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Despite the high incidence of brain injuries in children, we have yet to fully understand the unique vulnerability of a young brain to an injury and key determinants of long-term recovery. Here we consider how early life stress may influence recovery after an early age brain injury. Studies of early life stress alone reveal persistent structural and functional impairments at adulthood. We consider the interacting pathologies imposed by early life stress and subsequent brain injuries during early brain development as well as at adulthood. This review outlines how early life stress primes the immune cells of the brain and periphery to elicit a heightened response to injury. While the focus of this review is on early age traumatic brain injuries, there is also a consideration of preclinical models of neonatal hypoxia and stroke, as each further speaks to the vulnerability of the brain and reinforces those characteristics that are common across each of these injuries. Lastly, we identify a common mechanistic trend; namely, early life stress worsens outcomes independent of its temporal proximity to a brain injury.

Abstract Beta-4 (β4)-spectrin, encoded by the gene Sptbn4, is a cytoskeleton protein found at nodes and the axon initial segments (AIS). Sptbn4 mutations are associated with myopathy, neuropathy, and auditory deficits in humans. Related to auditory dysfunction, however, the expression and roles of β4-spectrin at axon segments along the myelinated axon in the developing auditory brain are not well explored. We found during postnatal development, β4-spectrin is critical for voltage-gated sodium channel (Nav) clustering at the heminode along the nerve terminal, but not for the formation of nodal and AIS structures in the auditory brainstem. Presynaptic terminal recordings in Sptbn4 geo mice, β4-spectrin null mice, showed an elevated threshold of action potential and increased failures during action potential train at high-frequency. Sptbn4 geo mice exhibited a slower central conduction and showed no startle responses, but had normal cochlear function. Taken together, the lack of β4-spectrin impairs Nav clustering at the heminode along the nerve terminal and the temporal fidelity and reliability of presynaptic spikes, leading to central auditory processing deficits during postnatal development.

ObjectivesE-cigarettes pose significant risks to youth, but smokers may benefit from switching to e-cigarettes by reducing their exposure to toxicants, which creates a challenge for the Food and Drug Administration (FDA) in regulating e-cigarettes to protect population health. This study aims to develop e-liquid product standards for nicotine form and concentration that reduce the appeal of e-cigarettes to young people while keeping e-cigarettes available as a safer alternative for smokers.Design and participantsA single-visit, double-blinded, randomized crossover design will be used to examine the effects of e-liquids with varying fractions of free-base nicotine (5%, 25%, 45%, 65%, 85%) among a sample of 66 young adult EC users and 66 older adult smokers, across ecologically valid total nicotine concentrations (20 mg or 50 mg/mL).Interventions and outcomesA 2-puff session will be conducted to test each of the 10 e-liquids in randomly assigned sequences, followed by a 10-minute washout period and participant ratings on appeal and sensory attributes such as throat hit and harshness, as well as behavioral intentions for continued use. Generalized linear mixed models will be used to determine a free-base nicotine level that has limited or no appeal to young adult e-cigarette users while remaining acceptable to smokers.ConclusionsThis study will provide the FDA with scientific evidence regarding the effect of product standards that mandate a minimum threshold for the fraction of free-base nicotine.Trial registrationThe study is registered on clinicaltrials.gov under the identifier NCT05864586.

μ-Crystallin, encoded by the CRYM gene, binds the thyroid hormones, T3 and T4. Because T3 and T4 are potent regulators of metabolism and gene expression, and CRYM levels in human skeletal muscle can vary widely, we investigated the effects of overexpression of Crym. We generated transgenic mice, Crym tg, that expressed Crym protein specifically in skeletal muscle at levels 2.6–147.5 fold higher than in controls. Muscular functions, Ca2+ transients, contractile force, fatigue, running on treadmills or wheels, were not significantly altered, although T3 levels in tibialis anterior (TA) muscle were elevated ~190-fold and serum T4 was decreased 1.2-fold. Serum T3 and thyroid stimulating hormone (TSH) levels were unaffected. Crym transgenic mice studied in metabolic chambers showed a significant decrease in the respiratory exchange ratio (RER) corresponding to a 13.7% increase in fat utilization as an energy source compared to controls. Female but not male Crym tg mice gained weight more rapidly than controls when fed high fat or high simple carbohydrate diets. Although labeling for myosin heavy chains showed no fiber type differences in TA or soleus muscles, application of machine learning algorithms revealed small but significant morphological differences between Crym tg and control soleus fibers. RNA-seq and gene ontology enrichment analysis showed a significant shift towards genes associated with slower muscle function and its metabolic correlate, β-oxidation. Protein expression showed a similar shift, though with little overlap. Our study shows that μ-crystallin plays an important role in determining substrate utilization in mammalian muscle and that high levels of μ-crystallin are associated with a shift toward greater fat metabolism.

Children born prematurely suffer from learning disabilities and exhibit reading, speech, and cognitive difficulties, which are associated with an auditory processing disorder. However, it is unknown whether gestational age at delivery and the unnatural auditory environment in neonatal intensive care units (NICU) collectively affect proper auditory development and neuronal circuitry in premature newborns. We morphologically characterized fetal development of the medial superior olivary nucleus (MSO), an area important for binaural hearing and sound localization, in the auditory brainstem of baboon neonates at different gestational ages. Axonal and synaptic structures and the tonotopic differentiation of ion channels in the MSO underwent profound refinements after hearing onset in the uterus. These developmental refinements of the MSO were significantly altered in preterm baboon neonates in the NICU. Thus, the maternal environment in uterus is critical for auditory nervous system development during the last trimester of pregnancy and critically affects the anatomic and functional formation of synapses and neural circuitry in the preterm newborn brain.

Over half of individuals infected with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HANDs), yet the molecular mechanisms leading to neuronal dysfunction are poorly understood. Feline immunodeficiency virus (FIV) naturally infects cats and shares its structure, cell tropism, and pathology with HIV, including wide-ranging neurological deficits. We employ FIV as a model to elucidate the molecular pathways underlying HIV-induced neuronal dysfunction, in particular, synaptic alteration. Among HIV-induced neuron-damaging products, HIV envelope glycoprotein gp120 triggers elevation of intracellular Ca2+ activity in neurons, stimulating various pathways to damage synaptic functions. We quantify neuronal Ca2+ activity using intracellular Ca2+ imaging in cultured hippocampal neurons and confirm that FIV envelope glycoprotein gp95 also elevates neuronal Ca2+ activity. In addition, we reveal that gp95 interacts with the chemokine receptor, CXCR4, and facilitates the release of intracellular Ca2+ by the activation of the endoplasmic reticulum (ER)-associated Ca2+ channels, inositol triphosphate receptors (IP3Rs), and synaptic NMDA receptors (NMDARs), similar to HIV gp120. This suggests that HIV gp120 and FIV gp95 share a core pathological process in neurons. Significantly, gp95's stimulation of NMDARs activates cGMP-dependent protein kinase II (cGKII) through the activation of the neuronal nitric oxide synthase (nNOS)-cGMP pathway, which increases Ca2+ release from the ER and promotes surface expression of AMPA receptors, leading to an increase in synaptic activity. Moreover, we culture feline hippocampal neurons and confirm that gp95-induced neuronal Ca2+ overactivation is mediated by CXCR4 and cGKII. Finally, cGKII activation is also required for HIV gp120-induced Ca2+ hyperactivation. These results thus provide a novel neurobiological mechanism of cGKII-mediated synaptic hyperexcitation in HAND.

We sought to characterize the population pharmacokinetic/pharmacodynamic (PK/PD) relationship of bococizumab (RN316/PF-04950615), a humanized IgG2Δa monoclonal antibody that binds to secreted human proprotein convertase subtilisin kexin type 9 (PCSK9), using data derived from 16 phase I, II, and III clinical studies (36,066 bococizumab observations, 46,790 low-density lipoprotein cholesterol [LDL-C] measurements, 3499 participants). A two-compartment disposition model with parallel linear and Michaelis-Menten elimination and an indirect response model was used to characterize the population PK and LDL-C response of bococizumab. Potential model parameters and covariate relationships were explored, and visual predictive checks were used for model assessment and validation. Key covariates included the effect of anti-drug antibodies (ADAs) on exposure through impact on clearance and bioavailability; impact of statins on bococizumab elimination (maximal rate of metabolism); and impact of statins, Asian race, and male sex on LDL-C efficacy (maximum effect). ADAs and neutralizing ADAs did not have additional effects on LDL-C beyond the influence on bococizumab exposure. In conclusion, the population PK/PD model adequately describes bococizumab concentration and LDL-C efficacy. The covariate effects are consistent with the presumed mechanism of action of PCSK9 inhibitors. With increasing availability of antibody-based therapeutics, improved understanding of the effect of ADAs and statins on bococizumab PK/PD adds to the literature and enhances our pharmacological understanding of how immunogenicity and concomitant medications may impact the PK/PD of biotherapeutics., (© 2023 Pfizer Inc. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30 , a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.

Adenosine triphosphate (ATP) released from injured or dying cells is a potent pro-inflammatory "danger" signal. Alkaline phosphatase (AP), an endogenous enzyme that de-phosphorylates extracellular ATP, likely plays an anti-inflammatory role in immune responses. We hypothesized that ilofotase alfa, a human recombinant AP, protects kidneys from ischemia-reperfusion injury (IRI), a model of acute kidney injury (AKI), by metabolizing extracellular ATP to adenosine, which is known to activate adenosine receptors. Ilofotase alfa (iv) with or without ZM241,385 (sc), a selective adenosine A 2A receptor (A 2A R) antagonist, was administered 1 h before bilateral IRI in WT, A 2A R KO ( Adora2a -/- ) or CD73 -/- mice. In additional studies recombinant alkaline phosphatase was given after IRI. In an AKI-on-chronic kidney disease (CKD) ischemic rat model, ilofotase alfa was given after the three instances of IRI and rats were followed for 56 days. Ilofotase alfa in a dose dependent manner decreased IRI in WT mice, an effect prevented by ZM241,385 and partially prevented in Adora2a -/- mice. Enzymatically inactive ilofotase alfa was not protective. Ilofotase alfa rescued CD73 -/- mice, which lack a 5'-ectonucleotidase that dephosphorylates AMP to adenosine; ZM241,385 inhibited that protection. In both rats and mice ilofotase alfa ameliorated IRI when administered after injury, thus providing relevance for therapeutic dosing of ilofotase alfa following established AKI. In an AKI-on-CKD ischemic rat model, ilofotase alfa given after the third instance of IRI reduced injury. These results suggest that ilofotase alfa promotes production of adenosine from liberated ATP in injured kidney tissue, thereby amplifying endogenous mechanisms that can reverse tissue injury, in part through A 2A R-and non-A 2A R-dependent signaling pathways., Competing Interests: JH, AB, KB, EM, JL, GH, NK and JG were employed by Pfizer Inc. AVE was employed by AM-Pharma B.V. MDO had a research grant from Pfizer Inc. AVE was consultant to AM-Pharma B.V. and a named inventor on patent filings related to ilofotase alfa. BM had research grants with AM-Pharma B.V. and Pfizer Inc. and was on a MAB for AM-Pharma B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rosin, Hall, Zheng, Huang, Campos-Bilderback, Sandoval, Bree, Beaumont, Miller, Larsen, Hariri, Kaila, Encarnacion, Gale, van Elsas, Molitoris and Okusa.)

Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al ], [ Nature ] [510], [288-292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al ], [ Nature ] [510], [288-292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [ J. Biol. Chem. ] [270], [29682-29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al ], [ Nature ] [510], [288-292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al ], [ Nat. Struct. Mol. Biol. ] [24], [507-514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies., Competing Interests: Competing interest statement: M.A.D, S.A., G.C.P.v.Z, A.V., and K.B. are employees of Schrödinger. A.V.T., N.K., K.D.K., and J.J.M. are employees of Nimbus Therapeutics., (Copyright © 2021 the Author(s). Published by PNAS.)

Introduction : Hematopoietic progenitor kinase (HPK1), a serine/threonine kinase, which is primarily expressed in hematopoietic cells is a negative regulator of T-cell receptor and B cell signaling. Studies using genetic disruption of HPK1 function show enhanced T-cell signaling, cytokine production, and in vivo tumor growth inhibition. This profile of enhanced immune response highlights small molecule inhibition of HPK1 as an attractive approach for the immunotherapy of cancer. Areas covered : This article summarizes the biological rationale for the inhibition of HPK1 as a potential adjunct to the current immuno-oncology (IO) therapies. The article primarily discloses the current state of development of HPK1 inhibitors. Expert Opinion : The rapid increase in the identification of small molecule inhibitors of HPK1 should translate into a fuller understanding of the role of HPK1 inhibition in the IO setting. This understanding will be of huge importance in determining whether HPK1 inhibition alone will be sufficient for tumor growth inhibition or if combination with current IO therapies will be required.

Background: Infusion-related reactions (IRRs) are the most common adverse event (AE) associated with infusion of rituximab, an anti-CD20 monoclonal antibody., Objective: Our objective was to evaluate the impact of dosing/infusion patterns and certain baseline characteristics on IRR occurrence during the first rituximab infusion administered as the biosimilar PF-05280586 (RTX-PF) or reference rituximab sourced from the EU (RTX-EU, MabThera ® ) in patients with CD20+ low-tumor-burden follicular lymphoma., Patients and Methods: Rituximab (RTX-PF, n=196; RTX-EU, n=198) was administered (375 mg/m 2 ) on days 1, 8, 15, and 22 (one cycle), with a follow-up period through 52 weeks. The relationships between infusion rate, drug exposure, and IRR incidence were assessed by logistic regression analysis and pharmacokinetic modeling and simulation. Baseline CD20 level, antidrug antibody (ADA) status, and tumor burden according to IRR occurrence (yes/no) were compared descriptively., Results: Median rituximab infusion duration on day 1 was 3.50 h for each of the two groups. There was a positive correlation between infusion rate and all-grade IRRs occurring within 24 h after infusion (p < 0.0001). Patients who developed IRRs had a higher median baseline CD20+ level. IRR incidence was unaffected by baseline ADA status. Drug exposure did not predict IRR incidence. Baseline tumor burden was similar between patients with and without IRRs., Conclusions: Results of this analysis provide a better understanding of IRRs after the first rituximab (RTX-PF or RTX-EU) infusion and demonstrate a potential correlation of infusion rate and other factors with IRR at the individual and population levels. Infusion-rate escalation steps continue to be needed to manage IRRs., Trial Registration (date of Registration): ClinicalTrials.gov Identifier: NCT02213263 (11 August 2014); and EudraCT: 2014-000132-41 (10 October 2014)., (© 2021. The Author(s).)

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.