Mesenchymal Stromal Cells Protect the Blood-Brain Barrier, Reduce Astrogliosis, and Prevent Cognitive and Behavioral Alterations in Surviving Septic Mice.

OBJECTIVES: Survivors of sepsis are frequently left with significant cognitive and behavioral impairments. These complications derive from nonresolving inflammation that persists following hospital discharge. To date, no study has investigated the effects of mesenchymal stromal cell therapy on the blood-brain barrier, astrocyte activation, neuroinflammation, and cognitive and behavioral alterations in experimental sepsis. DESIGN: Prospective, randomized, controlled experimental study. SETTING: Government-affiliated research laboratory. SUBJECTS: Male Swiss Webster mice (n = 309). INTERVENTIONS: Sepsis was induced by cecal ligation and puncture; sham-operated animals were used as control. All animals received volume resuscitation (1 mL saline/mouse subcutaneously) and antibiotics (meropenem 10 mg/kg intraperitoneally at 6, 24, and 48 hours). Six hours after surgery, mice were treated with mesenchymal stromal cells IV (1 × 10 cells in 0.05 mL of saline/mouse) or saline (0.05 mL IV). MEASUREMENTS AND MAIN RESULTS: At day 1, clinical score and plasma levels of inflammatory mediators were increased in cecal ligation and puncture mice. Mesenchymal stromal cells did not alter clinical score or survival rate, but reduced levels of systemic interleukin-1ß, interleukin-6, and monocyte chemoattractant protein-1. At day 15, survivor mice completed a battery of cognitive and behavioral tasks. Cecal ligation and puncture mice exhibited spatial and aversive memory deficits and anxiety-like behavior. These effects may be related to increased blood-brain barrier permeability, with altered tight-junction messenger RNA expression, increased brain levels of inflammatory mediators, and astrogliosis (induced at day 3). Mesenchymal stromal cells mitigated these cognitive and behavioral alterations, as well as reduced blood-brain barrier dysfunction, astrocyte activation, and interleukin-1ß, interleukin-6, tumor necrosis factor-α, and interleukin-10 levels in vivo. In cultured primary astrocytes stimulated with lipopolysaccharide, conditioned media from mesenchymal stromal cells reduced astrogliosis, interleukin-1ß, and monocyte chemoattractant protein-1, suggesting a paracrine mechanism of action. CONCLUSIONS: In mice who survived experimental sepsis, mesenchymal stromal cell therapy protected blood-brain barrier integrity, reduced astrogliosis and neuroinflammation, as well as improved cognition and behavior.

Comprehensive characterisation of polyphenols in leaves and stems of three anti-dengue virus type-2 active Brazilian Faramea species (Rubiaceae) by HPLC-DAD-ESI-MS/MS.

INTRODUCTION: The methanol (MeOH) leaf extracts of the species Faramea bahiensis, F. hyacinthina and F. truncata (Rubiaceae) have previously shown in vitro non-cytotoxic and anti-dengue virus serotype 2 (DENV2) activities in human hepatocarcinoma cell lineage (HepG2). Chemical studies have led to the isolation of major flavonoids, but quite complex fractions of phenolic compounds still remain. OBJECTIVE: To complete the study of phenolic compounds in the leaves and to access the presence of these compounds in the stems of these Faramea spp. by online high-performance liquid chromatography-diode array detector-electrospray ionisation tandem mass spectrometry (HPLC-DAD-ESI-MS/MS), as well as to evaluate the in vitro cytotoxic and anti-DENV2 activities of their MeOH stem extracts. METHODOLOGY: The identification was performed by comparing retention times, UV and mass spectra with those of available standards and by using the mechanisms and fragmentation patterns established in previous studies. The effects of the extracts in DENV2 infected HepG2 cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The virus titer was quantified by plaque assay. RESULTS: The study led to the characterisation of 31 phenolic compounds including flavonoid O- and C-glycosides, phenolic acids and one coumarin. The stem extracts from F. hyacinthina and F. bahiensis presented a similar bioactivity to those of their leaves but a loss of cytoprotective activity of F. bahiensis and a higher cytotoxicity of F. truncata were observed. CONCLUSIONS: This research allowed a detailed phenolic composition of three bioactive Faramea species to be achieved, thus contributing to the study of this genus and providing valuable information for further phytotherapeutic applications.

Antiviral Activity of Faramea hyacinthina and Faramea truncata Leaves on Dengue Virus Type-2 and Their Major Compounds.

The defatted fractions of the Faramea hyacinthina and F. truncata (Rubiaceae) leaf MeOH extracts showed in vitro non-cytotoxic and anti-dengue virus serotype 2 (DENV2) activity in human hepatocarcinoma cell lineage (HepG2). Submitting these fractions to the developed RP-SPE method allowed isolating the antiviral flavanone (2S)-isosakuranetin-7-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranoside (1) from both species and yielded less active sub-fractions. The new diastereoisomeric epimer pair (2S) + (2R) of 5,3',5'-trihydroxyflavanone-7-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranoside (2a/2b) from F. hyacinthina; the known narigenin-7-O-ß-d-apiofuranosyl-(1→6)-ß-d-glucopyranoside (3) from both species; rutin (4) and quercetin-4'-ß-d-O-glucopyranosyl-3-O-rutinoside (5) from F. hyacinthina, and kaempferol-3-O-rutinoside (6), erythroxyloside A (7) and asperuloside (8) from F. truncata have been isolated from these sub-fractions. Compounds 4 - 8 are reported for the first time in Faramea spp.

Different outcomes of neonatal and adult Zika virus infection on startle reflex and prepulse inhibition in mice.

Zika virus (ZIKV) infection causes severe neurological consequences in both gestationally-exposed infants and adults. Sensorial gating deficits strongly correlate to the motor, sensorial and cognitive impairments observed in ZIKV-infected patients. However, no startle response or prepulse inhibition (PPI) assessment has been made in patients or animal models. In this study, we identified different outcomes according to the age of infection and sex in mice: neonatally infected animals presented an increase in PPI and delayed startle latency. However, adult-infected male mice presented lower startle amplitude, while a PPI impairment was observed 14 days after infection in both sexes. Our data further the understanding of the functional impacts of ZIKV on the developing and mature nervous system, which could help explain other behavioral and cognitive alterations caused by the virus. With this study, we support the startle reflex testing in ZIKV-exposed patients, especially infants, allowing for early detection of functional neuromotor damage and early intervention.

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.

Yellow fever vaccine protects mice against Zika virus infection.

Zika virus (ZIKV) emerged as an important infectious disease agent in Brazil in 2016. Infection usually leads to mild symptoms, but severe congenital neurological disorders and Guillain-Barré syndrome have been reported following ZIKV exposure. Creating an effective vaccine against ZIKV is a public health priority. We describe the protective effect of an already licensed attenuated yellow fever vaccine (YFV, 17DD) in type-I interferon receptor knockout mice (A129) and immunocompetent BALB/c and SV-129 (A129 background) mice infected with ZIKV. YFV vaccination provided protection against ZIKV, with decreased mortality in A129 mice, a reduction in the cerebral viral load in all mice, and weight loss prevention in BALB/c mice. The A129 mice that were challenged two and three weeks after the first dose of the vaccine were fully protected, whereas partial protection was observed five weeks after vaccination. In all cases, the YFV vaccine provoked a substantial decrease in the cerebral viral load. YFV immunization also prevented hippocampal synapse loss and microgliosis in ZIKV-infected mice. Our vaccine model is T cell-dependent, with AG129 mice being unable to tolerate immunization (vaccination is lethal in this mouse model), indicating the importance of IFN-γ in immunogenicity. To confirm the role of T cells, we immunized nude mice that we demonstrated to be very susceptible to infection. Immunization with YFV and challenge 7 days after booster did not protect nude mice in terms of weight loss and showed partial protection in the survival curve. When we evaluated the humoral response, the vaccine elicited significant antibody titers against ZIKV; however, it showed no neutralizing activity in vitro and in vivo. The data indicate that a cell-mediated response promotes protection against cerebral infection, which is crucial to vaccine protection, and it appears to not necessarily require a humoral response. This protective effect can also be attributed to innate factors, but more studies are needed to strengthen this hypothesis. Our findings open the way to using an available and inexpensive vaccine for large-scale immunization in the event of a ZIKV outbreak.

Zika virus replicates in adult human brain tissue and impairs synapses and memory in mice.

Neurological complications affecting the central nervous system have been reported in adult patients infected by Zika virus (ZIKV) but the underlying mechanisms remain unknown. Here, we report that ZIKV replicates in human and mouse adult brain tissue, targeting mature neurons. ZIKV preferentially targets memory-related brain regions, inhibits hippocampal long-term potentiation and induces memory impairment in adult mice. TNF-α upregulation, microgliosis and upregulation of complement system proteins, C1q and C3, are induced by ZIKV infection. Microglia are found to engulf hippocampal presynaptic terminals during acute infection. Neutralization of TNF-α signaling, blockage of microglial activation or of C1q/C3 prevent synapse and memory impairment in ZIKV-infected mice. Results suggest that ZIKV induces synapse and memory dysfunction via aberrant activation of TNF-α, microglia and complement. Our findings establish a mechanism by which ZIKV affects the adult brain, and point to the need of evaluating cognitive deficits as a potential comorbidity in ZIKV-infected adults.

Co-protoporphyrin IX and Sn-protoporphyrin IX inactivate Zika, Chikungunya and other arboviruses by targeting the viral envelope.

The global situation of diseases transmitted by arthropod-borne viruses such as Dengue (DENV), Yellow Fever (YFV), Chikungunya (CHIKV) and Zika (ZIKV) viruses is alarming and treatment of human infection by these arboviruses faces several challenges. The discovery of broad-spectrum antiviral molecules, able to inactivate different groups of viruses, is an interesting approach. The viral envelope is a common structure among arboviruses, being a potential target for antivirals. Porphyrins are amphipathic molecules able to interact with membranes and absorb light, being widely used in photodynamic therapy. Previously, we showed that heme, Co-protoporphyrin IX (CoPPIX) and Sn-protoporphyrin IX (SnPPIX) directly inactivate DENV and YFV infectious particles. Here we demonstrate that the antiviral activity of these porphyrins can be broadened to CHIKV, ZIKV, Mayaro virus, Sindbis virus and Vesicular Stomatitis virus. Porphyrin treatment causes viral envelope protein loss, affecting viral morphology, adsorption and entry into target cells. Also, light-stimulation enhanced the SnPPIX activity against all tested arboviruses. In summary, CoPPIX and SnPPIX were shown to be efficient broad-spectrum compounds to inactivate medically and veterinary important viruses.

Acute and chronic neurological consequences of early-life Zika virus infection in mice.

Although congenital Zika virus (ZIKV) exposure has been associated with microcephaly and other neurodevelopmental disorders, long-term consequences of perinatal infection are largely unknown. We evaluated short- and long-term neuropathological and behavioral consequences of neonatal ZIKV infection in mice. ZIKV showed brain tropism, causing postnatal-onset microcephaly and several behavioral deficits in adulthood. During the acute phase of infection, mice developed frequent seizures, which were reduced by tumor necrosis factor-α (TNF-α) inhibition. During adulthood, ZIKV replication persisted in neonatally infected mice, and the animals showed increased susceptibility to chemically induced seizures, neurodegeneration, and brain calcifications. Altogether, the results show that neonatal ZIKV infection has long-term neuropathological and behavioral complications in mice and suggest that early inhibition of TNF-α-mediated neuroinflammation might be an effective therapeutic strategy to prevent the development of chronic neurological abnormalities.