Probes for INS

We previously confirmed that postprandial alterations in the circulation and metabolism after a single oral dose of flavan 3-ols (mixture of catechin and catechin oligomers) were involved in an increase in sympathetic nervous activity. However, it is well known that, in response to various stresses, activation of the hypothalamic-pituitary-adrenal (HPA) axis occurs together with sympathetic nerve activity, which is associated with activation of the sympathetic-adrenal-medullary (SAM) axis. In this study, we examined whether the HPA axis was activated after a single dose of flavan 3-ols. We administered an oral dose of 10 or 50 mg/kg flavan 3-ols to male ICR mice, removed the brains, and fixed them in paraformaldehyde-phosphate buffer. Other animals that were treated similarly were decapitated, and blood was collected. In the paraventricular nucleus (PVN), c-fos mRNA expression increased significantly at 15 min after administration of either 10 or 50 mg/kg flavan 3-ols. Corticotropin-releasing hormone (CRH) mRNA expression levels significantly increased at 240 min after administration of 10 mg/kg flavan 3-ols, and at 60 min after administration of 50 mg/kg flavan 3-ols. Plasma corticosterone levels were also significantly increased at 240 min after ingestion of 50 mg/kg flavan 3-ols. In this experiment, we confirmed that the ingestion of flavan 3-ols acted as a stressor in mammals with activation both the SAM and HPA axes.

The Zika virus (ZIKV) epidemic has resulted in congenital abnormalities in fetuses and neonates. Although some cross-reactive dengue virus (DENV)-specific antibodies can enhance ZIKV infection in mice, those recognizing the DENV E-dimer epitope (EDE) can neutralize ZIKV infection in cell culture. We evaluated the therapeutic activity of human monoclonal antibodies to DENV EDE for their ability to control ZIKV infection in the brains, testes, placentas, and fetuses of mice. A single dose of the EDE1-B10 antibody given 3 d after ZIKV infection protected against lethality, reduced ZIKV levels in brains and testes, and preserved sperm counts. In pregnant mice, wild-type or engineered LALA variants of EDE1-B10, which cannot engage Fcg receptors, diminished ZIKV burden in maternal and fetal tissues, and protected against fetal demise. Because neutralizing antibodies to EDE have therapeutic potential against ZIKV, in addition to their established inhibitory effects against DENV, it may be possible to develop therapies that control disease caused by both viruses.

Zika virus (ZIKV) has recently been associated with birth defects and pregnancy loss after maternal infection. Because dengue virus (DENV) and ZIKV co-circulate, understanding the role of antibody-dependent enhancement in the context of pregnancy is critical. Here, we showed that the presence of DENV-specific antibodies in ZIKV-infected pregnant mice significantly increased placental damage, fetal growth restriction, and fetal resorption. This was associated with enhanced viral replication in the placenta that coincided with an increased frequency of infected trophoblasts. ZIKV-infected human placental tissues also showed increased replication in the presence of DENV antibodies, which was reversed by FcγR blocking antibodies. Furthermore, ZIKV-mediated fetal pathogenesis was enhanced in mice in the presence of a DENV-reactive monoclonal antibody, but not in the presence of the LALA variant, indicating a dependence on FcγR engagement. Our data suggest a possible mechanism for the recent increase in severe pregnancy outcomes after ZIKV infection in DENV-endemic areas.

Abstract

PURPOSE:

The association of Zika virus (ZIKV) infection and development of neurological sequelae require a better understanding of the pathogenic mechanisms causing severe disease. The purpose of this study was to evaluate the ability and sensitivity of positron emission tomography (PET) imaging using [18F]DPA-714, a translocator protein (TSPO) 18 kDa radioligand, to detect and quantify neuroinflammation in ZIKV-infected mice.

PROCEDURES:

We assessed ZIKV-induced pathogenesis in wild-type C57BL/6 mice administered an antibody to inhibit type I interferon (IFN) signaling. [18F]DPA-714 PET imaging was performed on days 3, 6, and 10 post-infection (PI), and tissues were subsequently processed for histological evaluation, quantification of microgliosis, and detection of viral RNA by in situ hybridization (ISH).

RESULTS:

In susceptible ZIKV-infected mice, viral titers in the brain increased from days 3 to 10 PI. Over this span, these mice showed a two- to sixfold increase in global brain neuroinflammation using [18F]DPA-714 PET imaging despite limited, regional detection of viral RNA. No measurable increase in ionized calcium binding adaptor molecule 1 (Iba-1) expression was noted at day 3 PI; however, there was a modest increase at day 6 PI and an approximately significant fourfold increase in Iba-1 expression at day 10 PI in the susceptible ZIKV-infected group relative to controls.

CONCLUSIONS:

The results of the current study demonstrate that global neuroinflammation plays a significant role in the progression of ZIKV infection and that [18F]DPA-714 PET imaging is a sensitive tool relative to histology for the detection of neuroinflammation. [18F]DPA-714 PET imaging may be useful in dynamically characterizing the pathology associated with neurotropic viruses and the evaluation of therapeutics being developed for treatment of infectious diseases.

Social stress causes profound urinary bladder dysfunction in children that often continues into adulthood. We discovered that the intensity and duration of social stress influences whether bladder dysfunction presents as overactivity or underactivity. The transient receptor potential vanilloid type 1 (TRPV1) channel is integral in causing stress-induced bladder overactivity by increasing bladder sensory outflow, but little is known about the development of stress-induced bladder underactivity. We sought to determine if TRPV1 channels are involved in bladder underactivity caused by stress. Voiding function, sensory nerve activity, and bladder wall remodeling were assessed in C57Bl/6 and TRPV1 knockout mice exposed to intensified social stress, using conscious cystometry, ex vivo afferent nerve recordings, and histology. Intensified social stress increased void volume, intermicturition interval, bladder volume and bladder wall collagen content in C57Bl/6 mice, indicative of bladder wall remodeling and underactive bladder. However, afferent nerve activity was unchanged, and unaffected by the TRPV1 antagonist capsazepine. Interestingly, all indices of bladder function were unchanged in TRPV1 knockout mice in response to social stress, even though corticotrophin releasing hormone expression in Barrington's Nucleus still increased. These results suggest that TRPV1 channels in the periphery are a linchpin in the development of stress-induced bladder dysfunction, both with regard to increased sensory outflow that leads to overactive bladder, and bladder wall decompensation that leads to underactive bladder. TRPV1 channels represent an intriguing target to prevent the development of stress-induced bladder dysfunction in children.

Outcomes of Zika virus (ZIKV) infection in pregnant women vary from the birth of asymptomatic offspring to abnormal development and severe brain lesions in fetuses and infants. There are concerns that offspring affected in utero and born without apparent symptoms may develop mental illnesses. Therefore, animal models are important to test interventions against in utero infection and health sequelae in symptomatic and likely more widespread asymptomatic offspring. To partially reproduce in utero infection in humans, we directly inoculated selected porcine conceptuses with ZIKV. Inoculation resulted in rapid trans-fetal infections, persistent infection in conceptuses, molecular pathology in fetal brains, fetal antibody and type I interferon responses. Offspring infected in utero showed ZIKV in their fetal membranes collected after birth. Some in utero affected piglets were small, depressed, had undersized brains, showed seizures and potentially increased activity. Our data suggest that porcine model of persistent in utero ZIKV infection has a strong potential for translational research and can be used to test therapeutic interventions in vivo.

Tyro3, Axl, and Mertk (TAM) receptors are candidate entry receptors for infection with the Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl-/-, Mertk-/-, Axl-/-Mertk-/-, and Axl-/-Tyro3-/- mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR)-blocking (MAR1-5A3) antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection.

The Zika virus (ZIKV) epidemic is associated with fetal brain lesions and other serious birth defects classified as congenital ZIKV syndrome. Postnatal ZIKV infection in infants and children has been reported; however, data on brain anatomy, function, and behavioral outcomes following infection are absent. We show that postnatal ZIKV infection of infant rhesus macaques (RMs) results in persistent structural and functional alterations of the central nervous system compared to age-matched controls. We demonstrate ZIKV lymphoid tropism and neurotropism in infant RMs and histopathologic abnormalities in the peripheral and central nervous systems including inflammatory infiltrates, astrogliosis, and Wallerian degeneration. Structural and resting-state functional magnetic resonance imaging (MRI/rs-fMRI) show persistent enlargement of lateral ventricles, maturational changes in specific brain regions, and altered functional connectivity (FC) between brain areas involved in emotional behavior and arousal functions, including weakened amygdala-hippocampal connectivity in two of two ZIKV-infected infant RMs several months after clearance of ZIKV RNA from peripheral blood. ZIKV infection also results in distinct alterations in the species-typical emotional reactivity to acute stress, which were predicted by the weak amygdala-hippocampal FC. We demonstrate that postnatal ZIKV infection of infants in this model affects neurodevelopment, suggesting that long-term clinical monitoring of pediatric cases is warranted.