Spatially resolved distribution of pancreatic hormones proteoforms by MALDI-imaging mass spectrometry.

Zinc plays a crucial role both in the immune system and endocrine processes. Zinc restriction in the diet has been shown to lead to degeneration of the endocrine pancreas, resulting in hormonal imbalance within the ß-cells. Proteostasismay vary depending on the stage of a pathophysiological process, which underscores the need for tools aimed at directly analyzing biological status. Among proteomics methods, MALDI-ToF-MS can serve as a rapid peptidomics tool for analyzing extracts or by histological imaging. Here we report the optimization of MALDI imaging mass spectrometry analysis of histological thin sections from mouse pancreas. This optimization enables the identification of the major islet peptide hormones as well as the major accumulated precursors and/or proteolytic products of peptide hormones. Cross-validation of the identified peptide hormones was performed by LC-ESI-MS from pancreatic islet extracts. Mice subjected to a zinc-restricted diet exhibited a relatively lower amount of peptide intermediates compared to the control group. These findings provide evidence for a complex modulation of proteostasis by micronutrients imbalance, a phenomenon directly accessed by MALDI-MSI.

Antiviral, Cytoprotective, and Anti-Inflammatory Effect of Ampelozizyphus amazonicus Ducke Ethanolic Wood Extract on Chikungunya Virus Infection.

Chikungunya fever, a debilitating disease caused by Chikungunya virus (CHIKV), is characterized by a high fever of sudden onset and an intense arthralgia that impairs individual regular activities. Although most symptoms are self-limited, long-term persistent arthralgia is observed in 30-40% of infected individuals. Currently, there is no vaccine or specific treatment against CHIKV infection, so there is an urgent need for the discovery of new therapeutic options for CHIKF chronic cases. This present study aims to test the antiviral, cytoprotective, and anti-inflammatory activities of an ethanol extract (FF72) from Ampelozizyphus amazonicus Ducke wood, chemically characterized using mass spectrometry, which indicated the major presence of dammarane-type triterpenoid saponins. The major saponin in the extract, with a deprotonated molecule ion m/z 897 [M-H]-, was tentatively assigned as a jujubogenin triglycoside, a dammarane-type triterpenoid saponin. Treatment with FF72 resulted in a significant reduction in both virus replication and the production of infective virions in BHK-21-infected cells. The viability of infected cells was assessed using an MTT, and the result indicated that FF72 treatment was able to revert the toxicity mediated by CHIKV infection. In addition, FF72 had a direct effect on CHIKV, since the infectivity was completely abolished in the presence of the extract. FF72 treatment also reduced the expression of the major pro-inflammatory mediators overexpressed during CHIKV infection, such as IL-1ß, IL-6, IL-8, and MCP-1. Overall, the present study elucidates the potential of FF72 to become a promising candidate of herbal medicine for alphaviruses infections.

Peripheral nervous system is injured by neutrophil extracellular traps (NETs) elicited by nonstructural (NS) protein-1 from Zika virus.

The involvement of innate immune mediators to the Zika virus (ZIKV)-induced neuroinflammation is not yet well known. Here, we investigated whether neutrophil extracellular traps (NETs), which are scaffolds of DNA associated with proteins, have the potential to injure peripheral nervous. The tissue lesions were evaluated after adding NETs to dorsal root ganglia (DRG) explants and to DRG constituent cells or injecting them into mouse sciatic nerves. Identification of NET harmful components was achieved by pharmacological inhibition of NET constituents. We found that ZIKV inoculation into sciatic nerves recruited neutrophils and elicited the production of the cytokines CXCL1 and IL-1ß, classical NET inducers, but did not trigger NET formation. ZIKV blocked PMA- and CXCL8-induced NET release, but, in contrast, the ZIKV nonstructural protein (NS)-1 induced NET formation. NET-enriched supernatants were toxic to DRG explants, decreasing neurite area, length, and arborization. NETs were toxic to DRG constituent cells and affected myelinating cells. Myeloperoxidase (MPO) and histones were identified as the harmful component of NETs. NS1 injection into mouse sciatic nerves recruited neutrophils and triggered NET release and caspase-3 activation, events that were also elicited by the injection of purified MPO. In summary, we found that ZIKV NS1 protein induces NET formation, which causes nervous tissue damages. Our findings reveal new mechanisms leading to neuroinflammation by ZIKV.

Liver immunopathogenesis in fatal cases of dengue in children: detection of viral antigen, cytokine profile and inflammatory mediators.

Introduction: Dengue virus (DENV), the etiologic agent of dengue fever illness, represents a global public health concern, mainly in tropical and subtropical areas across the globe. It is well known that this acute viral disease can progress to severe hemorrhagic stages in some individuals, however, the immunopathogenic basis of the development of more severe forms by these patients is yet to be fully understood. Objective: In this context, we investigated and characterized the histopathological features as well as the cytokine profile and cell subpopulations present in liver tissues from three fatal cases of DENV in children. Methods: Hematoxylin and Eosin, Periodic Acid Schiff and Picro Sirius Red staining were utilized for the histopathological analysis. Immunohistochemistry assay was performed to characterize the inflammatory response and cell expression patterns. Results: Vascular dysfunctions such as hemorrhage, vascular congestion and edema associated with a mononuclear infiltrate were observedin all three cases. Liver tissues exhibited increased presence of CD68+ and TCD8+ cells as well as high expression of MMP-9, TNF-a, RANTES, VEGFR-2 mediators. Viral replication was confirmed by the detection of NS3 protein. Conclusion: Taken together, these results evidenced key factors that may be involved in the development of severe alterations in liver tissues of children in response to DENV infection.

Preparation of Hard Palm Seeds for Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry Analysis.

Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is applied to identify compounds in their native environments. Currently, MALDI-IMS is frequently used in clinical analysis. Still, an excellent perspective exists for better applying this technique to understand chemical compounds' physiological information in plant tissues. However, preparation may be challenging for specific samples from botanical materials, as MALDI-IMS requires thin slices (12-20 µm) for appropriate data acquisition and successful analysis. In this sense, previously, we developed a sample preparation protocol to obtain thin sections of Euterpe oleracea (açaí palm) hard seeds, enabling their molecular mapping by MALDI-IMS. Here, we show that the developed protocol is suitable for preparing other seeds from the same genus. Briefly, the protocol was based on submerging the seeds in deionized water for 24 h, embedding samples with gelatin, and sectioning them in an acclimatized cryostat. Then, for matrix deposition, an xy motion platform was coupled to an electrospray ionization (ESI) needle spray using a 1:1 (v/v) 2,5-dihydroxybenzoic acid (DHB) and methanol solution with 0.1% trifluoroacetic acid at 30 mg/mL. E. precatoria and E. edulis seed data were processed using software to map their metabolite patterns. Hexose oligomers were mapped within sample slices to prove the adequacy of the protocol for those samples, as it is known that those seeds contain large amounts of mannan, a polymer of the hexose mannose. As a result, peaks of hexose oligomers, represented by [M + K]+ adducts of (Δ = 162 Da), were identified. Thus, the sample preparation protocol, previously developed tailor-made for E. oleracea seeds, also enabled MALDI-IMS analysis of two other hard palm seeds. In short, the method could constitute a valuable tool for research in the morpho-anatomy and physiology of botanical materials, especially from cut-resistant samples.

Primary infection of BALB/c mice with a dengue virus type 4 strain leads to kidney injury.

BACKGROUND: Dengue is a disease caused by dengue virus (DENV-1 through -4). Among the four serotypes, DENV-4 remains the least studied. Acute kidney injury is a potential complication of dengue generally associated with severe dengue infection. OBJECTIVES: The goal of this study was to investigate the alterations caused by experimental dengue infection in the kidney of adult BALB/c mice. METHODS: In this study, BALB/c mice were infected through the intravenous route with a DENV-4 strain, isolated from a human patient. The kidneys of the mice were procured and subject to histopathological and ultrastructural analysis. FINDINGS: The presence of the viral antigen was confirmed through immunohistochemistry. Analysis of tissue sections revealed the presence of inflammatory cell infiltrate throughout the parenchyma. Glomerular enlargement was a common find. Necrosis of tubular cells and haemorrhage were also observed. Analysis of the kidney on a transmission electron microscope allowed a closer look into the necrotic tubular cells, which presented nuclei with condensed chromatin, and loss of cytoplasm. MAIN CONCLUSIONS: Even though the kidney is probably not a primary target of dengue infection in mice, the inoculation of the virus in the blood appears to damage the renal tissue through local inflammation.

Diabetogenic viruses: linking viruses to diabetes mellitus.

Diabetes Mellitus (DM) is a group of chronic metabolic diseases distinguished by elevated glycemia due to the alterations in insulin metabolism. DM is one of the most relevant diseases of the modern world, with high incidence and prevalence worldwide, associated with severe systemic complications and increased morbidity and mortality rates. Although genetic factors and lifestyle habits are two of the main factors involved in DM onset, viral infections, such as enteroviruses, cytomegalovirus, hepatitis C virus, human immunodeficiency virus, severe acute respiratory syndrome coronavirus 2, among others, have been linked as triggers of type 1 (T1DM) and type 2 (T2DM) diabetes. Over the years, various groups identified different mechanisms as to how viruses can promote these metabolic syndromes. However, this field is still poorly explored and needs further research, as millions of people live with these pathologies. Thus, this review aims to ex-plore the different processes of how viruses can induce DM and their contribution to the prevalence and incidence of DM worldwide.

Deep branch of the radial nerve: effect of pronation/supination on longitudinal nerve alignment.

OBJECTIVE: To evaluate the effect of maximal pronation and supination of the forearm on the alignment and anatomic relationship of the deep branch of the radial nerve (DBRN) at the superior arcade of the supinator muscle (SASM) by using high-resolution ultrasound (HRUS). MATERIALS AND METHODS: In this cross-sectional study, HRUS in the long axis of the DBRN was performed in asymptomatic participants enrolled from March to August 2021. DBRN alignment was evaluated by measuring angles of the nerve in maximal pronation and maximal supination of the forearm independently by two musculoskeletal radiologists. Forearm range of motion and biometric measurements were recorded. Student t, Shapiro-Wilk, Pearson correlation, reliability analyses, and Kruskal-Wallis test were used. RESULTS: The study population included 110 nerves from 55 asymptomatic participants (median age, 37.0 years; age range, 16-63 years; 29 [52.7%] women). There was a statistically significant difference between the DBRN angle in maximal supination and maximal pronation (Reader 1: 95% CI: 5.74, 8.21, p < 0.001, and Reader 2: 95% CI: 5.82, 8.37, p < 0.001). The mean difference between the angles in maximal supination and maximal pronation was approximately 7° for both readers. ICC was very good for intraobserver agreement (Reader1: r ≥ 0.92, p < 0.001; Reader 2: r ≥ 0.93, p < 0.001), as well as for interobserver agreement (phase 1: r ≥ 0.87, p < 0.001; phase 2: r ≥ 0.90, p < 0.001). CONCLUSION: The extremes of the rotational movement of the forearm affect the longitudinal morphology and anatomic relationships of the DBRN, primarily demonstrating the convergence of the nerve towards the SASM in maximal pronation and divergence in maximal supination.

The acquisition of thin sections of açaí (Euterpe oleracea Mart.) seed with elevated potassium content for molecular mapping by mass spectrometry imaging.

RATIONALE: Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) of tissues became popular in the last decade. Consequently, adapting sample preparation methods for different materials turned out to be a pivotal step for successful analysis due to the requirement of sample slices of 12-20 µm thickness. However, acquiring thin sections compatible with MALDI-IMS for unusual samples is challenging, as existing histological protocols may not be suitable, thus requiring new methods. Açaí (Euterpe oleracea Mart.) seed is an example of a challenging material due to its toughness and resistance to crack, therefore our goal was to develop a methodology to obtain thin (12-20 µm) and entire sections of açaí seeds for MALDI-IMS analysis. METHODS: Different strategies were evaluated for obtaining thin sections of seeds, and the combination of the following steps was found to be the most suitable option: (i) softening of seeds by water immersion for 24 h; (ii) transversal cut of seeds to obtain half-seeds using a razor blade and a hammer; (iii) half-seeds imbibition in gelatin; (iv) samples sectioning using a cryostat at -20°C to obtain samples with 12-20 µm thickness; (v) collection of samples in an indium tin oxide-coated glass slide covered by double-sided copper tape to avoid sample wrapping and ensure adhesion after unfreezing; and (vi) storage of samples in a -80°C freezer, if necessary. RESULTS: This adapted sample preparation method enabled the analysis of açaí seeds by MALDI-IMS, providing spatial distribution of carbohydrates in the endosperm. CONCLUSIONS: The adaptations developed for sample preparation will help investigate the metabolic and physiological properties of açaí seeds in future studies.

Primary infection of BALB/c mice with a dengue virus type 4 strain leads to kidney injury

BACKGROUND Dengue is a disease caused by dengue virus (DENV-1 through -4). Among the four serotypes, DENV-4 remains the least studied. Acute kidney injury is a potential complication of dengue generally associated with severe dengue infection. OBJECTIVES The goal of this study was to investigate the alterations caused by experimental dengue infection in the kidney of adult BALB/c mice. METHODS In this study, BALB/c mice were infected through the intravenous route with a DENV-4 strain, isolated from a human patient. The kidneys of the mice were procured and subject to histopathological and ultrastructural analysis. FINDINGS The presence of the viral antigen was confirmed through immunohistochemistry. Analysis of tissue sections revealed the presence of inflammatory cell infiltrate throughout the parenchyma. Glomerular enlargement was a common find. Necrosis of tubular cells and haemorrhage were also observed. Analysis of the kidney on a transmission electron microscope allowed a closer look into the necrotic tubular cells, which presented nuclei with condensed chromatin, and loss of cytoplasm. MAIN CONCLUSIONS Even though the kidney is probably not a primary target of dengue infection in mice, the inoculation of the virus in the blood appears to damage the renal tissue through local inflammation.

Phenolic Profile and Antioxidant Properties in Extracts of Developing Açaí (Euterpe oleracea Mart.) Seeds.

We investigated changes in the phenolic profile and antioxidant properties in the extracts of developing seeds of açaí (Euterpe oleracea). Four developmental stages were evaluated, with earlier stages displaying higher antioxidant activity and polyphenols content, while mass spectrometry analysis identified procyanidins (PCs) as the major components of the extracts in all stages. B-type PCs varied from dimers to decamers, with A-type linkages in a smaller number. Extracted PCs decreased in average length from 20.5 to 10.1 along seed development. PC composition indicated that (-)-epicatechin corresponded to over 95% of extension units in all stages, while (+)-catechin presence as the starter unit increased from 42 to 78.8% during seed development. This variation was correlated to the abundance of key enzymes for PC biosynthesis during seed development. This study is the first to report PC content and composition variations during açaí seed development, which can contribute to studies on the plant's physiology and biotechnological applications.

Immunopathology of Renal Tissue in Fatal Cases of Dengue in Children.

Dengue virus (DENV) infection represents a worldwide public health concern and can cause damage to multiple organs, including the kidney. In this work, we investigated the histopathological changes caused by dengue virus infection along with the detection of inflammatory mediators, cytokines, and cell expression patterns in the renal tissue of three fatal cases in children. Hematoxylin and Eosin staining was performed to analyze these histopathological changes. Immunohistochemistry allowed for the detection of immunological inflammatory markers in renal tissues that were quantified and further analyzed. Vascular congestion, edema and glomerular infiltrate were observed in the three cases, in addition to the thickening of the matrix area around the glomerular capillaries and mononuclear infiltrate associated with vascular congestion in the medullary region. The renal tissues exhibited collagen deposition and high expression of CD68+ Mø, CD8+ T, CD56+ cells and MMP-9, and the cytokine profile was mainly characterized by the expression of IFN-γ and TNF-α. Additionally, the expression of RANTES, VEGFR-2 and VCAM-1 were observed. The replication of DENV was evidenced by the detection of the NS3 protein. These results contributed to clarifying the main factors that may be involved in changes in the renal tissue of fatal cases of dengue in children.

Immunogenicity of SARS-CoV-2 Trimeric Spike Protein Associated to Poly(I:C) Plus Alum.

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid [Poly(I:C)] adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after two or three immunizations. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation [spike protein with Alum + Poly(I:C)] in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

Cytokines and inflammatory mediators: Markers involved in interstitial damage to the pancreas in two dengue fever cases associated with acute pancreatitis.

Dengue viral (DENV) infections can lead to acute pancreatitis and associated tissue damage. This study examined the pancreas from two fatal cases of DENV for histopathological changes as well as for the detection of cytokines, and other inflammatory mediators. Tissue sections were prepared for examination by ultrastructural and histopathological techniques. Sections from the pancreas of non-infected individuals were prepared in parallel as a control. The presence of viral replication in macrophages was detected by co-staining for the proteins NS3 and CD68 by immunofluorescence. Immunohistochemistry was used to detect cells that expressed cytokines and inflammatory mediators to characterize the inflammatory response. Edema, acinar necrosis and fibrosis areas associated with a mononuclear infiltrate were found in infected tissues. The major site of virus replication appeared to be macrophages based on their exclusive presentation of the viral protein NS3. Pancreatic tissues from the infected individuals also displayed increased levels of high mobility group box-1, caspase-3, gelatinase B and tumor necrosis factor alpha compared to controls. The presence of virus replicating macrophages in the pancreas was associated with multiple changes in tissue structure that included elevated levels of cytokines and inflammatory markers that may differentiate acute pancreatitis due to DENV infections from other causes.

Virus Propagation and Plaque Assay for Dengue Virus.

This chapter describes the methods of propagation and titration for DENV-1 to -4, which are required for most of the experiments using live viruses. DENV does not reach titers as high as those of other viruses or as high as desired for their use in biological assays. Although DENV grows in many different cell lines derived from both vertebrate and invertebrate cells, the most common cell lines used for virus isolation and propagation are mosquito cells C6/36 from Aedes albopictus. Amongst several methods for virus quantification, plaque assay stands out as being very practical and inexpensive. This technique is carried out essentially to estimate virion density in a particular material, being extremely important when evaluating the effect of an antiviral treatment or antibody neutralization capacity, for example. In this assay, viral particles are serially diluted and added onto confluent cell monolayers immersed in a semisolid medium, which is responsible for limiting virus spread throughout the culture. Therefore, regarding the medium consistency, once a virion successfully infects a cell, the newly produced particles can only infect neighboring cells, eventually leading them to death. This phenomenon can be observed as round gaps or plaques in the culture after staining live cells with a crystal violet solution. Then, plaques are counted and used to determine plaque-forming units per milliliter. Here, we describe a protocol established by our group for dengue virus (DENV) titration with porcine kidney (PS) cells.

Increased circulating levels of High Mobility Group Box 1 (HMGB1) in acute-phase Chikungunya virus infection: Potential disease biomarker.

BACKGROUND: Chikungunya virus (CHIKV) causes a febrile syndrome with intense and debilitating arthralgia that can persist for several months or years after complete virus clearance. As there is no specific antiviral treatment or vaccine against CHIKV, identification of serological markers that help clinical management of CHIKV patients is urgent. The High Mobility Group Box 1 (HMGB1) protein is secreted to extracellular milieu and triggers an intense inflammatory process by inducing the overexpression of pro-inflammatory cytokines. HMGB1 plays an important role in several virus diseases as well as in rheumatoid arthritis. OBJECTIVES: This study focus on the investigation of HMGB1 serum levels in a sera panel from CHIKV-infected patients in an attempt to assess its potential as a biomarker for chikungunya clinical management. STUDY DESIGN: Eighty CHIKV-positive samples and 32 samples from healthy donors were subjected to a quantitative HMGB1 ELISA assay to assess the HMGB1 circulating levels. RESULTS: HMGB1 levels were significantly higher in CHIKV-positive samples (516.12 ng/mL, SEM ± 48.83 ng/mL) compared to negative control (31.20 ng/mL, SEM ± 3.24 ng/mL, p < 0.0001). Circulating levels of HMGB1 persisted elevated during the whole acute-phase of disease and correlated with virus titer (p < 0.05). CONCLUSIONS: The present study is the first to describe increased serum levels of HMGB1 in CHIKV infection and its positive correlation with virus titer, suggesting its potential use as a biomarker for diagnosis and treatment of chikungunya fever.

Overview on Chikungunya Virus Infection: From Epidemiology to State-of-the-Art Experimental Models.

Chikungunya virus (CHIKV) is currently one of the most relevant arboviruses to public health. It is a member of the Togaviridae family and alphavirus genus and causes an arthritogenic disease known as chikungunya fever (CHIKF). It is characterized by a multifaceted disease, which is distinguished from other arbovirus infections by the intense and debilitating arthralgia that can last for months or years in some individuals. Despite the great social and economic burden caused by CHIKV infection, there is no vaccine or specific antiviral drugs currently available. Recent outbreaks have shown a change in the severity profile of the disease in which atypical and severe manifestation lead to hundreds of deaths, reinforcing the necessity to understand the replication and pathogenesis processes. CHIKF is a complex disease resultant from the infection of a plethora of cell types. Although there are several in vivo models for studying CHIKV infection, none of them reproduces integrally the disease signature observed in humans, which is a challenge for vaccine and drug development. Therefore, understanding the potentials and limitations of the state-of-the-art experimental models is imperative to advance in the field. In this context, the present review outlines the present knowledge on CHIKV epidemiology, replication, pathogenesis, and immunity and also brings a critical perspective on the current in vitro and in vivo state-of-the-art experimental models of CHIKF.

Morphological Aspects and Viremia Analysis of BALB/c Murine Model Experimentally Infected with Dengue Virus Serotype 4.

Ever since its brief introduction in the Brazilian territory in 1981, dengue virus serotype 4 (DENV-4) remained absent from the national epidemiological scenario for almost 25 years. The emergence of DENV-4 in 2010 resulted in epidemics in most Brazilian states. DENV-4, however, remains one of the least studied among the four DENV serotypes. Despite being known as a mild serotype, DENV-4 is associated with severe cases and deaths and deserves to be investigated; however, the lack of suitable experimental animal models is a limiting factor for pathogenesis studies. Here, we aimed to investigate the susceptibility and potential tropism of DENV-4 for liver, lung and heart of an immunocompetent mice model, and to evaluate and investigate the resulting morphological and ultrastructural alterations upon viral infection. BALB/c mice were inoculated intravenously with non-neuroadapted doses of DENV-4 isolated from a human case. The histopathological analysis of liver revealed typical alterations of DENV, such as microsteatosis, edema and vascular congestion, while in lung, widespread areas of hemorrhage and interstitial pneumonia were observed. While milder alterations were present in heart, characterized by limited hemorrhage and discrete presence of inflammatory infiltrate, the disorganization of the structure of the intercalated disc is of particular interest. DENV-4 RNA was detected in liver, lung, heart and serum of BALB/c mice through qRT-PCR, while the NS3 viral protein was observed in all of the aforementioned organs through immunohistochemistry. These findings indicate the susceptibility of the model to the serotype and further reinforce the usefulness of BALB/c mice in studying the many alterations caused by DENV.

The immunodominant antibody response to Zika virus NS1 protein is characterized by cross-reactivity to self.

Besides antigen-specific responses to viral antigens, humoral immune response in virus infection can generate polyreactive and autoreactive antibodies. Dengue and Zika virus infections have been linked to antibody-mediated autoimmune disorders, including Guillain-Barré syndrome. A unique feature of flaviviruses is the secretion of nonstructural protein 1 (NS1) by infected cells. NS1 is highly immunogenic, and antibodies targeting NS1 can have both protective and pathogenic roles. In the present study, we investigated the humoral immune response to Zika virus NS1 and found NS1 to be an immunodominant viral antigen associated with the presence of autoreactive antibodies. Through single B cell cultures, we coupled binding assays and BCR sequencing, confirming the immunodominance of NS1. We demonstrate the presence of self-reactive clones in germinal centers after both infection and immunization, some of which present cross-reactivity with NS1. Sequence analysis of anti-NS1 B cell clones showed sequence features associated with pathogenic autoreactive antibodies. Our findings demonstrate NS1 immunodominance at the cellular level as well as a potential role for NS1 in ZIKV-associated autoimmune manifestations.

GD3 synthase deletion alters retinal structure and impairs visual function in mice.

Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.