A mimetic peptide of ACE2 protects against SARS-CoV-2 infection and decreases pulmonary inflammation related to COVID-19.

Since human angiotensin-converting enzyme 2 (ACE2) serves as a primary receptor for SARS-CoV-2, characterizing ACE2 regions that allow SARS-CoV-2 to enter human cells is essential for designing peptide-based antiviral blockers and elucidating the pathogenesis of the virus. We identified and synthesized a 25-mer mimetic peptide (encompassing positions 22-46 of the ACE2 alpha-helix α1) implicated in the S1 receptor-binding domain (RBD)-ACE2 interface. The mimetic (wild-type, WT) ACE2 peptide significantly inhibited SARS-CoV-2 infection of human pulmonary Calu-3 cells in vitro. In silico protein modeling predicted that residues F28, K31, F32, F40, and Y41 of the ACE2 alpha-helix α1 are critical for the original, Delta, and Omicron strains of SARS-CoV-2 to establish the Spike RBD-ACE2 interface. Substituting these residues with alanine (A) or aspartic acid (D) abrogated the antiviral protective effect of the peptides, indicating that these positions are critical for viral entry into pulmonary cells. WT ACE2 peptide, but not the A or D mutated peptides, exhibited significant interaction with the SARS-CoV-2 S1 RBD, as shown through molecular dynamics simulations. Through identifying the critical amino acid residues of the ACE2 alpha-helix α1, which is necessary for the Spike RBD-ACE2 interface and mobilized during the in vitro viral infection of cells, we demonstrated that the WT ACE2 peptide protects susceptible K18-hACE2 mice against in vivo SARS-CoV-2 infection and is effective for the treatment of COVID-19.

STING recognition of viral dsDNA by nociceptors mediates pain in mice.

Pain is often one of the initial indicators of a viral infection, yet our understanding of how viruses induce pain is limited. Immune cells typically recognize viral nucleic acids, which activate viral receptors and signaling, leading to immunity. Interestingly, these viral receptors and signals are also present in nociceptors and are associated with pain. Here, we investigate the response of nociceptors to nucleic acids during viral infections, specifically focusing on the role of the viral signal, Stimulator of Interferon Genes (STING). Our research shows that cytosolic double-stranded DNA (dsDNA) from viruses, like herpes simplex virus 1 (HSV-1), triggers pain responses through STING expression in nociceptors. In addition, STING agonists alone can elicit pain responses. Notably, these responses involve the direct activation of STING in nociceptors through TRPV1. We also provided a proof-of-concept showing that STING and TRPV1 significantly contribute to the mechanical hypersensitivity induced by HSV-1 infection. These findings suggest that STING could be a potential therapeutic target for relieving pain during viral infections.

Probing the cw-Laser-Induced Fluorescence Enhancement in CsPbBr3 Nanocrystal Thin Films: An Interplay between Photo and Thermal Activation.

Perovskite nanocrystals hold significant promise for a wide range of applications, including solar cells, LEDs, photocatalysts, humidity and temperature sensors, memory devices, and low-cost photodetectors. Such technological potential stems from their exceptional quantum efficiency and charge carrier conduction capability. Nevertheless, the underlying mechanisms of photoexcitation, such as phase segregation, annealing, and ionic diffusion, remain insufficiently understood. In this context, we harnessed hyperspectral fluorescence microspectroscopy to advance our comprehension of fluorescence enhancement triggered by UV continuous-wave (cw) laser irradiation of CsPbBr3 colloidal nanocrystal thin films. Initially, we explored the kinetics of fluorescence enhancement and observed that its efficiency (φph) correlates with the laser power (P), following the relationship φph = 7.7⟨P⟩0.47±0.02. Subsequently, we estimated the local temperature induced by the laser, utilizing the finite-difference method framework, and calculated the activation energy (Ea) required for fluorescence enhancement to occur. Our findings revealed a very low activation energy, Ea ∼ 9 kJ/mol. Moreover, we mapped the fluorescence photoenhancement by spatial scanning and real-time static mode to determine its microscale length. Below a laser power of 60 µW, the photothermal diffusion length exhibited nearly constant values of approximately (22 ± 5) µm, while a significant increase was observed at higher laser power levels. These results were ascribed to the formation of nanocrystal superclusters within the film, which involves the interparticle spacing reduction, creating the so-called quantum dot solid configuration along with laser-induced annealing for higher laser powers.

Schwann cell TRPA1 elicits reserpine-induced fibromyalgia pain in mice.

BACKGROUND AND PURPOSE: Fibromyalgia is a complex clinical disorder with an unknown aetiology, characterized by generalized pain and co-morbid symptoms such as anxiety and depression. An imbalance of oxidants and antioxidants is proposed to play a pivotal role in the pathogenesis of fibromyalgia symptoms. However, the precise mechanisms by which oxidative stress contributes to fibromyalgia-induced pain remain unclear. The transient receptor potential ankyrin 1 (TRPA1) channel, known as both a pain sensor and an oxidative stress sensor, has been implicated in various painful conditions. EXPERIMENTAL APPROACH: The feed-forward mechanism that implicates reactive oxygen species (ROS) driven by TRPA1 was investigated in a reserpine-induced fibromyalgia model in C57BL/6J mice employing pharmacological interventions and genetic approaches. KEY RESULTS: Reserpine-treated mice developed pain-like behaviours (mechanical/cold hypersensitivity) and early anxiety-depressive-like disorders, accompanied by increased levels of oxidative stress markers in the sciatic nerve tissues. These effects were not observed upon pharmacological blockade or global genetic deletion of the TRPA1 channel and macrophage depletion. Furthermore, we demonstrated that selective silencing of TRPA1 in Schwann cells reduced reserpine-induced neuroinflammation (NADPH oxidase 1-dependent ROS generation and macrophage increase in the sciatic nerve) and attenuated fibromyalgia-like behaviours. CONCLUSION AND IMPLICATIONS: Activated Schwann cells expressing TRPA1 promote an intracellular pathway culminating in the release of ROS and recruitment of macrophages in the mouse sciatic nerve. These cellular and molecular events sustain mechanical and cold hypersensitivity in the reserpine-evoked fibromyalgia model. Targeting TRPA1 channels on Schwann cells could offer a novel therapeutic approach for managing fibromyalgia-related behaviours.

A novel diselenide attenuates the carrageenan-induced inflammation by reducing neutrophil infiltration and the resulting tissue damage in mice.

Selenium-containing compounds have emerged as promising treatment for redox-based and inflammatory diseases. This study aimed to investigate the in vitro and in vivo anti-inflammatory activity of a novel diselenide named as dibenzyl[diselanediyIbis(propane-3-1diyl)] dicarbamate (DD). DD reacted with HOCl (k = 9.2 x 107 M-1s-1), like glutathione (k = 1.2 x 108 M-1s-1), yielding seleninic and selenonic acid derivatives, and it also decreased HOCl formation by activated human neutrophils (IC50=4.6 µM) and purified myeloperoxidase (MPO) (IC50=3.8 µM). However, tyrosine, MPO-I and MPO-II substrates, did not restore HOCl formation in presence of DD. DD inhibited the oxidative burst in dHL-60 cells with no toxicity up to 25 µM for 48h. Next, an intraperitoneal administration of 25, 50, and 75 mg/kg DD decreased total leukocyte, neutrophil chemotaxis, and inflammation markers (MPO activity, lipid peroxidation, albumin exudation, nitrite, TNF-α, IL-1ß, CXCL1/KC, and CXCL2/MIP-2) on a murine model of carrageenan-induced peritonitis. Likewise, 50 mg/kg DD (i.p.) decreased carrageenan-induced paw edema over 5h. Histological and immunohistochemistry analyses of the paw tissue showed decreased neutrophil count, edema area, and MPO, carbonylated, and nitrated protein staining. Furthermore, DD treatment decreased the fMLP-induced chemotaxis of human neutrophils (IC50=3.7 µM) in vitro with no toxicity. Lastly, DD presented no toxicity in a single-dose model using mice (50 mg/kg, i.p.) over 15 days and in Artemia salina bioassay (50 to 2000 µM), corroborating findings from in silico toxicological study. Altogether, these results demonstrate that DD attenuates carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting damage from MPO-mediated oxidative burst.

[Ethics in the use of psychedelics: The definition of illicit drugs from the perspective of critical bioethics]. / Ética en el uso de psicodélicos: la definición de las drogas ilícitas bajo la óptica de la bioética crítica.

This essay, situated in the field of bioethics, examines the prohibition of psychedelic use, exploring arguments surrounding the growing evidence of their therapeutic potential and their millennia-long history of cultural and spiritual uses. It initially discusses the historical context of psychedelics and the various terms used to describe them. The essay problematizes the definition of "drugs," highlighting the lack of objective criteria for distinguishing between legal and illicit substances. Drawing on concepts and theoretical frameworks of critical bioethics, it analyzes how the prohibitionist moral discourse is sustained more by political and economic interests than by scientific justifications, leading to stigmatization and vulnerability. The essay advocates for the end of the prohibition of psychedelics based on ethical arguments, emphasizing their importance in reducing individual and collective suffering. The work contributes to a deeper reflection on this socially controversial topic, integrating interdisciplinary knowledge.

Este ensayo, ubicado en el campo de la bioética, analiza la prohibición del uso de psicodélicos, explorando argumentos sobre las crecientes evidencias de potenciales terapéuticos y su historia milenaria de usos culturales y espirituales. Discute inicialmente el contexto histórico de los psicodélicos y diferentes términos utilizados para nombrarlos. Problematiza la definición de "drogas", indicando falta de criterios objetivos para distinción entre lícitas e ilícitas. Bajo conceptos y referenciales teóricos de la bioética crítica, se analiza cómo el discurso moral prohibicionista se sostiene más por intereses políticos y económicos que por justificativas científicas, generando estigmatización y vulnerabilidad. Defiende el fin de la prohibición de los psicodélicos con base en argumentos éticos, resaltando su importancia para la reducción de sufrimientos individuales y colectivos. El trabajo contribuye a una reflexión profundizada sobre este tema socialmente controversial, articulando conocimientos interdisciplinarios.

Neutrophil extracellular traps mediate bone erosion in rheumatoid arthritis by enhancing RANKL-induced osteoclastogenesis.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. EXPERIMENTAL APPROACH: The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. KEY RESULTS: Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.

Profiles of interferon-stimulated genes in multiple tissues and circulating pregnancy-associated glycoproteins and their association with pregnancy loss in dairy cows†.

Pregnancy loss (PL) in lactating dairy cows disrupts reproductive and productive efficiency. We evaluated the expression of interferon-stimulated genes (ISG) in blood leukocytes, vaginal and cervical epithelial cells, luteolysis-related genes, progesterone, and pregnancy-associated glycoprotein (PAG) profiles in lactating dairy cows (n = 86) to gain insight about PL. Expression of ISG on d17, d19, and d21 was greater in cows that maintained the pregnancy (P33) compared to nonpregnant with no PL (NP). Greater ISG differences between groups were observed in the cervix (96.7-fold) than vagina (31.0-fold), and least in blood leukocytes (5.6-fold). Based on individual profiles of ISG and PAG, PL was determined to occur either before (~13%) or after (~25%) d22. For cows with PL before d22, ISG expression was similar on d17 but by d21 was lower and OXTR was greater than P33 cows and similar to NP; timing of luteolysis was similar compared to NP cows suggesting embryonic failure to promote luteal maintenance and to attach to the endometrium (no increase in PAG). For cows with PL after d22, ISG expression was similar to P33 cows on d17, d19, and d21 and luteolysis, when it occurred, was later than NP cows; delayed increase in PAG suggested later or inadequate embryonic attachment. In conclusion, PL before d22 occurred due to embryonic demise/failure to signal for luteal maintenance, as reflected in reduced ISG expression by d21. Alternatively, embryos with PL between d22 and 33 adequately signaled for luteal maintenance (ISG) but had delayed/inadequate embryonic attachment and/or inappropriate luteolysis causing PL.

Paediatric sepsis survivors are resistant to sepsis-induced long-term immune dysfunction.

BACKGROUND AND PURPOSE: Sepsis-surviving adult individuals commonly develop immunosuppression and increased susceptibility to secondary infections, an outcome mediated by the axis IL-33/ILC2s/M2 macrophages/Tregs. Nonetheless, the long-term immune consequences of paediatric sepsis are indeterminate. We sought to investigate the role of age in the genesis of immunosuppression following sepsis. EXPERIMENTAL APPROACH: Here, we compared the frequency of Tregs, the activation of the IL-33/ILC2s axis in M2 macrophages and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. Likewise, sepsis-surviving mice were inoculated intranasally with Pseudomonas aeruginosa or by subcutaneous inoculation of the B16 melanoma cell line. Finally, blood samples from sepsis-surviving patients were collected and the concentration of IL-33 and Tregs frequency were assessed. KEY RESULTS: In contrast to 6-week-old mice, 2-week-old mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistically, increased IL-33 levels, Tregs expansion, and activation of ILC2s and M2-macrophages were observed in 6-week-old but not 2-week-old post-septic mice. Moreover, impaired IL-33 production in 2-week-old post-septic mice was associated with increased DNA methylation in lung epithelial cells. Notably, IL-33 treatment boosted the expansion of Tregs and induced immunosuppression in 2-week-old mice. Clinically, adults but not paediatric post-septic patients exhibited higher counts of Tregs and seral IL-33 levels. CONCLUSION AND IMPLICATIONS: These findings demonstrate a crucial and age-dependent role for IL-33 in post-sepsis immunosuppression. Thus, a better understanding of this process may lead to differential treatments for adult and paediatric sepsis.

Salud bucal y vulnerabilidades en cuidados paliativos en Uruguay: perspectivas desde la Bioética Latinoamericana

Los pacientes en cuidados paliativos enfrentan múltiples dimensiones de vulnerabilidad (biológica, social, moral y programática) que impactan en su bienestar y generan diferentes conflictos bioéticos. A pesar de la prevalencia de problemas bucales entre estos pacientes, la odontología rara vez se integra en su atención paliativa. Objetivo: Examinar las diferentes formas de vulnerabilidad y como se abordan en cuidados paliativos la atención de la salud bucal en Uruguay teniendo como enfoques referenciales teóricos de la Bioética Latinoamericana. Metodología: Revisión narrativa. Conclusiones: Se resalta la necesidad de ampliar la agenda de investigaciones interdisciplinarias en esta intersección emergente, investigando las percepciones de profesionales y pacientes, para fundamentar pautas éticas que garanticen sus derechos y una atención odontológica integral, considerando las diferentes dimensiones de la vulnerabilidad.

Os pacientes em cuidados paliativos enfrentam múltiplas dimensões de vulnerabilidade (biológica, social, moral e programática) que impactam em seu bem-estar e geram diferentes conflitos bioéticos. Apesar da prevalência de problemas bucais entre esses pacientes, a odontologia raramente é integrada em sua atenção paliativa. Objetivo: Examinar as diferentes formas de vulnerabilidade e como são abordadas nos cuidados paliativos em relação à saúde bucal no Uruguai, tendo como referenciais teóricos a Bioética Latino-Americana. Metodologia: Revisão narrativa. Conclusões: Destaca-se a necessidade de ampliar a agenda de pesquisas interdisciplinares nesta interseção emergente, investigando as percepções de profissionais e pacientes, para fundamentar diretrizes éticas que garantam seus direitos e uma atenção odontológica integral, considerando as diferentes dimensões da vulnerabilidade.

Patients in palliative care face multiple dimensions of vulnerability (biological, social, moral and programmatic) that impact their well-being and generate different bioethical conflicts. Despite the prevalence of oral problems among these patients, dentistry is rarely integrated into their palliative care. Objective : Examine the different forms of vulnerability and how oral health care is addressed in palliative care in Uruguay, taking theoretical approaches from Latin American bioethics as reference approaches. Methodology: Narrative review. Conclusions: It concludes by highlighting the need to expand the interdisciplinary research agenda in this emerging intersection, investigating the perceptions of professionals and patients, to establish ethical guidelines that guarantee their rights and comprehensive dental care, considering the different dimensions of vulnerability.

Vulnerabilidade moral entre estranhos morais: limites do princípio da permissão / Moral vulnerability among moral strangers: limits of the principle of permission / Vulnerabilidad moral entre extraños morales: límites del principio de permiso

Resumo Este artigo apresenta uma análise crítica da obra de Tristram Engelhardt, com foco no princípio da permissão. Argumenta-se que, em contextos de intensas desigualdades sociais e negação de direitos, a aplicação da ética de procedimentos baseada apenas no princípio da permissão pode resultar na vulnerabilidade moral de indivíduos e grupos que não compartilham de determinada moralidade. Isso pode levá-los a serem expostos a diferentes formas de negação de direitos, violência, exploração, exclusão e estigmatização. Diante dessa realidade, destaca-se a importância de fortalecer uma bioética comprometida com a defesa da dignidade, da diversidade, dos direitos humanos e da justiça social.

Abstract This article presents a critical analysis of Tristram Engelhardt's work, focusing on the principle of permission. It is argued that, in a context of intense social inequalities and denial of rights, the application of procedural ethics based solely on the principle of permission can result in the moral vulnerability of individuals and groups who do not share a certain morality. This can expose them to different forms of denial of rights, violence, exploitation, exclusion, and stigmatization. Given this reality, the importance of strengthening a bioethics committed to defending dignity, diversity, human rights, and social justice is highlighted.

Resumen Este artículo presenta un análisis crítico de la obra de Tristram Engelhardt, centrándose en el principio de permiso. Se arguye que, en un contexto de intensas desigualdades sociales y negación de derechos, la aplicación de la ética de procedimientos basada solo en el principio de permiso puede generar vulnerabilidad moral en los individuos y grupos que no comparten cierta moralidad. Esto puede llevarlos a verse expuestos a diferentes formas de negación de derechos, violencia, explotación, exclusión y estigmatización. Ante esta realidad, se destaca la importancia de fortalecer una bioética comprometida con la defensa de la dignidad, la diversidad, los derechos humanos y la justicia social.

Ética en el uso de psicodélicos: la definición de las drogas ilícitas bajo la óptica de la bioética crítica / Ethics in the use of psychedelics: The definition of illicit drugs from the perspective of critical bioethics

RESUMEN Este ensayo, ubicado en el campo de la bioética, analiza la prohibición del uso de psicodélicos, explorando argumentos sobre las crecientes evidencias de potenciales terapéuticos y su historia milenaria de usos culturales y espirituales. Discute inicialmente el contexto histórico de los psicodélicos y diferentes términos utilizados para nombrarlos. Problematiza la definición de "drogas", indicando falta de criterios objetivos para distinción entre lícitas e ilícitas. Bajo conceptos y referenciales teóricos de la bioética crítica, se analiza cómo el discurso moral prohibicionista se sostiene más por intereses políticos y económicos que por justificativas científicas, generando estigmatización y vulnerabilidad. Defiende el fin de la prohibición de los psicodélicos con base en argumentos éticos, resaltando su importancia para la reducción de sufrimientos individuales y colectivos. El trabajo contribuye a una reflexión profundizada sobre este tema socialmente controversial, articulando conocimientos interdisciplinarios.

ABSTRACT This essay, situated in the field of bioethics, examines the prohibition of psychedelic use, exploring arguments surrounding the growing evidence of their therapeutic potential and their millennia-long history of cultural and spiritual uses. It initially discusses the historical context of psychedelics and the various terms used to describe them. The essay problematizes the definition of "drugs," highlighting the lack of objective criteria for distinguishing between legal and illicit substances. Drawing on concepts and theoretical frameworks of critical bioethics, it analyzes how the prohibitionist moral discourse is sustained more by political and economic interests than by scientific justifications, leading to stigmatization and vulnerability. The essay advocates for the end of the prohibition of psychedelics based on ethical arguments, emphasizing their importance in reducing individual and collective suffering. The work contributes to a deeper reflection on this socially controversial topic, integrating interdisciplinary knowledge.

Gasless laparoscopic renal biopsy in canine cadavers: a preclinical study.

In this study we aimed to analyze the feasibility of the gasless renal biopsy technique in canine cadavers. The cadavers were randomly divided into two groups: laparoscopic GCG, in which gasless laparoscopy was performed and GCP, laparoscopy with pneumoperitoneum was performed. The procedures were randomly performed on the right and left kidneys. The total surgical time, procedural steps, and intraoperative complications were recorded. The degree of difficulty of the surgical approaches was evaluated by the surgeon, assistant, and external evaluators. Renal samples were evaluated for quality, number of glomeruli, and proportion of renal cortex. The total operative time was higher in the GCG group than in the GCP group (p < 0.01). Additionally, positioning of the second portal and platform positioning took longer than the other steps. The surgical groups differed from each other in the Likert scale values for almost all the parameters regarding the difficulty of the surgical approaches (p < 0.05), with higher scores in the GCG group than in the GCP group. Based on the video recordings, the GCP group had higher scores than the GCG group for degrees of difficulty of the approach (p < 0.05). Renal histological parameters were similar between the surgical groups and surgical sides. Our study findings indicate that the proposed gasless renal biopsy technique is feasible. The longer operative time and technical difficulties in the gasless approach did not affect the quality of the renal specimens.

Neste estudo objetivamos analisar a viabilidade da técnica de biópsia renal gasless em cadáveres caninos. Os cadáveres foram divididos aleatoriamente em dois grupos: GCG laparoscópico, no qual foi realizada laparoscopia gasless e GCP, onde foi realizada laparoscopia com pneumoperitônio. Os procedimentos foram realizados aleatoriamente nos rins direito e esquerdo. O tempo cirúrgico total, etapas do procedimento e complicações intraoperatórias foram registrados. O grau de dificuldade das abordagens cirúrgicas foi avaliado pelo cirurgião, assistente e avaliadores externos. As amostras renais foram avaliadas quanto à qualidade, número de glomérulos e proporção do córtex renal. O tempo operatório total foi maior no grupo GCG do que no grupo GCP (p < 0,01). Além disso, o posicionamento do segundo portal e o posicionamento da plataforma demoraram mais do que as outras etapas. Os grupos cirúrgicos diferiram entre si nos valores da escala Likert para quase todos os parâmetros relativos à dificuldade das abordagens cirúrgicas (p < 0,05), com escores mais elevados no grupo GCG do que no grupo GCP. Com base nas gravações de vídeo, o grupo GCP obteve pontuações mais altas que o grupo GCG para graus de dificuldade da abordagem (p < 0,05). Os parâmetros histológicos renais foram semelhantes entre os grupos cirúrgicos e os lados cirúrgicos. Os resultados do nosso estudo indicam que a técnica proposta de biópsia renal gasless é viável. O maior tempo operatório e as dificuldades técnicas na abordagem sem gás não afetaram a qualidade das amostras renais.

Neutrophil virucidal activity against SARS-CoV-2 is mediated by NETs.

BACKGROUND: The inflammation in the lungs and other vital organs in COVID-19 are characterized by the presence of neutrophils and high concentration of neutrophil extracellular traps (NETs), which also seems to mediate host tissue damage. However, it is not known whether NETs could have virucidal activity against SARS-CoV-2. METHODS: We investigated whether NETs could prevent SARS-CoV-2 replication in neutrophils and epithelial cells, and what the consequence of NETs degradation in K18-humanized ACE2 transgenic mice infected with SARS-CoV-2. RESULTS: Here, by immunofluorescence microscopy we observed that viral particles co-localize with NETs in neutrophils isolated from COVID-19 patients or from healthy individuals and infected in vitro. The inhibition of NETs production increased virus replication in neutrophils. In parallel, we observed that NETs inhibited virus abilities to infect and replicate in epithelial cells after 24 h of infection. Degradation of NETs with DNase I prevented their virucidal effect in vitro. Using K18-humanized ACE2 transgenic mice we observed a higher viral load in animals treated with DNase I. On the other hand, the virucidal effect of NETs was not dependent on neutrophil elastase or myeloperoxidase activity. CONCLUSION: Our results provide evidence of the role of NETosis as a mechanism of SARS-CoV-2 viral capture and inhibition.

Enoxaparin improves COVID-19 by reducing Neutrophils Extracellular Traps (NETs) production.

BACKGROUND: COVID-19 causes consequences such as imbalance of the immune system and thrombotic events. During the infection process, NETs in excess induce a pro-inflammatory response and disseminated intravascular coagulation. We evaluated the role of enoxaparin as a potential inhibitor of NETs. METHODS: K18-hACE2 animals infected with the SARS-CoV-2 virus and a group of 23 individuals admitted to the hospital with COVID-19 treated with enoxaparin or without treatment and controls without the disease were included. RESULTS: Enoxaparin decreased the levels of NETs, reduced the signs of the disease and mitigated lung damage in the animals infected with SARS-CoV-2. These effects were partially associated with prevention of SARS-CoV-2 entry and NETs synthesis. Clinical data revealed that treatment with enoxaparin decreased the levels of inflammatory markers, the levels of NETs in isolated neutrophils and the organ dysfunction. CONCLUSION: This study provides evidence for the beneficial effects of enoxaparin in COVID-19 in addition to its anticoagulant role.

A clinically-relevant STING agonist restrains human TH17 cell inflammatory profile.

The STING signaling pathway has gained attention over the last few years due to its ability to incite antimicrobial and antitumoral immunity. Conversely, in mouse models of autoimmunity such as colitis and multiple sclerosis, where TH17 cells are implicated in tissue inflammation, STING activation has been associated with the attenuation of immunogenic responses. In this line, STING was found to limit murine TH17 pro-inflammatory program in vitro. Here we demonstrate that 2'3'-c-di-AM(PS)2(Rp,Rp), a STING agonist that has been undergoing clinical trials for antitumor immunotherapy, activates the STING signalosome in differentiating human TH17 cells. Of particular interest, 2'3'-c-di-AM(PS)2(Rp,Rp) reduces IL-17A production and IL23R expression by human TH17 cells while it favors the generation of regulatory T (Treg) cells. These findings suggest that STING agonists may be promising approaches for treating human TH17-mediated chronic inflammation.

The TIGIT+ T regulatory cells subset associates with nosocomial infection and fatal outcome in COVID-19 patients under mechanical ventilation.

The TIGIT+FOXP3+Treg subset (TIGIT+Tregs) exerts robust suppressive activity on cellular immunity and predisposes septic individuals to opportunistic infection. We hypothesized that TIGIT+Tregs could play an important role in intensifying the COVID-19 severity and hampering the defense against nosocomial infections during hospitalization. Herein we aimed to verify the association between the levels of the TIGIT+Tregs with the mechanical ventilation requirement, fatal outcome, and bacteremia during hospitalization. TIGIT+Tregs were immunophenotyped by flow cytometry from the peripheral blood of 72 unvaccinated hospitalized COVID-19 patients at admission from May 29th to August 6th, 2020. The patients were stratified during hospitalization according to their mechanical ventilation requirement and fatal outcome. COVID-19 resulted in a high prevalence of the TIGIT+Tregs at admission, which progressively increased in patients with mechanical ventilation needs and fatal outcomes. The prevalence of TIGIT+Tregs positively correlated with poor pulmonary function and higher plasma levels of LDH, HMGB1, FGL2, and TNF. The non-survivors presented higher plasma levels of IL-33, HMGB1, FGL2, IL-10, IL-6, and 5.54 times more bacteremia than survivors. Conclusions: The expansion of the TIGIT+Tregs in COVID-19 patients was associated with inflammation, lung dysfunction, bacteremia, and fatal outcome.

CRISPR Libraries and Whole-Genome Screening to Identify Essential Factors for Viral Infections.

The CRISPR-Cas9 system has revolutionized genetics and offers a simple and inexpensive way of generating perturbation that results in gene repression, activation, or editing. The advances in this technique make possible the development of CRISPR libraries which consist of a set of sgRNAs to cause perturbations in several genes in the same cell population. The use of libraries raised the CRISPR-Cas9 technique to a genomic scale and provides a powerful approach for identifying previously unknown molecular mechanisms and pathways involved in a specific phenotype or biological process. More specifically, the CRISPRko libraries (set of sgRNAs for gene knockout) and their high-throughput screenings are widely used in research with viral agents, and it was enlarged even more with the COVID-19 pandemic. With this chapter, we aim to point out how this tool helps in understanding virus-host relationships, such as the mechanisms of virus entry into the cell, the essential factors for its replication, and the cellular pathways involved in the response against the pathogen. The chapter also provided some practical considerations for each step of an experimentation using these tools that include choosing the library and screening type, the target cell, the viral strain, the library amplification and guaranteeing its coverage, the strategies for the gene screening pipeline by bioinformatics, and finally, target validation. To conclude, it was presented a table reviewing the last updates in the research for antiviral therapies using CRISPR libraries.

The metabolic function of pyruvate kinase M2 regulates reactive oxygen species production and microbial killing by neutrophils.

Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.