Clinical biomarkers for Lewy body diseases.

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.

Immune senescence in aged APP/PS1 mice.

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.

An open-label multiyear study of sargramostim-treated Parkinson's disease patients examining drug safety, tolerability, and immune biomarkers from limited case numbers.

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 .

Monocyte biomarkers define sargramostim treatment outcomes for Parkinson's disease.

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.

Defining the Innate Immune Responses for SARS-CoV-2-Human Macrophage Interactions.

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.

Safety, tolerability, and immune-biomarker profiling for year-long sargramostim treatment of Parkinson's 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.

Synthesis, biological evaluation, and metabolic stability of phenazine derivatives as antibacterial agents.

Drug-resistant pathogens are a major cause of hospital- and community-associated bacterial infections in the United States and around the world. These infections are increasingly difficult to treat due to the development of antibiotic resistance and the formation of bacterial biofilms. In the paper, a series of phenazines were synthesized and evaluated for their in vitro antimicrobial activity against Gram positive (methicillin resistant staphylococcus aureus, MRSA) and Gram negative (Escherichia coli, E. coli) bacteria. The compound 6,9-dichloro-N-(methylsulfonyl)phenazine-1-carboxamide (18c) proved to be the most active molecule (MIC = 16 µg/mL) against MRSA whereas 9-methyl-N-(methylsulfonyl)phenazine-1-carboxamide (30e) showed good activity against both MRSA (MIC = 32 µg/mL) and E. coli (MIC = 32 µg/mL). Molecule 18c also demonstrated significant biofilm dispersion and inhibition against S. aureus. Preliminary studies indicate the molecules do not disturb bacterial membranes and there activity is not directly linked to the generation of reactive oxygen species. Compound 18c displayed minor toxicity against mammalian cells. Metabolic stability studies of the most promising compounds indicate stability towards phase I and phase II metabolizing enzymes.