Popularity and Entropy in Friendship and Enmity Networks in Classrooms.

Looking for regular statistical trends of relations in schools, we constructed 42 independent weighted directed networks of simultaneous friendship and animosity from surveys we made in the Mexico City Metropolitan area in classrooms with students of different ages and levels by asking them to nominate and order five friends and five foes. However, the data show that older students nominated fewer than the five required five foes. Although each classroom was independent of the others, we found several general trends involving students of different ages and grade levels. In all classrooms, friendship entropy was found to be higher than enmity entropy, indicating that fewer students received enmity links than received friendship nominations. Popular agents exhibited more reciprocal nominations among themselves than less popular agents, and opposite-sex friendships increased with age.

Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.

Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of ß2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.

Neurodevelopmental Consequences of Dietary Zinc Deficiency: A Status Report.

Zinc is a tightly regulated trace mineral element playing critical roles in growth, immunity, neurodevelopment, and synaptic and hormonal signaling. Although severe dietary zinc deficiency is relatively uncommon in the United States, dietary zinc deficiency is a substantial public health concern in low- and middle-income countries. Zinc status may be a key determinant of neurodevelopmental processes. Indeed, limited cohort studies have shown that serum zinc is lower in people diagnosed with autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), and depression. These observations have sparked multiple studies investigating the mechanisms underlying zinc status and neurodevelopmental outcomes. Animal models of perinatal and adult dietary zinc restriction yield distinct behavioral phenotypes reminiscent of features of ASD, ADHD, and depression, including increased anxiety and immobility, repetitive behaviors, and altered social behaviors. At the cellular and molecular level, zinc has demonstrated roles in neurogenesis, regulation of cellular redox status, transcription factor trafficking, synaptogenesis, and the regulation of synaptic architecture via the Shank family of scaffolding proteins. Although mechanistic questions remain, the current evidence suggests that zinc status is important for adequate neuronal development and may be a yet overlooked factor in the pathogenesis of several psychiatric conditions. This review aims to summarize current knowledge of the role of zinc in the neurophysiology of the perinatal period, the many cellular targets of zinc in the developing brain, and the potential consequences of alterations in zinc homeostasis in early life.

Microglial reprogramming by Hv1 antagonism protects neurons from inflammatory and glutamate toxicity.

Although the precise mechanisms determining the neurotoxic or neuroprotective activation phenotypes in microglia remain poorly characterized, metabolic changes in these cells appear critical for these processes. As cellular metabolism can be tightly regulated by changes in intracellular pH, we tested whether pharmacological targeting of the microglial voltage-gated proton channel 1 (Hv1), an important regulator of intracellular pH, is critical for activated microglial reprogramming. Using a mouse microglial cell line and mouse primary microglia cultures, either alone, or co-cultured with rat cerebrocortical neurons, we characterized in detail the microglial activation profile in the absence and presence of Hv1 inhibition. We observed that activated microglia neurotoxicity was mainly attributable to the release of tumor necrosis factor alpha, reactive oxygen species, and zinc. Strikingly, pharmacological inhibition of Hv1 largely abrogated inflammatory neurotoxicity not only by reducing the production of cytotoxic mediators but also by promoting neurotrophic molecule production and restraining excessive phagocytic activity. Importantly, the Hv1-sensitive change from a pro-inflammatory to a neuroprotective phenotype was associated with metabolic reprogramming, particularly via a boost in NADH availability and a reduction in lactate. Most critically, Hv1 antagonism not only reduced inflammatory neurotoxicity but also promoted microglia-dependent neuroprotection against a separate excitotoxic injury. Our results strongly suggest that Hv1 blockers may provide an important therapeutic tool against a wide range of inflammatory neurodegenerative disorders.

Multisystem Inflammatory Syndrome in Children (MIS-C) temporally related to COVID-19: the experience at a pediatric reference hospital in Colombia / Síndrome inflamatória multissistêmica em criança associada à COVID-19: experiência em um hospital pediátrico de referência na Colômbia

Abstract Objective: This study aimed to describe the clinical characteristics and the different phenotypes of children with multisystem inflammatory syndrome in children (MIS-C) temporally related to COVID-19 and to evaluate the risk conditions that favored a greater severity of the disease during a 12-month period at a pediatric reference hospital in Colombia. Methods: A 12-month retrospective observational study of children under the age of 18 years who met criteria for MIS-C. Results: A total of 28 children presented MIS-C criteria. The median age was 7 years. Other than fever (100%) (onset 4 days prior to admission), the most frequent clinical features were gastrointestinal (86%) and mucocutaneous (61%). Notably, 14 (50%) children had Kawasaki-like symptoms. The most frequent echocardiographic abnormalities were pericardial effusion (64%), valvular involvement (68%), ventricular dysfunction (39%), and coronary artery abnormalities (29%). In addition, 75% had lymphopenia. All had at least one abnormal coagulation test. Most received intravenous immunoglobulin (89%), glucocorticoids (82%), vasopressors (54%), and antibiotics (64%). Notably, 61% had a more severe form of the disease and were admitted to an intensive care unit (median 4 days, mean 6 days); the severity predictors were patients with the inflammatory/MIS-C phenotype (OR 26.5; 95%CI 1.40-503.7; p=0.029) and rash (OR 14.7; 95%CI 1.2-178.7; p=0.034). Two patients had macrophage activation syndrome. Conclusions: Coronary artery abnormalities, ventricular dysfunction, and intensive care unit admission were frequent, which needs to highlight the importance of early clinical suspicion.

Resumo Objetivo: Descrever as características clínicas e os diferentes fenótipos de crianças com síndrome inflamatória multissistêmica na criança temporalmente relacionada com a COVID-19 (do inglês multisystem inflammatory syndrome in children — MIS-C) e avaliar as condições de risco que favorecem a maior gravidade da doença durante um período de 12 meses em um hospital pediátrico de referência na Colômbia. Métodos: Estudo retrospectivo de 12 meses de observação de crianças menores de 18 anos que cumprem os critérios para o MIS-C. Resultados: Vinte e oito crianças foram apresentadas com os critérios do MIS-C. A idade média era de sete anos, e 54% eram do sexo masculino. Para além da febre (100%) (com início quatro dias antes da admissão), as características clínicas mais frequentes eram gastrointestinais (86%) e mucocutâneas (61%). Quatorze crianças (50%) apresentavam sintomas semelhantes aos de Kawasaki. As anomalias ecocardiográficas mais frequentes foram derrame pericárdico (64%), envolvimento valvar (68%), disfunção ventricular (39%) e anomalias coronárias (29%). Tinham linfopenia 75% das crianças. Todas tinham algum teste de coagulação anormal. A maioria recebeu imunoglobulina intravenosa (89%), glucocorticoides (82%), vasopressores (54%) e antibióticos (64%). Tiveram envolvimento mais grave 61% dos pacientes, que precisaram ser internados em unidade de terapia intensiva (mediana de quatro dias, média de seis dias); os preditores de gravidade foram pacientes com fenótipo inflamatório/ MIS-C (odds ratio — OR 26,5; intervalo de confiança — IC95% 1,4-503,7; p=0,029) e erupção cutânea (OR 14,7; IC95% 1,2-178,7; p=0,034). Dois pacientes (7%) apresentavam síndrome de ativação macrofágica. Conclusões: Alteração da artéria coronária, disfunção ventricular e internação na unidade de terapia intensiva foram frequentes, o que nos alerta sobre a importância da suspeita clínica precoce.

Multisystem Inflammatory Syndrome in Children (MIS-C) temporally related to COVID-19: the experience at a pediatric reference hospital in Colombia.

OBJECTIVE: This study aimed to describe the clinical characteristics and the different phenotypes of children with multisystem inflammatory syndrome in children (MIS-C) temporally related to COVID-19 and to evaluate the risk conditions that favored a greater severity of the disease during a 12-month period at a pediatric reference hospital in Colombia. METHODS: A 12-month retrospective observational study of children under the age of 18 years who met criteria for MIS-C. RESULTS: A total of 28 children presented MIS-C criteria. The median age was 7 years. Other than fever (100%) (onset 4 days prior to admission), the most frequent clinical features were gastrointestinal (86%) and mucocutaneous (61%). Notably, 14 (50%) children had Kawasaki-like symptoms. The most frequent echocardiographic abnormalities were pericardial effusion (64%), valvular involvement (68%), ventricular dysfunction (39%), and coronary artery abnormalities (29%). In addition, 75% had lymphopenia. All had at least one abnormal coagulation test. Most received intravenous immunoglobulin (89%), glucocorticoids (82%), vasopressors (54%), and antibiotics (64%). Notably, 61% had a more severe form of the disease and were admitted to an intensive care unit (median 4 days, mean 6 days); the severity predictors were patients with the inflammatory/MIS-C phenotype (OR 26.5; 95%CI 1.40-503.7; p=0.029) and rash (OR 14.7; 95%CI 1.2-178.7; p=0.034). Two patients had macrophage activation syndrome. CONCLUSIONS: Coronary artery abnormalities, ventricular dysfunction, and intensive care unit admission were frequent, which needs to highlight the importance of early clinical suspicion.

Zika virus-like particle vaccine fusion loop mutation increases production yield but fails to protect AG129 mice against Zika virus challenge.

Zika virus (ZIKV) is a mosquito-borne flavivirus with maternal infection associated with preterm birth, congenital malformations, and fetal death, and adult infection associated with Guillain-Barré syndrome. Recent widespread endemic transmission of ZIKV and the potential for future outbreaks necessitate the development of an effective vaccine. We developed a ZIKV vaccine candidate based on virus-like-particles (VLPs) generated following transfection of mammalian HEK293T cells using a plasmid encoding the pre-membrane/membrane (prM/M) and envelope (E) structural protein genes. VLPs were collected from cell culture supernatant and purified by column chromatography with yields of approximately 1-2mg/L. To promote increased particle yields, a single amino acid change of phenylalanine to alanine was made in the E fusion loop at position 108 (F108A) of the lead VLP vaccine candidate. This mutation resulted in a modest 2-fold increase in F108A VLP production with no detectable prM processing by furin to a mature particle, in contrast to the lead candidate (parent). To evaluate immunogenicity and efficacy, AG129 mice were immunized with a dose titration of either the immature F108A or lead VLP (each alum adjuvanted). The resulting VLP-specific binding antibody (Ab) levels were comparable. However, geometric mean neutralizing Ab (nAb) titers using a recombinant ZIKV reporter were significantly lower with F108A immunization compared to lead. After virus challenge, all lead VLP-immunized groups showed a significant 3- to 4-Log10 reduction in mean ZIKV RNAemia levels compared with control mice immunized only with alum, but the RNAemia reduction of 0.5 Log10 for F108A groups was statistically similar to the control. Successful viral control by the lead VLP candidate following challenge supports further vaccine development for this candidate. Notably, nAb titer levels in the lead, but not F108A, VLP-immunized mice inversely correlated with RNAemia. Further evaluation of sera by an in vitro Ab-dependent enhancement assay demonstrated that the F108A VLP-induced immune sera had a significantly higher capacity to promote ZIKV infection in FcγR-expressing cells. These data indicate that a single amino acid change in the fusion loop resulted in increased VLP yields but that the immature F108A particles were significantly diminished in their capacity to induce nAbs and provide protection against ZIKV challenge.

Interferon receptor-deficient mice are susceptible to eschar-associated rickettsiosis.

Arthropod-borne rickettsial pathogens cause mild and severe human disease worldwide. The tick-borne pathogen Rickettsia parkeri elicits skin lesions (eschars) and disseminated disease in humans; however, inbred mice are generally resistant to infection. We report that intradermal infection of mice lacking both interferon receptors (Ifnar1-/-;Ifngr1-/-) with as few as 10 R. parkeri elicits eschar formation and disseminated, lethal disease. Similar to human infection, eschars exhibited necrosis and inflammation, with bacteria primarily found in leukocytes. Using this model, we find that the actin-based motility factor Sca2 is required for dissemination from the skin to internal organs, and the outer membrane protein OmpB contributes to eschar formation. Immunizing Ifnar1-/-;Ifngr1-/- mice with sca2 and ompB mutant R. parkeri protects against rechallenge, revealing live-attenuated vaccine candidates. Thus, Ifnar1-/-;Ifngr1-/- mice are a tractable model to investigate rickettsiosis, virulence factors, and immunity. Our results further suggest that discrepancies between mouse and human susceptibility may be due to differences in interferon signaling.

Tick bites allow disease-causing microbes, including multiple species of Rickettsia bacteria, to pass from arthropods to humans. Being exposed to Rickettsia parkeri, for example, can cause a scab at the bite site, fever, headache and fatigue. To date, no vaccine is available against any of the severe diseases caused by Rickettsia species. Modelling human infections in animals could help to understand and combat these illnesses. R. parkeri is a good candidate for such studies, as it can give insight into more severe Rickettsia infections while being comparatively safer to handle. However, laboratory mice are resistant to this species of bacteria, limiting their use as models. To explore why this is the case, Burke et al. probed whether an immune mechanism known as interferon signalling protects laboratory rodents against R. parkeri. During infection, the immune system releases molecules called interferons that stick to 'receptors' at the surface of cells, triggering defense mechanisms that help to fight off an invader. Burke et al. injected R. parkeri into the skin of mice that had or lacked certain interferon receptors, showing that animals without two specific receptors developed scabs and saw the disease spread through their body. Further investigation showed that two R. parkeri proteins, known as OmpB or Sca2, were essential for the bacteria to cause skin lesions and damage internal organs. Burke et al. then used R. parkeri that lacked OmpB or Sca2 to test whether these modified, inoffensive microbes could act as 'vaccines'. And indeed, vulnerable laboratory mice which were first exposed to the mutant bacteria were then able to survive the 'normal' version of the microbe. Together, this work reveals that interferon signalling protects laboratory mice against R. parkeri infections. It also creates an animal model that can be used to study disease and vaccination.

Structural basis for antibody inhibition of flavivirus NS1-triggered endothelial dysfunction.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the ß-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

Impact of pulmonary hypertension and congenital heart disease with hemodynamic repercussion on the severity of acute respiratory infections in children under 5 years of age at a pediatric referral center in Colombia, South America.

BACKGROUND: Acute respiratory infection is one of the main causes of morbidity in children. Some studies have suggested that pulmonary hypertension and congenital heart disease with haemodynamic repercussion increase the severity of respiratory infections, but there are few publications in developing countries. METHODS: This was a prospective cohort study evaluating the impact of pulmonary hypertension and congenital heart disease (CHD) with haemodynamic repercussion as predictors of severity in children under 5 years of age hospitalised for acute respiratory infection. RESULTS: Altogether, 217 children hospitalised for a respiratory infection who underwent an echocardiogram were evaluated; 62 children were diagnosed with CHD with haemodynamic repercussion or pulmonary hypertension. Independent predictors of admission to intensive care included: pulmonary hypertension (RR 2.14; 95% CI 1.06-4.35, p = 0.034), respiratory syncytial virus (RR 2.52; 95% CI 1.29-4.92, p = 0.006), and bacterial pneumonia (RR 3.09; 95% CI 1.65-5.81, p = 0.000). A significant difference was found in average length of hospital stay in children with the cardiovascular conditions studied (p = 0.000). CONCLUSIONS: Pulmonary hypertension and CHD with haemodynamic repercussion as well as respiratory syncytial virus and bacterial pneumonia were predictors of severity in children with respiratory infections in this study. Early recognition of cardiovascular risks in paediatric populations is necessary to lessen the impact on respiratory infections.

Avian anti-NS1 IgY antibodies neutralize dengue virus infection and protect against lethal dengue virus challenge.

Dengue is the most prevalent arboviral disease in humans and a continually increasing global public health burden. To date, there are no approved antiviral therapies against dengue virus (DENV) and the only licensed vaccine, Dengvaxia, is exclusively indicated for individuals with prior DENV infection. Endothelial hyperpermeability and vascular leak, pathogenic hallmarks of severe dengue disease, can be directly triggered by DENV non-structural protein 1 (NS1). As such, anti-NS1 antibodies can prevent NS1-triggered endothelial dysfunction in vitro and pathogenesis in vivo. Recently, goose-derived anti-DENV immunoglobulin Y (IgY) antibodies were shown to neutralize DENV and Zika virus (ZIKV) infection without adverse effects, such as antibody-dependent enhancement (ADE). In this study, we used egg yolks from DENV-immunized geese to purify IgY antibodies specific to DENV NS1 epitopes. We determined that 2 anti-NS1 IgY antibodies, NS1-1 and NS1-8, were capable of neutralizing DENV infection in vitro. In addition, these antibodies did not cross-react with the DENV Envelope (E) protein nor enhance DENV or ZIKV infection in vitro. Intriguingly, NS1-8, but not NS1-1, partially blocked NS1-induced endothelial dysfunction in vitro while neither antibody blocked binding of soluble NS1 to cells. Finally, prophylactic treatment of mice with NS1-8 conferred significant protection against lethal DENV challenge. Although further research is needed to define the mechanism of action of these antibodies, our findings highlight the potential of anti-NS1 IgY as a promising prophylactic approach against DENV infection.

Identification of Dengue Virus Serotype 3 Specific Antigenic Sites Targeted by Neutralizing Human Antibodies.

The rational design of dengue virus (DENV) vaccines requires a detailed understanding of the molecular basis for antibody-mediated immunity. The durably protective antibody response to DENV after primary infection is serotype specific. However, there is an incomplete understanding of the antigenic determinants for DENV type-specific (TS) antibodies, especially for DENV serotype 3, which has only one well-studied, strongly neutralizing human monoclonal antibody (mAb). Here, we investigated the human B cell response in children after natural DENV infection in the endemic area of Nicaragua and isolated 15 DENV3 TS mAbs recognizing the envelope (E) glycoprotein. Functional epitope mapping of these mAbs and small animal prophylaxis studies revealed a complex landscape with protective epitopes clustering in at least 6-7 antigenic sites. Potently neutralizing TS mAbs recognized sites principally in E glycoprotein domains I and II, and patterns suggest frequent recognition of quaternary structures on the surface of viral particles.

In silico drug repurposing for the identification of potential candidate molecules against arboviruses infection.

Arboviral diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses represent a major public health problem worldwide, especially in tropical areas where millions of infections occur every year. The aim of this research was to identify candidate molecules for the treatment of these diseases among the drugs currently available in the market, through in silico screening and subsequent in vitro evaluation with cell culture models of DENV and ZIKV infections. Numerous pharmaceutical compounds from antibiotics to chemotherapeutic agents presented high in silico binding affinity for the viral proteins, including ergotamine, antrafenine, natamycin, pranlukast, nilotinib, itraconazole, conivaptan and novobiocin. These five last compounds were tested in vitro, being pranlukast the one that exhibited the best antiviral activity. Further in vitro assays for this compound showed a significant inhibitory effect on DENV and ZIKV infection of human monocytic cells and human hepatocytes (Huh-7 cells) with potential abrogation of virus entry. Finally, intrinsic fluorescence analyses suggest that pranlukast may have some level of interaction with three viral proteins of DENV: envelope, capsid, and NS1. Due to its promising results, suitable accessibility in the market and reduced restrictions compared to other pharmaceuticals; the anti-asthmatic pranlukast is proposed as a drug candidate against DENV, ZIKV, and CHIKV, supporting further in vitro and in vivo assessment of the potential of this and other lead compounds that exhibited good affinity scores in silico as therapeutic agents or scaffolds for the development of new drugs against arboviral diseases.

Reporte de un caso: eritema discrómico perstans en paciente con diagnóstico de virus de inmunodeficiencia humana / Case report: dyschromic erythema perstans in a patient diagnosed with human immunodeficiency virus

El eritema discrómico perstans o dermatitis cenicienta, es un trastorno pigmentario de la piel poco común, de etiología desconocida. Se describe el caso de un adulto de 35 años con antecedente de VIH, quien consulta por aparición de lesiones irregulares, bien definidas café-grises localizadas en cuello, área mandibular inferior, espalda y brazos, de borde levemente eritematoso. El diagnóstico de eritema discrómico perstans se realizó con base en los hallazgos clínicos e histopatológicos.

Erythema dyschromicum perstans also known as ashy dermatosis is a rare skin pigmentary disorder of unknown etiology. We describe the case of a 35-year-old man HIV positive who presented irregular, well defined brown-gray lesions with slightly erythematous border located in neck, lower jaw, back and arms. Diagnosis of erythema dyschromicum perstans in this patient was made based on clinical and histopathological criteria.

Increased serum sialic acid is associated with morbidity and mortality in a murine model of dengue disease.

Dengue virus (DENV) causes the most prevalent arboviral infection of humans, resulting in a spectrum of outcomes, ranging from asymptomatic infection to dengue fever to severe dengue characterized by vascular leakage and shock. Previously, we determined that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability, disrupts the endothelial glycocalyx layer (EGL) in vitro and triggers shedding of structural components, including sialic acid (Sia) and heparan sulfate. Here, using a murine model of dengue disease disease, we found high levels of Sia and NS1 circulating in mice with DENV-induced morbidity and lethal DENV infection. Further, we developed a liquid chromatography/mass spectrometry-based method for quantifying free Sia in serum and determined that the levels of free N-glycolylneuraminic acid were significantly higher in DENV-infected mice than in uninfected controls. These data provide additional evidence that DENV infection disrupts EGL components in vivo and warrant further research assessing Sia as a biomarker of severe dengue disease.

Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation.

Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.

El papel de las especies reactivas de oxígeno y de nitrógeno en algunas enfermedades neurodegenerativas / The role of reactive oxygen and nitrogen species in some neurodegenerative diseases

Resumen Las especies reactivas de oxígeno y nitrógeno son moléculas que se generan a partir del metabolismo celular fisiológico; sin embargo, cuando existe un desequilibrio entre la producción de radicales libres y los mecanismos antioxidantes se genera estrés oxidante. El estrés oxidante se ha asociado con el desarrollo y progresión de enfermedades neurodegenerativas como Alzheimer, Parkinson y Huntington. Dado que el inicio del estrés oxidante es imperceptible y aún no se cuenta con estudios de laboratorio que determinen el impacto de los radicales libres en pacientes con enfermedades neurodegenerativas, es importante dilucidar el papel de estos en los procesos neurodegenerativos con el fin de tener indicios sólidos sobre las posibles dianas de tratamiento y prevenir el daño progresivo en este tipo de enfermedades.

Abstract The reactive oxygen and nitrogen species are molecules that are generated from the physiological cellular metabolism. However, when there is an imbalance between the production of free radicals and the antioxidant mechanisms, oxidative stress is generated. Oxidative stress has been associated with the development and progression of neurodegenerative diseases such as Alzheimer, Parkinson and Huntington, given that the onset of oxidative stress is imperceptible and that there are still no laboratory studies that can determine the impact of free radicals in patients with neurodegenerative diseases. It is important to elucidate the role of free radicals in neurodegenerative processes in order to have solid indications about the possible treatment targets and to prevent the progressive damage in this type of diseases.

Role of NADPH oxidase-2 in the progression of the inflammatory response secondary to striatum excitotoxic damage.

BACKGROUND: During excitotoxic damage, neuronal death results from the increase in intracellular calcium, the induction of oxidative stress, and a subsequent inflammatory response. NADPH oxidases (NOX) are relevant sources of reactive oxygen species (ROS) during excitotoxic damage. NADPH oxidase-2 (NOX-2) has been particularly related to neuronal damage and death, as well as to the resolution of the subsequent inflammatory response. As ROS are crucial components of the regulation of inflammatory response, in this work, we evaluated the role of NOX-2 in the progression of inflammation resulting from glutamate-induced excitotoxic damage of the striatum in an in vivo model. METHODS: The striata of wild-type C57BL/6 J and NOX-2 KO mice (gp91Cybbtm1Din/J) were stereotactically injected with monosodium glutamate either alone or in combination with IL-4 or IL-10. The damage was evaluated in histological sections stained with cresyl violet and Fluoro-Jade B. The enzymatic activity of caspase-3 and NOX were also measured. Additionally, the cytokine profile was identified by ELISA and motor activity was verified by the tests of the cylinder, the adhesive tape removal, and the inverted grid. RESULTS: Our results show a neuroprotective effect in mice with a genetic inhibition of NOX-2, which is partially due to a differential response to excitotoxic damage, characterized by the production of anti-inflammatory cytokines. In NOX-2 KO animals, the excitotoxic condition increased the production of interleukin-4, which could contribute to the production of interleukin-10 that decreased neuronal apoptotic death and the magnitude of striatal injury. Treatment with interleukin-4 and interleukin-10 protected from excitotoxic damage in wild-type animals. CONCLUSIONS: The release of proinflammatory cytokines during the excitotoxic event promotes an additional apoptotic death of neurons that survived the initial damage. During the subsequent inflammatory response to excitotoxic damage, ROS generated by NOX-2 play a decisive role in the extension of the lesion and consequently in the severity of the functional compromise, probably by regulating the anti-inflammatory cytokines production.

Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism.

Flaviviruses cause systemic or neurotropic-encephalitic pathology in humans. The flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein involved in viral replication, immune evasion, and vascular leakage during dengue virus infection. However, the contribution of secreted NS1 from related flaviviruses to viral pathogenesis remains unknown. Here, we demonstrate that NS1 from dengue, Zika, West Nile, Japanese encephalitis, and yellow fever viruses selectively binds to and alters permeability of human endothelial cells from lung, dermis, umbilical vein, brain, and liver in vitro and causes tissue-specific vascular leakage in mice, reflecting the pathophysiology of each flavivirus. Mechanistically, each flavivirus NS1 leads to differential disruption of endothelial glycocalyx components, resulting in endothelial hyperpermeability. Our findings reveal the capacity of a secreted viral protein to modulate endothelial barrier function in a tissue-specific manner both in vitro and in vivo, potentially influencing virus dissemination and pathogenesis and providing targets for antiviral therapies and vaccine development.

Cyclic Dinucleotide-Adjuvanted Dengue Virus Nonstructural Protein 1 Induces Protective Antibody and T Cell Responses.

Endothelial dysfunction and vascular leak, pathogenic hallmarks of severe dengue disease, are directly triggered by dengue virus (DENV) nonstructural protein 1 (NS1). Previous studies have shown that immunization with NS1, as well as passive transfer of NS1-immune serum or anti-NS1 mAb, prevent NS1-mediated lethality in vivo. In this study, we evaluated the immunogenicity and protective capacity of recombinant DENV NS1 administered with cyclic dinucleotides (CDNs), potent activators of innate immune pathways and highly immunogenic adjuvants. Using both wild-type C57BL/6 mice and IFN-α/ß receptor-deficient mice, we show that NS1-CDN immunizations elicit serotype-specific and cross-reactive Ab and T cell responses. Furthermore, NS1-CDN vaccinations conferred significant homotypic and heterotypic protection from DENV2-induced morbidity and mortality. In addition, we demonstrate that high anti-NS1 Ab titers are associated with protection, supporting the role of humoral responses against DENV NS1 as correlates of protection. These findings highlight the potential of CDN-based adjuvants for inducing Ab and T cell responses and validate NS1 as an important candidate for dengue vaccine development.