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Shantanu Balkundi1, Ari S Nowacek1, Ram S Veerubhotla1, Han Chen2, Andrea Martinez-Skinner1, Upal Roy1, R Lee Mosley1,3, Georgette Kanmogne1, Xinming Liu1,3,4, Alexander V Kabanov3,4, Tatiana Bronich3,4, JoEllyn McMillan1, Howard E Gendelman1,31Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA; 2Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE, USA; 3Center for Drug Delivery and Nanomedicine, University of Nebraska Medical Center, Omaha, NE, USA; 4Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE, USAAbstract: Nanoformulations of crystalline indinavir, ritonavir, atazanavir, and efavirenz were manufactured by wet milling, homogenization or sonication with a variety of excipients. The chemical, biological, immune, virological, and toxicological properties of these formulations were compared using an established monocyte-derived macrophage scoring indicator system. Measurements of drug uptake, retention, release, and antiretroviral activity demonstrated differences amongst preparation methods. Interestingly, for drug cell targeting and antiretroviral responses the most significant difference among the particles was the drug itself. We posit that the choice of drug and formulation composition may ultimately affect clinical utility.Keywords: human immunodeficiency virus type one, nanotoxicology, monocyte-derived macrophage, nanoformulated antiretroviral therapy, manufacturing techniques

Costimulation mediated by the CD28 receptor has been shown to play an important role in the development of a vigorous T cell immune response. Nevertheless, CD28-deficient mice can mount effective T cell-dependent immune responses. These data suggest that other costimulatory molecules may play a role in T cell activation. In a search for other costimulatory receptors on T cells, we have characterized a monoclonal antibody (mAb) that can costimulate T cells in the absence of accessory cells. Similar to CD28 antibodies, this mAb, R2/60, was found to synergize with T cell receptor engagement in inducing proliferation. Independent ligation of CD3 and the ligand recognized by R2/60 results in T cell proliferation, suggesting that the two molecules do not have to colocalize to activate the R2/60 costimulatory pathway. R2/60 does not react with CD28, and furthermore, R2/60 costimulates in a CD28-independent fashion since the mAb costimulates T cells from the CD28-deficient mice as well as wild-type mice. Expression cloning of the R2/60 antigen identified the ligand as murine CD43. Together, these data demonstrate that CD43 can serve as a receptor on T cells that can provide CD28-independent costimulation.

Lack of adherence, inaccessibility to viral reservoirs, long-term drug toxicities, and treatment failures are limitations of current antiretroviral therapy (ART). These limitations lead to increased viral loads, medicine resistance, immunocompromise, and comorbid conditions. To this end, we developed long-acting nanoformulated ART (nanoART) through modifications of existing atazanavir, ritonavir, and efavirenz suspensions in order to establish cell and tissue drug depots to achieve sustained antiretroviral responses. NanoART's abilities to affect immune and antiviral responses, before or following human immunodeficiency virus type 1 infection were tested in nonobese severe combined immune-deficient mice reconstituted with human peripheral blood lymphocytes. Weekly subcutaneous injections of drug nanoformulations at doses from 80 mg/kg to 250 mg/kg, 1 day before and/or 1 and 7 days after viral exposure, elicited drug levels that paralleled the human median effective concentration, and with limited toxicities. NanoART treatment attenuated viral replication and preserved CD4(+) Tcell numbers beyond that seen with orally administered native drugs. These investigations bring us one step closer toward using long-acting antiretrovirals in humans. [ABSTRACT FROM AUTHOR]

Background: Regulatory T cells (Tregs) maintain immune tolerance. While Treg-mediated neuroprotective activities are now well-accepted, the lack of defined antigen specificity limits their therapeutic potential. This is notable for neurodegenerative diseases where cell access to injured brain regions is required for disease-specific therapeutic targeting and improved outcomes. To address this need, amyloid-beta (Aβ) antigen specificity was conferred to Treg responses by engineering the T cell receptor (TCR) specific for Aβ (TCR A β ). The TCR Ab were developed from disease-specific T cell effector (Teff) clones. The ability of Tregs expressing a transgenic TCR Aβ (TCR Aβ -Tregs) to reduce Aβ burden, transform effector to regulatory cells, and reverse disease-associated neurotoxicity proved beneficial in an animal model of Alzheimer's disease., Methods: TCR A β -Tregs were generated by CRISPR-Cas9 knockout of endogenous TCR and consequent incorporation of the transgenic TCR Ab identified from Aβ reactive Teff monoclones. Antigen specificity was confirmed by MHC-Aβ-tetramer staining. Adoptive transfer of TCR Aβ -Tregs to mice expressing a chimeric mouse-human amyloid precursor protein and a mutant human presenilin-1 followed measured behavior, immune, and immunohistochemical outcomes., Results: TCR Aβ -Tregs expressed an Aβ-specific TCR. Adoptive transfer of TCR Aβ -Tregs led to sustained immune suppression, reduced microglial reaction, and amyloid loads. 18 F-fluorodeoxyglucose radiolabeled TCR Aβ -Treg homed to the brain facilitating antigen specificity. Reduction in amyloid load was associated with improved cognitive functions., Conclusions: TCR Aβ -Tregs reduced amyloid burden, restored brain homeostasis, and improved learning and memory, supporting the increased therapeutic benefit of antigen specific Treg immunotherapy for AD., (© 2023. The Author(s).)

Synucleinopathies are a group of neurodegenerative disorders characterized by pathologic aggregates of neural and glial α-synuclein (α-syn) in the form of Lewy bodies (LBs), Lewy neurites, and cytoplasmic inclusions in both neurons and glia. Two major classes of synucleinopathies are LB disease and multiple system atrophy. LB diseases include Parkinson's disease (PD), PD with dementia, and dementia with LBs. All are increasing in prevalence. Effective diagnostics, disease-modifying therapies, and therapeutic monitoring are urgently needed. Diagnostics capable of differentiating LB diseases are based on signs and symptoms which might overlap. To date, no specific diagnostic test exists despite disease-specific pathologies. Diagnostics are aided by brain imaging and cerebrospinal fluid evaluations, but more accessible biomarkers remain in need. Mechanisms of α-syn evolution to pathologic oligomers and insoluble fibrils can provide one of a spectrum of biomarkers to link complex neural pathways to effective therapies. With these in mind, we review promising biomarkers linked to effective disease-modifying interventions., (© 2023. The Author(s).)

Objectives: To evaluate the linkage between age and deficits in innate and adaptive immunity which heralds both Alzheimer's disease (AD) onset and progression. The pathobiological events which underlie and tie these outcomes remain not fully understood., Methods: To investigate age-dependent immunity in AD, we evaluated innate and adaptive immunity in coordinate studies of regulatory T cell (Treg) function, T cell frequencies, and microglial integrity. These were assessed in blood, peripheral lymphoid tissues, and the hippocampus of transgenic (Tg) amyloid precursor protein/presenilin 1 (APP/PS1) against non-Tg mice. Additionally, immune arrays of hippocampal tissue were performed at 4, 6, 12, and 20 months of age., Results: APP/PS1 mice showed progressive impairment of Treg immunosuppressive function with age. There was partial restoration of Treg function in 20-month-old mice. Ingenuity pathway analyses of hippocampal tissues were enriched in inflammatory, oxidative, and cellular activation pathways that paralleled advancing age and AD-pathobiology. Operative genes in those pathways included, but were not limited to triggering receptor on myeloid cells 1 (TREM1), T helper type 1 (Th1), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways. Interleukin-17 (IL-17), nitric oxide, acute phase, and T cell receptor signaling pathways were also perturbed. Significant inflammation was observed at 6- and 12-months. However, at 20-months, age associated partial restoration of Treg function reduced inflammatory phenotype., Conclusions: Impaired Treg function, inflammation and oxidative stress were associated with AD pathology. Age associated partial restoration of Treg function in old mice reduced the hippocampal inflammatory phenotype. Restoring Treg suppressive function can be a therapeutic modality for AD., Competing Interests: Competing interests: No conflicts of interest., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Background: The clinical utility and safety of sargramostim has previously been reported in cancer, acute radiation syndrome, autoimmune disease, inflammatory conditions, and Alzheimer's disease. The safety, tolerability, and mechanisms of action in Parkinson's disease (PD) during extended use has not been evaluated., Methods: As a primary goal, safety and tolerability was assessed in five PD patients treated with sargramostim (Leukine ® , granulocyte-macrophage colony-stimulating factor) for 33 months. Secondary goals included numbers of CD4 + T cells and monocytes and motor functions. Hematologic, metabolic, immune, and neurological evaluations were assessed during a 5-day on, 2-day off therapeutic regimen given at 3 μg/kg. After 2 years, drug use was discontinued for 3 months. This was then followed by an additional 6 months of treatment., Results: Sargramostim-associated adverse events included injection-site reactions, elevated total white cell counts, and bone pain. On drug, blood analyses and metabolic panels revealed no untoward side effects linked to long-term treatment. Unified Parkinson's Disease Rating Scale scores remained stable throughout the study while regulatory T cell number and function were increased. In the initial 6 months of treatment, transcriptomic and proteomic monocyte tests demonstrated autophagy and sirtuin signaling. This finding paralleled anti-inflammatory and antioxidant activities within both the adaptive and innate immune profile arms., Conclusions: Taken together, the data affirmed long-term safety as well as immune and anti-inflammatory responses reflecting clinical stability in PD under the sargramostim treatment. Confirmation in larger patient populations is planned in a future phase II evaluation., Trial Registration: ClinicalTrials.gov: NCT03790670, Date of Registration: 01/02/2019, URL: https://clinicaltrials.gov/ct2/show/NCT03790670?cond=leukine+parkinson%27s&draw=2&rank=2 ., (© 2023. The Author(s).)

Treatment of HIV-1 ADA -infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4 + T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.

While inflammation may not be the cause of disease, it is well known that it contributes to disease pathogenesis across a multitude of peripheral and central nervous system disorders. Chronic and overactive inflammation due to an effector T-cell-mediated aberrant immune response ultimately leads to tissue damage and neuronal cell death. To counteract peripheral and neuroinflammatory responses, research is being focused on regulatory T cell enhancement as a therapeutic target. Regulatory T cells are an immunosuppressive subpopulation of CD4+ T helper cells essential for maintaining immune homeostasis. The cells play pivotal roles in suppressing immune responses to maintain immune tolerance. In so doing, they control T cell proliferation and pro-inflammatory cytokine production curtailing autoimmunity and inflammation. For nervous system pathologies, Treg are known to affect the onset and tempo of neural injuries. To this end, we review recent findings supporting Treg's role in disease, as well as serving as a therapeutic agent in multiple sclerosis, myasthenia gravis, Guillain-Barre syndrome, Parkinson's and Alzheimer's diseases, and amyotrophic lateral sclerosis. An ever-broader role for Treg in the control of neurologic disease has been shown for traumatic brain injury, stroke, neurotrophic pain, epilepsy, and psychiatric disorders. To such ends, this review serves to examine the role played by Tregs in nervous system diseases with a focus on harnessing their functional therapeutic role(s)., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transformation of CD4+ T cell effector to regulatory (Teff to Treg) cells have been shown to attenuate disease progression by restoring immunological balance during the onset and progression of neurodegenerative diseases. In our prior studies, we defined a safe and effective pathway to restore this balance by restoring Treg numbers and function through the daily administration of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). These studies were conducted as a proof-of-concept testing in Parkinson's disease (PD) preclinical models and early phase I clinical investigations. In both instances, they served to ameliorate disease associated signs and symptoms. However, despite the recorded efficacy, the cytokine's short half-life, low bioavailability, and injection site reactions proved to be limitations for any broader use. To overcome these limitations, mRNA lipid nanoparticles encoding an extended half-life albumin-GM-CSF fusion protein were developed for both mouse (Msa-GM-CSF) and rat (Rsa-GM-CSF). These formulations were tested for immunomodulatory and neuroprotective efficacy using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and human wild-type alpha-synuclein (αSyn) overexpression preclinical models of PD. A single dose of the extended half-life mouse and rat mRNA lipid nanoparticles generated measurable GM-CSF plasma cytokine levels up to four days. Increased Treg frequency and function were associated with a resting microglial phenotype, nigrostriatal neuroprotection, and restoration of brain tissue immune homeostasis. These findings were substantively beyond the recorded efficacy of daily recombinant wild-type GM-CSF with a recorded half-life of six hours. Mechanistic evaluation of neuropathological transcriptional profiles performed in the disease-affected nigral brain region demonstrated an upregulation of neuroprotective CREB and synaptogenesis signaling and neurovascular coupling pathways. These findings highlight the mRNA-encoded albumin GM-CSF fusion protein modification linked to improvements in therapeutic efficacy. The improvements achieved were associated with the medicine's increased bioavailability. Taken together, the data demonstrate that mRNA LNP encoding the extended half-life albumin-GM-CSF fusion protein can serve as a benchmark for PD immune-based therapeutics. This is especially notable for improving adherence of drug regimens in a disease-affected patient population with known tremors and gait abnormalities., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Dysregulation of innate and adaptive immunity heralds both the development and progression of Parkinson's disease (PD). Deficits in innate immunity in PD are defined by impairments in monocyte activation, function, and pro-inflammatory secretory factors. Each influences disease pathobiology., Methods and Results: To define monocyte biomarkers associated with immune transformative therapy for PD, changes in gene and protein expression were evaluated before and during treatment with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim, Leukine ® ). Monocytes were recovered after leukapheresis and isolation by centrifugal elutriation, before and 2 and 6 months after initiation of treatment. Transcriptome and proteome biomarkers were scored against clinical motor functions. Pathway enrichments from single cell-RNA sequencing and proteomic analyses from sargramostim-treated PD patients demonstrate a neuroprotective signature, including, but not limited to, antioxidant, anti-inflammatory, and autophagy genes and proteins (LRRK2, HMOX1, TLR2, TLR8, RELA, ATG7, and GABARAPL2)., Conclusions: This monocyte profile provides an "early" and unique biomarker strategy to track clinical immune-based interventions, but requiring validation in larger case studies., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Background: Pharmacological approaches that boost neuroprotective regulatory T cell (Treg) number and function lead to neuroprotective activities in neurodegenerative disorders., Objectives: We investigated whether low-dose interleukin 2 (IL-2) expands Treg populations and protects nigrostriatal dopaminergic neurons in a model of Parkinson's disease (PD)., Methods: IL-2 at 2.5 × 10 4 IU/dose/mouse was administered for 5 days. Lymphocytes were isolated and phenotype determined by flow cytometric analyses. To 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice, 0.5 × 10 6 of enriched IL-2-induced Tregs were adoptively transferred to assess the effects on nigrostriatal neuron survival., Results: IL-2 increased frequencies of CD4 + CD25 + CD127 low FoxP3 + Tregs that express ICOS and CD39 in blood and spleen. Adoptive transfer of IL-2-induced Tregs to MPTP-treated recipients increased tyrosine hydroxylase (TH) + nigral dopaminergic neuronal bodies by 51% and TH + striatal termini by 52% compared to control MPTP-treated animal controls., Conclusions: IL-2 expands numbers of neuroprotective Tregs providing a vehicle for neuroprotection of nigrostriatal dopaminergic neurons in a pre-clinical PD model., Competing Interests: Competing interests: Authors state no conflict of interest., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Pathologically, the disease is characterized by the deposition of amyloid beta (Aβ) plaques and the presence of neurofibrillary tangles. These drive microglia neuroinflammation and consequent neurodegeneration. While the means to affect Aβ plaque accumulation pharmacologically was achieved, how it affects disease outcomes remains uncertain. Cerium oxide (CeO 2 ) reduces Aβ plaques, oxidative stress, inflammation, and AD signs and symptoms. In particular, CeO 2 nanoparticles (CeO 2 NPs) induce free-radical-scavenging and cell protective intracellular signaling. This can ameliorate the pathobiology of an AD-affected brain. To investigate whether CeO 2 NPs affect microglia neurotoxic responses, a novel formulation of europium-doped CeO 2 NPs (EuCeO 2 NPs) was synthesized. We then tested EuCeO 2 NPs for its ability to generate cellular immune homeostasis in AD models. EuCeO 2 NPs attenuated microglia BV2 inflammatory activities after Aβ 1-42 exposure by increasing the cells' phagocytic and Aβ degradation activities. These were associated with increases in the expression of the CD36 scavenger receptor. EuCeO 2 NPs facilitated Aβ endolysosomal trafficking and abrogated microglial inflammatory responses. We posit that EuCeO 2 NPs may be developed as an AD immunomodulator.

Prodrugs are bioreversible drug derivatives which are metabolized into a pharmacologically active drug following chemical or enzymatic modification. This approach is designed to overcome several obstacles that are faced by the parent drug in physiological conditions that include rapid drug metabolism, poor solubility, permeability, and suboptimal pharmacokinetic and pharmacodynamic profiles. These suboptimal physicochemical features can lead to rapid drug elimination, systemic toxicities, and limited drug-targeting to disease-affected tissue. Improving upon these properties can be accomplished by a prodrug design that includes the careful choosing of the promoiety, the linker, the prodrug synthesis, and targeting decorations. We now provide an overview of recent developments and applications of prodrugs for treating neurodegenerative, inflammatory, and infectious diseases. Disease interplay reflects that microbial infections and consequent inflammation affects neurodegenerative diseases and vice versa, independent of aging. Given the high prevalence, personal, social, and economic burden of both infectious and neurodegenerative disorders, therapeutic improvements are immediately needed. Prodrugs are an important, and might be said a critical tool, in providing an avenue for effective drug therapy.

Dysregulation of innate and adaptive immunity can lead to alpha-synuclein (α-syn) misfolding, aggregation, and post-translational modifications in Parkinson's disease (PD). This process is driven by neuroinflammation and oxidative stress, which can contribute to the release of neurotoxic oligomers that facilitate dopaminergic neurodegeneration. Strategies that promote vaccines and antibodies target the clearance of misfolded, modified α-syn, while gene therapy approaches propose to deliver intracellular single chain nanobodies to mitigate α-syn misfolding, or to deliver neurotrophic factors that support neuronal viability in an otherwise neurotoxic environment. Additionally, transformative immune responses provide potential targets for PD therapeutics. Anti-inflammatory drugs represent one strategy that principally affects innate immunity. Considerable research efforts have focused on transforming the balance of pro-inflammatory effector T cells (Teffs) to favor regulatory T cell (Treg) activity, which aims to attenuate neuroinflammation and support reparative and neurotrophic homeostasis. This approach serves to control innate microglial neurotoxic activities and may facilitate clearance of α-syn aggregates accordingly. More recently, changes in the intestinal microbiome have been shown to alter the gut-immune-brain axis leading to suppressed leakage of bacterial products that can promote peripheral inflammation and α-syn misfolding. Together, each of the approaches serves to interdict chronic inflammation associated with disordered immunity and neurodegeneration. Herein, we examine research strategies aimed at improving clinical outcomes in PD.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to coronavirus disease 2019 (COVID-19). Virus-specific immunity controls infection, transmission and disease severity. With respect to disease severity, a spectrum of clinical outcomes occur associated with age, genetics, comorbidities and immune responses in an infected person. Dysfunctions in innate and adaptive immunity commonly follow viral infection. These are heralded by altered innate mononuclear phagocyte differentiation, activation, intracellular killing and adaptive memory, effector, and regulatory T cell responses. All of such affect viral clearance and the progression of end-organ disease. Failures to produce effective controlled antiviral immunity leads to life-threatening end-organ disease that is typified by the acute respiratory distress syndrome. The most effective means to contain SARS-CoV-2 infection is by vaccination. While an arsenal of immunomodulators were developed for control of viral infection and subsequent COVID-19 disease, further research is required to enable therapeutic implementation., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by pathological deposition of misfolded self-protein amyloid beta (Aβ) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as a key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered that begin early and persist throughout the disease. How these occur and whether they can be harnessed to halt disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teffs) that drive pro-inflammatory and neurodestructive immunity leading to cognitive impairments. Here, we investigated the role of effector immunity and how it could affect cellular-level disease pathobiology in an AD animal model., Methods: In this report, we developed and characterized cloned lines of amyloid beta (Aβ) reactive type 1 T helper (Th1) and type 17 Th (Th17) cells to study their role in AD pathogenesis. The cellular phenotype and antigen-specificity of Aβ-specific Th1 and Th17 clones were confirmed using flow cytometry, immunoblot staining and Aβ T cell epitope loaded haplotype-matched major histocompatibility complex II IA b (MHCII-IA b -KLVFFAEDVGSNKGA) tetramer binding. Aβ-Th1 and Aβ-Th17 clones were adoptively transferred into APP/PS1 double-transgenic mice expressing chimeric mouse/human amyloid precursor protein and mutant human presenilin 1, and the mice were assessed for memory impairments. Finally, blood, spleen, lymph nodes and brain were harvested for immunological, biochemical, and histological analyses., Results: The propagated Aβ-Th1 and Aβ-Th17 clones were confirmed stable and long-lived. Treatment of APP/PS1 mice with Aβ reactive Teffs accelerated memory impairment and systemic inflammation, increased amyloid burden, elevated microglia activation, and exacerbated neuroinflammation. Both Th1 and Th17 Aβ-reactive Teffs progressed AD pathology by downregulating anti-inflammatory and immunosuppressive regulatory T cells (Tregs) as recorded in the periphery and within the central nervous system., Conclusions: These results underscore an important pathological role for CD4+ Teffs in AD progression. We posit that aberrant disease-associated effector T cell immune responses can be controlled. One solution is by Aβ reactive Tregs., (© 2021. The Author(s).)

Background: A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery., Methods: A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat 1-2 /rev 1-2 /gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels., Findings: Virus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing., Interpretation: These results provide "proof of concept' for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing., Funding: The work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239., Competing Interests: Declaration of Competing Interest J.H., M.H., and H.E.G. are named inventors on provisional patents for the CRISPR therapy described in this report (62/985,392; 62/986,216). J.H., M.H., B.K., and H.E.G hold a patent on a virus-like particle-based delivery for HIV-1 CRISPR therapeutics (Docket No. 19040PCT; Serial No. PCT/US2020/016126; International Publication No. WO 2020/160418 A1). H.E.G is a member of the scientific advisory board at Longevity Biotech and a co-founder of Exavir Therapeutics, Inc., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end-organ malfunctions. All follow the presence of persistent viral components and virions without evidence of viral replication. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation, it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways, specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding end-organ tissue damage., Competing Interests: Author HEG is a co-founder of Exavir Therapeutics, Inc. who is developing antiviral and elimination therapies for HIV/AIDS and other viral infections. 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 © 2021 Abdelmoaty, Yeapuri, Machhi, Olson, Shahjin, Kumar, Zhou, Liang, Pandey, Acharya, Byrareddy, Mosley and Gendelman.)

Humanized mice model human disease and as such are used commonly for research studies of infectious, degenerative and cancer disorders. Recent models also reflect hematopoiesis, natural immunity, neurobiology, and molecular pathways that influence disease pathobiology. A spectrum of immunodeficient mouse strains permit long-lived human progenitor cell engraftments. The presence of both innate and adaptive immunity enables high levels of human hematolymphoid reconstitution with cell susceptibility to a broad range of microbial infections. These mice also facilitate investigations of human pathobiology, natural disease processes and therapeutic efficacy in a broad spectrum of human disorders. However, a bridge between humans and mice requires a complete understanding of pathogen dose, co-morbidities, disease progression, environment, and genetics which can be mirrored in these mice. These must be considered for understanding of microbial susceptibility, prevention, and disease progression. With known common limitations for access to human tissues, evaluation of metabolic and physiological changes and limitations in large animal numbers, studies in mice prove important in planning human clinical trials. To these ends, this review serves to outline how humanized mice can be used in viral and pharmacologic research emphasizing both current and future studies of viral and neurodegenerative diseases. In all, humanized mouse provides cost-effective, high throughput studies of infection or degeneration in natural pathogen host cells, and the ability to test transmission and eradication of disease.

Background: Neuroinflammation plays a pathogenic role in Parkinson's disease (PD). Immunotherapies that restore brain homeostasis can mitigate neurodegeneration by transforming T cell phenotypes. Sargramostim has gained considerable attention as an immune transformer through laboratory bench to bedside clinical studies. However, its therapeutic use has been offset by dose-dependent adverse events. Therefore, we performed a reduced drug dose regimen to evaluate safety and to uncover novel disease-linked biomarkers during 5 days/week sargramostim treatments for one year., Methods: Five PD subjects were enrolled in a Phase 1b, unblinded, open-label study to assess safety and tolerability of 3 μg/kg/day sargramostim. Complete blood counts and chemistry profiles, physical examinations, adverse events (AEs), immune profiling, Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) scores, T cell phenotypes/function, DNA methylation, and gene and protein patterns were evaluated., Findings: Sargramostim administered at 3 μg/kg/day significantly reduced numbers and severity of AEs/subject/month compared to 6 μg/kg/day treatment. While MDS-UPDRS Part III score reductions were recorded, peripheral blood immunoregulatory phenotypes and function were elevated. Hypomethylation of upstream FOXP3 DNA elements was also increased., Interpretation: Long-term sargramostim treatment at 3 μg/kg/day is well-tolerated and effective in restoring immune homeostasis. There were decreased numbers and severity of AEs and restored peripheral immune function coordinate with increased numbers and function of Treg. MDS-UPDRS Part III scores did not worsen. Larger patient numbers need be evaluated to assess conclusive drug efficacy (ClinicalTrials.gov NCT03790670)., Funding: The research was supported by community funds to the University of Nebraska Foundation and federal research support from 5 R01NS034239-25., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Published by Elsevier B.V.)

Restoring numbers and function of regulatory T cells (Tregs) is a novel therapeutic strategy for neurodegenerative disorders. Whether Treg function is boosted by adoptive cell transfer, pharmaceuticals, or immune modulators, the final result is a robust anti-inflammatory and neuronal sparing response. Herein, a newly developed lipid nanoparticle (LNP) containing mRNA encoding granulocyte-macrophage colony-stimulating factor (Gm-csf mRNA) was developed to peripherally induce Tregs and used for treatment in preclinical Parkinson's disease (PD) models. Administration of Gm-csf mRNA to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and rats overexpressing alpha-synuclein produced dose-dependent increases in plasma GM-CSF levels and peripheral CD4+CD25+FoxP3+ Treg populations. This upregulation paralleled nigrostriatal neuroprotection, upregulated immunosuppression-associated mRNAs that led to the detection of a treatment-induced CD4+ T cell population, and decreased reactive microgliosis. The current findings strengthen prior works utilizing immune modulation by harnessing Gm-csf mRNA to augment adaptive immune function by employing a new delivery platform to treat PD and potentially other neurodegenerative disorders., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Loss of dopaminergic neurons along the nigrostriatal axis, neuroinflammation, and peripheral immune dysfunction are the pathobiological hallmarks of Parkinson's disease (PD). Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been successfully tested for PD treatment. GM-CSF is a known immune modulator that induces regulatory T cells (Tregs) and serves as a neuronal protectant in a broad range of neurodegenerative diseases. Due to its short half-life, limited biodistribution, and potential adverse effects, alternative long-acting treatment schemes are of immediate need. A long-acting mouse GM-CSF (mPDM608) was developed through Calibr, a Division of Scripps Research. Following mPDM608 treatment, complete hematologic and chemistry profiles and T-cell phenotypes and functions were determined. Neuroprotective and anti-inflammatory capacities of mPDM608 were assessed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice that included transcriptomic immune profiles. Treatment with a single dose of mPDM608 resulted in dose-dependent spleen and white blood cell increases with parallel enhancements in Treg numbers and immunosuppressive function. A shift in CD4 + T-cell gene expression towards an anti-inflammatory phenotype corresponded with decreased microgliosis and increased dopaminergic neuronal cell survival. mPDM608 elicited a neuroprotective peripheral immune transformation. The observed phenotypic shift and neuroprotective response was greater than observed with recombinant GM-CSF (rGM-CSF) suggesting human PDM608 as a candidate for PD treatment.

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB's effectiveness.

Emerging evidence demonstrates that adaptive immunity influences the pathobiology of neurodegenerative disorders. Misfolded aggregated self-proteins can break immune tolerance leading to the induction of autoreactive effector T cells (Teffs) with associated decreases in anti-inflammatory neuroprotective regulatory T cells (Tregs). An imbalance between Teffs and Tregs leads to microglial activation, inflammation and neuronal injury. The cascade of such a disordered immunity includes the drainage of the aggregated protein antigens into cervical lymph nodes serving to amplify effector immune responses. Both preclinical and clinical studies demonstrate transformation of this altered immunity for therapeutic gain. We posit that the signs and symptoms of common neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, and stroke can be attenuated by boosting Treg activities.

With the increasing prevalence of Parkinson's disease (PD), there is an immediate need to interdict disease signs and symptoms. In recent years this need was met through therapeutic approaches focused on regenerative stem cell replacement and alpha-synuclein clearance. However, neither have shown long-term clinical benefit. A novel therapeutic approach designed to affect disease is focused on transforming the brain's immune microenvironment. As disordered innate and adaptive immune functions are primary components of neurodegenerative disease pathogenesis, this has emerged as a clear opportunity for therapeutic development. Interventions that immunologically restore the brain's homeostatic environment can lead to neuroprotective outcomes. These have recently been demonstrated in both laboratory and early clinical investigations. To these ends, efforts to increase the numbers and function of regulatory T cells over dominant effector cells that exacerbate systemic inflammation and neurodegeneration have emerged as a primary research focus. These therapeutics show broad promise in affecting disease outcomes beyond PD, such as for Alzheimer's disease, stroke and traumatic brain injuries, which share common neurodegenerative disease processes., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Human immunodeficiency virus theranostics facilitates the development of long acting (LA) antiretroviral drugs (ARVs) by defining drug-particle cell depots. Optimal drug formulations are made possible based on precise particle composition, structure, shape and size. Through the creation of rod-shaped particles of defined sizes reflective of native LA drugs, theranostic probes can be deployed to measure particle-cell and tissue biodistribution, antiretroviral activities and drug retention. Methods : Herein, we created multimodal rilpivirine (RPV) 177 lutetium labeled bismuth sulfide nanorods ( 177 LuBSNRs) then evaluated their structure, morphology, configuration, chemical composition, biological responses and adverse reactions. Particle biodistribution was analyzed by single photon emission computed tomography (SPECT/CT) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging. Results : Nanoformulated RPV and BSNRs-RPV particles showed comparable physicochemical and cell biological properties. Drug-particle pharmacokinetics (PK) and biodistribution in lymphoid tissue macrophages proved equivalent, one with the other. Rapid particle uptake and tissue distribution were observed, without adverse reactions, in primary blood-derived and tissue macrophages. The latter was seen within the marginal zones of spleen. Conclusions : These data, taken together, support the use of 177 LuBSNRs as theranostic probes as a rapid assessment tool for PK LA ARV measurements., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

A paradigm shift has emerged in Parkinson's disease (PD) highlighting the prominent role of CD4 + Tregs in pathogenesis and treatment. Bench to bedside research, conducted by others and our own laboratories, advanced a neuroprotective role for Tregs making pharmacologic transformation of immediate need. Herein, a vasoactive intestinal peptide receptor-2 (VIPR2) peptide agonist, LBT-3627, was developed as a neuroprotectant for PD-associated dopaminergic neurodegeneration. Employing both 6-hydroxydopamine (6-OHDA) and α-synuclein (α-Syn) overexpression models in rats, the sequential administration of LBT-3627 increased Treg activity without altering cell numbers both in naïve animals and during progressive nigrostriatal degeneration. LBT-3627 administration was linked to reductions of inflammatory microglia, increased survival of dopaminergic neurons, and improved striatal densities. While α-Syn overexpression resulted in reduced Treg activity, LBT-3627 rescued these functional deficits. This occurred in a dose-dependent manner closely mimicking neuroprotection. Taken together, these data provide the basis for the use of VIPR2 agonists as potent therapeutic immune modulating agents to restore Treg activity, attenuate neuroinflammation, and interdict dopaminergic neurodegeneration in PD. The data underscore an important role of immunity in PD pathogenesis., (Copyright © 2019 Mosley, Lu, Olson, Machhi, Yan, Namminga, Smith, Shandler and Gendelman.)

Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.

Antiretroviral therapy (ART) has changed the outcome of human immunodeficiency virus type one (HIV-1) infection from certain death to a life free of disease co-morbidities. However, infected people must remain on life-long daily ART. ART reduces but fails to eliminate the viral reservoir. In order to improve upon current treatment regimens, our laboratory created long acting slow effective release (LASER) ART nanoformulated prodrugs from native medicines. LASER ART enables antiretroviral drugs (ARVs) to better reach target sites of HIV-1 infection while, at the same time, improve ART's half-life and potency. However, novel ARV design has been slowed by prolonged pharmacokinetic testing requirements. To such ends, tri-modal theranostic nanoparticles were created with single-photon emission computed tomography (SPECT/CT), magnetic resonance imaging (MRI) and fluorescence capabilities to predict LASER ART biodistribution. The created theranostic ARV probes were then employed to monitor drug tissue distribution and potency. Intrinsically 111 Indium ( 111 In) radiolabeled, europium doped cobalt-ferrite particles and rilpivirine were encased in a polycaprolactone core surrounded by a lipid shell ( 111 InEuCF-RPV). Particle cell and tissue distribution, and antiretroviral activities were sustained in macrophage tissue depots. 111 InEuCF-PCL/RPV particles injected into mice demonstrated co-registration of MRI and SPECT/CT tissue signals with RPV and cobalt. Cell and animal particle biodistribution paralleled ARV activities. We posit that particle selection can predict RPV distribution and potency facilitated by multifunctional theranostic nanoparticles., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The widespread use of antiretroviral therapy for treatment of human immunodeficiency virus (HIV) infections has dramatically improved the quality and duration of life for HIV-positive individuals. Despite this success, HIV persists for the life of an infected person in tissue reservoirs including the nervous system. Thus, whether HIV exacerbates age-related brain disorders such as Parkinson's disease (PD) is of concern. In support of this idea, HIV infection can be associated with motor and gait abnormalities that parallel late-stage manifestations of PD including dopaminergic neuronal loss. With these findings in hand, we investigated whether viral infection could affect nigrostriatal degeneration or exacerbate chemically induced nigral degeneration. We now demonstrate an additive effect of EcoHIV on dopaminergic neuronal loss and neuroinflammation induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication. HIV-1-infected humanized mice failed to recapitulate these EcoHIV results suggesting species-specific neural signaling. The results demonstrate a previously undefined EcoHIV-associated neurodegenerative response that may be used to model pathobiological aspects of PD.

We investigated the effects of granulocyte-macrophage colony stimulating factor (GM-CSF) on behavioral and pathological outcomes in Alzheimer's disease (AD) and non-transgenic mice. GM-CSF treatment in AD mice reduced brain amyloidosis, increased plasma Aβ, and rescued cognitive impairment with increased hippocampal expression of calbindin and synaptophysin and increased levels of doublecortin-positive cells in the dentate gyrus. These data extend GM-CSF pleiotropic neuroprotection mechanisms in AD and include regulatory T cell-mediated immunomodulation of microglial function, Aβ clearance, maintenance of synaptic integrity, and induction of neurogenesis. Together these data support further development of GM-CSF as a neuroprotective agent for AD., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases regulatory T cell (Treg) number and function with control of neuroinflammation and neuronal protection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease (PD). Recently, we demonstrated in an early phase 1 clinical trial that GM-CSF also improves motor skills in PD patients. However, the mechanisms of Treg induction and its effects on neuroprotective responses remain unknown. As GM-CSF induces tolerogenic dendritic cells (DCs) that in turn convert conventional T cells to Tregs, the pathways for DC induction of Tregs were assessed., Methods: Following differentiation, bone marrow-derived dendritic cells (BMDCs) were cultured in media or GM-CSF with or without post-culture stimulation with nitrated α-synuclein (N-α-Syn). Expression of cell surface co-stimulatory molecules and proinflammatory cytokines, and induction of Tregs were evaluated. The neuroprotective capacity of tolerogenic BMDCs was assessed by adoptive transfer to MPTP-intoxicated mice. The extent of neuroinflammation and numbers of surviving dopaminergic neurons were assessed in relation to Treg numbers., Results: Co-culture of differentiated BMDCs with conventional T cells led to Treg induction. Stimulation of BMDCs with N-α-Syn increased expression of co-stimulatory molecules and proinflammatory cytokines, with modest increases in Treg numbers. In contrast, continued culture of BMDCs with GM-CSF modestly altered expression of co-stimulatory molecules and proinflammatory cytokines and chemokines, but decreased Treg induction. Continued culture in GM-CSF and combined stimulation with N-α-Syn reduced Treg induction to the lowest levels. Adoptive transfer of tolerogenic BMDCs to MPTP-intoxicated mice increased splenic Tregs, attenuated neuroinflammatory responses, and protected nigrostriatal dopaminergic neurons., Conclusions: GM-CSF acts broadly to differentiate DCs and affect immune transformation from effector to regulatory immune responses. DCs skew such immune responses by increasing Treg numbers and activities that serve to attenuate proinflammatory responses and augment neuroprotection.

Background: The mixed lineage kinase type 3 inhibitor URMC-099 facilitates amyloid-beta (Aβ) clearance and degradation in cultured murine microglia. One putative mechanism is an effect of URMC-099 on Aβ uptake and degradation. As URMC-099 promotes endolysosomal protein trafficking and reduces Aβ microglial pro-inflammatory activities, we assessed whether these responses affect Aβ pathobiogenesis. To this end, URMC-099's therapeutic potential, in Aβ precursor protein/presenilin-1 (APP/PS1) double-transgenic mice, was investigated in this model of Alzheimer's disease (AD)., Methods: Four-month-old APP/PS1 mice were administered intraperitoneal URMC-099 injections at 10 mg/kg daily for 3 weeks. Brain tissues were examined by biochemical, molecular and immunohistochemical tests., Results: URMC-099 inhibited mitogen-activated protein kinase 3/4-mediated activation and attenuated β-amyloidosis. Microglial nitric oxide synthase-2 and arginase-1 were co-localized with lysosomal-associated membrane protein 1 (Lamp1) and Aβ. Importatly, URMC-099 restored synaptic integrity and hippocampal neurogenesis in APP/PS1 mice., Conclusions: URMC-099 facilitates Aβ clearance in the brain of APP/PS1 mice. The multifaceted immune modulatory and neuroprotective roles of URMC-099 make it an attractive candidate for ameliorating the course of AD. This is buttressed by removal of pathologic Aβ species and restoration of the brain's microenvironment during disease.

T cells use the endocytic pathway for key cell biological functions, including receptor turnover and maintenance of the immunological synapse. Some of the established players include the Rab GTPases, the SNARE complex proteins, and others, which function together with EPS-15 homology domain-containing (EHD) proteins in non-T cell systems. To date, the role of the EHD protein family in T cell function remains unexplored. We generated conditional EHD1/3/4 knockout mice using CD4-Cre and crossed these with mice bearing a myelin oligodendrocyte glycoprotein-specific TCR transgene. We found that CD4 + T cells from these mice exhibited reduced Ag-driven proliferation and IL-2 secretion in vitro. In vivo, these mice exhibited reduced severity of experimental autoimmune encephalomyelitis. Further analyses showed that recycling of the TCR-CD3 complex was impaired, leading to increased lysosomal targeting and reduced surface levels on CD4 + T cells of EHD1/3/4 knockout mice. Our studies reveal a novel role of the EHD family of endocytic recycling regulatory proteins in TCR-mediated T cell functions., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Long-acting parenteral (LAP) antiretroviral drugs have generated considerable interest for treatment and prevention of HIV-1 infection. One new LAP is cabotegravir (CAB), a highly potent integrase inhibitor, with a half-life of up to 54 days, allowing for every other month parenteral administrations. Despite this excellent profile, high volume dosing, injection site reactions and low body fluid drug concentrations affect broad use for virus infected and susceptible people. To improve the drug delivery profile, we created a myristoylated CAB prodrug (MCAB). MCAB formed crystals that were formulated into nanoparticles (NMCAB) of stable size and shape facilitating avid monocyte-macrophage entry, retention and reticuloendothelial system depot formulation. Drug release kinetics paralleled sustained protection against HIV-1 challenge. After a single 45 mg/kg intramuscular injection to BALB/cJ mice, the NMCAB pharmacokinetic profiles was 4-times greater than that recorded for CAB LAP. These observations paralleled replicate measurements in rhesus macaques. The results coupled with improved viral restriction in human adult lymphocyte reconstituted NOD/SCID/IL2Rγc -/- mice led us to conclude that NMCAB can improve biodistribution and viral clearance profiles upon current CAB LAP formulations., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A potential therapeutic role for immune transformation in Parkinson's disease evolves from more than a decade of animal investigations demonstrating regulatory T cell (Treg) nigrostriatal neuroprotection. To bridge these results to human disease, we conducted a randomized, placebo-controlled double-blind phase 1 trial with a well-studied immune modulator, sargramostim (granulocyte-macrophage colony-stimulating factor). We enrolled 17 age-matched non-Parkinsonian subjects as non-treated controls and 20 Parkinson's disease patients. Both Parkinson's disease patients and controls were monitored for 2 months for baseline profiling. Parkinson's disease patients were then randomized into two equal groups to self-administer placebo (saline) or sargramostim subcutaneously at 6 μg/kg/day for 56 days. Adverse events for the sargramostim and placebo groups were 100% (10/10) and 80% (8/10), respectively. These included injection site reactions, increased total white cell counts, and upper extremity bone pain. One urticarial and one vasculitis reaction were found to be drug and benzyl alcohol related, respectively. An additional patient with a history of cerebrovascular disease suffered a stroke on study. Unified Parkinson's disease rating scale, Part III scores in the sargramostim group showed modest improvement after 6 and 8 weeks of treatment when compared with placebo. This paralleled improved magnetoencephalography-recorded cortical motor activities and Treg numbers and function compared with pretreated Parkinson's disease patients and non-Parkinsonian controls. Peripheral Treg transformation was linked to serum tryptophan metabolites, including L-kynurenine, quinolinic acid, and serotonin. These data offer a potential paradigm shift in modulating immune responses for potential therapeutic gain for Parkinson's disease. Confirmation of these early study results requires larger numbers of enrolled patients and further clinical investigation.

Background: Despite improved clinical outcomes seen following antiretroviral therapy (ART), resting CD4+ T cells continue to harbor latent human immunodeficiency virus type one (HIV-1). However, such cells are not likely the solitary viral reservoir and as such defining where and how others harbor virus is imperative for eradication measures. To such ends, we used HIV-1 ADA -infected NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice reconstituted with a human immune system to explore two long-acting ART regimens investigating their abilities to affect viral cell infection and latency. At 6 weeks of infection animals were divided into four groups. One received long-acting (LA) cabotegravir (CAB) and rilpivirine (RVP) (2ART), a second received LA CAB, lamivudine, abacavir and RVP (4ART), a third were left untreated and a fourth served as an uninfected control. After 4 weeks of LA ART treatment, blood, spleen and bone marrow (BM) cells were collected then phenotypically characterized. CD4+ T cell subsets, macrophages and hematopoietic progenitor cells were analyzed for HIV-1 nucleic acids by droplet digital PCR., Results: Plasma viral loads were reduced by two log 10 or to undetectable levels in the 2 and 4ART regimens, respectively. Numbers and distributions of CD4+ memory and regulatory T cells, macrophages and hematopoietic progenitor cells were significantly altered by HIV-1 infection and by both ART regimens. ART reduced viral DNA and RNA in all cell and tissue compartments. While memory cells were the dominant T cell reservoir, integrated HIV-1 DNA was also detected in the BM and spleen macrophages in both regimen-treated mice., Conclusion: Despite vigorous ART regimens, HIV-1 DNA and RNA were easily detected in mature macrophages supporting their potential role as an infectious viral reservoir.

Long-acting anti-HIV products can substantively change the standard of care for patients with HIV/AIDS. To this end, hydrophobic antiretroviral drugs (ARVs) were recently developed for parenteral administration at monthly or longer intervals. While shorter-acting hydrophilic drugs can be made into nanocarrier-encased prodrugs, the nanocarrier encasement must be boosted to establish long-acting ARV depots. The mixed-lineage kinase 3 (MLK-3) inhibitor URMC-099 provides this function by affecting autophagy. Here, we have shown that URMC-099 facilitates ARV sequestration and its antiretroviral responses by promoting the nuclear translocation of the transcription factor EB (TFEB). In monocyte-derived macrophages, URMC-099 induction of autophagy led to retention of nanoparticles containing the antiretroviral protease inhibitor atazanavir. These nanoparticles were localized within macrophage autophagosomes, leading to a 4-fold enhancement of mitochondrial and cell vitality. In rodents, URMC-099 activation of autophagy led to 50-fold increases in the plasma drug concentration of the viral integrase inhibitor dolutegravir. These data paralleled URMC-099-mediated induction of autophagy and the previously reported antiretroviral responses in HIV-1-infected humanized mice. We conclude that pharmacologic induction of autophagy provides a means to extend the action of a long-acting, slow, effective release of antiretroviral therapy.

Neuroprotective immunity is defined by transformation of T-cell polarity for therapeutic gain. For neurodegenerative disorders and specifically for Parkinson's disease (PD), granulocyte-macrophage colony stimulating factor or vasoactive intestinal peptide receptor 2 (VIPR2) agonists elicit robust anti-inflammatory microglial responses leading to neuronal sparing in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. While neurotherapeutic potential was demonstrated for PD, there remain inherent limitations in translating these inventions from the laboratory to patients. One obstacle in translating such novel neurotherapeutics centers on the availability of suitable noninvasive methods to track disease progression and therapeutic efficacy. To this end, we developed manganese-enhanced magnetic resonance imaging (MEMRI) assays as a way to track a linkage between glial activation and VIPR2 agonist (LBT-3627)-induced neuroprotective immunity for MPTP-induced nigrostriatal degeneration. Notably, LBT-3627-treated, MPTP-intoxicated mice show reduced MEMRI brain signal intensities. These changes paralleled reduced astrogliosis and resulted in sparing of nigral tyrosine hydroxylase neurons. Most importantly, the data suggest that MEMRI can be developed as a biomarker tool to monitor neurotherapeutic responses that are relevant to common neurodegenerative disorders used to improve disease outcomes.

Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection.

Vasoactive intestinal peptide (VIP) mediates a broad range of biological responses by activating two related receptors, VIP receptor 1 and 2 (VIPR1 and VIPR2). Although the use of native VIP facilitates neuroprotection, clinical application of the hormone is limited due to VIP's rapid metabolism and inability to distinguish between VIPR1 and VIPR2 receptors. In addition, activation of both receptors by therapeutics may increase adverse secondary toxicities. Therefore, we developed metabolically stable and receptor-selective agonists for VIPR1 and VIPR2 to improve pharmacokinetic and pharmacodynamic therapeutic end points. Selective agonists were investigated for their abilities to protect mice against MPTP-induced neurodegeneration used to model Parkinson's disease (PD). Survival of tyrosine hydroxylase neurons in the substantia nigra was determined by stereological tests after MPTP intoxication in mice pretreated with either VIPR1 or VIPR2 agonist or after adoptive transfer of splenic cell populations from agonist-treated mice administered to MPTP-intoxicated animals. Treatment with VIPR2 agonist or splenocytes from agonist-treated mice resulted in increased neuronal sparing. Immunohistochemical tests showed that agonist-treated mice displayed reductions in microglial responses, with the most pronounced effects in VIPR2 agonist-treated, MPTP-intoxicated mice. In parallel studies, we observed reductions in proinflammatory cytokine release that included IL-17A, IL-6, and IFN-γ and increases in GM-CSF transcripts in CD4(+) T cells recovered from VIPR2 agonist-treated animals. Moreover, a phenotypic shift of effector to regulatory T cells was observed. These results support the use of VIPR2-selective agonists as neuroprotective agents for PD treatment., Significance Statement: Vasoactive intestinal peptide receptor 2 can elicit immune transformation in a model of Parkinson's disease (PD). Such immunomodulatory capabilities can lead to neuroprotection by attenuating microglial activation and by slowing degradation of neuronal cell bodies and termini in MPTP-intoxicated mice. The protective mechanism arises from altering a Th1/Th2 immune cytokine response into an anti-inflammatory and neuronal sparing profile. These results are directly applicable for the development of novel PD therapies., (Copyright © 2015 the authors 0270-6474/15/3516463-16$15.00/0.)

Neurodegenerative and neuropsychological disorders are becoming a greater proportion of the global disease burden; however the pathogenic mechanisms by which these disorders originate and contribute to disease progression are not well-described. Increasing evidence supports neuroinflammation as a common underlying component associated with the neuropathological processes that effect disease progression. This collection of articles explores the role of adaptive immunity in autoimmunity, neurodegeneration, neurotrauma, and psychological disorders. The section emphasizes the interactions of T cells with innate cellular responses within the CNS and the effects on neurological functions. One recurrent theme is that modified and aggregated self-proteins upregulate innate-mediated inflammation and provide a permissive environment for polarization of T cells to proinflammatory effector cells. Moreover, infiltration and reactivation of those T effector cells exacerbate neuroinflammation and oxidative stress to greater neurotoxic levels. Another recurrent theme in these disorders promotes diminished regulatory functions that reduce control over activated T effector cells and microglia, and ultimately augment proinflammatory conditions. Augmentation of regulatory control is discussed as therapeutic strategies to attenuate neuroinflammation, mitigate neurodegeneration or neuronal dysfunction, and lessen disease progression.

Aberrant innate and adaptive immune responses are neurodegenerative disease effectors. Disease is heralded by a generalized, but subtle immune activation orchestrated by the release of extracellular prion-like aggregated and oxidized or otherwise modified proteins. These are responsible for an inflammatory neurotoxic cascade. The perpetrators of such events include effector T cells and activated microglia. What ensues are Alzheimer's and Parkinson's disease, amyotrophic lateral sclerosis and stroke with changed frequencies of effector T cell and reduced numbers or function of regulatory lymphocytes. The control of such immune responses could lead to new therapeutic strategies and the means to effectively combat a composite of diseases that have quite limited therapeutic options.

Long-acting nanoformulated antiretroviral therapy (nanoART) can sustain plasma drug levels and improve its biodistribution. Cell targeted-nanoART can achieve this and bring drug efficiently to viral reservoirs. However, whether such improvements affect antiretroviral responses remains unknown. To these ends, we tested folic acid (FA)-linked poloxamer407-coated ritonavir-boosted atazanavir (FA-nanoATV/r) nanoparticles for their ability to affect chronic HIV-1 infection in humanized mice. Following three, 100mg/kg FA-nanoATV/r intramuscular injections administered every other week to infected animals, viral RNA was at or below the detection limit, cell-associated HIV-1p24 reduced and CD4+ T cell counts protected. The dosing regimen improved treatment outcomes more than two fold from untargeted nanoATV/r. We posit that these nanoformulations have potential for translation to human use., (Copyright © 2015 Elsevier B.V. All rights reserved.)