No abstract

BackgroundExosomes are a subset of extracellular vesicles 30–200 nm in diameter secreted from cells, which contain functional mRNAs and microRNAs. Cerebrospinal fluid (CSF) is the primary source for liquid biopsy to examine diseases in central nervous system. To date, there is no available method to analyze exosomal mRNAs comprehensively in human CSF.MethodsThe main purpose of this study is to established the methodology of comprehensive analysis of exosomal mRNAs in CSF by a highly sensitive next-generation sequencing. The signatures of CSF exosomal mRNAs were then compared between four normal healthy donors and four sporadic amyotrophic lateral sclerosis patients to identify disease-related biomarkers. Differentially expressed genes were identified by DESeq2.ResultsRNA sequencing from CSF exosomes was successfully performed, that was demonstrated by the high pearson’s product-moment correlation coefficient (r = 0.993) in the technical replicates. Also, position coverage analysis revealed that most detected mRNAs retained their integrity throughout their full-length in CSF exosomes. In CSF exosomes from normal healthy donors, an average of 14,807 genes were detected, of which 4580 genes were commonly detected among four individuals, including neuron-enriched genes such as TUBB3 and CAMK2A. In comparison with exosomal mRNAs in CSF from four patients with amyotrophic lateral sclerosis, 543 genes were significantly changed, as represented by CUEDC2. Gene Ontology analysis and pathway analysis with these genes revealed functional enrichment of ubiquitin-proteasome pathway, oxidative stress response, and unfolded protein response. These pathways are related to pathomechanisms of amyotrophic lateral sclerosis.ConclusionWe successfully established the methodology of comprehensive analysis of exosomal mRNAs in human CSF. It was shown to be useful to identify disease biomarkers for central nervous system. Several genes, such as CUEDC2, in CSF exosomes were suggested to be candidate disease biomarkers for amyotrophic lateral sclerosis.Electronic supplementary materialThe online version of this article (10.1186/s12920-019-0473-z) contains supplementary material, which is available to authorized users.

We describe novel humanized anti-CD20 monoclonal antibodies (mAbs) developed for therapeutic use on the basis of their physicochemical properties and cellular cytotoxicity. A distinct correlation between apparent dissociation constants (K d ) and apoptotic activity for eight murine anti-CD20 mAbs (OUBM1-OUBM8) and previously-developed murine anti-CD20 mAbs enabled us to categorize anti-CD20 mAbs into two groups. Group A mAbs had lower K d values and did not induce definite apoptosis, while Group B mAbs had greater K d values and did induce definite apoptosis. A murine version mAb of rituximab, 2B8, belongs to Group B. An epitope analysis showed that the epitope of two murine mAbs, OUBM3 and OUBM6, differed from that of 2B8 or 2F2 (ofatumumab). Two mAbs, OUBM3 from Group A and OUBM6 from Group B, were selected and humanized. As expected, the humanized OUBM3 with the lower K d did not induce apoptosis, while the humanized OUBM6 (hOUBM6) with the greater K d did. Both hOU-BM3 and hOUBM6 induced highly-effective, complement-dependent cytotoxicity and antibody-dependent, cell-mediated cytotoxicity against Burkitt's and follicular lymphomas. Importantly, hOUBM6 exhibited cellular cytotoxicity against diffuse, large B cells that are less effectively depleted by rituximab and also exhibited effective cytotoxicity against tumor cells from human CD20(+) leukemia and lymphoma patients. These results suggest the potential impact of the further development of our anti-CD20 mAbs. Our study shows that the selection of mAbs based on their physicochemical parameters, followed by the biological activity assessment for the selected mAbs, is a rational and efficient approach for pharmaceutical mAb development. (Cancer Sci 2010; 101: 201-209) R ecently, the application of immunotherapy using monoclonal antibodies (mAbs) has increased considerably, and numerous mAb candidates are under development or are being assessed in clinical trials. In the case of mAb development using murine mAbs, the degree of complement-dependent cytotoxicity (CDC) and antibody-dependent, cell-mediated cytotoxicity (ADCC) activity can be measured after humanization, which implies that it is difficult to evaluate the clinical effectiveness of these antibodies (Abs) before humanization. The humanization of large numbers of murine mAbs without data to support future clinical use requires massive investments in terms of time, labor, and funding. Furthermore, the evaluation of bioactivity is often dependent on the assay systems themselves. Therefore, it is highly desirable to select candidates for humanization on the basis of the intrinsic parameters of the mAb itself, such as its dissociation constant and epitope, at an early stage of mAb development. In order to establish an efficient and rational method of developing mAbs, we focused on anti-CD20 mAbs as the ideal study target for studying the intrinsic properties of mAbs, because data on these Abs is available.CD20 is a B-cell-specific tetra-transmembrane protein with a molecular weight of 33-37 kDa. It i...

There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA (CD63, CD81 and albumin), and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability.

No abstract