A multiadjuvant polysaccharide-amino acid-lipid (PAL) subunit nanovaccine generates robust systemic and lung-specific mucosal immune responses against SARS-CoV-2 in mice.

Existing parenteral SARS-CoV-2 vaccines produce only limited mucosal responses, which are essential for reducing transmission and achieving sterilizing immunity. Appropriately designed mucosal boosters could overcome the shortcomings of parenteral vaccines and enhance pre- existing systemic immunity. Here we present a new protein subunit nanovaccine using multiadjuvanted (e.g. RIG-I: PUUC, TLR9: CpG) polysaccharide-amino acid-lipid nanoparticles (PAL-NPs) that can be delivered both intramuscularly (IM) and intranasally (IN) to generate balanced mucosal-systemic SARS-CoV-2 immunity. Mice receiving IM-Prime PUUC+CpG PAL- NPs, followed by an IN-Boost, developed high levels of IgA, IgG, and cellular immunity in the lung, and showed robust systemic humoral immunity. Interestingly, as a purely intranasal vaccine (IN-Prime/IN-Boost), PUUC+CpG PAL-NPs induced stronger lung-specific T cell immunity than IM-Prime/IN-Boost, and a comparable IgA and neutralizing antibodies, although with a lower systemic antibody response, indicating that a fully mucosal delivery route for SARS-CoV-2 vaccination may also be feasible. Our data suggest that PUUC+CpG PAL-NP subunit vaccine is a promising candidate for generating SARS-CoV-2 specific mucosal immunity.

Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.

Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-ß-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.

Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability remain. Molecular adjuvants targeting pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulates various PRRs, including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors. We hypothesized that targeting PRRs using molecular adjuvants on nanoparticles (NPs) along with a stabilized spike protein antigen could stimulate broad and efficient immune responses. Adjuvants targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer NPs were combined with the S1 subunit of spike protein and assessed in vitro with isogeneic mixed lymphocyte reactions (isoMLRs). For in vivo studies, the adjuvant-NPs were combined with stabilized spike protein or spike-conjugated NPs and assessed using a two-dose intranasal or intramuscular vaccination model in mice. Combination adjuvant-NPs simultaneously targeting TLR and RIG-I receptors (MPLA+PUUC, CpG+PUUC, and R848+PUUC) differentially induced T cell proliferation and increased proinflammatory cytokine secretion by APCs in vitro. When delivered intranasally, MPLA+PUUC NPs enhanced CD4+CD44+ activated memory T cell responses against spike protein in the lungs while MPLA NPs increased anti-spike IgA in the bronchoalveolar (BAL) fluid and IgG in the blood. Following intramuscular delivery, PUUC NPs induced strong humoral immune responses, characterized by increases in anti-spike IgG in the blood and germinal center B cell populations (GL7+ and BCL6+ B cells) in the draining lymph nodes (dLNs). MPLA+PUUC NPs further boosted spike protein-neutralizing antibody titers and T follicular helper cell populations in the dLNs. These results suggest that protein subunit vaccines with particle-delivered molecular adjuvants targeting TLR4 and RIG-I could lead to robust and unique route-specific adaptive immune responses against SARS-CoV-2.

Monitoring the Sialome on Human Immune Cells.

The sialome or display of sialic acids on the surface of human immune cells can vary according to immune response and activation state. Here, human peripheral blood mononuclear cells (PBMCs) were isolated and activated with anti-CD3 antibody and the cell surface sialome was quantified using a combination of click chemistry, confocal microscopy and flow cytometry techniques. Carbohydrate click chemistry was used to detect and measure the incorporation of an azido-m65odified sialic acid precursor molecule, N-acetylmannosamine (ManNaz) sugar into the PBMC surface sialome. Incorporation of sialic acid into the PBMC glycocalyx was visualized using copper-catalyzed click conjugation of Alexa 488 alkyne and confocal microscopy and further quantified using flow cytometry. The use of these methods indicate that regulating the sialome content on the surface of activated immune cells may be monitored during immunomodulatory responses and anti-inflammatory therapies.

Effects of route of administration on neural exposure to platinum-based chemotherapy treatment: a pharmacokinetic study in rat.

The persisting need for effective clinical treatment of chemotherapy-induced neurotoxicity (CIN) motivates critical evaluation of preclinical models of CIN for their translational relevance. The present study aimed to provide the first quantitative evaluation of neural tissue exposed in vivo to a platinum-based anticancer compound, oxaliplatin (OX) during and after two commonly used dosing regimens: slow IV infusion used clinically and bolus IP injection used preclinically. Inductively-coupled plasma mass spectrometry analysis of dorsal root ganglia indicated that while differences in the temporal dynamics of platinum distribution exist, key drivers of neurotoxicity, e.g. peak concentrations and exposure, were not different across the two routes of administration. We conclude that the IP route of OX administration achieves clinically relevant pharmacokinetic exposure of neural tissues in a rodent model of CIN.

STAR particles for enhanced topical drug and vaccine delivery.

Drug delivery to the skin is highly constrained by the stratum corneum barrier layer1. Here, we developed star-shaped particles, termed STAR particles, to dramatically increase skin permeability. STAR particles are millimeter-scale particles made of aluminum oxide or stainless steel with micron-scale projections designed to create microscopic pores across the stratum corneum. After gentle topical application for 10 s to porcine skin ex vivo, delivery of dermatological drugs and macromolecules, including those that cannot be given topically, was increased by 1 to 2 orders of magnitude. In mice treated with topical 5-fluorouracil, use of STAR particles increased the efficacy of the drug in suppressing the growth of subcutaneous melanoma tumors and prolonging survival. Moreover, topical delivery of tetanus toxoid vaccine to mice using STAR particles generated immune responses that were at least as strong as delivery of the vaccine by intramuscular injection, albeit at a higher dose for topical than intramuscular vaccine administration. STAR particles were well tolerated and effective at creating micropores when applied to the skin of human participants. Use of STAR particles provides a simple, low-cost and well-tolerated method for increasing drug and vaccine delivery to the skin and could widen the range of compounds that can be topically administered.

Pharmaceutical jewelry: Earring patch for transdermal delivery of contraceptive hormone.

Lack of adherence to medication dosing schedules is a significant cause of morbidity and mortality with large associated financial costs. This is especially true for contraceptive hormones, which provide almost perfect prevention of pregnancy when used correctly, but have significant failure rates in typical use, due largely to poor adherence. To increase medication acceptability and adherence, we introduce pharmaceutical jewelry, in which a transdermal patch is incorporated into jewelry worn on skin. To demonstrate the approach, we incorporated transdermal patches containing the contraceptive hormone levonorgestrel (LNG) into an earring, a ring, a necklace, and a wrist watch. Transdermal delivery of LNG from earring patches across porcine skin ex vivo achieved a steady state flux of 1.7 µg/cm2·h. Pharmacokinetic analysis in hairless rats yielded LNG delivery rates that maintained serum LNG levels near 1500 pg/ml throughout the 1-week patch application period, which is well above the human contraceptive threshold concentration of 200 pg/ml. When patches were applied cyclically for 16 h on and 8 h off to simulate earring removal at night, serum LNG concentrations dipped during off periods, but remained well above the human contraceptive threshold. Earring patches were well tolerated by the rats. We conclude that pharmaceutical jewelry can provide a novel method of drug delivery, especially for contraceptive hormones, that has the potential to improve acceptability and increase medication adherence.

Osteoarthritis-associated basic calcium phosphate crystals induce pro-inflammatory cytokines and damage-associated molecules via activation of Syk and PI3 kinase.

The pro-inflammatory cytokines, TNFα, IL-1 and IL-18, amplify cartilage destruction associated with osteoarthritis (OA). Current data suggest that basic calcium phosphate (BCP) crystals are potent drivers of inflammatory mediator and matrix metalloprotease expression in the OA joint. It has previously been demonstrated that synovial macrophages play a role in initiating and driving BCP-induced inflammation. However, the molecular mechanisms by which BCP crystals exert their effects remain unclear. Here we demonstrate that exposure of macrophages to BCP crystals leads to activation of Syk and PI3 kinase. Furthermore, we show that production of pro-inflammatory cytokines and phosphorylation of the downstream kinase, ERK, are suppressed following treatment with Syk and PI3 kinase inhibitors. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule, S100A8, in a Syk dependent manner. We therefore identify Syk and PI3 kinase as potential novel targets for the treatment of BCP-related pathologies.

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons.

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.

Social anxiety in Cornelia de Lange syndrome.

In this study we assessed the behavioral presentation of social anxiety in Cornelia de Lange syndrome (CdLS) using a contrast group of Cri du Chat syndrome (CdCS). Behaviors indicative of social anxiety were recorded in twelve children with CdLS (mean age = 11.00; SD = 5.15) and twelve children with CdCS (8.20; SD = 2.86) during social interaction. Lag sequential analysis revealed that participants with CdLS were significantly more likely to evidence behavior indicative of anxiety in close temporal proximity to the point at which they maintained eye contact or spoke. Individuals with CdLS demonstrate a heightened probability of anxiety related behavior during social interaction but only at the point at which social demand is high.

CK-MM autoantibodies: prevalence, immune complexes, and effect on CK clearance.

Although the blood level of creatine kinase (CK) is the most commonly used marker of muscle injury, there is marked interindividual variability in this measure. Part of this variability may be attributed to variability in the rate of CK clearance from the circulation. In this study, we assessed the possibility that CK-MM autoantibodies form immune complexes with CK following muscle injury and subsequently affect the CK clearance rate. Using an enzyme-linked immunosorbent assay, CK-MM autoantibodies were detected in all 25 human subjects studied but the levels varied greatly. Using protein A-sepharose, the percentage of the plasma CK activity found in immune complexes was determined to be correlated with the CK-MM autoantibody level at lower CK levels (<1,022 U/L). When CK-MM antibodies were administered to mice, plasma CK activity following a bolus CK injection was reduced by 11%-32%. We conclude that CK-MM autoantibodies can modulate the rate of CK clearance from the circulation. Thus, the relatively low blood CK levels seen in some individuals following injury may be attributed partly or entirely to an autoantibody-enhanced clearance of CK.

Chemokine receptor CCR2 involvement in skeletal muscle regeneration.

Chemokines, signaling through the CCR2 receptor, are highly expressed in injured skeletal muscle. Their target specificity depends on the cellular expression of the specific receptors. Here we demonstrate that, in freeze-injured muscle, CCR2 co-localized with Mac-3, a marker of activated macrophages as well as with myogenin, a marker of activated muscle precursor cells. The degeneration/regeneration process in skeletal muscle of CCR2-/- and wild-type mice was not significantly different at day 3. However in contrast to the regenerated muscle of the wild-type mice, the muscle from CCR2-/- mice was characterized by impaired regeneration, inflammation, and fibrotic response at day 14, increased fat infiltration, fibrosis, and calcification at day 21, and impaired strength recovery until at least 28 days post-injury. Consistently, the increased expression of Mac-1 and TNF-alpha was prolonged in the injured muscle of CCR2-/- mice. The expression pattern of the myogenic factors MyoD and myogenin was similar for both types of mice, while NCAM, which is associated with the initiation of fusion of muscle precursor cells, was more increased in the injured muscle of CCR2-/- mice. In conclusion, the study delineates that signaling through CCR2 is involved in muscle precursor cell activities necessary for complete and rapid regeneration of injured skeletal muscle.

The neurobehavioral effects of phytoestrogens in male Syrian hamsters.

We used a phytoestrogen (PE) and a phytoestrogen-free (PE-Free) diet to determine whether or not diet can have neurobehavioral effects on intermale aggression in Syrian hamsters (Mesocricetus auratus). In Experiment 1, 20 adult male hamsters were pre-tested for aggression and then placed on a PE (n=10) or a PE-Free diet (n=10) for 4 weeks in isolation. During week 5, experimental hamsters were exposed to a group-housed, nonaggressive opponent (NAO) for 5 min in a neutral cage arena. PE-fed hamsters exhibited more attacks (33.4+/-6.1) toward the NAO compared to the PE-Free-fed hamsters (18.1+/-4) (p<0.05). Interestingly, testosterone in the blood serum was higher in the PE-fed group (11.01+/-1.48 ng/ml) compared to the PE-Free group (6.5+/-0.87 ng/ml). In Experiment 2, 16 juvenile hamsters were weaned onto a PE (n=8) or a PE-Free diet (n=8). After 7 weeks on the diet, experimental hamsters were exposed to a NAO for 5 min in a neutral cage arena. Although the PE group exhibited higher levels of aggressive behavior, there were no statistically significant differences between groups. However, the PE group had higher levels of testosterone (9.0+/-0.95 ng/ml) compared to the PE-Free group (4.6+/-0.98 ng/ml) (p<0.05). In addition, analysis of the brains from both experiments revealed differences in binding for vasopressin 1A (V1A) receptors. Optical densities were converted to disintegrating units per min/mg. The PE-Free group had higher levels of V1A receptor binding (2689.93+/-254.8 dpm/mg) compared to the PE group (1907.32+/-136.3 dpm/mg) in the lateral septum (p<0.05). In addition, there were differences in the lateral hypothalamus, but the PE group had higher receptor binding (2550.9+/-63.59 dpm/mg) when compared to the PE-Free group (2011.9+/-174.14 dpm/mg) (p<0.05). In sum, these data present the first evidence that phytoestrogens can affect aggressive behavior and, concurrently, alter hormonal status and stimulate changes in the brain of male hamsters.

Role of CC chemokines in skeletal muscle functional restoration after injury.

The purpose of this study was to determine whether certain chemokines, which are highly expressed in injured skeletal muscle, are involved in the repair and functional recovery of the muscle after traumatic injury. In wild-type control mice, mRNA transcripts of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and monocyte chemoattractant protein (MCP)-1 as well as their major receptors, CCR5 and CCR2, increased after freeze injury and gradually returned to control (uninjured) levels by 14 days. Muscle function and histological characteristics were monitored in injured mice that were genetically deficient for the CCR5 receptor (a major receptor for MIP-1alpha and MIP-1beta) and also rendered MCP-1 deficient with neutralizing antibodies. To dissect the role of these chemokines, additional studies were conducted in CCR5- and CCR2-deficient mice. CCR5-/- mice injected with MCP-1 antiserum for the first 3 days after injury exhibited a twofold greater maximal isometric tetanic torque deficit at 14 days after injury than did controls (i.e., 33% vs. 17%; P = 0.002). The impaired functional recovery was accompanied with an increased fat infiltration within the regenerating muscle without a significant difference in the influx of inflammatory cells, including macrophages. Strength recovery was also impaired in mice deficient for the receptor of MCP-1, CCR2, but not in CCR5-/- mice that were not injected with MCP-1 antiserum. The data suggest that MCP-1/CCR2 plays a role in the regeneration and recovery of function after traumatic muscle injury.