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.

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).

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.

Bacterial and parasite co-infection in Mexican golden trout (Oncorhynchus chrysogaster) by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator.

BACKGROUND: Bacterial infections are responsible of high economic losses in aquaculture. Mexican golden trout (Oncorhynchus chrysogaster) is a threatened native trout species that has been introduced in aquaculture both for species conservation and breeding for production and for which no studies of bacterial infections have been reported. CASE PRESENTATION: Fish from juvenile stages of Mexican golden trout showed an infectious outbreak in a farm in co-culture with rainbow trout (Oncorhynchus mykiss), showing external puntiform red lesions around the mouth and caudal pedunculus resembling furuncles by Aeromonas spp. and causing an accumulated mortality of 91%. Isolation and molecular identification of bacteria from lesions and internal organs showed the presence of Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator isolated from a single individual. All bacterial isolates were resistant to amoxicillin-clavulanic acid and cefazoline. P. shigelloides was resistant to third generation ß-lactamics. CONCLUSIONS: This is the first report of coinfection by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator in an individual of Mexican golden trout in co-culture with rainbow trout. Resistance to ß-lactams suggests the acquisition of genetic determinants from water contamination by human- or livestock-associated activities.

Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutive enzyme implicated in the regulation of cytokine expression and the inflammatory response during bacterial infections. Mammals have two GSK3 isoforms named GSK3α and GSK3ß that plays different but often overlapping functions. Although the role of GSK3ß in cytokine regulation during the inflammatory response caused by bacteria is well described, GSK3α has not been found to participate in this process. Therefore, we tested if GSK3α may act as a regulatory isoform in the cytokine expression by bovine endothelial cells infected with Staphylococcus aureus because this bacterium is one of the major pathogens that cause tissue damage associated with inflammatory dysfunction. Interestingly, although both isoforms were phosphorylated-inactivated, we consistently observed a higher phosphorylation of GSK3α at Ser21 than that of GSK3ß at Ser9 after bacterial challenge. During a temporal course of infection, we characterized a molecular switch from pro-inflammatory cytokine expression (IL-8), promoted by nuclear factor-kappa B (NF-κB), at an early stage (2 h) to an anti-inflammatory cytokine expression (IL-10), promoted by cAMP response element binding (CREB), at a later stage (6 h). We observed an indirect effect of GSK3α activity on NF-κB activation that resulted in a low phosphorylation of CREB at Ser133, a decreased interaction between CREB and the co-activator CREB-binding protein (CBP), and a lower expression level of IL-10. Gene silencing of GSK3α and GSK3ß with siRNA indicated that GSK3α knockout promoted the interaction between CREB and CBP that, in turn, increased the expression of IL-10, reduced the interaction of NF-κB with CBP, and reduced the expression of IL-8. These results indicate that GSK3α functions as the primary isoform that regulates the expression of IL-10 in endothelial cells infected with S. aureus.

The CSL proteins, versatile transcription factors and context dependent corepressors of the notch signaling pathway.

The Notch signaling pathway is a reiteratively used cell to cell communication pathway that triggers pleiotropic effects. The correct regulation of the pathway permits the efficient regulation of genes involved in cell fate decision throughout development. This activity relies notably on the CSL proteins, (an acronym for CBF-1/RBPJ-κ in Homo sapiens/Mus musculus respectively, Suppressor of Hairless in Drosophila melanogaster, Lag-1 in Caenorhabditis elegans) which is the unique transcription factor and DNA binding protein involved in this pathway. The CSL proteins have the capacity to recruit activation or repression complexes according to the cellular context. The aim of this review is to describe the different co-repressor proteins that interact directly with CSL proteins to form repression complexes thereby regulating the Notch signaling pathway in animal cells to give insights into the paralogous evolution of these co-repressors in higher eumetazoans and their subsequent effects at developmental processes.

The Glycogen Synthase Kinase 3α and ß Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3ß is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3ß activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3ß at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3ß. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3ß with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3ß. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3ß gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

Collaborative action of Toll-like and NOD-like receptors as modulators of the inflammatory response to pathogenic bacteria.

Early sensing of pathogenic bacteria by the host immune system is important to develop effective mechanisms to kill the invader. Microbial recognition, activation of signaling pathways, and effector mechanisms are sequential events that must be highly controlled to successfully eliminate the pathogen. Host recognizes pathogens through pattern-recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs). Some of these PRRs include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), retinoic acid-inducible gene-I- (RIG-I-) like receptors (RLRs), and C-type lectin receptors (CLRs). TLRs and NLRs are PRRs that play a key role in recognition of extracellular and intracellular bacteria and control the inflammatory response. The activation of TLRs and NLRs by their respective ligands activates downstream signaling pathways that converge on activation of transcription factors, such as nuclear factor-kappaB (NF-κB), activator protein-1 (AP-1) or interferon regulatory factors (IRFs), leading to expression of inflammatory cytokines and antimicrobial molecules. The goal of this review is to discuss how the TLRs and NRLs signaling pathways collaborate in a cooperative or synergistic manner to counteract the infectious agents. A deep knowledge of the biochemical events initiated by each of these receptors will undoubtedly have a high impact in the design of more effective strategies to control inflammation.

Evaluation of DNA extraction methods of rumen microbial populations.

The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.

Role of glycogen synthase kinase-3 beta in the inflammatory response caused by bacterial pathogens.

Glycogen synthase kinase 3ß (GSK3ß) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3ß may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3ß and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

The phosphoinositide-3-kinase-Akt signaling pathway is important for Staphylococcus aureus internalization by endothelial cells.

Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)-Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3ß on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3ß, and NF-κB.

Gluconacetobacter diazotrophicus levansucrase is involved in tolerance to NaCl, sucrose and desiccation, and in biofilm formation.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane, which expresses levansucrase, a fructosyltransferase exoenzyme with sucrose hydrolytic and levan biosynthetic activities. As a result of their physical properties, the levan can provide protection against stress caused by abiotic or biotic factors and participate in the formation of biofilms. In this study, we investigated the construction and function of a levansucrase-defective mutant of G. diazotrophicus. The lsdA mutant showed a decreased tolerance (65.5%) to 50-150 mM NaCl and a decrease of 89% in 876 mM (30%) sucrose, a reduction (99%) in tolerance to desiccation after 18 h, and a decrease (36.9-58.5%) in the ability to form cell aggregates on abiotic surfaces. Complementation of the mutant with the complete lsdA gene leads to a recovery of the ability to grow on sucrose-containing medium and to form slimy colonies, the ability to form the cell aggregates on abiotic surfaces and the tolerance to NaCl. This report demonstrates the importance of levansucrase in environmental adaptation of G. diazotrophicus under high osmotic stress and in biofilm formation.

TNF-alpha reduces the level of Staphylococcus epidermidis internalization by bovine endothelial cells.

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha). Therefore, this work was designed to explore the effect of TNF-alpha on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-alpha caused a marked reduction in the number of intracellular S. epidermidis, suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-kappaB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-kappaB inhibitors, SN50 or BAY 11-7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis. These results suggest that activation of the signaling pathway initiated by TNF-alpha could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-kappaB was shown not to be important for the internalization process of S. epidermidis.

Structural analysis of point mutations in the Hairless gene and their association with the activity of the Hairless protein.

The Notch signaling pathway (NSP) is an important intercellular communication mechanism that regulates embryo development and adult physiological functions. The Hairless (H) protein is a powerful antagonist of the NSP by its interaction with the Suppressor of Hairless (Su[H]) protein, recruiting the corepressors Gro and CtBP. In the present work, we examined the role of several important amino acids in different H protein domains analyzing four mutant lines of Drosophila melanogaster. The mutant alleles were evaluated by single-strand conformational polymorphism (SSCP) analysis and we located mutated regions at different positions along the sequence of the Hairless gene.

The capacity of bovine endothelial cells to eliminate intracellular Staphylococcus aureus and Staphylococcus epidermidis is increased by the proinflammatory cytokines TNF-alpha and IL-1beta.

Staphylococcus aureus is a pathogenic bacterium causing clinical and subclinical bovine mastitis. Infections of the udder by S. aureus are frequently associated with the presence of Staphylococcus epidermidis, an opportunistic pathogen. We reported previously that the capacity of bovine endothelial cells (BEC) to endocytize S. aureus is associated with the activation of NF-kappaB and modulated by the proinflammatory cytokines TNF-alpha and IL-1beta. In this work, we explore the ability of BEC to eliminate intracellular S. aureus and S. epidermidis and their response to these cytokines. Time-kinetics survival experiments indicated that BEC eliminate intracellular S. epidermidis more efficiently. Replication of S. aureus, but not S. epidermidis, inside BEC was evident by an increase in intracellular bacteria recovered at 2 h postinfection. Afterwards, the intracellular number of staphylococci decreased gradually, reaching the lowest value at 24 h. Treatment of BEC with TNF-alpha or IL-1beta potentiated the capacity of BEC to eliminate both Staphylococcus species at the times tested. These results indicate that activation of the intrinsic antistaphylococcal response in BEC, enhanced by TNF-alpha and IL-1beta, is effective to eliminate S. aureus and S. epidermidis and suggest that endothelial cells may play a prominent role in the defense against infections caused by these bacteria.

Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis.

Mastitis (mammary gland inflammation) is one of the most important bovine diseases causing economic losses to dairy producers. Mammary gland inflammation is a consequence of the activity of a number of cell and soluble factors that function together to eliminate invading microorganisms. The factors involved in this inflammatory response differ depending on the infectious agent. This review analyzes the factors involved in the immunologic mechanisms against the main pathogenic bacteria causing mastitis, and emphasizes the innate immune response of the mammary gland. Knowledge, at the molecular level, of the mammary gland immune response during infection by pathogenic bacteria is fundamental to the design of effective therapies to control and eradicate bovine mastitis.

Hairless-mediated repression of notch target genes requires the combined activity of Groucho and CtBP corepressors.

Notch signal transduction centers on a conserved DNA-binding protein called Suppressor of Hairless [Su(H)] in Drosophila species. In the absence of Notch activation, target genes are repressed by Su(H) acting in conjunction with a partner, Hairless, which contains binding motifs for two global corepressors, CtBP and Groucho (Gro). Usually these corepressors are thought to act via different mechanisms; complexed with other transcriptional regulators, they function independently and/or redundantly. Here we have investigated the requirement for Gro and CtBP in Hairless-mediated repression. Unexpectedly, we find that mutations inactivating one or the other binding motif can have detrimental effects on Hairless similar to those of mutations that inactivate both motifs. These results argue that recruitment of one or the other corepressor is not sufficient to confer repression in the context of the Hairless-Su(H) complex; Gro and CtBP need to function in combination. In addition, we demonstrate that Hairless has a second mode of repression that antagonizes Notch intracellular domain and is independent of Gro or CtBP binding.