An observational study investigating soluble immune checkpoints as indicators of severe COVID-19.

This study aimed to investigate the immunomodulatory behavior of soluble immune checkpoints (sICPs) and other biomarkers in the pathophysiology of SARS-CoV-2 infection. The study included 59 adult participants, 43 of whom tested positive for SARS-CoV-2. Patients were divided into three cohorts: those with moderate disease (n = 16), recovered patients with severe disease (n = 13), and deceased patients with severe disease (n = 16). In addition, 16 participants were pre-pandemic subjects negative for SARS-CoV-2. The relative activity of neutralizing antibodies (rNAbs) against SARS-CoV-2 and the values of 14 sICPs in peripheral blood were compared between the four groups. Because the increase of markers values of inflammation [NLR > 12; CRP > 150 mg/L] and venous thromboembolism [D-dimer > 0.5 mg/L] has been associated with mortality from COVID-19, the total and differential leukocyte counts, the NLR, and CRP and D-dimer values were obtained in patients with severe disease. No differences in rNAbs were observed between the cohorts. Only the levels of five sICPs, sCD27, sHVEM sTIM-3, sPD-1, and sPDL-1, were significantly higher in patients with severe rather than moderate disease. The sPDL-2 level and NLR were higher in deceased patients than in recovered patients. However, there was no difference in CRP and D-dimer values between the two groups. Of the five soluble biomarkers compared among patients with severe disease, only sPDL-2 was higher in deceased patients than in recovered patients. This suggests that immuno-inhibitory sICPs might be used as indicators for severe COVID-19, with sPDL-2 used to assess individual risk for fatality.IMPORTANCECOVID-19, the disease caused by a SARS-CoV-2 infection, generates a broad spectrum of clinical symptoms, progressing to multiorgan failure in the most severe cases. As activation of the immune system is pivotal to eradicating the virus, future research should focus on identifying reliable biomarkers to efficiently predict the outcome in severe COVID-19 cases. Soluble immune checkpoints represent the function of the immune system and are easily determined in peripheral blood. This research could lead to implementing more effective severity biomarkers for COVID-19, which could increase patients' survival rate and quality of life.

Incubation in shaded hatcheries biases sex-determination but preserves Lepidochelys olivacea hatchling physiology.

Some studies have associated ex situ conservation with cerebral and gonadal developmental delay, as well as decreased motor performance in Lepidochelys olivacea offspring. Ex situ management is also related to a more mature spleen and a differential leukocyte count in newly emerged Lepidochelys olivacea hatchlings. The physiological relevance of a more mature spleen is unknown in sea turtles, but studies in birds suggest an increased immune response. Because egg relocation to hatcheries is a common conservation practice, it is imperative to know its impact on hatchling physiology. Herein, plasma activity of superoxide dismutase, alkaline phosphatase and the alternative complement pathway, as well as total antioxidant capacity and hydrogen peroxide concentrations were quantified in hatchlings from in situ and ex situ nests under basal conditions at nest emergence. Toll-like receptor 4 (tlr4), heat shock proteins (hsp) 70 and hsp90 expression were quantified in the spleen and liver of the hatchlings. Hepatocyte density and nuclear area were quantified in histological sections of the liver and all turtles were sexed by histological sectioning of the gonads. Total antioxidant capacity and hydrogen peroxide concentrations in plasma were lower in turtles from ex situ nests, while tlr4 and hsp70 mRNA expression was higher in the spleen but not in the liver. Ex situ incubation produced 98% male hatchlings, whereas in situ incubation produced 100% females. There were no other differences in the attributes sampled between hatchlings emerging from ex situ and in situ treatments. The results suggest that ex situ relocated turtles may be less prone to oxidative stress than in situ incubated hatchlings and could have more mature splenic function. Together, the data suggest that ex situ relocation to shaded hatcheries biased sex determination but preserved the general physiological condition of sea turtle hatchlings.

Response of lymphocytes from pigs naturally infected with porcine respiratory disease complex at 3 different stages of development.

The objective of this study was to analyze the response of lymphocytes from pigs naturally infected with porcine respiratory disease complex (PRDC) at 3 different stages of development. Porcine respiratory disease complexes were isolated from 2 groups: The infected group, consisting of pigs with PRDC and no vaccination against any virus (n = 24), and the control group, consisting of vaccinated and noninfected piglets (n = 24). Both groups were sampled at 3 stages of development: Weaning (WEA) (n = 8), initiation (INI) (n = 8), and growth (GRO) (n = 8). The PRDC status was confirmed by serological testing against porcine circovirus type 2 (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (H1N1), and Mycoplasma hyopneumoniae. PCV-2+ cells were quantified by flow cytometry. Weight gain was registered at each stage. PCV-2+ cells, CD4+ cells, monocytes and lymphocytes populations were measured. Gene expression in CD4+ cells was quantified for interferon-γ (IFN-γ), GATA binding protein 3 (GATA3), T-box transcription factor (T-bet), interleukin-10 (IL-10), and IL-4. Control piglets gained approximately 35% more weight than those infected with PRDC. Specifically, PCV-2+ cells were detected in piglets from the infected group in the following proportions: WEA ≤ INI ≤ GRO. In infected piglets, the CD4+ count increased at WEA and decreased at GRO, CD4+ expression profile showed an overexpression of T-bet at INI and GRO, and the expression of IFN-γ was lower at WEA and GRO. In contrast, IL-4 was overexpressed at all 3 stages. GATA3 was overexpressed at INI and GRO. The infected piglets showed lymphopenia and less CD4+ cells. CD4+ cells showed a different expression profile than the control group, in which IFN-γ was less expressed, whereas IL-4 and T-bet were overexpressed.

L'objectif de cette étude était d'analyser la réponse des lymphocytes de porcs naturellement infectés par le complexe respiratoire porcin (PRDC) à trois stades de développement différents. Des PRDC ont été isolés à partir de deux groupes : le groupe infecté, composé de porcs atteints de PRDC et non vaccinés contre un virus (n = 24), et le groupe témoin, composé de porcelets vaccinés et non infectés (n = 24). Les deux groupes ont été échantillonnés à trois stades de développement : sevrage (WEA) (n = 8), initiation (INI) (n = 8) et croissance (GRO) (n = 8). Le statut de PRDC a été confirmé par des tests sérologiques contre le circovirus porcin de type 2 (PCV-2), le virus du syndrome reproducteur et respiratoire porcin (PRRSV), le virus de la grippe porcine (H1N1) et Mycoplasma hyopneumoniae. Les cellules PCV-2+ ont été quantifiées par cytométrie en flux. Un gain de poids a été enregistré à chaque étape. Les populations de cellules PCV-2+, de cellules CD4+, de monocytes et de lymphocytes ont été mesurées. L'expression génique dans les cellules CD4+ a été quantifiée pour l'interféron-γ (IFN-γ), la protéine de liaison GATA 3 (GATA3), le facteur de transcription T-box (T-bet), l'interleukine-10 (IL-10) et l'IL-4. Les porcelets témoins ont pris environ 35 % de poids en plus que ceux infectés par le PRDC. Plus précisément, des cellules PCV-2+ ont été détectées chez les porcelets du groupe infecté dans les proportions suivantes : WEA ≤ INI ≤ GRO. Chez les porcelets infectés, le nombre de CD4+ a augmenté à WEA et diminué à GRO, le profil d'expression de CD4+ a montré une surexpression de T-bet à INI et GRO, et l'expression d'IFN-γ était plus faible à WEA et GRO. En revanche, l'IL-4 était surexprimée aux trois stades. GATA3 était surexprimé à INI et GRO. Les porcelets infectés présentaient une lymphopénie et moins de cellules CD4+. Les cellules CD4+ ont montré un profil d'expression différent de celui du groupe témoin, dans lequel l'IFN-γ était moins exprimé, tandis que l'IL-4 et le T-bet étaient surexprimés.(Traduit par Docteur Serge Messier).

Neuroendocrine Differentiation of Lung Cancer Cells Impairs the Activation of Antitumor Cytotoxic Responses in Mice.

Lung cancer has the highest mortality among all types of cancer; during its development, cells can acquire neural and endocrine properties that affect tumor progression by releasing several factors, some acting as immunomodulators. Neuroendocrine phenotype correlates with invasiveness, metastasis, and low survival rates. This work evaluated the effect of neuroendocrine differentiation of adenocarcinoma on the mouse immune system. A549 cells were treated with FSK (forskolin) and IBMX (3-Isobutyl-1-methylxanthine) for 96 h to induce neuroendocrine differentiation (NED). Systemic effects were assessed by determining changes in circulating cytokines and immune cells of BALB/c mice immunized with PBS, undifferentiated A549 cells, or neuroendocrine A549NED cells. A549 cells increased circulating monocytes, while CD4+CD8- and CD4+CD8+ T cells increased in mice immunized with neuroendocrine cells. IL-2 and IL-10 increased in mice that received untreated A549 cells, suggesting that the immune system mounts a regulated response against adenocarcinoma, which did not occur with A549NED cells. Cocultures demonstrated the cytotoxic capacity of PBMCs when confronted with A549 cells, while in the presence of neuroendocrine cells they not only were unable to show cytolytic activity, but also lost viability. Neuroendocrine differentiation seems to mount less of an immune response when injected in mice, which may contribute to the poor prognosis of cancer patients affected by this pathology.

Glycoprotein 5-Derived Peptides Induce a Protective T-Cell Response in Swine against the Porcine Reproductive and Respiratory Syndrome Virus.

We analyzed the T-cell responses induced by lineal epitopes of glycoprotein 5 (GP5) from PRRSV to explore the role of this protein in the immunological protection mediated by T-cells. The GP5 peptides were conjugated with a carrier protein for primary immunization and booster doses. Twenty-one-day-old pigs were allocated into four groups (seven pigs per group): control (PBS), vehicle (carrier), PTC1, and PTC2. Cytokine levels were measured at 2 days post-immunization (DPI) from serum samples. Cytotoxic T-lymphocytes (CTLs, CD8+) from peripheral blood were quantified via flow cytometry at 42 DPI. The cytotoxicity was evaluated by co-culturing primed lymphocytes with PRRSV derived from an infectious clone. The PTC2 peptide increased the serum concentrations of pro-inflammatory cytokines (i.e., TNF-α, IL-1ß, IL-8) and cytokines that activate the adaptive cellular immunity associated with T-lymphocytes (i.e., IL-4, IL-6, IL-10, and IL-12). The concentration of CTLs (CD8+) was significantly higher in groups immunized with the peptides, which suggests a proliferative response in this cell population. Primed CTLs from immunized pigs showed cytolytic activity in PRRSV-infected cells in vitro. PTC1 and PTC2 peptides induced a protective T-cell-mediated response in pigs immunized against PRRSV, due to the presence of T epitopes in their sequences.

Ultrastructural changes associated to the neuroendocrine transdifferentiation of the lung adenocarcinoma cell line A549.

The neuroendocrine transdifferentiation has been found in many cancer cell types, such as prostate, lung and gastrointestinal cells and is accompanied by a lower patient life expectancy. The transdifferentiation process has been induced in vitro by the exposure to different stimuli in human lung adenocarcinoma. The aim of this work was to identify the morphological characteristics of the neuroendocrine phenotype in a human lung cancer cell line, induced by two cAMP elevating agents (IBMX and FSK). Our results showed two phenotypes, one produced by IBMX with higher volume, cell size and increased number of secondary projections, and the other produced by FSK with higher area, roughness of the membrane, cell neurite percentage, number of outgrowths per cell and increased number of primary projections. In conclusion, we describe some morphological and ultrastructural characteristics of the neuroendocrine phenotype in A549 human lung cancer cell line promoted by IBMX and FSK to contribute to the understanding of the autocrine or paracrine signaling within the tumor microenvironment.

Behavior of Eosinophil Counts in Recovered and Deceased COVID-19 Patients over the Course of the Disease.

Knowledge about the immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, particularly regarding the function of eosinophils, has been steadily emerging recently. There exists controversy regarding the implications of eosinophils in the coronavirus disease 2019 (COVID-19)'s pathology. We report a retrospective cohort study including the comparison of leukocyte counts in COVID-19 patients, considering the outcomes of recovery (n = 59) and death (n = 60). Among the different types of leukocytes, the eosinophil counts were those that showed the greatest difference between recovered and deceased patients. Eosinopenia (eosinophil count < 0.01 × 109/L) was more frequently observed in deceased than recovered patients (p = 0.0012). The eosinophil counts more rapidly increased and showed a greater proportion over the course of the disease in the recovered than deceased patients. Furthermore, the estimated survival rate was greater in patients without eosinopenia than in patients with eosinopenia (p = 0.0070) during hospitalization. Importantly, recovered but not deceased patients showed high negative correlations of the eosinophils with the neutrophil-to-lymphocyte ratio (NLR) and neutrophil counts at Day 9 of the onset of clinical symptoms (p ≤ 0.0220). Our analysis suggests that eosinopenia may be associated with unfavorable disease outcomes and that the eosinophils have a beneficial function in COVID-19 patients, probably contributing by controlling the exacerbated inflammation induced by neutrophils.

Toxicity and antimicrobial effect of silver nanoparticles in swine sperms.

Bacterial contamination in swine semen affects the quality and longevity of sperm and consequently fertility is reduced. Antibiotics have been used to prevent bacterial growth, but the frequency of bacterial resistance to various antibiotics are increasing. Silver nanoparticles (AgNPs) of 10-20 nm in size have shown a biocide effect in bacteria and fungi microorganisms without toxicity to certain mammalian cells. The goal of this study was to analyze both, antimicrobial activity against Staphylococcus aureus and toxicity in swine sperms after 10-20 nm AgNPs treatment. S. aureus proliferation decreased when concentrations from 0.4 to 10 mM AgNPs were assayed. Also, sperm viability measured by mitochondrial metabolism after AgNPs treatment up to a concentration of 10 mM, was viable. In addition, viability determined by membrane integrity of sperms showed that AgNPs treatment up to a concentration of 10 mM was safe. Sperm morphology was evaluated by automated quantification of proximal and distal drops and whiptails. Data indicated that AgNPs treatment up to a concentration of 4 mM were harmless. Finally, sperm capacitation and acrosome reactions were determined by (chlortetracycline) CTC assay. Data showed that no changes in sperm capacitation were observed when sperms were treated with 2 mM of AgNPs, but data showed increased calcium mobilization when treated with 10 mM AgNPs, which suggested sperm capacitation. Finally, there were no significant changes encountered on sperm acrosome reaction for any of the treatments after AgNPs treatment. Taken together, these results show the potential of AgNPs as an alternative to conventional antimicrobial agents that are currently used in extenders to preserve semen required for storage. ABBREVIATIONS: AgNPs: silver nanoparticles; AMK: amikacin; AMP: adenosine monophosphate; AR: acrosome reaction; C: capacitation; CF: cefallotin; CFU: colony-forming unit; CTC: chlortetracycline; CXM: cefuroxime; DMSO: dimethyl sulfoxide; NC: non-capacitation; NOM: Norma Oficial Mexicana; PBS: phosphate buffered saline; RLUs: relative light units; ROS: reactive oxygen species; SQS: Seminal Quality System.

Effect of A549 neuroendocrine differentiation on cytotoxic immune response.

The present study was designed to determine the effects of factors secreted by the lung adenocarcinoma cell line with the neuroendocrine phenotype, A549NED, on cytotoxic T lymphocytes (CTLs) activity in vitro A perspective that integrates the nervous, endocrine and immune system in cancer research is essential to understand the complexity of dynamic interactions in tumours. Extensive clinical research suggests that neuroendocrine differentiation (NED) is correlated with worse patient outcomes; however, little is known regarding the effects of neuroendocrine factors on the communication between the immune system and neoplastic cells. The human lung cancer cell line A549 was induced to NED (A549NED) using cAMP-elevating agents. The A549NED cells showed changes in cell morphology, an inhibition of proliferation, an overexpression of chromogranin and a differential pattern of biogenic amine production (decreased dopamine and increased serotonin [5-HT] levels). Using co-cultures to determine the cytolytic CTLs activity on target cells, we showed that the acquisition of NED inhibits the decrease in the viability of the target cells and release of fluorescence. Additionally, the conditioned medium of A549NED and 5-HT considerably decreased the viability and proliferation of the Jurkat cells after 24 h. Thus, our study successfully generated a neuroendocrine phenotype from the A549 cell line. In co-cultures with CTLs, the pattern of secretion by A549NED impaired the proliferation and cytotoxic activity of CTLs, which might be partly explained by the increased release of 5-HT.

Toxicology of antimicrobial nanoparticles for prosthetic devices.

Advances in nanotechnology are producing an accelerated proliferation of new nanomaterial composites that are likely to become an important source of engineered health-related products. Nanoparticles with antifungal effects are of great interest in the formulation of microbicidal materials. Fungi are found as innocuous commensals and colonize various habitats in and on humans, especially the skin and mucosa. As growth on surfaces is a natural part of the Candida spp. lifestyle, one can expect that Candida organisms colonize prosthetic devices, such as dentures. Macromolecular systems, due to their properties, allow efficient use of these materials in various fields, including the creation of reinforced nanoparticle polymers with antimicrobial activity. This review briefly summarizes the results of studies conducted during the past decade and especially in the last few years focused on the toxicity of different antimicrobial polymers and factors influencing their activities, as well as the main applications of antimicrobial polymers in dentistry. The present study addresses aspects that are often overlooked in nanotoxicology studies, such as careful time-dependent characterization of agglomeration and ion release.

Requirement of phosphorylatable endothelial nitric oxide synthase at Ser-1177 for vasoinhibin-mediated inhibition of endothelial cell migration and proliferation in vitro.

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide is a major vasorelaxing factor and a mediator of vasopermeability and angiogenesis. Vasoinhibins, a family of antiangiogenic prolactin fragments that include 16 K prolactin, block most eNOS-mediated vascular effects. Vasoinhibins activate protein phosphatase 2A, causing eNOS inactivation through dephosphorylation of eNOS at serine residue 1179 in bovine endothelial cells and thereby blocking vascular permeability. In this study, we examined whether human eNOS phosphorylation at S1177 (analogous to bovine S1179) influences other actions of vasoinhibins. Bovine umbilical vein endothelial cells were stably transfected with human wild-type eNOS (WT) or with phospho-mimetic (S1177D) or non-phosphorylatable (S1177A) eNOS mutants. Vasoinhibins inhibited the increases in eNOS activity, migration, and proliferation following the overexpression of WT eNOS but did not affect these responses in cells expressing S1177D and S1177A eNOS mutants. We conclude that eNOS inhibition by dephosphorylation of S1177 is fundamental for the inhibition of endothelial cell migration and proliferation by vasoinhibins.

Cytocompatible antifungal acrylic resin containing silver nanoparticles for dentures.

BACKGROUND: Inhibition of Candida albicans on denture resins could play a significant role in preventing the development of denture stomatitis. The safety of a new dental material with antifungal properties was analyzed in this work. METHODS: Poly(methyl methacrylate) [PMMA] discs and PMMA-silver nanoparticle discs were formulated, with the commercial acrylic resin, Nature-Cryl™, used as a control. Silver nanoparticles were synthesized and characterized by ultraviolet-visible spectroscopy, dispersive Raman spectroscopy, and transmission electron microscopy. The antifungal effect was assessed using a luminescent microbial cell viability assay. Biocompatibility tests were carried out using NIH-3T3 mouse embryonic fibroblasts and a Jurkat human lymphocyte cell line. Cells were cultured for 24 or 72 hours in the presence or absence of the polymer formulations and analyzed using three different tests, ie, cellular viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell proliferation by enzyme-linked immunosorbent assay BrdU, and genomic DNA damage (Comet assay). Finally, the samples were evaluated mechanically, and the polymer-bearing silver nanoparticles were analyzed microscopically to evaluate dispersion of the nanoparticles. RESULTS: The results show that PMMA-silver nanoparticle discs significantly reduce adherence of C. albicans and do not affect metabolism or proliferation. They also appear not to cause genotoxic damage to cells. CONCLUSION: The present work has developed a new biocompatible antifungal PMMA denture base material.