction of AD-related anomalies caused in this system. CONCLUSIONS: BK is an important immunomodulatory agent and enhances the immunological changes identified in transgenic neurospheres carrying the genetic load of AD. Bradykinin treatments modulate the expression rates of genes related to microglia-mediated neuroinflammation. Inhibiting bradykinin activity in Alzheimer's disease may slow disease progression.

Oxidative profile, inflammatory responses and δ-aminolevulinate dehydratase enzyme activity in influenza B virus infection.

The aim of the current study was to determine the activity of the delta-aminolevulinate dehydratase (δ-ALA-D) enzyme, oxidative stress biomarkers and the expression of cytokines in those infected with influenza B virus (IBV). To evaluate the activity of the δ-ALA-D enzyme, lipid peroxidation was estimated as levels of thiobarbituric acid reactive substances, protein and non-protein thiol groups, ferric-reducing antioxidant power (FRAP), vitamin C concentration and cytokine levels in IBV-infected individuals (n = 50) and a control group (n = 30). δ-ALA-D activity was significantly lower in IBV-infected individuals compared with controls, as well as levels of thiols, vitamin C and FRAP. Lipid peroxidation and cytokine levels of IL-6, IL-10, IL-17A and IFN-y were statistically higher in the IBV group. In conclusion, we found evidence of the generation of oxidants, the depletion of the antioxidant system, decrease in the activity of the δ-ALA-D enzyme and an increase in the synthesis of cytokines, thus contributing to a better understanding of oxidative and inflammatory pathways during IBV infection.

Use of recombinant S1 protein with hFc for analysis of SARS-CoV-2 adsorption and evaluation of drugs that inhibit entry into VERO E6 cells.

The significant number of deaths and infection caused by the new coronavirus SARS-CoV-2 has created an urgent demand for effective and readily available drugs for the treatment of COVID-19. However, the requirements for biosafety level 3 (NB-3) laboratories for experiments with the virus has made it very challenging for such research to meet this demand. It is known that angiotensin-converting enzyme 2 (ACE2), located on the surface of host cells, serves as the viral receptor for the spike (S) protein of SARS-CoV-2. This protein is a tetramer subdivided into S1 and S2 regions, with the former containing the receptor-binding domain (RBD). Therefore, drugs that interfere with the interaction between the spike and the receptor (as well as accessory proteins) or suppress their expression could inhibit the entry and spread of SARS-CoV-2 between cells. In this context, we standardized the use of recombinant SARS-CoV-2 S1 Protein with hFc (human Fc) for the analysis of binding in VERO E6 cells by flow cytometry, aiming to provide a new tool for identifying drugs and neutralizing antibodies, thus eliminating the need for NB-3 laboratories. Because minocycline (MCL), nimesulide (NMS), and berberine (BBR) have effects related to the ACE2 receptor, inhibit inflammation, and do not suppress the adaptive immune response (crucial for patient recovery), we investigated whether these drugs prevent the absorption of the spike protein into the host cell. For this purpose, we used VERO E6 cells under control conditions, pre-treated with these drugs and exposed to recombinant SARS-CoV-2 S1 Protein with hFC. We found that an exposure time of 30 min and a concentration of 10 µg/mL of spike S1 caused a strong signal detected by flow cytometry, using the secondary anti-hFc antibody conjugated with Alexa Fluor 647. Pre-treatment of cells with BBR for 30 min suppressed the signal from spike-positive cells, suggesting that this alkaloid interferes with spike adsorption on ACE2. The pre-incubation of spike protein with BBR did not alter its adsorption and internalization, indicating that BBR does not directly interact with spike protein. The ACE2 inactivation with a specific antibody inhibited spike protein adsorption and internalization. Furthermore, the pharmacological treatments did not alter the expression of ACE2. Exposure to spike protein increased IFNγ levels and the treatments with MCL and NMS were effective in inhibiting this increase. Taken together, we standardized a technique for analyzing the adsorption of SARS-CoV-2 and studying molecules that inhibit this process. Additionally, we demonstrated that BBR blocks spike entry bypre-binding to the host cell,and that the ACE2 receptor inactivation prevents Spike protein adsorption and penetration into cells.

Differentiated Embryonic Neurospheres from Familial Alzheimer's Disease Model Show Innate Immune and Glial Cell Responses.

Proteins involved in the Alzheimer's disease (AD), such as amyloid precursor protein (APP) and presenilin-1 (PS1), play critical roles in early development of the central nervous system (CNS), as well as in innate immune and glial cell responses. Familial AD is associated with the presence of APPswe and PS1dE9 mutations. However, it is still unknown whether these mutations cause deficits in CNS development of carriers. We studied genome-wide gene expression profiles of differentiated neural progenitor cells (NPCs) from wild-type and APPswe/PS1dE9 mouse embryo telencephalon. The occurrence of strong innate immune and glial cell responses in APPswe/PS1dE9 neurospheres mainly involves microglial activation, inflammatory mediators and chemokines. APPswe/PS1dE9 neurospheres augmented up to 100-fold CCL12, CCL5, CCL3, C3, CX3CR1, TLR2 and TNF-alpha expression levels, when compared to WT neurospheres. Expression levels of the glia cell marker GFAP and microglia marker Iba-1 were up to 20-fold upregulated in APPswe/PS1dE9 neurospheres. The secretome of differentiated APPswe/PS1dE9 NPCs revealed enhanced chemoattraction of peripheral blood mononuclear cells. When evaluating the inferred protein interaction networks constructed from the array data, an improvement in astrocyte differentiation in APPswe/PS1dE9 neurospheres was evident in view of increased GFAP expression. Transgenic NPCs differentiated into neural phenotypes presented expression patterns of cytokine, glial cells, and inflammatory mediators characteristic of APPswe/PS1dE9 adult animals. Consequently, the neurogenic niche obtained from differentiation of embryonic APPswe/PS1dE9 neurospheres spontaneously presents several alterations observed in adult AD brains. Finally, our data strengthen pathophysiological hypotheses that propose an early neurodevelopmental origin for familial AD.

Kinin B1 and B2 receptors mediate cancer pain associated with both the tumor and oncology therapy using aromatase inhibitors.

Pain caused by the tumor or aromatase inhibitors (AIs) is a disabling symptom in breast cancer survivors. Their mechanisms are unclear, but pro-algesic and inflammatory mediators seem to be involved. Kinins are endogenous algogenic mediators associated with various painful conditions via B1 and B2 receptor activation, including chemotherapy-induced pain and breast cancer proliferation. We investigate the involvement of the kinin B1 and B2 receptors in metastatic breast tumor (4T1 breast cancer cells)-caused pain and in aromatase inhibitors (anastrozole or letrozole) therapy-associated pain. A protocol associating the tumor and antineoplastic therapy was also performed. Kinin receptors' role was investigated via pharmacological antagonism, receptors protein expression, and kinin levels. Mechanical and cold allodynia and muscle strength were evaluated. AIs and breast tumor increased kinin receptors expression, and tumor also increased kinin levels. AIs caused mechanical allodynia and reduced the muscle strength of mice. Kinin B1 (DALBk) and B2 (Icatibant) receptor antagonists attenuated these effects and reduced breast tumor-induced mechanical and cold allodynia. AIs or paclitaxel enhanced breast tumor-induced mechanical hypersensitivity, while DALBk and Icatibant prevented this increase. Antagonists did not interfere with paclitaxel's cytotoxic action in vitro. Thus, kinin B1 or B2 receptors can be a potential target for treating the pain caused by metastatic breast tumor and their antineoplastic therapy.

Perspectives of purinergic signaling in stem cell differentiation and tissue regeneration.

Replacement of lost or dysfunctional tissues by stem cells has recently raised many investigations on therapeutic applications. Purinergic signaling has been shown to regulate proliferation, differentiation, cell death, and successful engraftment of stem cells originated from diverse origins. Adenosine triphosphate release occurs in a controlled way by exocytosis, transporters, and lysosomes or in large amounts from damaged cells, which is then subsequently degraded into adenosine. Paracrine and autocrine mechanisms induced by immune responses present critical factors for the success of stem cell therapy. While P1 receptors generally exert beneficial effects including anti-inflammatory activity, P2 receptor-mediated actions depend on the subtype of stimulated receptors and localization of tissue repair. Pro-inflammatory actions and excitatory tissue damages mainly result from P2X7 receptor activation, while other purinergic receptor subtypes participate in proliferation and differentiation, thereby providing adequate niches for stem cell engraftment and novel mechanisms for cell therapy and endogenous tissue repair. Therapeutic applications based on regulation of purinergic signaling are foreseen for kidney and heart muscle regeneration, Clara-like cell replacement for pulmonary and bronchial epithelial cells as well as for induction of neurogenesis in case of neurodegenerative diseases.