Exosomes are 50‐ to 150‐nm‐diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation‐ and column‐based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek^®) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream^®). High‐performance liquid chromatography followed by nano‐flow liquid chromatography–mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5‐fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Pre‐analytic fluid collection and processingBasic Protocol 2: Exosome isolation by ultracentrifugationAlternate Protocol 1: Exosome isolation by precipitationBasic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysisAlternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometryBasic Protocol 4: Downstream analysis of exosomes using high‐performance liquid chromatographyBasic Protocol 5: Downstream analysis of the exosome proteome using nano‐flow liquid chromatography–mass spectrometry

Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer's disease (AD). While it has been commonly assumed that RA patients' usage of non-steroidal anti-inflammatory drugs (NSAIDs) helped prevent onset and progression of

Introduction: Inflammatory markers have long been observed in the brain, cerebrospinal fluid (CSF), and plasma of Alzheimer's disease (AD) patients, suggesting that inflammation contributes to AD and might be a therapeutic target. However, non‐steroidal anti‐inflammatory drug trials in AD and mild cognitive impairment (MCI) failed to show benefit. Our previous work seeking to understand why people with the inflammatory disease rheumatoid arthritis are protected from AD found that short‐term treatment of transgenic AD mice with the pro‐inflammatory cytokine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) led to an increase in activated microglia, a 50% reduction in amyloid load, an increase in synaptic area, and improvement in spatial memory to normal. These results called into question the consensus view that inflammation is solely detrimental in AD. Here, we tested our hypothesis that modulation of the innate immune system might similarly be used to treat AD in humans by investigating the ability of GM‐CSF/sargramostim to safely ameliorate AD symptoms/pathology. Methods: A randomized, double‐blind, placebo‐controlled trial was conducted in mild‐to‐moderate AD participants (NCT01409915). Treatments (20 participants/group) occurred 5 days/week for 3 weeks plus two follow‐up (FU) visits (FU1 at 45 days and FU2 at 90 days) with neurological, neuropsychological, blood biomarker, and imaging assessments. Results: Sargramostim treatment expectedly changed innate immune system markers, with no drug‐related serious adverse events or amyloid‐related imaging abnormalities. At end of treatment (EOT), the Mini‐Mental State Examination score of the sargramostim group increased compared to baseline ( P = .0074) and compared to placebo ( P = .0370); the treatment effect persisted at FU1 ( P = .0272). Plasma markers of amyloid beta (Aβ40 [decreased in AD]) increased 10% ( P = .0105); plasma markers of neurodegeneration (total tau and UCH‐L1) decreased 24% ( P = .0174) and 42% ( P = .0019), respectively, after sargramostim treatment compared to placebo. Discussion: The innate immune system is a viable target for therapeutic intervention in AD. An extended treatment trial testing the long‐term safety and efficacy of GM‐CSF/sargramostim in AD is warranted.