Isabella Zanella

Antiretroviral therapy (ART) has significantly increased life expectancy of people living with HIV (PLWH). Nonetheless, despite effective virological control, PLWH are faced with accelerated aging, characterized by higher prevalence of age-related comorbidities than the general population. Persistent inflammation and activation of innate immunity seem to drive this immunosenescent phenotype. The cyclic GMP–AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is increasingly recognized as a key player in the aging process and in age-related diseases, like cardiovascular and metabolic disorders, cancer, neurological diseases and cognitive decline. The human gene encoding for STING (STING1/TMEM173) exhibit significant heterogeneity, with some common single nucleotide polymorphisms (SNPs) variably affecting its immune functions (R232H and R71H, G230A, R293Q, frequently co-segregating as HAQ haplotype). This study aims to investigate the impact of those hypomorphic variants on immune control, comorbidities, and cognitive and functional outcomes in a cohort of 60 aged (> 50 years) chronic PLWH on stable ART. Patients were genotyped and differences in comorbidities, HIV-related clinical parameters, and cognitive and motor performances were assessed across genotype groups. Here we show that carriers of R71H, G230A and R293Q alleles were associated with a lower prevalence of dyslipidemia, while carriers of the R232H polymorphism show higher CD4⁺ T cell counts. Our results suggest that the common STING1/TMEM173 polymorphisms may influence immunological and clinical outcomes, modulating lipid metabolism and the dynamics of immune recovery in these people.

Therapeutic resistance remains a critical obstacle in rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma. Increasing evidence implicates redox adaptation in tumor survival and treatment failure. Here, we investigated the functional role and clinical relevance of catalase, a primary hydrogen peroxide-detoxifying enzyme, in modulating RMS therapeutic response. Integrated transcriptomic analyses revealed that while catalase is overall downregulated in RMS compared to healthy skeletal muscle, elevated expression strongly correlates with high-risk, metastatic disease and poor overall survival. In human RMS cell lines, catalase was detectable, and its pharmacological inhibition using 3-amino-1,2,4-triazole (3-ATA) promoted reactive oxygen species (ROS) accumulation, sensitizing cells to standard chemotherapeutics. Notably, robust catalase upregulation was observed in RMS cell models characterized by intrinsic and acquired radioresistance, with strong immunoreactivity validated on cell-block sections. Immunohistochemical validation across patient specimens revealed generally weak catalase expression in RMS tumors, whereas strong reactivity was observed in a secondary embryonal RMS (ERMS) arisen following chemoradiotherapy for nasopharyngeal carcinoma. Functionally, targeting catalase with 3-ATA restored both radio- and chemosensitivity in radioresistant RMS lines. Furthermore, co-targeting catalase and Akt using sub-toxic doses of 3-ATA and MK-2206 cooperatively enhanced oxidative stress-mediated cytotoxicity in resistant cells. Together, these findings identify catalase as a pivotal driver of adaptive radioresistance and establish dual catalase/Akt targeting as a promising pro-oxidant strategy to overcome radiotherapy resistance in RMS.

The term frontotemporal dementia (FTD) comprises a group of neurodegenerative disorders characterized by the progressive degeneration of the frontal and temporal lobes of the brain with language impairment and changes in cognitive, behavioral and executive functions, and in some cases motor manifestations. A high proportion of FTD cases are due to genetic mutations and inherited in an autosomal-dominant manner with variable penetrance depending on the implicated gene. Iron is a crucial microelement that is involved in several cellular essential functions in the whole body and plays additional specialized roles in the central nervous system (CNS) mainly through its redox-cycling properties. Such a feature may be harmful under aerobic conditions, since it may lead to the generation of highly reactive hydroxyl radicals. Dysfunctions of iron homeostasis in the CNS are indeed involved in several neurodegenerative disorders, although it is still challenging to determine whether the dyshomeostasis of this essential but harmful metal is a direct cause of neurodegeneration, a contributor factor or simply a consequence of other neurodegenerative mechanisms. Unlike many other neurodegenerative disorders, evidence of the dysfunction in brain iron homeostasis in FTD is still scarce; nonetheless, the recent literature intriguingly suggests its possible involvement. The present review aims to summarize what is currently known about the contribution of iron dyshomeostasis in FTD based on clinical, imaging, histological, biochemical and molecular studies, further suggesting new perspectives and offering new insights for future investigations on this underexplored field of research.

Several anti-viral drugs like those used to treat human immunodeficiency virus (HIV) infections have been associated with neurotoxicity or neurodevelopmental impairments following in utero exposures. The aim of this special issue was to provide an updated overview on this topic. The published articles explored the argument using classical in vitro and animal models, and new emerging tools like cerebral organoids, covering several aspects of the proposed topic.Monnerie and colleagues explored the possibility that elvitegravir (EGV), belonging to the integrase strand transfer inhibitor (INSTI) class of antiretroviral drugs, may contribute to white matter damage in people living with HIV. The authors used mature rat cerebral cortical oligodendrocyte cultures as an in vitro model and found that EVG reduced oligodendrocyte differentiation, particularly at later stages. They investigated the involved mechanisms and found that EVG modulated the expression of sterol regulatory elementbinding protein-1 and -2 (SREBP-1 and SREBP-2), major regulators of cellular lipid metabolism. Their modulation was accompanied by reduced expression of key enzymes involved in fatty acid synthesis, while cholesterol pathways seemed unaffected. In contrast, the INSTI raltegravir (RAL) had no effect on SREBP expression. Unexpectedly, the addition of palmitate, a fatty acid, to oligodendrocyte cultures did not prevent EVG-induced inhibition of oligodendrocyte differentiation, while addition of serum albumin did prevent these effects.Rodent models are often used to evaluate neuronal toxicity of antiretroviral drugs or HIV-associated insults, however experimental observations may consistently coincide with results from human neuronal models.To overcome this problem, Starr and colleagues established an in vitro model of human induced pluripotent stem cell (iPSC)-derived cortical glutamatergic neurons. The authors first characterized their model, then assessed the effects of EVG and RAL exposure. The authors previously demonstrated that EVG but not RAL was neurotoxic in primary rat neuroglial cultures and that its toxicity was driven by the activation of the integrated stress response (ISR) (Stern et al., 2018). Here, they did not observe toxicity with either drug, measured by microtubule-associated protein 2 (MAP2) staining. However, electrophysiological measures suggested sub-lethal damage. RNA expression analysis further revealed that both INSTIs downregulated genes involved in extracellular matrix interaction, cell motility and morphogenesis.LaNoce and colleagues described the use of human iPSC-based forebrain organoids as models of human early cortical development to explore the effect of the INSTIs dolutegravir (DTG) and RAL. Previously, this model was successfully used to study the effects of Zika virus on neurodevelopment (Qian et al., 2016).Researchers treated early-stage cortical organoids with DTG or RAL within the range of cord blood concentrations. DTG exposure resulted in reduced organoid size and density of neural rosette structures and increased caspase-3 staining, while RAL had no appreciable effects. The authors performed RNA expression analyses on treated organoids and found that DTG exposure resulted in the upregulation of cellular stress, response to glucose starvation and amino acid stimulus pathways and downregulation of pathways involved in neuron differentiation and migration, CNS development and neuroblast proliferation.Immuno-histological analysis revealed reduced neurogenesis with fewer neural stem cells and postmitotic neurons. Further, they demonstrated that DTG induced ISR activation with the upregulation of downstream effectors, which may disrupt cell cycle progression and impair neurogenesis.A three-dimensional stem cell-derived brain organoid developmental model was also employed by Caiaffa and colleagues to analyze the effect of DTG with or without folic acid (FA) exposure in the earliest maturation stages. RNA expression analysis revealed that DTG exposure modulated the expression of genes involved in neurodevelopment, cell cycle, extracellular matrix, and membrane and motor proteins, as well as inducing the expression of the folate receptor FOLR1. FA exposure in the presence of DTG attenuated the alteration of gene expression, suggesting a protective role of FA. Using multimodal optical instruments, the authors found that organoids exposed to DTG presented biomechanical alterations like higher surface stiffness, lower internal stiffness and decreased volume during maturation, that were not rescued or mitigated by FA supplementation.Foster and colleagues evaluated safety and benefits of injectable long-acting DTG nano-formulation (NDTG) on fetal neurodevelopment compared to daily oral administration of native DTG in pregnant mice. The authors found that intramuscular injection of NDTG achieved therapeutic plasma DTG concentrations in pregnant dams. These plasma drug concentrations were comparable to levels observed in dams following daily oral DTG administration. Of note, NDTG injections resulted in significantly lower levels of DTG biodistribution to the embryo brain compared to daily oral DTG administration. T1 mapping evaluations using in utero magnetic resonance imaging (MRI) indicated that lower drug exposure to the embryo brain using injectable NDTG can prevent native drug induced oxidative stress. Further, non-targeted proteomic profiling of whole embryo brain tissues from the oral native DTG group showed oxidative stress, mitochondrial dysfunction, reduced energy production, cell damage and impaired neuronal or synaptic development. These drug-induced adverse effects on embryo brain were prevented or attenuated when NDTG was administered, signifying that delivery of DTG as injectable long acting nanoformulations (NDTG) can prevent DTG-associated neurodevelopmental impairments and improve safety of DTG during pregnancy.Finally, Dhume and colleagues explored the long-term...

Neuroinflammatory conditions in the central nervous system (CNS) are implicated in the pathogenesis of several neuroimmune disorders such as acquired demyelinating syndromes, autoimmune encephalopathies, acute or chronic bacterial and viral CNS infections as well as multiple sclerosis (MS)

Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 μM) and observed the disruption of the anterior–posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (ntl, pax2a, ngn1, neurod1) and by in vivo visualization of the transgenic Tg(ngn1:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain–hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of ngn1 expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)⁺ dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.

In the past, one of the most widely used non-nucleoside reverse transcriptase inhibitors (NNRTI) in first-line antiretroviral therapy (ART) of HIV infection was efavirenz (EFV), which is already used as a cost-effective treatment in developing countries due to its efficacy, tolerability, and availability. However, EFV also demonstrates several adverse effects, like hepatotoxicity, altered lipid profile, neuropsychological symptoms, and behavioral effects in children after in utero exposure. In 2018, another NNRTI, doravirine (DOR), was approved due to its similar efficacy but better safety profile. Preclinical safety studies demonstrated that DOR is not genotoxic and exhibits no developmental toxicity or effects on fertility in rats. Zebrafish (Danio rerio) embryos have been widely accepted as a vertebrate model for pharmacological and developmental studies. We used zebrafish embryos as an in vivo model to investigate the developmental toxicity of DOR compared to EFV. After exposure of the embryos to the drugs from the gastrula stage up to different developmental stages (30 embryos for each arm, in three independent experiments), we assessed their survival, morphology, hatching rate, apoptosis in the developing head, locomotion behavior, vasculature development, and neutral lipid distribution. Overall, DOR showed a better safety profile than EFV in our model. Therapeutic and supra-therapeutic doses of DOR induced very low mortality [survival rates: 92, 90, 88, 88, and 81% at 1, 5, 10, 25, and 50 μM, respectively, at 24 h post fecundation (hpf), and 88, 85, 88, 89, and 75% at the same doses, respectively, at 48 hpf] and mild morphological alterations compared to EFV exposure also in the sub-therapeutic ranges (survival rates: 80, 77, 69, 63, and 44% at 1, 5, 10, 25, and 50 μM, respectively, at 24 hpf and 72, 70, 63, 52, and 0% at the same doses, respectively, at 48 hpf). Further, DOR only slightly affected the hatching rate at supra-therapeutic doses (97, 98, 96, 87, and 83% at 1, 5, 10, 25, and 50 μM, respectively, at 72 hpf), while EFV already strongly reduced hatching at sub-therapeutic doses (83, 49, 11, 0, and 0% at 1, 5, 10, 25, and 50 μM, respectively, at the same time endpoint). Both DOR at therapeutic doses and most severely EFV at sub-therapeutic doses enhanced apoptosis in the developing head during crucial phases of embryo neurodevelopment and perturbed the locomotor behavior. Furthermore, EFV strongly affected angiogenesis and disturbed neutral lipid homeostasis even at sub-therapeutic doses compared to DOR at therapeutic concentrations. Our findings in zebrafish embryos add further data confirming the higher safety of DOR with respect to EFV regarding embryo development, neurogenesis, angiogenesis, and lipid metabolism. Further studies are needed to explore the molecular mechanisms underlying the better pharmacological safety profile of DOR, and further human studies are required to confirm these results in the zebrafish animal model.

As most new medications, Cabotegravir (CAB) was recently approved as an antiretroviral treatment of HIV infection without in-depth safety information on in utero exposure. Although no developmental toxicity in rats and rabbits was reported, recent studies demonstrated that CAB decreases pluripotency of human embryonic stem cells. CAB exposure effects during development were assessed in zebrafish embryos by the Fish Embryo Toxicity test after exposure at subtherapeutic concentrations up to 25× the human C_max. Larvae behavior was assessed by the light–dark locomotion test. The expression of factors involved in neurogenesis was evaluated by whole-mount in situ hybridization. CAB did not cause gross morphological defects at low doses, although pericardial edema, uninflated swim bladder, decreased heartbeats, growth delay, and decreased hatching rate were observed at the highest concentrations. Decreased locomotion was observed even at the subtherapeutic dose, suggesting alterations of nervous system integrity. This hypothesis was supported by the observation of decreased expression of crucial factors involved in early neuronal differentiation in diencephalic and telencephalic dopaminergic areas, midbrain/hindbrain boundary, and craniofacial ganglia. These findings support CAB effects on neurogenesis in zebrafish embryos and suggest long-term follow-up of exposed infants to provide data on drug safety during pregnancy.

Background: Nirmatrelvir, in combination with ritonavir, is one of the first orally available antiviral treatment for coronavirus disease 2019 (COVID‐19). Symptomatic pregnant women are at increased risk for severe illness and complications that can affect the developing baby. No malformations or lower embryo‐fetal survival have been observed when nirmatrelvir were administered to pregnant rats and rabbits. Safety evaluation of drugs used for treating COVID‐19 also in pregnancy is urgent for public health, then in this study we further investigated nirmatrelvir developmental toxicity using zebrafish as in vivo model.Material and Methods: Using the standardized Fish Embryo Toxicity (FET) test, we first determined the lethal concentration 50 (LC50), exposing embryos from gastrula stage up to 120 hr post fertilization (hpf) and daily recording lethality. Then, we exposed embryos to five doses comprising the human therapeutic one and up to the LC50 (25 μM). Morphology was evaluated at 72 and 120 hpf.Results: Nirmatrelvir did not affect survival rate and did not induce morphological defects up to the human therapeutic dose. Exposure at higher doses (2.4× and 3× the human C_max) however resulted in decreased hatching rate, reduced growth, slower heartbeat with pericardial edema, reduction of eye dimension, absence of the swim bladder and disruption of the anterior–posterior axis, with lack of tail detachment, spinal curvature and straight and smaller head.Conclusions: Our findings in zebrafish embryos add further information about developmental nirmatrelvir safety. Further studies are needed for pharmacological safety assessment of nirmatrelvir exposure during pregnancy.