High-Throughput Screening for the Prevalence of Neutralizing Antibodies against Human Adenovirus Serotype 5.

Adenoviral vectors based on the human adenovirus species C serotype 5 (HAdV-C5) are commonly used for vector-based gene therapies and vaccines. In the preclinical stages of development, their safety and efficacy are often validated in suitable animal models. However, pre-existing neutralizing antibodies may severely influence study outcomes. Here, we generated a new HAdV-C5-based reporter vector and established a high-throughput screening assay for the multivalent detection of HAdV-C5-neutralizing antibodies in serum. We screened the sera of rhesus macaques at different primate centers, and of rabbits, horses, cats, and dogs, showing that HAdV-C5-neutralizing antibodies can be found in all species, albeit at different frequencies. Our results emphasize the need to prescreen model animals in HAdV-C5-based studies.

Early-in-life isoflurane exposure alters resting-state functional connectivity in juvenile non-human primates.

BACKGROUND: Clinical studies suggest that anaesthesia exposure early in life affects neurobehavioural development. We designed a non-human primate (NHP) study to evaluate cognitive, behavioural, and brain functional and structural alterations after isoflurane exposure during infancy. These NHPs displayed decreased close social behaviour and increased astrogliosis in specific brain regions, most notably in the amygdala. Here we hypothesise that resting-state functional connectivity MRI can detect alterations in connectivity of brain areas that relate to these social behaviours and astrogliosis. METHODS: Imaging was performed in 2-yr-old NHPs under light anaesthesia, after early-in-life (postnatal days 6-12) exposure to 5 h of isoflurane either one or three times, or to room air. Brain images were segmented into 82 regions of interest; the amygdala and the posterior cingulate cortex were chosen for a seed-based resting-state functional connectivity MRI analysis. RESULTS: We found differences between groups in resting-state functional connectivity of the amygdala and the auditory cortices, medial premotor cortex, and posterior cingulate cortex. There were also alterations in resting-state functional connectivity between the posterior cingulate cortex and secondary auditory, polar prefrontal, and temporal cortices, and the anterior insula. Relationships were identified between resting-state functional connectivity alterations and the decrease in close social behaviour and increased astrogliosis. CONCLUSIONS: Early-in-life anaesthesia exposure in NHPs is associated with resting-state functional connectivity alterations of the amygdala and the posterior cingulate cortex with other brain regions, evident at the juvenile age of 2 yr. These changes in resting-state functional connectivity correlate with the decrease in close social behaviour and increased astrogliosis. Using resting-state functional connectivity MRI to study the neuronal underpinnings of early-in-life anaesthesia-induced behavioural alterations could facilitate development of a biomarker for anaesthesia-induced developmental neurotoxicity.

TRPV2, a novel player in the human ovary and human granulosa cells.

The cation channel 'transient receptor potential vanilloid 2' (TRPV2) is activated by a broad spectrum of stimuli, including mechanical stretch, endogenous and exogenous chemical compounds, hormones, growth factors, reactive oxygen species, and cannabinoids. TRPV2 is known to be involved in inflammatory and immunological processes, which are also of relevance in the ovary. Yet, neither the presence nor possible roles of TRPV2 in the ovary have been investigated. Data mining indicated expression, for example, in granulosa cells (GCs) of the human ovary in situ, which was retained in cultured GCs derived from patients undergoing medical reproductive procedures. We performed immunohistochemistry of human and rhesus monkey ovarian sections and then cellular studies in cultured GCs, employing the preferential TRPV2 agonist cannabidiol (CBD). Immunohistochemistry showed TRPV2 staining in GCs of large antral follicles and corpus luteum but also in theca, endothelial, and stromal cells. TRPV2 transcript and protein levels increased upon administration of hCG or forskolin. Acutely, application of the agonist CBD elicited transient Ca2+ fluxes, which was followed by the production and secretion of several inflammatory factors, especially COX2, IL6, IL8, and PTX3, in a time- and dose-dependent manner. CBD interfered with progesterone synthesis and altered both the proteome and secretome, as revealed by a proteomic study. While studies are somewhat hampered by the lack of highly specific TRPV2 agonist or antagonists, the results pinpoint TRPV2 as a modulator of inflammation with possible roles in human ovarian (patho-)physiology. Finally, as TRPV2 is activated by cannabinoids, their possible ovarian actions should be further evaluated.

Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells.

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.

Astrogliosis in juvenile non-human primates 2 years after infant anaesthesia exposure.

BACKGROUND: Infant anaesthesia causes acute brain cell apoptosis, and later in life cognitive deficits and behavioural alterations, in non-human primates (NHPs). Various brain injuries and neurodegenerative conditions are characterised by chronic astrocyte activation (astrogliosis). Glial fibrillary acidic protein (GFAP), an astrocyte-specific protein, increases during astrogliosis and remains elevated after an injury. Whether infant anaesthesia is associated with a sustained increase in GFAP is unknown. We hypothesised that GFAP is increased in specific brain areas of NHPs 2 yr after infant anaesthesia, consistent with prior injury. METHODS: Eight 6-day-old NHPs per group were exposed to 5 h isoflurane once (1×) or three times (3×), or to room air as a control (Ctr). Two years after exposure, their brains were assessed for GFAP density changes in the primary visual cortex (V1), perirhinal cortex (PRC), hippocampal subiculum, amygdala, and orbitofrontal cortex (OFC). We also assessed concomitant microglia activation and hippocampal neurogenesis. RESULTS: Compared with controls, GFAP densities in V1 were increased in exposed groups (Ctr: 0.208 [0.085-0.427], 1×: 0.313 [0.108-0.533], 3×: 0.389 [0.262-0.652]), whereas the density of activated microglia was unchanged. In addition, GFAP densities were increased in the 3× group in the PRC and the subiculum, and in both exposure groups in the amygdala, but there was no increase in the OFC. There were no differences in hippocampal neurogenesis among groups. CONCLUSIONS: Two years after infant anaesthesia, NHPs show increased GFAP without concomitant microglia activation in specific brain areas. These long-lasting structural changes in the brain caused by infant anaesthesia exposure may be associated with functional alterations at this age.

Brain pathology caused in the neonatal macaque by short and prolonged exposures to anticonvulsant drugs.

Barbiturates and benzodiazepines are potent GABAA receptor agonists and strong anticonvulsants. In the developing brain they can cause neuronal and oligodendroglia apoptosis, impair synaptogenesis, inhibit neurogenesis and trigger long-term neurocognitive sequelae. In humans, the vulnerable period is projected to extend from the third trimester of pregnancy to the third year of life. Infants with seizures and epilepsies may receive barbiturates, benzodiazepines and their combinations for days, months or years. How exposure duration affects neuropathological sequelae is unknown. Here we investigated toxicity of phenobarbital/midazolam (Pb/M) combination in the developing nonhuman primate brain. Neonatal rhesus monkeys received phenobarbital intravenously, followed by infusion of midazolam over 5 (n = 4) or 24 h (n = 4). Animals were euthanized at 8 or 36 h and brains examined immunohistochemically and stereologically. Treatment was well tolerated, physiological parameters remained at optimal levels. Compared to naïve controls, Pb/M exposed brains displayed widespread apoptosis affecting neurons and oligodendrocytes. Pattern and severity of cell death differed depending on treatment-duration, with more extensive neurodegeneration following longer exposure. At 36 h, areas of the brain not affected at 8 h displayed neuronal apoptosis, while oligodendroglia death was most prominent at 8 h. A notable feature at 36 h was degeneration of neuronal tracts and trans-neuronal death of neurons, presumably following their disconnection from degenerated presynaptic partners. These findings demonstrate that brain toxicity of Pb/M in the neonatal primate brain becomes more severe with longer exposures and expands trans-synaptically. Impact of these sequelae on neurocognitive outcomes and the brain connectome will need to be explored.

Infant isoflurane exposure affects social behaviours, but does not impair specific cognitive domains in juvenile non-human primates.

BACKGROUND: Clinical studies show that children exposed to anaesthetics for short times at young age perform normally on intelligence tests, but display altered social behaviours. In non-human primates (NHPs), infant anaesthesia exposure for several hours causes neurobehavioural impairments, including delayed motor reflex development and increased anxiety-related behaviours assessed by provoked response testing. However, the effects of anaesthesia on spontaneous social behaviours in juvenile NHPs have not been investigated. We hypothesised that multiple, but not single, 5 h isoflurane exposures in infant NHPs are associated with impairments in specific cognitive domains and altered social behaviours at juvenile age. METHODS: Eight Rhesus macaques per group were anaesthetised for 5 h using isoflurane one (1×) or three (3×) times between postnatal days 6 and 12 or were exposed to room air (control). Cognitive testing, behavioural assessments in the home environment, and provoked response testing were performed during the first 2 yr of life. RESULTS: The cognitive functions tested did not differ amongst groups. However, compared to controls, NHPs in the 3× group showed less close social behaviour (P=0.016), and NHPs in the 1× group displayed increased anxiety-related behaviours (P=0.038) and were more inhibited towards novel objects (P<0.001). CONCLUSIONS: 5 h exposures of NHPs to isoflurane during infancy are associated with decreased close social behaviour after multiple exposures and more anxiety-related behaviours and increased behavioural inhibition after single exposure, but they do not affect the cognitive domains tested. Our findings are consistent with behavioural alterations in social settings reported in clinical studies, which may guide future research.

Novel WT1 Target Genes: IL-2, IL-2RB, and IL-2RG Discovered during WT1 Silencing Using Lentiviral-Based RNAi in Myeloid Leukemia Cells.

Wilms' tumor 1 (WT1) is a transcription factor which plays a major role in cell proliferation, differentiation, survival, and apoptosis. WT1 was first identified as a tumor suppressor gene in Wilms' tumor. However, overexpression of WT1 has been detected in several types of malignancy including some types of leukemia. To investigate the molecular mechanism underlying WT1-mediated leukemogenesis, lentiviral-based siRNA was employed as a tool to suppress WT1 expression in the myeloid leukemia cell line, K562. Successfully, both WT1 RNA and protein levels were downregulated in the leukemia cells. The silencing of WT1 resulted in significant growth inhibition in WT1-siRNA-treated cells for 40 ± 7.0%, 44 ± 9.5%, and 88 ± 9.1% at 48, 72, and 96 hours posttransduction as compared with the control cells, respectively. By using apoptosis detection assays (caspase-3/7 activity and Annexin V-FITC/PI assays), WT1 silencing induced a higher degree of early and late apoptosis in siRNA-treated K562 as compared with the control cells. Interestingly, the expression of survival signaling genes, IL-2, IL-2RB, and IL-2RG, was also suppressed after WT1-siRNA treatment. In addition, the WT1 silencing also inhibited the S phase of the cell cycle and induced cell death. Our results indicated that WT1 silencing by siRNA can suppress cellular proliferation, induce apoptosis, and reduce S phase fraction of K562 cells. Moreover, transcriptional modulation of IL-2, IL-2RB, and IL2-2RG expression by WT1 was likely involved in this phenotypic change. Overall, this study confirmed the oncogenic role of WT1 in myeloid leukemia and discovered the new target genes of WT1 which are likely involved in WT1-mediated leukemogenesis.

Adeno-associated virus-binding antibodies detected in cats living in the Northeastern United States lack neutralizing activity.

Cats are a critical pre-clinical model for studying adeno-associated virus (AAV) vector-mediated gene therapies. A recent study has described the high prevalence of anti-AAV neutralizing antibodies among domestic cats in Switzerland. However, our knowledge of pre-existing humoral immunity against various AAV serotypes in cats is still limited. Here, we show that, although antibodies binding known AAV serotypes (AAV1 to AAV11) are prevalent in cats living in the Northeastern United States, these antibodies do not necessarily neutralize AAV infectivity. We analyzed sera from 35 client-owned, 20 feral, and 30 specific pathogen-free (SPF) cats for pre-existing AAV-binding antibodies against the 11 serotypes. Antibody prevalence was 7 to 90% with an overall median of 50%. The AAV-binding antibodies showed broad reactivities with other serotypes. Of 44 selected antibodies binding AAV2, AAV6 or AAV9, none exhibited appreciable neutralizing activities. Instead, AAV6 or AAV9-binding antibodies showed a transduction-enhancing effect. AAV6-binding antibodies were highly prevalent in SPF cats (83%), but this was primarily due to cross-reactivity with preventive vaccine-induced anti-feline panleukopenia virus antibodies. These results indicate that prevalent pre-existing immunity in cats is not necessarily inhibitory to AAV and highlight a substantial difference in the nature of AAV-binding antibodies in cats living in geographically different regions.

FGF21 decreases body weight without reducing food intake or bone mineral density in high-fat fed obese rhesus macaque monkeys.

OBJECTIVE: Administration of FGF21 and FGF21 analogues reduce body weight; improve insulin sensitivity and dyslipidemia in animal models of obesity and in short term clinical trials. However potential adverse effects identified in mice have raised concerns for the development of FGF21 therapeutics. Therefore, this study was designed to address the actions of FGF21 on body weight, glucose and lipid metabolism and importantly its effects on bone mineral density (BMD), bone markers, and plasma cortisol in high-fat fed obese rhesus macaque monkeys. METHODS: Obese non-diabetic rhesus macaque monkeys (five males and five ovariectomized (OVX) females) were maintained on a high-fat diet and treated for 12 weeks with escalating doses of FGF21. Food intake was assessed daily and body weight weekly. Bone mineral content (BMC) and BMD were measured by DEXA scanning prior to the study and on several occasions throughout the treatment period as well as during washout. Plasma glucose, glucose tolerance, insulin, lipids, cortisol, and bone markers were likewise measured throughout the study. RESULTS: On average, FGF21 decreased body weight by 17.6 ± 1.6% after 12 weeks of treatment. No significant effect on food intake was observed. No change in BMC or BMD was observed, while a 2-fold increase in CTX-1, a marker of bone resorption, was seen. Overall glucose tolerance was improved with a small but significant decrease in HbA1C. Furthermore, FGF21 reduced concentrations of plasma triglycerides and very low density lipoprotein cholesterol. No adverse changes in clinical chemistry markers were demonstrated, and no alterations in plasma cortisol were observed during the study. CONCLUSION: In conclusion, FGF21 reduced body weight in obese rhesus macaque monkeys without reducing food intake. Furthermore, FGF21 had beneficial effects on body composition, insulin sensitivity, and plasma triglycerides. No adverse effects on bone density or plasma cortisol were observed after 12 weeks of treatment.