Improved Cryopreservation of Human Induced Pluripotent Stem Cell (iPSC) and iPSC-derived Neurons Using Ice-Recrystallization Inhibitors.

Human induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iPSC-Ns) represent a differentiated modality toward developing novel cell-based therapies for regenerative medicine. However, the successful application of iPSC-Ns in cell-replacement therapies relies on effective cryopreservation. In this study, we investigated the role of ice recrystallization inhibitors (IRIs) as novel cryoprotectants for iPSCs and terminally differentiated iPSC-Ns. We found that one class of IRIs, N-aryl-D-aldonamides (specifically 2FA), increased iPSC post-thaw viability and recovery with no adverse effect on iPSC pluripotency. While 2FA supplementation did not significantly improve iPSC-N cell post-thaw viability, we observed that 2FA cryopreserved iPSC-Ns re-established robust neuronal network activity and synaptic function much earlier compared to CS10 cryopreserved controls. The 2FA cryopreserved iPSC-Ns retained expression of key neuronal specific and terminally differentiated markers and displayed functional electrophysiological and neuropharmacological responses following treatment with neuroactive agonists and antagonists. We demonstrate how optimizing cryopreservation media formulations with IRIs represents a promising strategy to improve functional cryopreservation of iPSCs and post-mitotic iPSC-Ns, the latter of which have been challenging to achieve. Developing IRI enabling technologies to support an effective cryopreservation and an efficiently managed cryo-chain is fundamental to support the delivery of successful iPSC-derived therapies to the clinic.

Cross protection to SARS-CoV-2 variants in hamsters with naturally-acquired immunity.

Since SARS-CoV-2 was first reported in late 2019, multiple variations of the original virus have emerged. Each variant harbors accumulations of mutations, particularly within the spike glycoprotein, that are associated with increased viral transmissibility and escape immunity. The different mutations in the spike protein of different variants shape the subsequent antibody and T cell responses, such that exposure to different spike proteins can result in reduced or enhanced responses to heterologous variants further down the line. Globally, people have been exposed and re-exposed to multiple variations of the Ancestral strain, including the five variants of concerns. Studies have shown that the protective immune response of an individual is influenced by which strain or combination of strains they are exposed to. The initial exposure to a specific strain may also shape their subsequent immune patterns and response to later infections with a heterologous virus. Most immunological observations were carried out early during the pandemic when the Ancestral strain was circulating. However, SARS-CoV-2 variants exhibit varying patterns of disease severity, waning immunity, immune evasion and sensitivity to therapeutics. Here we investigated the cross-protection in hamsters previously infected with a variant of concern (VOC) and subsequently re-infected with a heterologous variant. We also determined if cross-protection and immunity were dependent on the specific virus to which the hamster was first exposed. We further profiled the host cytokine response induced by each SARS-CoV-2 variants as well as subsequent to re-infection. A comparative analysis of the three VOCs revealed that Alpha variant was the most pathogenic VOC to emerge. We showed that naturally acquired immunity protected hamsters from subsequent re-infection with heterologous SARS-CoV-2 variant, regardless which variant the animal was first exposed to. Our study supports observations that heterologous infection of different SARS-CoV-2 variants do not exacerbate disease in subsequent re-infections. The continual emergence of new SARS-CoV-2 variants mandates a better understanding of cross-protection and immune imprinting in infected individuals. Such information is essential to guide vaccine strategy and public policy to emerging SARS-CoV-2 VOCs and future novel pandemic coronaviruses.

In Vitro Topical Delivery of Chlorhexidine to the Cornea: Enhancement Using Drug-Loaded Contact Lenses and ß-Cyclodextrin Complexation, and the Importance of Simulating Tear Irrigation.

Microbial keratitis is a severe, sight-threatening condition caused by various pathogens. Eyedrops are the standard delivery modality for treating these disorders; however, blinking reflex, elevated tear production, and nasolacrimal drainage eliminate much of the instilled dose within a few seconds. Therefore, eyedrops must be applied repeatedly for prolonged periods. The present study aimed to probe more effective ocular delivery of chlorhexidine based upon drug-loaded hydrogel contact lenses and ß-cyclodextrin (ß-CD), while also determining the effect of constant irrigation with simulated tear fluid (STF) in in vitro experiments. Chlorhexidine digluconate (as 0.2 and 2% solutions, ß-CD inclusion complexes, and loaded hydrogel contact lenses) were applied to enucleated porcine eyes as single or multiple 10 µL doses, or as drug-loaded contact lenses, with and without ß-CD. The corneas were then excised and drug-extracted quantified by high-performance liquid chromatography (HPLC). The effect of constant irrigation by STF was evaluated to test the effect of increased tear production on corneal delivery. Potential antimicrobial activity of the delivered drug was also assessed. Results showed that drug-loaded contact lenses delivered the greatest amount of chlorhexidine into the cornea over a 24 h period, while the eyedrop solution comparator delivered the least. The ß-CD significantly enhanced chlorhexidine delivery to the cornea from eyedrop solution, although contact lenses loaded with chlorhexidine-ß-CD failed to enhance delivery. ß-CD within the hydrogel matrix impeded drug release. Constant irrigation with STF significantly reduced the amount of drug delivered to the cornea in all cases. Chlorhexidine retained antimicrobial activity in all delivery methods. Hydrogel contact lenses loaded with chlorhexidine delivered significantly higher levels to the cornea compared to eyedrops, either multiple hourly doses or a single dose. They also offer reduced application, in particular, to a nonulcerated corneal infection. Finally, the importance of fully accounting for tear production in in vitro ocular delivery experiments was highlighted.

The lived experience of military beneficiaries with amputations at the hip and pelvic level.

BACKGROUND: Hip- and pelvic-level amputations are devastating injuries that drastically alter patient function and quality of life. This study examined the experience of military beneficiaries with a hip- or pelvic-level amputation to better characterize their challenges and specific needs and to optimize treatment in the future. METHODS: We conducted a retrospective review of the Military Health System and identified 118 patients with a history of one or more amputation(s) at the hip or pelvic level between October 2001 and September 2017. Surviving participants (n = 97) were mailed a letter which explained the details of the study and requested participation in a telephonic interview. A total of six individuals (one female, five males) participated in structured interviews. RESULTS: The study group included four participants with hip disarticulations and two participants with hemipelvectomies (one internal, one external). All six participants reported significant challenges with activities related to prosthetic use, mobility, residual limb health, pain, gastrointestinal and genitourinary function, psychiatric health, and sexual function. CONCLUSIONS: These interviews highlight the unique needs of individuals with hip- and pelvic-level amputations and may improve access to higher echelons of care that would enhance the function and quality of life for these participants.

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM-1) protein binds ß-amyloid (Aß1-42 ) oligomers.

We have recently reported that a ~19-kDa polypeptide, rPK-4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171-1323 amino acid region of the 228-kDa human pericentriolar material-1 (PCM-1) protein (Chakravarthy et al. 2012). We have now discovered that rPK-4 binds oligomeric amyloid-ß peptide (Aß)1-42 with high affinity. Most importantly, a PCM-1-selective antibody co-precipitated Aß and amyloid ß precursor protein (AßPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AßPP and Aß1-42 , suggesting that PCM-1 may interact with amyloid precursor protein/Aß in vivo. We have identified rPK-4's Aß-binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot-blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)-p4-5 binds Aß1-42 at nM levels. Most importantly, ABP-p4-5, like rPK-4, appears to preferentially bind Aß1-42 oligomers, believed to be the toxic AD-drivers. As expected from these observations, ABP-p4-5 prevented Aß1-42 from killing human SH-SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP-p4-5 is a possible candidate therapeutic for AD.

Effectiveness of VSV vectored SARS-CoV-2 spike when administered through intranasal, intramuscular or a combination of both.

A critical feature of the VSV vector platform is the ability to pseudotype the virus with different glycoproteins from other viruses, thus altering cellular tropism of the recombinant virus. The route of administration is critical in triggering local and systemic immune response and protection. Most of the vaccine platforms used at the forefront are administered by intramuscular injection. However, it is not known at what level ACE2 is expressed on the surface of skeletal muscle cells, which will have a significant impact on the efficiency of a VSV-SARS-CoV-2 spike vaccine to mount a protective immune response when administered intramuscularly. In this study, we investigate the immunogenicity and efficacy of a prime-boost immunization regimen administered intranasally (IN), intramuscularly (IM), or combinations of the two. We determined that the prime-boost combinations of IM followed by IN immunization (IM + IN) or IN followed by IN immunization (IN + IN) exhibited strong spike-specific IgG, IgA and T cell response in vaccinated K18 knock-in mice. Hamsters vaccinated with two doses of VSV expressing SARS-CoV-2 spike, both delivered by IN or IM + IN, showed strong protection against SARS-CoV-2 variants of concern Alpha and Delta. This protection was also observed in aged hamsters. Our study underscores the highly crucial role immunization routes have with the VSV vector platform to elicit a strong and protective immune response.

Why Parents Say No to Having Their Children Vaccinated against Measles: A Systematic Review of the Social Determinants of Parental Perceptions on MMR Vaccine Hesitancy.

Ongoing outbreaks of measles threaten its elimination status in the United States. Its resurgence points to lower parental vaccine confidence and local pockets of unvaccinated and undervaccinated individuals. The geographic clustering of hesitancy to MMR indicates the presence of social drivers that shape parental perceptions and decisions on immunization. Through a qualitative systematic review of published literature (n = 115 articles; 7 databases), we determined major themes regarding parental reasons for MMR vaccine hesitancy, social context of MMR vaccine hesitancy, and trustworthy vaccine information sources. Fear of autism was the most cited reason for MMR hesitancy. The social drivers of vaccine hesitancy included primary care/healthcare, education, economy, and government/policy factors. Social factors, such as income and education, exerted a bidirectional influence, which facilitated or hindered vaccine compliance depending on how the social determinant was experienced. Fear of autism was the most cited reason for MMR hesitancy. Vaccine hesitancy to MMR and other childhood vaccines clustered in middle- to high-income areas among mothers with a college-level education or higher who preferred internet/social media narratives over physician-based vaccine information. They had low parental trust, low perceived disease susceptibility, and were skeptical of vaccine safety and benefits. Combating MMR vaccine misinformation and hesitancy requires intersectoral and multifaceted approaches at various socioecological levels to address the social drivers of vaccine behavior.

Preconditioning induces tolerance by suppressing glutamate release in neuron culture ischemia models.

This study determined how preconditioned neurons responded to oxygen-glucose deprivation (OGD) to result in neuroprotection instead of neurotoxicity. Neurons preconditioned using chronically elevated synaptic activity displayed suppressed elevations in extracellular glutamate ([glutamateex ]) and intracellular Ca(2+) (Ca(2+) in ) during OGD. The glutamate uptake inhibitor TBOA induced neurotoxicity, but at a longer OGD duration for preconditioned cultures, suggestive of delayed up-regulation of transporter activity relative to non-preconditioned cultures. This delay was attributed to a critically attenuated release of glutamate, based on tolerance observed against insults mimicking key neurotoxic signaling during OGD (OGD-mimetics). Specifically, in the presence of TBOA, preconditioned neurons displayed potent protection to the OGD-mimetics: ouabain (a Na(+) /K(+) ATPase inhibitor), high 55 mM KCl extracellular buffer (plasma membrane depolarization), veratridine (a Na(+) ionophore), and paraquat (intracellular superoxide producer), which correlated with suppressed [glutamateex ] elevations in the former two insults. Tolerance by preconditioning was reversed by manipulations that increased [glutamateex ], such as by exposure to TBOA or GABAA receptor agonists during OGD, or by exposure to exogenous NMDA or glutamate. Pre-synaptic suppression of neuronal glutamate release by preconditioning, possibly via suppressed exocytic release, represents a key convergence point in neuroprotection during exposure to OGD and OGD-mimetics.

Brain Delivery of IGF1R5, a Single-Domain Antibody Targeting Insulin-like Growth Factor-1 Receptor.

The ability of drugs and therapeutic antibodies to reach central nervous system (CNS) targets is greatly diminished by the blood-brain barrier (BBB). Receptor-mediated transcytosis (RMT), which is responsible for the transport of natural protein ligands across the BBB, was identified as a way to increase drug delivery to the brain. In this study, we characterized IGF1R5, which is a single-domain antibody (sdAb) that binds to insulin-like growth factor-1 receptor (IGF1R) at the BBB, as a ligand that triggers RMT and could deliver cargo molecules that otherwise do not cross the BBB. Surface plasmon resonance binding analyses demonstrated the species cross-reactivity of IGF1R5 toward IGF1R from multiple species. To overcome the short serum half-life of sdAbs, we fused IGF1R5 to the human (hFc) or mouse Fc domain (mFc). IGF1R5 in both N- and C-terminal mFc fusion showed enhanced transmigration across a rat BBB model (SV-ARBEC) in vitro. Increased levels of hFc-IGF1R5 in the cerebrospinal fluid and vessel-depleted brain parenchyma fractions further confirmed the ability of IGF1R5 to cross the BBB in vivo. We next tested whether this carrier was able to ferry a pharmacologically active payload across the BBB by measuring the hypothermic and analgesic properties of neurotensin and galanin, respectively. The fusion of IGF1R5-hFc to neurotensin induced a dose-dependent reduction in the core temperature. The reversal of hyperalgesia by galanin that was chemically linked to IGF1R5-mFc was demonstrated using the Hargreaves model of inflammatory pain. Taken together, our results provided a proof of concept that appropriate antibodies, such as IGF1R5 against IGF1R, are suitable as RMT carriers for the delivery of therapeutic cargos for CNS applications.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models.

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

COVID-19: A catalyst for change in virtual health care utilization for persons with limb loss.

The dramatic uptake of virtual care, or telehealth, utilization because of COVID-19 restrictions for persons with limb loss has led to a much greater understanding of this health care delivery method for this complex patient population. However, much is still unknown. Therefore, the authors provide a comprehensive literature review of existing evidence for virtual care delivery across the phases of amputation rehabilitation, as well as anecdotal evidence, to provide a platform for further discussion and development of research and innovative opportunities. Evidence reveals that virtual care serves as a complement to in-person health care for individuals with limb loss because it allows for increased accessibility to these services. The authors conclude that continued use of telehealth beyond the COVID-19 restrictions to optimize outcomes across the continuum of care for persons with limb loss is warranted.

IFN-alpha receptor deficiency enhances host resistance to oral Salmonella enterica serovar Typhimurium infection during murine pregnancy.

PROBLEM: Maternal tolerance during pregnancy increases the risk of infection with certain intracellular pathogens. Systemic Salmonella enterica serovar Typhimurium (S.Tm) infection during pregnancy in normally resistant 129X1/SvJ mice leads to severe placental infection, as well as fetal and maternal deaths. However, the effect of oral infection with S.Tm in pregnant mice and the roles of infection-induced inflammation and cell death pathways in contributing to susceptibility to infection are unclear. METHOD OF STUDY: Non-pregnant and pregnant C57BL/6J wild-type (WT) and cell death pathway-altered mice (IFNAR1-/- , Caspase-1, 11-/- , RIP3-/- ) were infected orally with S.Tm. Host survival and fetal resorption were determined. Bacterial burden in mesenteric lymph nodes (MLNs), spleen, liver, and placentas was enumerated at various time points post-infection. Serum cytokine expression was measured through cytometric bead array. RESULTS: Oral infection of WT mice with S.Tm on days 9-10 of gestation resulted in systemic dissemination of the bacteria, substantial placental colonization, and fetal loss 5 days post-infection. Histopathological examination of the placentas indicated that infection-induced widespread focal necrosis and neutrophil infiltration throughout the spongiotrophoblast (SpT) layer. In the non-pregnant state, IFNAR1-/- mice exhibited increased survival following oral S.Tm infection relative to Caspase-1, 11-/- , RIP3-/- , and WT mice. The increased resistance to S.Tm infection in IFNAR1-/- mice was seen during pregnancy as well, with decreased bacterial burden within MLNs, spleen, and placenta, which correlated with the decreased resorptions relative to WT and Caspase-1, 11-/- mice. CONCLUSION: Oral S.Tm exposure leads to placental infection, inflammation, and resorption, whereas IFNAR1 deficiency enhances host resistance both in the non-pregnant and pregnant states.

Resilience of network activity in preconditioned neurons exposed to 'stroke-in-a-dish' insults.

Exposing cultured cortical neurons to stimulatory agents - the K+ channel blocker 4-aminopyridine (4-ap), and the GABAA receptor antagonist bicuculline (bic) - for 48 h induces down-regulated synaptic scaling, and preconditions neurons to withstand subsequent otherwise lethal 'stroke-in-a-dish' insults; however, the degree to which usual neuronal function remains is unknown. As a result, multi-electrode array and patch-clamp electrophysiological techniques were employed to characterize hallmarks of spontaneous synaptic activity over a 12-day preconditioning/insult experiment. Spiking frequency increased 8-fold immediately upon 4-ap/bic treatment but declined within the 48 h treatment window to sub-baseline levels that persisted long after washout. Preconditioning resulted in key markers of network activity - spiking frequency, bursting and avalanches - being impervious to an insult. Surprisingly, preconditioning resulted in higher peak NMDA mEPSC amplitudes, resulting in a decrease in the ratio of AMPA:NMDA mEPSC currents, suggesting a relative increase in synaptic NMDA receptors. An investigation of a broad mRNA panel of excitatory and inhibitory signaling mediators indicated preconditioning rapidly up-regulated GABA synthesis (GAD67) and BDNF, followed by up-regulation of neuronal activity-regulated pentraxin and down-regulation of presynaptic glutamate release (VGLUT1). Preconditioning also enhanced surface expression of GLT-1, which persisted following an insult. Overall, preconditioning resulted in a reduced spiking frequency which was impervious to subsequent exposure to 'stroke-in-a-dish' insults, a phenotype initiated predominantly by up-regulation of inhibitory neurotransmission, a lower neuronal postsynaptic AMPA: NMDA receptor ratio, and trafficking of GLT-1 to astrocyte plasma membranes.

Analysis and Simulation of Glioblastoma Cell Lines-Derived Extracellular Vesicles Metabolome.

Glioblastoma (GBM) is one of the most aggressive cancers of the central nervous system. Despite current advances in non-invasive imaging and the advent of novel therapeutic modalities, patient survival remains very low. There is a critical need for the development of effective biomarkers for GBM diagnosis and therapeutic monitoring. Extracellular vesicles (EVs) produced by GBM tumors have been shown to play an important role in cellular communication and modulation of the tumor microenvironment. As GBM-derived EVs contain specific "molecular signatures" of their parental cells and are able to transmigrate across the blood-brain barrier into biofluids such as the blood and cerebrospinal fluid (CSF), they are considered as a valuable source of potential diagnostic biomarkers. Given the relatively harsh extracellular environment of blood and CSF, EVs have to endure and adapt to different conditions. The ability of EVs to adjust and function depends on their lipid bilayer, metabolic content and enzymes and transport proteins. The knowledge of EVs metabolic characteristics and adaptability is essential for their utilization as diagnostic and therapeutic tools. The main aim of this study was to determine the metabolome of small EVs or exosomes derived from different GBM cells and compare to the metabolic profile of their parental cells using NMR spectroscopy. In addition, a possible flux of metabolic processes in GBM-derived EVs was simulated using constraint-based modeling from published proteomics information. Our results showed a clear difference between the metabolic profiles of GBM cells, EVs and media. Machine learning analysis of EV metabolomics, as well as flux simulation, supports the notion of active metabolism within EVs, including enzymatic reactions and the transfer of metabolites through the EV membrane. These results are discussed in the context of novel GBM diagnostics and therapeutic monitoring.

Controlled in vitro delivery of voriconazole and diclofenac to the cornea using contact lenses for the treatment of Acanthamoeba keratitis.

Acanthamoeba keratitis is caused by a protozoal infection of the cornea, with 80% of cases involving the improper use of contact lenses. The infection causes intense pain and is potentially blinding. However, early diagnosis improves treatment efficacy and the chances of healing. Despite the apparent accessibility of the cornea, patients do not always respond well to current eye drop treatments largely due to rapid dose loss due to blinking and nasolacrimal drainage. Here, the topical drug delivery of voriconazole alone and in combination with diclofenac via drug-loaded contact lenses, were investigated in vitro. The contact lenses were applied onto excised porcine eyeballs and maintained at 32 °C under constant irrigation, with simulated tear fluid applied to mimic in vivo conditions. The drug delivered to the corneas was quantified by HPLC analysis. The system was further tested in terms of cytotoxicity and a scratch wound repopulation model, using resident cell types. Sustained drug delivery to the cornea was achieved and for voriconazole, the MIC against Acanthamoeba castellanii was attained alone and in combination with diclofenac. MTT and scratch wound data showed reasonable cell proliferation and wound repopulation at the drug doses used, supporting further development of the system to treat Acanthamoeba keratitis.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human.

Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.

Simplified Admix Archaeal Glycolipid Adjuvanted Vaccine and Checkpoint Inhibitor Therapy Combination Enhances Protection from Murine Melanoma.

Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that when used as adjuvants induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigens. However, traditional entrapped archaeosome formulations have only low entrapment efficiency, therefore we have developed a novel admixed formulation which offers many advantages, including reduced loss of antigen, consistency of batch-to-batch production as well as providing the option to formulate the vaccine immediately before use, which is beneficial for next generation cancer therapy platforms that include patient specific neo-antigens or for use with antigens that are less stable. Herein, we demonstrate that, when used in combination with anti-CTLA-4 and anti-PD-1 checkpoint therapy, this novel admixed archaeosome formulation, comprised of preformed sulfated lactosyl archaeol (SLA) archaeosomes admixed with OVA antigen (SLA-OVA (adm)), was as effective at inducing strong CD8+ T cell responses and protection from a B16-OVA melanoma tumor challenge as the traditionally formulated archaeosomes with encapsulated OVA protein. Furthermore, archaeosome vaccine formulations combined with anti-CTLA-4 and anti-PD-1 therapy, induced OVA-CD8+ T cells within the tumor and immunohistochemical analysis revealed the presence of CD8+ T cells associated with dying or dead tumor cells as well as within or around tumor blood vessels. Overall, archaeosomes constitute an attractive option for use with combinatorial checkpoint inhibitor cancer therapy platforms.

Analysis of the pharmacological properties of JWH-122 isomers and THJ-2201, RCS-4 and AB-CHMINACA in HEK293T cells and hippocampal neurons.

Synthetic cannabinoids are marketed as legal alternatives to Δ9-THC, and are a growing worldwide concern as these drugs are associated with severe adverse effects. Unfortunately, insufficient information regarding the physiological and pharmacological effects of emerging synthetic cannabinoids (ESCs) makes their regulation by government authorities difficult. One strategy used to evade regulation is to distribute isomers of regulated synthetic cannabinoids. This study characterized the pharmacological properties of a panel of ESCs in comparison to Δ9-THC, as well as six JWH-122 isomers relative to its parent compound (JWH-122-4). Two cell-based assays were used to determine the potency and efficacy of ESCs and a panel of reference cannabinoids. HEK293T cells were transfected with human cannabinoid receptor 1 (CB1) and pGloSensor-22F, and the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live cells. All ESCs examined were classified as agonists, with the following rank order of potency: Win 55,212-2 > CP 55,940 > JWH-122-4 > Δ9-THC ≈ RCS-4 ≈ THJ-2201 > JWH-122-5 > JWH-122-7 > JWH-122-2 ≈ AB-CHMINACA > JWH-122-8 > JWH-122-6 > JWH-122-3. Evaluation of ESC-stimulated Ca2+ transients in cultured rat primary hippocampal neurons confirmed the efficacy of four of the most potent ESCs (JWH-122-4, JWH-122-5, JWH-122-7 and AB-CHMINACA). This work helps regulatory agencies make informed decisions concerning these poorly characterized recreational drugs.

Competing approaches to excitotoxic neuroprotection by inert and catalytic antioxidant porphyrins.

The goal of this study was to determine if novel porphyrins protect cultured cortical neurons from excitotoxic NMDA exposure or oxygen-glucose deprivation (OGD), which model key aspects of cerebral ischemia. Porphyrins were chosen based on conventional and unconventional criteria. Metalloporphyrin catalytic antioxidants possessing a redox-sensitive metal core can exhibit potent and wide-ranging catalytic antioxidant abilities, which are conventionally believed to underlie neuroprotection. We report here that a recent-generation potent peroxynitrite decomposition catalyst, FP-15, protected a majority of neurons against OGD and NMDA toxicity, without suppressing NMDA-mediated intracellular Ca2+ (Cai2+) elevations or whole-cell currents. We have previously shown that neuroprotection against OGD and NMDA toxicity correlated with an ability to suppress neurotoxic Cai2+ elevations and not antioxidant ability. We now evaluate if this unconventional mechanism extends to inert metal-free porphyrins. Neuron cultures were completely protected against OGD and NMDA toxicity by H2-meso-tetrakis(3-benzoic acid)porphyrin (H2-TBAP(3)) or H2-meso-tetrakis(4-sulfonatophenyl)porphyrin (H2-TPPS(4)), although only H2-TPPS(4) suppressed (completely) NMDA-induced Cai2+ rises. H2-meso-tetrakis(3,3'-benzoic acid)porphyrin (H2-TBAP(3,3')) or H2-meso-tetrakis(N-methylpyridynium-4-yl)porphyrin (H2-TM-PyP(4)) provided at least partial protection against OGD and NMDA toxicity and partially suppressed NMDA-induced Cai2+ elevations. Despite the complexity of Ca2+-independent and -dependent based mechanisms, the inventory of porphyrins demonstrating neuroprotection in ischemia-relevant insults is now expanded to include FP-15 and inert metal-free compounds, although with no apparent advantage gained by using FP-15.

Failure and rescue of preconditioning-induced neuroprotection in severe stroke-like insults.

Preconditioning is a well established neuroprotective modality. However, the mechanism and relative efficacy of neuroprotection between diverse preconditioners is poorly defined. Cultured neurons were preconditioned by 4-aminopyridine and bicuculline (4-AP/bic), rendering neurons tolerant to normally lethal (sufficient to kill most neurons) oxygen-glucose deprivation (OGD) or a chemical OGD-mimic, ouabain/TBOA, by suppression of extracellular glutamate (glutamateex) elevations. However, subjecting preconditioned neurons to longer-duration supra-lethal insults caused neurotoxic glutamateex elevations, thereby identifying a 'ceiling' to neuroprotection. Neuroprotective 'rescue' of neurons could be obtained by administration of an NMDA receptor antagonist, MK-801, just before glutamateex rose during these supra-lethal insults. Next, we evaluated if these concepts of glutamateex suppression during lethal OGD, and a neuroprotective ceiling requiring MK-801 rescue under supra-lethal OGD, extended to the preconditioning field. In screening a panel of 42 diverse putative preconditioners, neuroprotection against normally lethal OGD was observed in 12 cases, which correlated with glutamateex suppression, both of which could be reversed, either by the inclusion of a glutamate uptake inhibitor (TBOA, to increase glutamateex levels) during OGD or by exposure to supra-lethal OGD. Administrating MK-801 during the latter stages of supra-lethal OGD again rescued neurons, although to varying degrees dependent on the preconditioning agent. Thus, 'stress-testing' against the harshest ischemic-like insults yet tested identifies the most efficacious preconditioners, which dictates how early MK-801 needs to be administered during the insult in order to maintain neuroprotection. Preconditioning delays a neurotoxic rise in glutamateex levels, thereby 'buying time' for acute anti-excitotoxic pharmacologic rescue.