Volume 2, Issue 1

Objectives Zika virus (ZIKV) has become an epidemic in several countries and was declared a major public health issue by the WHO. Although ZIKV infection is asymptomatic or shows mild fever-related symptoms in most people, the virus can be transmitted from a pregnant mother to the fetus, resulting in severe brain developmental abnormalities, including microcephaly. Multiple groups have identified developmental neuronal and neuronal progenitor compromise during ZIKV infection within the fetal brain, but little is known about whether ZIKV could infect human astrocytes and its effect on the developing brain. Thus, our objective was to determine astrocyte ZiKV infection in a developmental-dependent manner. Methods We analyze infection of pure cultures of astrocytes and mixed cultures of neurons and astrocytes in response to ZIKV using plaque assays, confocal, and electron microscopy to identify infectivity, ZIKV accumulation and intracellular distribution as well as apoptosis and interorganelle dysfunction. Results Here, we demonstrated that ZIKV enters, infects, replicates, and accumulates in large quantities in human fetal astrocytes in a developmental-dependent manner. Astrocyte infection and intracellular viral accumulation resulted in neuronal apoptosis, and we propose astrocytes are a ZIKV reservoir during brain development. Conclusions Our data identify astrocytes in different stages of development as major contributors to the devastating effects of ZIKV in the developing brain.

Objectives Opioids including morphine and DAMGO activate mu-opioid receptors (MOR), increase intracellular reactive oxygen species (ROS) levels, and induce cell death. Ferrous iron (Fe 2+ ) through Fenton-like chemistry increases ROS levels and endolysosomes are “master regulators of iron metabolism” and contain readily-releasable Fe 2+ stores. However, mechanisms underlying opioid-induced changes in endolysosome iron homeostasis and downstream-signaling events remain unclear. Methods We used SH-SY5Y neuroblastoma cells, flow cytometry, and confocal microscopy to measure Fe 2+ and ROS levels and cell death. Results Morphine and DAMGO de-acidified endolysosomes, decreased endolysosome Fe 2+ levels, increased cytosol and mitochondria Fe 2+ and ROS levels, depolarized mitochondrial membrane potential, and induced cell death; effects blocked by the nonselective MOR antagonist naloxone and the selective MOR antagonist β-funaltrexamine (β-FNA). Deferoxamine, an endolysosome-iron chelator, inhibited opioid agonist-induced increases in cytosolic and mitochondrial Fe 2+ and ROS. Opioid-induced efflux of endolysosome Fe 2+ and subsequent Fe 2+ accumulation in mitochondria were blocked by the endolysosome-resident two-pore channel inhibitor NED-19 and the mitochondrial permeability transition pore inhibitor TRO. Conclusions Opioid agonist-induced increases in cytosolic and mitochondrial Fe 2+ and ROS as well as cell death appear downstream of endolysosome de-acidification and Fe 2+ efflux from the endolysosome iron pool that is sufficient to affect other organelles.

Objectives We aimed to compare brain white matter integrity in participants with post-COVID-19 conditions (PCC) and healthy controls. Methods We compared cognitive performance (NIH Toolbox ® ), psychiatric symptoms and diffusion tensor imaging (DTI) metrics between 23 PCC participants and 24 controls. Fractional anisotropy (FA), axial (AD), radial (RD), and mean (MD) diffusivities were measured in 9 white matter tracts and 6 subcortical regions using MRICloud. Results Compared to controls, PCC had similar cognitive performance, but greater psychiatric symptoms and perceived stress, as well as higher FA and lower diffusivities in multiple white matter tracts (ANCOVA-p-values≤0.001–0.048). Amongst women, PCC had higher left amygdala-MD than controls (sex-by-PCC p=0.006). Regardless of COVID-19 history, higher sagittal strata-FA predicted greater fatigue (r=0.48-0.52, p<0.001) in all participants, and higher left amygdala-MD predicted greater fatigue (r=0.61, p<0.001) and anxiety (r=0.69, p<0.001) in women, and higher perceived stress (r=0.45, p=0.002) for all participants. Conclusions Microstructural abnormalities are evident in PCC participants averaged six months after COVID-19. The restricted diffusivity (with reduced MD) and higher FA suggest enhanced myelination or increased magnetic susceptibility from iron deposition, as seen in stress conditions. The higher amygdala-MD in female PCC suggests persistent neuroinflammation, which might contribute to their fatigue, anxiety, and perceived stress.

Objectives HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory autoimmune disease characterized by high levels of infected immortalized T cells in circulation, which makes it difficult for antiretroviral (ART) drugs to work effectively. In previous studies, we established that Apigenin, a flavonoid, can exert immunomodulatory effects to reduce neuroinflammation. Flavonoids are natural ligands for the aryl hydrocarbon receptor (AhR), which is a ligand activated endogenous receptor involved in the xenobiotic response. Consequently, we tested Apigenin’s synergy in combination with ART against the survival of HTLV-1-infected cells. Methods First, we established a direct protein-protein interaction between Apigenin and AhR. We then demonstrated that Apigenin and its derivative VY-3-68 enter activated T cells, drive nuclear shuttling of AhR, and modulate its signaling both at RNA and protein level. Results In HTLV-1 producing cells with high AhR expression, Apigenin cooperates with ARTs such as Lopinavir (LPN) and Zidovudine (AZT), to impart cytotoxicity by exhibiting a major shift in IC 50 that was reversed upon AhR knockdown. Mechanistically, Apigenin treatment led to an overall downregulation of NF-κB and several other pro-cancer genes involved in survival. Conclusions This study suggest the potential combinatorial use of Apigenin with current first-line antiretrovirals for the benefit of patients affected by HTLV-1 associated pathologies.

Objectives Spatial–temporal biodistribution of antiretroviral drugs (ARVs) can now be achieved using MRI by utilizing chemical exchange saturation transfer (CEST) contrasts. However, the presence of biomolecules in tissue limits the specificity of current CEST methods. To overcome this limitation, a Lorentzian line-shape fitting algorithm was developed that simultaneously fits CEST peaks of ARV protons on its Z-spectrum. Case presentation This algorithm was tested on the common first line ARV, lamivudine (3TC), that has two peaks resulting from amino (–NH 2 ) and hydroxyl (–OH) protons in 3TC. The developed dual-peak Lorentzian function fitted these two peaks simultaneously, and used the ratio of –NH 2 and –OH CEST contrasts as a constraint parameter to measure 3TC presence in brains of drug-treated mice. 3TC biodistribution calculated using the new algorithm was compared against actual drug levels measured using UPLC-MS/MS. In comparison to the method that employs the –NH 2 CEST peak only, the dual-peak Lorentzian fitting algorithm showed stronger correlation with brain tissue 3TC levels, signifying estimation of actual drug levels. Conclusions We concluded that 3TC levels can be extracted from confounding CEST effects of tissue biomolecules resulting in improved specificity for drug mapping. This algorithm can be expanded to measure a variety of ARVs using CEST MRI.

Background We recently reveal that anti-CD4 autoantibodies contribute to blunted CD4+ T cell reconstitution in HIV+ individuals on antiretroviral therapy (ART). Cocaine use is common among HIV+ individuals and is associated with accelerated disease progression. However, the mechanisms underlying cocaine-induced immune perturbations remain obscure. Methods We evaluated plasma levels of anti-CD4 IgG and markers of microbial translocation, as well as B-cell gene expression profiles and activation in HIV+ chronic cocaine users and non-users on suppressive ART, as well as uninfected controls. Plasma purified anti-CD4 IgGs were assessed for antibody-dependent cytotoxicity (ADCC). Results HIV+ cocaine users had increased plasma levels of anti-CD4 IgGs, lipopolysaccharide (LPS), and soluble CD14 (sCD14) versus non-users. An inverse correlation was observed in cocaine users, but not non-drug users. Anti-CD4 IgGs from HIV+ cocaine users mediated CD4+ T cell death through ADCC in vitro . B cells from HIV+ cocaine users exhibited activation signaling pathways and activation (cycling and TLR4 expression) related to microbial translocation versus non-users. Conclusions This study improves our understanding of cocaine associated B cell perturbations and immune failure and the new appreciation for autoreactive B cells as novel therapeutic targets.

Human immunodeficiency virus type 1 (HIV-1) is a chronic disease that afflicts over 38 million people worldwide without a known cure. The advent of effective antiretroviral therapies (ART) has significantly decreased the morbidity and mortality associated with HIV-1 infection in people living with HIV-1 (PWH), thanks to durable virologic suppression. Despite this, people with HIV-1 experience chronic inflammation associated with co-morbidities. While no single known mechanism accounts for chronic inflammation, there is significant evidence to support the role of the NLRP3 inflammasome as a key driver. Numerous studies have demonstrated therapeutic impact of cannabinoids, including exerting modulatory effects on the NLRP3 inflammasome. Given the high rates of cannabinoid use in PWH, it is of great interest to understand the intersecting biology of the role of cannabinoids in HIV-1-associated inflammasome signaling. Here we describe the literature of chronic inflammation in people with HIV, the therapeutic impact of cannabinoids in PWH, endocannabinoids in inflammation, and HIV-1-associated inflammation. We describe a key interaction between cannabinoids, the NLRP3 inflammasome, and HIV-1 viral infection, which supports further investigation of the critical role of cannabinoids in HIV-1 infection and inflammasome signaling.