Journal articles on the topic 'CSF single chain library'
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Tapryal, Suman, Yogender Pal khasa, and K. J. Mukherjee. "Single chain Fv fragment specific for human GM-CSF: Selection and expression using a bacterial expression library." Biotechnology Journal 5, no. 10 (September 3, 2010): 1078–89. http://dx.doi.org/10.1002/biot.201000043.
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Piper, Clinton, Achia Khatun, Yao Chen, Ryan Zander, Weiguo Cui, and William R. Drobyski. "Single Cell Immune Profiling of Colitogenic T Cells during Acute Graft Versus Host Disease Reveals Two Novel Transcriptionally Distinct CD4+ GM-CSF+ Lineages." Blood 134, Supplement_1 (November 13, 2019): 197. http://dx.doi.org/10.1182/blood-2019-127095.
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Gastrointestinal (GI) tract involvement is the major cause of morbidity and mortality in acute graft versus host disease (GVHD) and pathological damage is largely attributable to inflammatory cytokine production. Recently, we and others identified GM-CSF as a cytokine that is produced primarily by donor-derived CD4+ T cells and mediates inflammation in the GI tract. However, the precise mechanism by which GM-CSF induces pathological damage and the transcriptional profile of this novel colitogenic CD4+ GM-CSF+ T cell population have not been defined. To address these questions, we employed a well-defined murine model of GVHD [C57BL/6 (H-2b)→Balb/c (H-2d)] and demonstrated that GM-CSF induces inflammation by enhancing the activation of donor-derived dendritic cells in the colon as evidenced by increased expression of costimulatory molecules (i.e. CD80 and CD86) and the production of IL-23. In addition, GM-CSF linked adaptive to innate immunity by promoting indirect alloantigen presentation in the mesenteric lymph nodes which was IL-23 dependent and characterized by an increased number of CD103+ CD11b+ dendritic cells and donor CD4+ T cells with a proinflammatory cytokine phenotype. Unexpectedly, we observed two distinct CD4+ GM-CSF+ populations in the GI tract that were distinguishable by the presence or absence of IFN-γ production by intracellular cytokine staining (i.e. CD4+ GM-CSF+ IFN-γ+ and CD4+ GM-CSF+ IFN-γ-). Notably, CD4+ GM-CSF+ IFN-γ- cells were largely absent from other target organs (e.g. liver, lung), suggesting that this population had a unique role in the biology of GVHD in the GI tract. To determine whether these two populations represented transcriptionally distinct lineages or reflected TH1-biased lineage plasticity, we performed single cell RNA sequencing and immunological profiling on donor-derived sort-purified T cells from the colons of GVHD mice one and three weeks post-transplant using the 10X Genomics platform. After selecting only high quality reads, we recovered 6315 unique barcodes corresponding to individual cells and identified several transcriptionally distinct cell clusters that spatially segregated following Seurat and UMAP analysis. Colonic T cells obtained on days 7 and 21 post transplantation completely separated, indicating that the transcriptional profile of these cells changes dramatically between early and later time points. Detectable transcription of GM-CSF was observed in two distinct populations of CD4+ T cells only at the 21-day timepoint. Notably, only one of these clusters co-expressed IFN-γ, confirming our flow-based results, and indicating that CD4+ GM-CSF+ IFN-γ+ and GM-CSF+ IFN-γ- T cells represented distinct populations. Further analysis revealed that CD4+ GM-CSF+ IFN-γ+ T cells were T-bet+ and differentially expressed high levels of costimulatory molecules (CD137, OX40, and CD81) and PD-1, indicative of an activated T cell phenotype. In contrast, CD4+ GM-CSF+ IFN-γ- T cells were distinguishable by the co-expression of T-bet and Gata-3, which is a TH2-defining transcription factor, as well as by the IL-7R and a series of interferon stimulated genes (IFITM1, IFITM2, and IFITM3), supporting the premise that these cells constitute a discrete TH cell lineage. To further characterize these CD4+ T cell populations, we examined the T cell repertoire (TCR) using a targeted sequencing analysis approach of our barcoded cDNA library. We identified 444 unique clonotypes among CD4+ GM-CSF+ T cells based on sequencing of CDR3 regions of TCR alpha and beta chains. Notably, only 5 clonotypes were shared between CD4+ GM-CSF+ IFN-γ+ and CD4+ GM-CSF+ IFN-γ-T cells, representing 58 of 1154 (~5%) of the total cells in both clusters. Thus, this minimal overlap suggested that these T cells were responding to non-overlapping antigens within the GI tract. Analysis of V beta TCR gene usage revealed that CD4+ GM-CSF+ IFN-γ- cells had a highly-biased repertoire with approximately half of cells utilizing a single V beta gene, Vbeta3. In contrast, CD4+ GM-CSF+ IFN-γ+ T cells had a much more evenly distributed Vbeta receptor profile with no predominant Vbeta usage. Collectively, these studies demonstrate the existence of two transcriptionally distinct CD4+ GM-CSF+ T cell populations that accumulate within the GI tract, possess non-overlapping T cell repertoires, promote indirect alloantigen presentation, and mediate pathological damage during GVHD. Disclosures No relevant conflicts of interest to declare.
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Ning, Liangxia, and Bin Wang. "Neurofilament light chain in blood as a diagnostic and predictive biomarker for multiple sclerosis: A systematic review and meta-analysis." PLOS ONE 17, no. 9 (September 14, 2022): e0274565. http://dx.doi.org/10.1371/journal.pone.0274565.
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Background Neurofilament light chain (NfL) in cerebrospinal fluid (CSF) is a biomarker of multiple sclerosis (MS). However, CSF sampling is invasive and has limited the clinical application. With the development of highly sensitive single-molecule assay, the accurate quantification of the very low NfL levels in blood become feasible. As evidence being accumulated, we performed a meta-analysis to evaluate the diagnostic and predictive value of blood NfL in MS patients. Methods We performed literature search on PubMed, EMBASE, Web of Science and Cochrane Library from inception to May 31, 2022. The blood NfL differences between MS vs. controls, MS vs. clinically isolated syndrome (CIS), progressive MS (PMS) vs. relapsing-remitting MS (RRMS), and MS in relapse vs. MS in remission were estimated by standard mean difference (SMD) and corresponding 95% confidence interval (CI). Pooled hazard ratio (HR) and 95%CI were calculated to predict time to reach Expanded Disability Status Scale (EDSS) score≥4.0 and to relapse. Results A total of 28 studies comprising 6545 MS patients and 2477 controls were eligible for meta-analysis of diagnosis value, and 5 studies with 4444 patients were synthesized in analysis of predictive value. Blood NfL levels were significantly higher in MS patients vs. age-matched controls (SMD = 0.64, 95%CI 0.44–0.85, P<0.001), vs. non-matched controls (SMD = 0.76, 95%CI 0.56–0.96, P<0.001) and vs. CIS patients (SMD = 0.30, 95%CI 0.18–0.42, P<0.001), in PMS vs. RRMS (SMD = 0.56, 95%CI 0.27–0.85, P<0.001), and in relapsed patients vs. remitted patients (SMD = 0.54, 95%CI 0.16–0.92, P = 0.005). Patients with high blood NfL levels had shorter time to reach EDSS score≥4.0 (HR = 2.36, 95%CI 1.32–4.21, P = 0.004) but similar time to relapse (HR = 1.32, 95%CI 0.90–1.93, P = 0.155) compared to those with low NfL levels. Conclusion As far as we know, this is the first meta-analysis evaluating the diagnosis and predictive value of blood NfL in MS. The present study indicates blood NfL may be a useful biomarker in diagnosing MS, distinguishing MS subtypes and predicting disease worsening in the future.
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Tashiro, Haruko, Ryosuke Shirasaki, Yoko Oka, Tadashi Yamamoto, Nobu Akiyama, Kazuo Kawasugi, and Naoki Shirafuji. "Interleukin-1β Promotes the Expression of CD34 and Granulocyte Colony-Stimulating Factor-Receptor in Adult Dermal Fibroblasts." Blood 118, no. 21 (November 18, 2011): 4815. http://dx.doi.org/10.1182/blood.v118.21.4815.4815.
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Abstract Abstract 4815 Background and Aims: We reported that acute myelogenous leukemia blasts and chronic myelogenous leukemia cells converted to stromal myofibroblasts to create an environment for the proliferation of leukemic cells in vitro and also in a non-obese diabetes/ severe combined immunodeficiency (NOD/SCID) murine bone-marrow in vivo. In normal hematopoiesis, hematopoietic stem cell (HSC) and stromal immature mesenchymal stem cell (MSC) are speculated to have a cross-talk, and some reports indicate that the HSC generates MSC, and also a specific fraction of MSC shares similar molecular expressions to that of HSC. We made a hypothesis that HSC might be generated from MSC. To make clear this issue, expression cloning was performed to isolate a molecule that stimulated bone-marrow stromal myofibroblasts to express hematopoietic stem cell marker, CD34. And, we also observed the effect of the isolated molecule to an adult human dermal fibroblast (HDF). Materials and Methods: cDNA-expression library was constructed using PHA-P-stimulated normal human blood lymphocytes, and the prepared plasmids were transfected to COS7 cells. After 3 days of culture, supernatants were added to the normal human bone-marrow-derived myofibroblasts (final 10%), and cells were further cultured for one week. RNA was extracted from the cultured myofibroblasts, and cDNA was synthesized. Positive clones were selected on CD34-expression with reverse transcription-polymerase chain reaction, and a single clone was isolated. The purified protein from the isolated single clone was added to HDF-culture, and the morphological changes and the expression of specific hematopoiesis-related proteins were analyzed. Results and Discussion: Isolated single clone was human interleukin 1β (IL-1β). When the purified IL-1β protein was added to the bone-marrow-derived myofibroblast cultures, cell growth was increased, and up-regulation of the expression of several hematopoietic specific proteins, including cytokine receptors and transcription factor SCL, was observed. Based on these observations, we determined the effect of IL-1β to HDF. When HDFs were cultured with human IL-1β for 3 weeks, the expression of granulocyte colony-stimulating factor (G-CSF)-receptor, and SCL was increased. When these IL-1β-stimulated cells were cultured in a non-coated dish, cells were floating, and budding of the cells was also observed. When HDF were cultured with IL-1β for 3 weeks, and then G-CSF and erythropoietin were added to the cultures, expression of transcription factor GATA-1 and CEBPA was significantly increased after one week. These observations indicate that IL-1β can stimulate to induce HDF toward hematopoietic cells. Now we determine the precise actions of human IL-1β to HDF using NOD/SCID transplantation model in vivo. Disclosures: No relevant conflicts of interest to declare.
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Smith, Eric L., Sham Mailankody, Arnab Ghosh, Reed Masakayan, Mette Staehr, Terence J. Purdon, Elizabeth Halton, et al. "Development and Evaluation of a Human Single Chain Variable Fragment (scFv) Derived Bcma Targeted CAR T Cell Vector Leads to a High Objective Response Rate in Patients with Advanced MM." Blood 130, Suppl_1 (December 7, 2017): 742. http://dx.doi.org/10.1182/blood.v130.suppl_1.742.742.
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Abstract Patients with relapsed/refractory MM (RRMM) rarely obtain durable remissions with available therapies. Clinical use of BCMA targeted CAR T cell therapy was first reported in 12/2015 for RRMM, and based on small numbers, preliminary results appear promising. Given that host immune anti-murine CAR responses have limited the efficacy of repeat dosing (Turtle C. Sci Trans Med 2016), our goal was to develop a human BCMA targeted CAR T cell vector for clinical translation. We screened a human B cell derived scFv phage display library containing 6x1010 scFvs with BCMA expressing NIH 3T3 cells, and validated results on human MM cell lines. 57 unique and diverse BCMA specific scFvs were identified containing light and heavy chain CDR's each covering 6 subfamilies, with HCDR3 length ranges from 5-18 amino acids. 17 scFvs met stringent specificity criteria, and a diverse set was cloned into CAR vectors with either a CD28 or a 4-1BB co-stimulatory domain. Donor T cells transduced with BCMA targeted CAR vectors that conveyed particularly desirable properties over multiple in vitro assays, including: cytotoxicity on human MM cell lines at low E:T ratios (>90% lysis, 1:1, 16h), robust proliferation after repeat antigen stimulation (up to 700 fold, stimulation q3-4d for 14d), and active cytokine profiling, were selected for in vivo studies using a marrow predominant human MM cell line model in NSG mice. A single IV injection of CAR T cells, either early (4d) or late (21d) after MM engraftment was evaluated. In both cases survival was increased when treated with BCMA targeted CAR T cells vs CD19 targeted CAR T cells (median OS at 60d NR vs 35d p<0.05). Tumor and CAR T cells were imaged in vivo by taking advantage of luciferase constructs with different substrates. Results show rapid tumor clearance, peak (>10,000 fold) CAR T expansion at day 6, followed by contraction of CAR T cells after MM clearance, confirming the efficacy of the anti-BCMA scFv/4-1BB containing construct. Co-culture with primary cells from a range of normal tissues did not activate CAR T cells as noted by a lack of IFN release. Co-culture of 293 cells expressing this scFv with those expressing a library of other TNFRSF or Ig receptor members demonstrated specific binding to BCMA. GLP toxicity studies in mice showed no unexpected adverse events. We generated a retroviral construct for clinical use including a truncated epithelial growth factor receptor (EGFRt) elimination gene: EGFRt/hBCMA-41BBz. Clinical investigation of this construct is underway in a dose escalation, single institution trial. Enrollment is completed on 2/4 planned dose levels (DL). On DL1 pts received cyclophosphamide conditioning (3g/m2 x1) and 72x106 mean CAR+ T cells. On DL2 pts received lower dose cyclophosphamide/fludarabine (300/30 mg/m2 x3) and 137x106 mean CAR+ T cells. All pts screened for BCMA expression by IHC were eligible. High risk cytogenetics were present in 4/6 pts. Median prior lines of therapy was 7; all pts had IMiD, PI, high dose melphalan, and CD38 directed therapies. With a data cut off of 7/20/17, 6 pts are evaluable for safety. There were no DLT's. At DL1, grade 1 CRS, not requiring intervention, occurred in 1/3 pts. At DL2, grade 1/2 CRS occurred in 2/3 pts; both received IL6R directed Tocilizumab (Toci) with near immediate resolution. In these 2 pts time to onset of fever was a mean 2d, Tmax was 39.4-41.1 C, peak CRP was 25-27mg/dl, peak IL6 level pre and post Toci were 558-632 and 3375-9071 pg/ml, respectively. Additional serum cytokines increased >10 fold from baseline in both pts include: IFNg, GM CSF, Fractalkine, IL5, IL8, and IP10. Increases in ferritin were limited, and there were no cases of hypofibrinogenemia. There were no grade 3-5 CRS and no neurotoxicities or cerebral edema. No pts received steroids or Cetuximab. Median time to count recovery after neutropenia was 10d (range 6-15d). Objective responses by IMWG criteria after a single dose of CAR T cells were observed across both DLs. At DL1, of 3 pts, responses were 1 VGPR, 1 SD, and 1 pt treated with baseline Mspike 0.46, thus not evaluable by IMWG criteria, had >50% reduction in Mspike, and normalization of K/L ratio. At DL2, 2/2 pts had objective responses with 1 PR and 1 VGPR (baseline 95% marrow involvement); 1 pt is too early to evaluate. As we are employing a human CAR, the study was designed to allow for an optional second dose in pts that do not reach CR. We have treated 2 pts with a second dose, and longer follow up data is pending. Figure 1 Figure 1. Disclosures Smith: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: BCMA targeted CAR T cells, Research Funding. Almo: Cue Biopharma: Other: Founder, head of SABequity holder; Institute for Protein Innovation: Consultancy; AKIN GUMP STRAUSS HAUER & FELD LLP: Consultancy. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xu: Eureka Therapeutics, Inc: Employment, Equity Ownership. Park: Amgen: Consultancy. Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Dogan: Celgene: Consultancy; Peer Review Institute: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
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Short, M. K., T. Housel, and intro by P. T. Jubinsky. "Selection of single chain antibody fragments against the gm-csf receptor." Experimental Hematology 28, no. 7 (July 2000): 86. http://dx.doi.org/10.1016/s0301-472x(00)00355-6.
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Hanna, Rachel, Lia Cardarelli, Nish Patel, Levi L. Blazer, Jarrett J. Adams, and Sachdev S. Sidhu. "A phage‐displayed single‐chain Fab library optimized for rapid production of single‐chain IgGs." Protein Science 29, no. 10 (September 15, 2020): 2075–84. http://dx.doi.org/10.1002/pro.3931.
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Schneider, Ruth, Barbara Bellenberg, Barbara Gisevius, Sarah Hirschberg, Roman Sankowski, Marco Prinz, Ralf Gold, Carsten Lukas, and Aiden Haghikia. "Chitinase 3–like 1 and neurofilament light chain in CSF and CNS atrophy in MS." Neurology - Neuroimmunology Neuroinflammation 8, no. 1 (November 10, 2020): e906. http://dx.doi.org/10.1212/nxi.0000000000000906.
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ObjectiveTo investigate cross-sectional associations of CSF levels of neurofilament light chain (NfL) and of the newly emerging marker chitinase 3–like protein 1 (CHI3L1) with brain and spinal cord atrophy, which are established MRI markers of disease activity in MS, to study CHI3L1 and NfL in relapsing (RMS) and progressive MS (PMS), and to assess the expression of CHI3L1 in different cell types.MethodsIn a single-center study, 131 patients with MS (42 RMS and 89 PMS) were assessed for NfL and CHI3L1 concentrations in CSF, MRI-based spinal cord and brain volumetry, MS subtype, age, disease duration, and disability. We included 42 matched healthy controls receiving MRI. CHI3L1 expression of human brain cell types was examined in 2 published single-cell RNA sequencing data sets.ResultsCHI3L1 was associated with spinal cord volume (B = −1.07, 95% CI −2.04 to −0.11, p = 0.029) but not with brain volumes. NfL was associated with brain gray matter (B = −7.3, 95% CI −12.0 to −2.7, p = 0.003) but not with spinal cord volume. CHI3L1 was suitable to differentiate between progressive or relapsing MS (p = 0.015, OR 1.0103, CI for OR 1.002–1.0187), and its gene expression was found in MS-associated microglia and macrophages and in astrocytes of MS brains.ConclusionsNfL and CHI3L1 in CSF were differentially related to brain and spinal cord atrophy. CSF CHI3L1 was associated with spinal cord volume loss and was less affected than NfL by disease duration and age, whereas CSF NfL was associated with brain gray matter atrophy. CSF NfL and CHI3L1 measurement provides complementary information regarding brain and spinal cord volumes.Classification of evidenceThis study provides Class II evidence that CSF CHI3L1 is associated with spinal cord volume loss and that CSF NfL is associated with gray matter atrophy.
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Kamiya, Toshio, Osamu Saitoh, and Hiroyasu Nakata. "Functional Expression of Single-Chain Heterodimeric G-Protein-Coupled Receptors for Adenosine and Dopamine." Cell Structure and Function 29, no. 5,6 (2005): 139–45. http://dx.doi.org/10.1247/csf.29.139.
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Rojas, Julio C., Jee Bang, Iryna V. Lobach, Richard M. Tsai, Gil D. Rabinovici, Bruce L. Miller, and Adam L. Boxer. "CSF neurofilament light chain and phosphorylated tau 181 predict disease progression in PSP." Neurology 90, no. 4 (December 27, 2017): e273-e281. http://dx.doi.org/10.1212/wnl.0000000000004859.
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ObjectiveTo determine the ability of CSF biomarkers to predict disease progression in progressive supranuclear palsy (PSP).MethodsWe compared the ability of baseline CSF β-amyloid1–42, tau, phosphorylated tau 181 (p-tau), and neurofilament light chain (NfL) concentrations, measured by INNO-BIA AlzBio3 or ELISA, to predict 52-week changes in clinical (PSP Rating Scale [PSPRS] and Schwab and England Activities of Daily Living [SEADL]), neuropsychological, and regional brain volumes on MRI using linear mixed effects models controlled for age, sex, and baseline disease severity, and Fisher F density curves to compare effect sizes in 50 patients with PSP. Similar analyses were done using plasma NfL measured by single molecule arrays in 141 patients.ResultsHigher CSF NfL concentration predicted more rapid decline (biomarker × time interaction) over 52 weeks in PSPRS (p = 0.004, false discovery rate–corrected) and SEADL (p = 0.008), whereas lower baseline CSF p-tau predicted faster decline on PSPRS (p = 0.004). Higher CSF tau concentrations predicted faster decline by SEADL (p = 0.004). The CSF NfL/p-tau ratio was superior for predicting change in PSPRS, compared to p-tau (p = 0.003) or NfL (p = 0.001) alone. Higher NfL concentrations in CSF or blood were associated with greater superior cerebellar peduncle atrophy (fixed effect, p ≤ 0.029 and 0.008, respectively).ConclusionsBoth CSF p-tau and NfL correlate with disease severity and rate of disease progression in PSP. The inverse correlation of p-tau with disease severity suggests a potentially different mechanism of tau pathology in PSP as compared to Alzheimer disease.
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Cui, H., X. H. Chang, B. Liu, J. Feng, Y. Li, X. Ye, S. J. Cao, et al. "The anti-tumor immune responses induced by a fusion protein of ovarian carcinoma anti-idiotypic antibody 6B11ScFv and murine GM-CSF in BALB/c mice." International Journal of Gynecologic Cancer 14, no. 2 (2004): 234–41. http://dx.doi.org/10.1136/ijgc-00009577-200403000-00009.
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Ovarian carcinoma anti-idiotypic antibody 6B11 was murine derived; we previously have cloned 6B11 single-chain Fv antibody (6B11ScFv) and constructed the 6B11ScFv/human granulocyte-macrophage colony stimulating factor (GM-CSF) fusion protein (designated as 6B11GM) to enhance the immunogenecity of the single-chain Ab2. Because of the difference in species specificity between human GM-CSF and murine GM-CSF, there is no immune competent animal model on which the effect and metabolism of 6B11GM as a vaccine could be observed. In this study, 6B11mGM fusion gene was constructed by the fusing murine GM-CSF cDNA gene with 6B11ScFv. The fusion gene was cloned and expressed. The product of this gene is a fusion protein. It could specifically interact with the primary anti-ovarian carcinoma monoclonal antibody (COC166-9) and rat anti-mouse GM-CSF monoclonal antibody, respectively, and stimulate the growth of NFS-60 cells (a murine GM-CSF-dependent cell line). The specific anti-tumor immune response could be induced in BALB/c mice after immunized with anti-idiotypic fusion protein instead of ovarian carcinoma antigen without carrier proteins and adjuvant. Ab3 could be detected in the sera of immunized mice with 6B11mGM by enzyme-linked immunoadsorbent assay test. Moreover, the fusion protein stimulated proliferation of CD4+ T cell from the spleen of BALB/c mice and proliferation of CD8+ T cell to a lesser degree. Therefore, 6B11mGM probably induces both humoral and cellular immunity against ovarian carcinoma in vivo.
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Piehl, Fredrik, Ingrid Kockum, Mohsen Khademi, Kaj Blennow, Jan Lycke, Henrik Zetterberg, and Tomas Olsson. "Plasma neurofilament light chain levels in patients with MS switching from injectable therapies to fingolimod." Multiple Sclerosis Journal 24, no. 8 (June 19, 2017): 1046–54. http://dx.doi.org/10.1177/1352458517715132.
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Background: Neurofilament light chain (NFL) is a cerebrospinal fluid (CSF) marker of neuroaxonal damage in multiple sclerosis (MS). Objective: To determine the correlation of NFL in CSF and serum/plasma, and in plasma after switching from injectable MS therapies to fingolimod. Methods: A first cohort consisted of MS patients ( n = 39) and neurological disease controls ( n = 27) where CSF and plasma/serum had been collected for diagnostic purposes. A second cohort ( n = 243) consisted of patients from a post-marketing study of fingolimod. NFL was determined with Single Molecule Array (Simoa™) technology (detection threshold 1.95 pg/mL). Results: Mean NFL pg/mL (standard deviation ( SD)) was 341 (267) and 1475 (2358) in CSF and 8.2 (3.58) and 17.0 (16.94) in serum from controls and MS, respectively. CSF/serum and plasma/serum levels were highly correlated ( n = 66, rho = 0.672, p < 0.0001 and n = 16, rho = 0.684, p = 0.009, respectively). In patients starting fingolimod ( n = 243), mean NFL pg/mL ( SD) in plasma was reduced between baseline (20.4 (10.7)) and at 12 months (13.5 (7.3), p < 3 × 10−6), and levels remained stable at 24 months (13.2 (6.2)). Conclusion: NFL in serum and CSF are highly correlated and plasma NFL levels decrease after switching to highly effective MS therapy. Blood NFL measurement can be considered as a biomarker for MS therapy response.
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Ang, S. L., J. G. Seidman, G. M. Peterman, A. D. Duby, D. Benjamin, S. J. Lee, and D. A. Hafler. "Functional gamma chain-associated T cell receptors on cerebrospinal fluid-derived natural killer-like T cell clones." Journal of Experimental Medicine 165, no. 5 (May 1, 1987): 1453–58. http://dx.doi.org/10.1084/jem.165.5.1453.
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We have derived 33 independent T cell clones from the cerebrospinal fluid (CSF) of a patient with subacute sclerosing panencephalitis using a single T cell cloning method. 6% (2 of 33) of these clones express the T cell receptor gamma (TCR-gamma) protein and are called CSF TCR-gamma clones. Phenotypic analyses of the CSF TCR-gamma clones indicate that they are WT-31-, CD3+, CD4-, and CD8-. The TCR-gamma protein exists on the cell surface as part of an 85-kD disulphide-linked dimer noncovalently associated with the CD3 polypeptides. The CSF TCR-gamma clones have NK-like activity that can be inhibited by anti-CD3 mAbs. Both CSF TCR-gamma clones proliferated in response to anti-CD3 mAbs coupled to Sepharose beads and/or IL-2. Furthermore, stimulation of one of these clones with anti-CD3 mAbs results in a rapid rise in intracellular calcium. These data suggest that T cells bearing the CD3-TCR-gamma protein complex are functional and play a role in the human immune response.
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McClure, Barbara J., Timothy R. Hercus, Bronwyn A. Cambareri, Joanna M. Woodcock, Christopher J. Bagley, Geoff J. Howlett, and Angel F. Lopez. "Molecular assembly of the ternary granulocyte-macrophage colony-stimulating factor receptor complex." Blood 101, no. 4 (February 15, 2003): 1308–15. http://dx.doi.org/10.1182/blood-2002-06-1903.
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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the production and functional activity of granulocytes and macrophages, properties that have encouraged its clinical use in bone marrow transplantation and in certain infectious diseases. Despite the importance of GM-CSF in regulating myeloid cell numbers and function, little is known about the exact composition and mechanism of assembly of the GM-CSF receptor complex. We have now produced soluble forms of the GM-CSF receptor α chain (sGMRα) and β chain (sβc) and utilized GM-CSF, the GM-CSF antagonist E21R (Glu21Arg), and the βc-blocking monoclonal antibody BION-1 to define the molecular assembly of the GM-CSF receptor complex. We found that GM-CSF and E21R were able to form low-affinity, binary complexes with sGMRα, each having a stoichiometry of 1:1. Importantly, GM-CSF but not E21R formed a ternary complex with sGMRα and sβc, and this complex could be disrupted by E21R. Significantly, size-exclusion chromatography, analytical ultracentrifugation, and radioactive tracer experiments indicated that the ternary complex is composed of one sβc dimer with a single molecule each of sGMRα and of GM-CSF. In addition, a hitherto unrecognized direct interaction between βc and GM-CSF was detected that was absent with E21R and was abolished by BION-1. These results demonstrate a novel mechanism of cytokine receptor assembly likely to apply also to interleukin-3 (IL-3) and IL-5 and have implications for our molecular understanding and potential manipulation of GM-CSF activation of its receptor.
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Yan, Xiyun, and Bo Tian. "Human antibodies from a single-chain Fv fusion phage library." Chinese Science Bulletin 42, no. 15 (August 1997): 1300–1303. http://dx.doi.org/10.1007/bf02882765.
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Paul, CC, M. Tolbert, S. Mahrer, A. Singh, MJ Grace, and MA Baumann. "Cooperative effects of interleukin-3 (IL-3), IL-5, and granulocyte- macrophage colony-stimulating factor: a new myeloid cell line inducible to eosinophils." Blood 81, no. 5 (March 1, 1993): 1193–99. http://dx.doi.org/10.1182/blood.v81.5.1193.1193.
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Abstract The cytokines interleukin-3 (IL-3); IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to contribute to the proliferation and differentiation of eosinophil progenitors. Recently, it was determined that the cellular receptors for these three cytokines share a common beta-chain while having unique alpha-chains. Thus, there is considerable interest in how these cytokines and their receptors interact in promoting production of eosinophils. We have established a cell line (AML14) from a patient with acute myelogenous leukemia that will consistently exhibit eosinophilic differentiation in suspension in response to IL-3, IL-5, and GM-CSF. Proliferation with only modest differentiative effects was observed in response to a single cytokine. Combinations of two cytokines gave variable results, with GM-CSF + IL-3 and IL-3 + IL-5 causing more proliferation than a single cytokine but little more differentiation. The combination of GM-CSF + IL-5 caused marked enhancement of eosinophilic differentiation with only modest augmentation of proliferation. The combination of all three cytokines was most effective in stimulating both proliferation and eosinophilic differentiation (up to 70% of cells) of AML14 cells. Specific binding of GM-CSF and IL-5 to AML14 cells can be conveniently studied by flow cytometric methods, and cross-competition of these two cytokines for their respective receptors was demonstrated. IL-3 was shown to partially compete for IL-5 binding on AML14 cells. Although specific IL- 3 binding could not be demonstrated by flow cytometry, mRNA for the alpha-chains of the IL-3, IL-5, and GM-CSF receptors and the beta-chain common to all three receptors was detected in AML14 cells. The AML14 cell line may be a useful model for the study of cooperative interactions of IL-3, IL-5, GM-CSF, and their respective receptors in the promotion of eosinophil progenitor growth and differentiation.
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Paul, CC, M. Tolbert, S. Mahrer, A. Singh, MJ Grace, and MA Baumann. "Cooperative effects of interleukin-3 (IL-3), IL-5, and granulocyte- macrophage colony-stimulating factor: a new myeloid cell line inducible to eosinophils." Blood 81, no. 5 (March 1, 1993): 1193–99. http://dx.doi.org/10.1182/blood.v81.5.1193.bloodjournal8151193.
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The cytokines interleukin-3 (IL-3); IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are known to contribute to the proliferation and differentiation of eosinophil progenitors. Recently, it was determined that the cellular receptors for these three cytokines share a common beta-chain while having unique alpha-chains. Thus, there is considerable interest in how these cytokines and their receptors interact in promoting production of eosinophils. We have established a cell line (AML14) from a patient with acute myelogenous leukemia that will consistently exhibit eosinophilic differentiation in suspension in response to IL-3, IL-5, and GM-CSF. Proliferation with only modest differentiative effects was observed in response to a single cytokine. Combinations of two cytokines gave variable results, with GM-CSF + IL-3 and IL-3 + IL-5 causing more proliferation than a single cytokine but little more differentiation. The combination of GM-CSF + IL-5 caused marked enhancement of eosinophilic differentiation with only modest augmentation of proliferation. The combination of all three cytokines was most effective in stimulating both proliferation and eosinophilic differentiation (up to 70% of cells) of AML14 cells. Specific binding of GM-CSF and IL-5 to AML14 cells can be conveniently studied by flow cytometric methods, and cross-competition of these two cytokines for their respective receptors was demonstrated. IL-3 was shown to partially compete for IL-5 binding on AML14 cells. Although specific IL- 3 binding could not be demonstrated by flow cytometry, mRNA for the alpha-chains of the IL-3, IL-5, and GM-CSF receptors and the beta-chain common to all three receptors was detected in AML14 cells. The AML14 cell line may be a useful model for the study of cooperative interactions of IL-3, IL-5, GM-CSF, and their respective receptors in the promotion of eosinophil progenitor growth and differentiation.
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Alagaratnam, Jasmini, Sophia von Widekind, Davide De Francesco, Jonathan Underwood, Paul Edison, Alan Winston, Henrik Zetterberg, and Sarah Fidler. "Correlation between CSF and blood neurofilament light chain protein: a systematic review and meta-analysis." BMJ Neurology Open 3, no. 1 (June 2021): e000143. http://dx.doi.org/10.1136/bmjno-2021-000143.
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ObjectiveTo assess the overall pooled correlation coefficient estimate between cerebrospinal fluid (CSF) and blood neurofilament light (NfL) protein.MethodsWe searched Medline, Embase and Web of Science for published articles, from their inception to 9 July 2019, according to Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Studies reporting the correlation between CSF and blood NfL in humans were included. We conducted a random-effects meta-analysis to calculate the overall pooled correlation coefficient estimate, accounting for correlation technique and assay used. Heterogeneity was assessed using the I2 statistic test. In sensitivity analyses, we calculated the pooled correlation coefficient estimate according to blood NfL assay: single-molecule array digital immunoassay (Simoa), electrochemiluminescence (ECL) assay or ELISA.ResultsData were extracted from 36 articles, including 3961 paired CSF and blood NfL samples. Overall, 26/36 studies measured blood NfL using Simoa, 8/36 ECL, 1/36 ELISA and 1 study reported all three assay results. The overall meta-analysis demonstrated that the pooled correlation coefficient estimate for CSF and blood NfL was r=0.72. Heterogeneity was significant: I2=83%, p<0.01. In sensitivity analyses, the pooled correlation coefficient was similar for studies measuring blood NfL using Simoa and ECL (r=0.69 and r=0.68, respectively) but weaker for ELISA (r=0.35).ConclusionModerate correlations are demonstrated between CSF and blood NfL, especially when blood NfL was measured using Simoa and ECL. Given its high analytical sensitivity, Simoa is the preferred assay for measuring NfL, especially at low or physiological concentrations, and this meta-analysis supports its use as the current most advanced surrogate measure of CSF NfL.PROSPERO registration numberCRD42019140469
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Sano, Tomoya, Yasushi Masuda, Hironobu Yasuno, Tadahiro Shinozawa, Takeshi Watanabe, and Masaaki Kakehi. "Blood Neurofilament Light Chain as a Potential Biomarker for Central and Peripheral Nervous Toxicity in Rats." Toxicological Sciences 185, no. 1 (October 22, 2021): 10–18. http://dx.doi.org/10.1093/toxsci/kfab122.
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Abstract Neurotoxicity is a principal concern in nonclinical drug development. However, standardized and universally accepted fluid biomarkers for evaluating neurotoxicity are lacking. Increasing clinical evidence supports the potential use of neurofilament light (NfL) chain as a biomarker of several neurodegenerative diseases; therefore, we investigated changes in the cerebrospinal fluid (CSF) and serum levels of NfL in Sprague Dawley rats treated with central nervous system (CNS) toxicants (trimethyltin [TMT, 10 mg/kg po, single dose], kainic acid [KA, 12 mg/kg sc, single dose], MK-801 [1 mg/kg sc, single dose]), and a peripheral nervous system (PNS) toxicant (pyridoxine, 1200 mg/kg/day for 3 days). Animals were euthanized 1 (day 2), 3 (day 4), or 7 days after administration (day 8). Increased serum NfL was observed in TMT- and KA-treated animals, which indicated neuronal cell death in the brain on days 2, 4, and/or 8. MK-801-treated animals exhibited no changes in the serum and CSF levels of NfL and no histopathological changes in the brain at any time point. Pyridoxine-induced chromatolysis of the dorsal root ganglion on day 2 and degeneration of peripheral nerve fiber on day 4; additionally, serum NfL was increased. A strong correlation was observed between the serum and CSF levels of NfL and brain lesions caused by TMT and KA, indicating that NfL could be a useful biomarker for detecting CNS toxicity. Additionally, PNS changes were correlated with serum NfL levels. Therefore, serum NfL could serve as a useful peripheral biomarker for detecting both CNS and PNS toxicity in rats.
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Okuda, Keiko, Lorie Smith, James D. Griffin, and Rosemary Foster. "Signaling Functions of the Tyrosine Residues in the βc Chain of the Granulocyte-Macrophage Colony-Stimulating Factor Receptor." Blood 90, no. 12 (December 15, 1997): 4759–66. http://dx.doi.org/10.1182/blood.v90.12.4759.
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Abstract The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR βc-chain (GMRβ), but not the GMR α-chain (GMRα). To examine the role of GMRβ tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRβ was mutated to phenylalanine (GMRβ-F8), and this mutant receptor was expressed with wild-type GMRα in the interleukin-3–dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRβ-F8 , including JAK2 and STAT5. However, GM-CSF–induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF–dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF–inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRβ-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRβ, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRβ tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.
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Okuda, Keiko, Lorie Smith, James D. Griffin, and Rosemary Foster. "Signaling Functions of the Tyrosine Residues in the βc Chain of the Granulocyte-Macrophage Colony-Stimulating Factor Receptor." Blood 90, no. 12 (December 15, 1997): 4759–66. http://dx.doi.org/10.1182/blood.v90.12.4759.4759_4759_4766.
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The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. Binding of GM-CSF activates at least one receptor-associated tyrosine kinase, JAK2, and rapidly induces tyrosine phosphorylation of the GMR βc-chain (GMRβ), but not the GMR α-chain (GMRα). To examine the role of GMRβ tyrosine phosphorylaiton, each of the 8 tyrosine residues in the cytoplasmic domain of the human GMRβ was mutated to phenylalanine (GMRβ-F8), and this mutant receptor was expressed with wild-type GMRα in the interleukin-3–dependent murine hematopoietic cell line, Ba/F3. GM-CSF induced tyrosine phosphorylation of multiple cellular proteins in cells expressing GMRβ-F8 , including JAK2 and STAT5. However, GM-CSF–induced tyrosine phosphorylation of both SHP2 and SHC was reduced or absent compared with wild-type. Next, a series of 8 receptors were generated, each containing only a single, restored, tyrosine residue. Tyrosine 577 was found to be sufficient to regenerate GM-CSF–dependent phosphorylation of SHC, and any of Y577, Y612, or Y695 was sufficient to regenerate GM-CSF–inducible phosphorylation of SHP2. Despite the signaling defect to SHC and SHP2, Ba/F3 cells expressing GMRβ-F8 were still able to proliferate in response to 10 ng/mL of human GM-CSF, although mitogenesis was impaired compared with wild-type GMRβ, and this effect was even more prominent at lower concentrations of GM-CSF (1 ng/mL). Overall, these results indicate that GMRβ tyrosine residues are not necessary for activation of the JAK/STAT pathway or for proliferation, viability, or adhesion signaling in Ba/F3 cells, although tyrosine residues significantly affect the magnitude of the response. However, specific tyrosine residues are needed for activation of SHC and SHP2.
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Engel, Sinah, Michaela Friedrich, Muthuraman Muthuraman, Falk Steffen, Alicia Poplawski, Sergiu Groppa, Stefan Bittner, Frauke Zipp, and Felix Luessi. "Intrathecal B-cell accumulation and axonal damage distinguish MRI-based benign from aggressive onset in MS." Neurology - Neuroimmunology Neuroinflammation 6, no. 5 (July 19, 2019): e595. http://dx.doi.org/10.1212/nxi.0000000000000595.
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ObjectiveWe explored the incremental value of adding multiple disease activity biomarkers in CSF and serum for distinguishing MRI-based benign from aggressive MS in early disease course.MethodsNinety-three patients diagnosed with clinically isolated syndrome (CIS) or early MS were divided into 3 nonoverlapping severity groups defined by objective MRI criteria. Ninety-seven patients with noninflammatory neurologic disorders and 48 patients with other inflammatory neurologic diseases served as controls. Leukocyte subsets in the CSF were analyzed by flow cytometry. CSF neurofilament light chain (NfL) and chitinase-3-like protein 1 (CHI3L1) levels were measured by ELISA. Serum NfL levels were examined using single molecule array technology.ResultsCSF CD20+/CD14+ ratios and NfL levels in CSF and serum were significantly different between high and low MRI severity groups, whereas no difference was found for CSF CHI3L1 levels. NfL levels in CSF and serum highly correlated. Receiver operating characteristic analysis demonstrated that the cumulative sums combining CSF CD20+/CD14+ ratios and NfL levels in serum or CSF considerably improved diagnostic accuracy. A composite score built from these 2 cumulative sums best distinguished MRI severity. These findings were validated by support vector machine analysis, which confirmed that the accuracy of the cumulative sums and composite score outperforms single biomarkers.ConclusionPatients with extreme manifestations of CIS or early MS defined by strict MRI parameters can be best distinguished by combining markers of intrathecal B-cell accumulation and axonal damage. This could stratify individual treatment decisions toward a more personalized immunotherapy.
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Tao, Jie, Benqiang Li, Jinghua Cheng, Ying Shi, Xiaohui Shen, and Huili Liu. "Development and neutralization analysis of recombinant BVDVs expressing a CSF single chain antibody in vitro." Biologicals 70 (April 2021): 38–43. http://dx.doi.org/10.1016/j.biologicals.2021.01.004.
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Shanmugam, Arulkumaran, Robert Suriano, Neha Goswami, Devyani Chaudhuri, Badithe T. Ashok, Shilpi Rajoria, Andrea L. George, Abraham Mittelman, and Raj K. Tiwari. "Identification of peptide mimotopes of gp96 using single-chain antibody library." Cell Stress and Chaperones 16, no. 2 (October 16, 2010): 225–34. http://dx.doi.org/10.1007/s12192-010-0234-6.
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Matsuo, Osamu, Hideharu Fukao, Seiichi Izaki, Chikami Matsuo, and Shigeru Ueshima. "Production and characterization of single-chain tissue-type plasminogen activator produced by an established cell line from human uterine muscle." Cell Structure and Function 14, no. 1 (1989): 45–60. http://dx.doi.org/10.1247/csf.14.45.
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Rich, Kelly A., Ashley Fox, Mehmet Yalvac, Sarah Heintzman, Marco Tellez, Amy Bartlett, Steven Severyn, et al. "Neurofilament Levels in CSF and Serum in an Adult SMA Cohort Treated with Nusinersen." Journal of Neuromuscular Diseases 9, no. 1 (January 4, 2022): 111–19. http://dx.doi.org/10.3233/jnd-210735.
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Objective: To retrospectively evaluate the utility of serum and cerebrospinal fluid (CSF) levels of neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) as biomarkers for spinal muscular atrophy (SMA) progression and response to nusinersen treatment. Methods: NfL and pNfH levels were quantified using single molecular array (SIMOA) in CSF of 33 adult SMA patients (SMN copy number 3–5) before and in response to nusinersen treatment. In 11 of the patients, blood serum samples were also collected. CSF NfL and pNfH from patients were compared to CSF Nfs from age-matched controls without neurological disease (n = 6). For patients, pearson correlation coefficients (r) were calculated to investigate associations between Nf levels and other functional outcome measures. Results: Nf levels were similar between SMA and control adults and showed no change in response to nusinersen treatment in CSF or serum. Cross-sectional analyses showed an increase in CSF NfL and pNfH with age in patients (NfL p = 0.0013; pNfH p = 0.0035) and an increase in CSF NfL in controls (p = 0.002). In non-ambulatory patients, baseline serum pNfH showed a negative correlation with multiple strength and functional assessment metrics including Revised Upper Limb Module (r = –0.822, p = 0.04), upper extremity strength (r = –0.828, p = 0.042), lower extremity strength (r = –0.860, p = 0.028), and total strength (r = –0.870, p = 0.024). Conclusions: Nf levels did not change in response to nusinersen in adults with SMA and were not different from controls. In patients and controls, we detected an age-related increase in baseline CSF NfL and pNfH levels. Though some associations were identified, our results suggest Nf levels are not preditive or prognostic biomarkers in this population.
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van Dijk, Thamar B., Belinda Baltus, Eric Caldenhoven, Hiroshi Handa, Jan A. M. Raaijmakers, Jan-Willem J. Lammers, Leo Koenderman, and Rolf P. de Groot. "Cloning and Characterization of the Human Interleukin-3 (IL-3)/IL-5/ Granulocyte-Macrophage Colony-Stimulating Factor Receptor βc Gene: Regulation by Ets Family Members." Blood 92, no. 10 (November 15, 1998): 3636–46. http://dx.doi.org/10.1182/blood.v92.10.3636.
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Abstract High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common β chain (βc). Whereas the mouse has two homologous β subunits (βc and βIL-3), in humans, only a single β chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor β subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine βc/βIL-3 genes. Surprisingly, we also found the remnants of a second βc chain gene directly downstream of βc. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from −103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5′ element specifically binds PU.1, whereas the 3′ element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances βc promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.
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van Dijk, Thamar B., Belinda Baltus, Eric Caldenhoven, Hiroshi Handa, Jan A. M. Raaijmakers, Jan-Willem J. Lammers, Leo Koenderman, and Rolf P. de Groot. "Cloning and Characterization of the Human Interleukin-3 (IL-3)/IL-5/ Granulocyte-Macrophage Colony-Stimulating Factor Receptor βc Gene: Regulation by Ets Family Members." Blood 92, no. 10 (November 15, 1998): 3636–46. http://dx.doi.org/10.1182/blood.v92.10.3636.422k45_3636_3646.
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High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common β chain (βc). Whereas the mouse has two homologous β subunits (βc and βIL-3), in humans, only a single β chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor β subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine βc/βIL-3 genes. Surprisingly, we also found the remnants of a second βc chain gene directly downstream of βc. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from −103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5′ element specifically binds PU.1, whereas the 3′ element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances βc promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.
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Rival, Manon, Eric Thouvenot, Lucile Du Trieu de Terdonck, Sabine Laurent-Chabalier, Christophe Demattei, Ugur Uygunoglu, Giovanni Castelnovo, et al. "Neurofilament Light Chain Levels Are Predictive of Clinical Conversion in Radiologically Isolated Syndrome." Neurology - Neuroimmunology Neuroinflammation 10, no. 1 (October 24, 2022): e200044. http://dx.doi.org/10.1212/nxi.0000000000200044.
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Background and ObjectivesTo evaluate the predictive value of serum neurofilament light chain (sNfL) and CSF NfL (cNfL) in patients with radiologically isolated syndrome (RIS) for evidence of disease activity (EDA) and clinical conversion (CC).MethodssNfL and cNfL were measured at RIS diagnosis by single-molecule array (Simoa). The risk of EDA and CC according to sNfL and cNfL was evaluated using the Kaplan-Meier analysis and multivariate Cox regression models including age, spinal cord (SC) or infratentorial lesions, oligoclonal bands, CSF chitinase 3–like protein 1, and CSF white blood cells.ResultsSixty-one patients with RIS were included. At diagnosis, sNfL and cNfL were correlated (Spearman r = 0.78,p< 0.001). During follow-up, 47 patients with RIS showed EDA and 36 patients showed CC (median time 12.6 months, 1–86). When compared with low levels, medium and high cNfL (>260 pg/mL) and sNfL (>5.0 pg/mL) levels were predictive of EDA (log rank,p< 0.01 andp= 0.02, respectively). Medium-high cNfL levels were predictive of CC (log rank,p< 0.01). In Cox regression models, cNfL and sNfL were independent factors of EDA, while SC lesions, cNfL, and sNfL were independent factors of CC.DiscussioncNfL >260 pg/mL and sNfL >5.0 pg/mL at diagnosis are independent predictive factors of EDA and CC in RIS. Although cNfL predicts disease activity better, sNfL is more accessible than cNfL and can be considered when a lumbar puncture is not performed.Classification of EvidenceThis study provides Class II evidence that in people with radiologic isolated syndrome (RIS), initial serum and CSF NfL levels are associated with subsequent evidence of disease activity or clinical conversion.
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Kaewchim, Kanasap, Kittirat Glab-ampai, Kodchakorn Mahasongkram, Monrat Chulanetra, Watee Seesuay, Wanpen Chaicumpa, and Nitat Sookrung. "Engineered Fully Human Single-Chain Monoclonal Antibodies to PIM2 Kinase." Molecules 26, no. 21 (October 25, 2021): 6436. http://dx.doi.org/10.3390/molecules26216436.
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Proviral integration site of Moloney virus-2 (PIM2) is overexpressed in multiple human cancer cells and high level is related to poor prognosis; thus, PIM2 kinase is a rational target of anti-cancer therapeutics. Several chemical inhibitors targeting PIMs/PIM2 or their downstream signaling molecules have been developed for treatment of different cancers. However, their off-target toxicity is common in clinical trials, so they could not be advanced to official approval for clinical application. Here, we produced human single-chain antibody fragments (HuscFvs) to PIM2 by using phage display library, which was constructed in a way that a portion of phages in the library carried HuscFvs against human own proteins on their surface with the respective antibody genes in the phage genome. Bacterial derived-recombinant PIM2 (rPIM2) was used as an antigenic bait to fish out the rPIM2-bound phages from the library. Three E. coli clones transfected with the HuscFv genes derived from the rPIM2-bound phages expressed HuscFvs that bound also to native PIM2 from cancer cells. The HuscFvs presumptively interact with the PIM2 at the ATP binding pocket and kinase active loop. They were as effective as small chemical drug inhibitor (AZD1208, which is an ATP competitive inhibitor of all PIM isoforms for ex vivo use) in inhibiting PIM kinase activity. The HuscFvs should be engineered into a cell-penetrating format and tested further towards clinical application as a novel and safe pan-anti-cancer therapeutics.
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Ni, Ming, Bing Yu, Yu Huang, Zhenjie Tang, Ping Lei, Xin Shen, Wei Xin, Huifen Zhu, and Guanxin Shen. "Homology modelling and bivalent single-chain Fv construction of anti-HepG2 single-chain immunoglobulin Fv fragments from a phage display library." Journal of Biosciences 33, no. 5 (December 2008): 691–97. http://dx.doi.org/10.1007/s12038-008-0089-5.
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Ramirez, Arturo B., Christian M. Loch, Yuzheng Zhang, Yan Liu, Xiaohong Wang, Elizabeth A. Wayner, Jonathon E. Sargent, et al. "Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery." Molecular & Cellular Proteomics 9, no. 7 (May 13, 2010): 1449–60. http://dx.doi.org/10.1074/mcp.m900496-mcp200.
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Gao, Changshou, Oliver Brümmer, Shenlan Mao, and Kim D. Janda. "Selection of Human Metalloantibodies from a Combinatorial Phage Single-Chain Antibody Library." Journal of the American Chemical Society 121, no. 27 (July 1999): 6517–18. http://dx.doi.org/10.1021/ja990966e.
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Yakushiji, Hiromasa, Kazuko Kobayashi, Fumiaki Takenaka, Yoshiro Kishi, Midori Shinohara, Masaru Akehi, Takanori Sasaki, Eiji Ohno, and Eiji Matsuura. "Novel single‐chain variant of antibody against mesothelin established by phage library." Cancer Science 110, no. 9 (August 28, 2019): 2722–33. http://dx.doi.org/10.1111/cas.14150.
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Nishihori, Taiga, Jun Zhou, Kenneth H. Shain, Rachid Baz, Melissa Alsina, Daniel Sullivan, Ryan Hillgruber, Elizabeth M. Sagatys, Lynn C. Moscinski, and Ling Zhang. "Cerebrospinal Fluid (CSF) Involvement By Plasma Cell Dyscrasias – Single Center Experience." Blood 124, no. 21 (December 6, 2014): 5686. http://dx.doi.org/10.1182/blood.v124.21.5686.5686.
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Abstract Introduction: Central nervous system (CNS) involvement by plasma cell dyscrasias (PCD) is uncommon but poses significant clinical challenges and has a dismal prognosis. Lumbar puncture (LP) is typically performed only for patients with neurologic signs or symptoms and data on patients with CNS involvement are rather scarce. Here, we report a retrospective single institution review of clinicopathological features and treatment outcomes in the setting of cerebrospinal fluid (CSF) involvement by PCD. Methods: We identified consecutive patients with plasma cell disorders who had abnormal cytology or flow cytometry results in the CSF in the Department of Hematopathology database at Moffitt Cancer Center from 1997 to 2014. Cytology slides [Wright-Giemsa (WG) and Papanicolaou (Pap) stained preparations] and the corresponding flow cytometry were reviewed to confirm the diagnosis. Four-color flow cytometry was performed using antibodies against CD38, CD138, CD56, CD117, CD19, and cytoplasmic kappa and lambda light chains, withadditional markers added when necessary. Clinical variables were abstracted from the patient medical records. Overall survival was estimated from the time CSF involvement was identified using the Kaplan-Meier method. Results: Sixty-seven Pap-stained cytology smeas/cytospins and WG stained cytospins from 65 patients who underwent LP for clinical suspicion of CSF involvement were reviewed. Flow cytometry was preformed on 48 cases positive for atypical plasma cells by cytology. Sixteen of 67 (23.9%) were suspicious or diagnostic for PCD (median age of 58 years (range 44 – 75), 56% were male). However, only 4 of 16 cases (25%) were diagnosed as PCD by cytology without additional flow cytometry study. Median tumor load of PCD by flow cytometry was 81% (range 4 - 99%). PCD included 14 patients (88%) with multiple myeloma [MM; 1 patient progressed to secondary plasma cell leukemia (PCL)], 1 with primary PCL, and 1 with Waldenström macroglobulinemia (Neel-Bing syndrome). Of the 14 MM patients, 57% had high-risk disease by cytogenetics/FISH, and immunophenotypes were IgA (50%), IgG (29%), and light chain (21%). All MM patients had Durie-Salmon stage 3 disease. Median number of prior therapies was 2 (1-4), and 44% received stem cell transplant prior to CSF involvement. Median time from diagnosis to CSF involvement was 23 (range, 6 – 78) months. Presenting symptoms included diplopia/vision loss (31%), headache (25%), and leg weakness (cauda equina/cord compression) (19%). Two patients presented with gross orbital involvement and new/enlarging scalp lesions. On radiologic imaging, 5 (31%) had leptomeningeal, 4 (25%) had epi- or extra-dural, and 2 (13%) had dural enhancement/lesions. None had CSF involvement at the time of initial PCD diagnosis. Treatment included intrathecal chemotherapy (methotrexate, cytarabine or triple regimen; 86%), radiation therapy (including whole brain, craniospinal, radiosurgery or involved field; 63%) and systemic chemotherapy (65%). One patient did not receive treatment due to poor performance status. For 5 patients who had repeat CSF analyses, only one had no evidence of disease on cytology but flow cytometry remained positive. Six-month overall survival rate was 20.2% (95% CI 4.4 – 43.5). At the time of data review, only 2 patients were alive. Conclusions: CSF involvement by PCD carries extremely limited prognosis and represents advanced stage of the disease. Patients may be treated with systemic therapy as well as CNS-directed therapy, though the outcomes are dismal. Careful assessment of patients’ neurologic symptoms and low threshold for performing LP is required for early detection of CSF involvement. Application of flow cytometry appears to be a useful tool in the diagnosis of CSF involvement by PCD; improving sensitivity and specificity over cytology alone, particularly when the tumor load is low or cytologically equivocal for atypical plasma cells. Further research is needed to improve the outcomes of these patients. Disclosures No relevant conflicts of interest to declare.
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Kremer, E., E. Baker, RJ D'Andrea, R. Slim, H. Phillips, PA Moretti, AF Lopez, C. Petit, MA Vadas, and GR Sutherland. "A cytokine receptor gene cluster in the X-Y pseudoautosomal region?" Blood 82, no. 1 (July 1, 1993): 22–28. http://dx.doi.org/10.1182/blood.v82.1.22.bloodjournal82122.
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The receptors for interleukin-3 (IL-3), IL-5, and granulocyte- macrophage colony-stimulating factor (GM-CSF) are heterodimers comprised of ligand specific alpha chains and a common beta chain. The genes encoding the IL-5 receptor alpha chain and the common beta chain reside on chromosome 3 and 22 respectively, while the GM-CSF receptor alpha chain gene (CSF2RA) has been mapped to the pseudoautosomal region (PAR) of the sex chromosomes, which is a 2.6-Mb stretch of homologous sequence at the tips of the short arms within which a single obligatory recombination occurs during male meiosis. We have mapped the gene encoding the IL-3 receptor alpha chain (IL3RA) to the sex chromosomes by polymerase chain reaction (PCR) analysis of human-mouse or human- chinese hamster cell hybrids, and to Yp13.3 and Xp22.3 using fluorescence in situ hybridization. To explore the possibility that IL3RA is located within the pseudoautosomal region we screened the Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees for an informative-restriction fragment-length polymorphism (RFLP) that showed male meiotic recombination. Two informative CEPH pedigrees were identified that displayed this phenomenon, confirming the psuedoautosomal location of IL3RA. Using long-range restriction mapping we have found that IL3RA maps to the same 190-kb restriction fragment as CSF2RA, suggesting that a cytokine receptor gene cluster may reside in the PAR.
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Ramesh, Akshaya, Ryan D. Schubert, Ariele L. Greenfield, Ravi Dandekar, Rita Loudermilk, Joseph J. Sabatino, Matthew T. Koelzer, et al. "A pathogenic and clonally expanded B cell transcriptome in active multiple sclerosis." Proceedings of the National Academy of Sciences 117, no. 37 (August 28, 2020): 22932–43. http://dx.doi.org/10.1073/pnas.2008523117.
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Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; n = 12), other neurologic diseases (ONDs; n = 1), and healthy controls (HCs; n = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS (n = 4), clinically isolated syndrome (n = 2), and OND (n = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; n = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-κB) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-β1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood–brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein–Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype.
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Guba, SC, CA Sartor, R. Hutchinson, LA Boxer, and SG Emerson. "Granulocyte colony-stimulating factor (G-CSF) production and G-CSF receptor structure in patients with congenital neutropenia." Blood 83, no. 6 (March 15, 1994): 1486–92. http://dx.doi.org/10.1182/blood.v83.6.1486.1486.
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Abstract Congenital neutropenia (Kostmann's syndrome [KS]) is an autosomal recessive syndrome that is characterized by profound neutropenia, resulting in major clinical infections and death. Since the neutropenia and symptoms in KS improve in response to exogenous administration of granulocyte colony-stimulating factor (G-CSF), we studied bone marrow cytokine (G-CSF, granulocyte-macrophage CSF [GM-CSF], and interleukin- 6) production under both basal and stimulated conditions. No differences in G-CSF, GM-CSF, or IL-6 gene expression were found in bone marrow stromal cells between normal controls and KS patients, and all three cytokines were detected by enzyme-linked immunosorbent assay (ELISA) in medium conditioned by bone marrow stromal cells from normal donors and patients with KS. Each KS patient tested had detectable, functional G-CSF in their own serum before exogenous G-CSF administration. Since G-CSF production appeared normal in KS patients, we then asked whether we could detect structural defects in the signaling portion of G-CSF receptor genes. Polymerase chain reaction (PCR) amplification of the G-CSF receptor transmembrane region alone, and of the transmembrane plus cytosolic portions of the receptor, yielded the size products predicted from the sequences of the normal G- CSF receptor. Single-strand conformational polymorphism (SSCP) analysis of G-CSF receptor PCR products demonstrated no variance in structural conformation between KS patients and normal subjects. These results demonstrate that bone marrow stromal cells in patients with KS secrete normal concentrations of functional G-CSF and suggest that the neutropenia in KS patients is caused by an inability of neutrophilic progenitor and precursor cells to respond to normal, physiologic levels of G-CSF. Such a defect, clinically responsive to pharmacologic doses of G-CSF, might be caused by defects in the post-G-CSF receptor signal transduction pathway.
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Guba, SC, CA Sartor, R. Hutchinson, LA Boxer, and SG Emerson. "Granulocyte colony-stimulating factor (G-CSF) production and G-CSF receptor structure in patients with congenital neutropenia." Blood 83, no. 6 (March 15, 1994): 1486–92. http://dx.doi.org/10.1182/blood.v83.6.1486.bloodjournal8361486.
Full textAbstract:
Congenital neutropenia (Kostmann's syndrome [KS]) is an autosomal recessive syndrome that is characterized by profound neutropenia, resulting in major clinical infections and death. Since the neutropenia and symptoms in KS improve in response to exogenous administration of granulocyte colony-stimulating factor (G-CSF), we studied bone marrow cytokine (G-CSF, granulocyte-macrophage CSF [GM-CSF], and interleukin- 6) production under both basal and stimulated conditions. No differences in G-CSF, GM-CSF, or IL-6 gene expression were found in bone marrow stromal cells between normal controls and KS patients, and all three cytokines were detected by enzyme-linked immunosorbent assay (ELISA) in medium conditioned by bone marrow stromal cells from normal donors and patients with KS. Each KS patient tested had detectable, functional G-CSF in their own serum before exogenous G-CSF administration. Since G-CSF production appeared normal in KS patients, we then asked whether we could detect structural defects in the signaling portion of G-CSF receptor genes. Polymerase chain reaction (PCR) amplification of the G-CSF receptor transmembrane region alone, and of the transmembrane plus cytosolic portions of the receptor, yielded the size products predicted from the sequences of the normal G- CSF receptor. Single-strand conformational polymorphism (SSCP) analysis of G-CSF receptor PCR products demonstrated no variance in structural conformation between KS patients and normal subjects. These results demonstrate that bone marrow stromal cells in patients with KS secrete normal concentrations of functional G-CSF and suggest that the neutropenia in KS patients is caused by an inability of neutrophilic progenitor and precursor cells to respond to normal, physiologic levels of G-CSF. Such a defect, clinically responsive to pharmacologic doses of G-CSF, might be caused by defects in the post-G-CSF receptor signal transduction pathway.
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Messerschmidt, Katrin, Meina Neumann-Schaal, and Katja Heilmann. "Use of antibody gene library for the isolation of specific single chain antibodies by ampicillinantigen conjugates (P3378)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 135.15. http://dx.doi.org/10.4049/jimmunol.190.supp.135.15.
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Abstract Isolation of recombinant antibodies from antibody libraries is commonly performed by different molecular display formats including phage display and ribosome display or different cellsurface display formats. We describe a new method which allows the selection of Escherichia coli cells producing the required single chain antibody by cultivation in presence of ampicillin conjugated to the antigen of interest. The method utilizes the neutralization of the conjugate by the produced single chain antibody which is secreted to the periplasm. Therefore, a new expression system based on the pET26b vector was designed and a library was constructed. The method was successfully established first for the selection of E. coli BL21 Star (DE3) cells expressing a model single chain antibody (antifluorescein) by a simple selection assay on LB-agar plates. Using this selection assay, we could identify a new single-chain antibody binding biotin by growing E. coli BL21 Star (DE3) containing the library in presence of a biotin-ampicillin conjugate. In contrast to methods as molecular or cell surface display our selection system applies the soluble single chain antibody molecule and thereby avoids undesired effects e.g. by the phage particle or the yeast fusion protein. By selecting directly in an expression strain, production and characterization of the selected single chain antibody is possible without any further cloning or transformation steps.
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Durrani, Naveed Ur Rehman, Sourabh Dutta, Niels Rochow, Salhab el Helou, and Enas el Gouhary. "C-reactive protein as a predictor of meningitis in early onset neonatal sepsis: a single unit experience." Journal of Perinatal Medicine 48, no. 8 (October 25, 2020): 845–51. http://dx.doi.org/10.1515/jpm-2019-0420.
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AbstractObjectivesTo determine whether there is a cut off value of serum C-reactive protein (CRP) associated with a higher risk of meningitis in suspected early onset sepsis (EOS) (onset birth to 7 days of life).MethodsA retrospective cohort study on neonates admitted in neonatal intensive care unit at McMaster Children’s Hospital from January 2010 to 2017 and had lumbar puncture (LP) and CRP for workup of EOS. Included subjects had either (a) non-traumatic LP or (b) traumatic LP with cerebral spinal fluid (CSF) polymerase chain reaction or gram stain or culture-positive or had received antimicrobials for 21 days. Excluded were CSF done for metabolic errors, before cytomegalovirus (CMV) treatment; from ventriculo-peritoneal (VP) shunts; missing data and contamination. Neonates were classified into definite and probable meningitis and on the range of CRP. We calculated sensitivity, specificity, and likelihood ratios for CRP values; and area under the receiver operating characteristic (AUROC) curve.ResultsOut of 609 CSF samples, 184 were eligible (28 cases of definite or probable meningitis and 156 controls). Sensitivity, specificity, predictive values, likelihood ratios, and AUROC were too low to be of clinical significance to predict meningitis in EOS.ConclusionsSerum CRP values have poor discriminatory power to distinguish between subjects with and without meningitis, in symptomatic EOS.
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Tweardy, DJ, K. Anderson, LA Cannizzaro, RA Steinman, CM Croce, and K. Huebner. "Molecular cloning of cDNAs for the human granulocyte colony-stimulating factor receptor from HL-60 and mapping of the gene to chromosome region 1p32-34." Blood 79, no. 5 (March 1, 1992): 1148–54. http://dx.doi.org/10.1182/blood.v79.5.1148.1148.
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Abstract Early studies examining the effects of purified or recombinant granulocyte colony-stimulating factor (G-CSF) on human leukemia cell lines demonstrated that some cell lines, such as HL-60, could be induced to differentiate in response to G-CSF. In two recent studies reporting the cloning of the human G-CSF receptor (hGCSFR), four classes of receptor cDNA were identified and, surprisingly, the message for this receptor was reportedly expressed by HL-60 at either very low levels or not at all. Using a mouse G-CSF receptor probe, we cloned and sequenced a cDNA for hGCSFR from an HL-60 cDNA library in plasmid and used it to identify 31 additional clones from an HL-60 cDNA library in phage. Polymerase chain reaction analysis of the 31 phage clones established that 29 were derived from class I hGCSFR mRNA, one was derived from class III mRNA, and one was derived from class IV mRNA. In addition, the hGCSFR gene was chromosomally localized by Southern blot analysis of its segregation pattern in a panel of rodent-human hybrid DNAs using the radiolabeled cDNA probe. The hGCSFR locus was present in hybrids retaining the distal short arm of human chromosome 1 and absent in hybrids that did not retain this region. Chromosomal in situ hybridization refined the localization of the hGCSFR gene to region 1p32-p34. The combination of hybrid DNA analysis and in situ hybridization places the hGCSFR gene telomeric to the CSF1, JUN, and TCL-5 loci.
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Tweardy, DJ, K. Anderson, LA Cannizzaro, RA Steinman, CM Croce, and K. Huebner. "Molecular cloning of cDNAs for the human granulocyte colony-stimulating factor receptor from HL-60 and mapping of the gene to chromosome region 1p32-34." Blood 79, no. 5 (March 1, 1992): 1148–54. http://dx.doi.org/10.1182/blood.v79.5.1148.bloodjournal7951148.
Full textAbstract:
Early studies examining the effects of purified or recombinant granulocyte colony-stimulating factor (G-CSF) on human leukemia cell lines demonstrated that some cell lines, such as HL-60, could be induced to differentiate in response to G-CSF. In two recent studies reporting the cloning of the human G-CSF receptor (hGCSFR), four classes of receptor cDNA were identified and, surprisingly, the message for this receptor was reportedly expressed by HL-60 at either very low levels or not at all. Using a mouse G-CSF receptor probe, we cloned and sequenced a cDNA for hGCSFR from an HL-60 cDNA library in plasmid and used it to identify 31 additional clones from an HL-60 cDNA library in phage. Polymerase chain reaction analysis of the 31 phage clones established that 29 were derived from class I hGCSFR mRNA, one was derived from class III mRNA, and one was derived from class IV mRNA. In addition, the hGCSFR gene was chromosomally localized by Southern blot analysis of its segregation pattern in a panel of rodent-human hybrid DNAs using the radiolabeled cDNA probe. The hGCSFR locus was present in hybrids retaining the distal short arm of human chromosome 1 and absent in hybrids that did not retain this region. Chromosomal in situ hybridization refined the localization of the hGCSFR gene to region 1p32-p34. The combination of hybrid DNA analysis and in situ hybridization places the hGCSFR gene telomeric to the CSF1, JUN, and TCL-5 loci.
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Seto, Y., R. Fukunaga, and S. Nagata. "Chromosomal gene organization of the human granulocyte colony-stimulating factor receptor." Journal of Immunology 148, no. 1 (January 1, 1992): 259–66. http://dx.doi.org/10.4049/jimmunol.148.1.259.
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Abstract The human chromosomal gene for the granulocyte CSF (G-CSF) receptor was molecularly cloned from a human gene library. The gene is about 16.5 kb long, and present in a single copy per haploid human genome. The human G-CSF receptor gene consists of 17 exons, and the sequences of exons are completely identical to those of cDNAs isolated from human U-937 myeloid leukemia or placenta cDNA libraries. The G-CSF receptor can be subdivided into several regions: an Ig-like domain, a cytokine receptor homologous domain, three fibronectin type III domains, a transmembrane domain, and a cytoplasmic region. Exons 3-17 code for the G-CSF receptor protein, and each subdomain of the receptor is encoded by a set of exons. Primer extension analysis of the G-CSF receptor mRNA identified major and minor transcription start sites. There is no canonical "TATA" box upstream of the CAP site. About 110 nucleotides upstream of the transcription initiation site of the gene, there is an element of 18 nucleotides that is homologous to the sequences found in the promoter of human myeloperoxidase and neutrophil elastase genes.
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Dehal, P. K., M. J. Embleton, J. T. Kemshead, and R. E. Hawkins. "Targeted cytokine delivery to neuroblastoma." Biochemical Society Transactions 30, no. 4 (August 1, 2002): 518–20. http://dx.doi.org/10.1042/bst0300518.
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The aim of this study was to construct a fusion protein from the cytokine granulocyte/macrophage colony-stimulating factor (GM-CSF) and a single-chain Fv fragment (scFv D29) and to investigate its potential to activate cells of the immune system against neuroblastoma cells expressing neural cell adhesion molecule (NCAM). Mammalian cell expression of the scFv D29-GM-CSF fusion protein was compared using a number of vectors, including retroviral and adenoviral vectors. The resultant fusion protein, expressed by HeLa cells, was found by ELISA to bind immobilized recombinant NCAM. Moreover, FACS analysis confirmed binding to the human neuroblastoma cell line SKNBE and a murine neuroblastoma cell line engineered to express the glycosylphosphatidylinositol form of human NCAM (N2A-rKNIE). The fusion protein was also found to stimulate the proliferation of the FDC-P1 haemopoietic cell line, which is dependent on GM-CSF (or interleukin 3) for continued growth. In vitro clonogenic assays indicated that scFv-GM-CSF could selectively induce growth inhibition of SKNBE cells by murine lymphoid cells.
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Goeral, Katharina, Annalisa Hauck, Andrew Atkinson, Michael B. Wagner, Birgit Pimpel, Renate Fuiko, Katrin Klebermass-Schrehof, et al. "Early life serum neurofilament dynamics predict neurodevelopmental outcome of preterm infants." Journal of Neurology 268, no. 7 (February 10, 2021): 2570–77. http://dx.doi.org/10.1007/s00415-021-10429-5.
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Abstract Background and purpose To determine whether neurofilament light chain (NfL), a promising serum and cerebrospinal fluid (CSF) biomarker of neuroaxonal damage, predicts functional outcome in preterm infants with neonatal brain injury. Methods Our prospective observational study used a sensitive single-molecule array assay to measure serum and CSF NfL concentrations in preterm infants with moderate to severe peri/intraventricular hemorrhage (PIVH). We determined temporal serum and CSF NfL profiles from the initial diagnosis of PIVH until term-equivalent age and their association with clinical and neurodevelopmental outcome until 2 years of age assessed by Bayley Scales of Infant Development (3rd edition). We fitted univariate and multivariate logistic regression models to determine risk factors for poor motor and cognitive development. Results The study included 48 infants born at < 32 weeks of gestation. Median serum NfL (sNfL) at PIVH diagnosis was 251 pg/mL [interquartile range (IQR) 139–379], decreasing markedly until term-equivalent age to 15.7 pg/mL (IQR 11.1–33.5). CSF NfL was on average 113-fold higher (IQR 40–211) than corresponding sNfL values. Additional cerebral infarction (n = 25)-but not post-hemorrhagic hydrocephalus requiring external ventricular drainage (n = 29) nor any other impairment-was independently associated with sNfL. Multivariate logistic regression models identified sNfL as an independent predictor of poor motor outcome or death at 1 and 2 years. Conclusions Serum neurofilament light chain dynamics in the first weeks of life predict motor outcome in preterm infants with PIVH.
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De Schaepdryver, Maxim, Andreas Jeromin, Benjamin Gille, Kristl G. Claeys, Victor Herbst, Britta Brix, Philip Van Damme, and Koen Poesen. "Comparison of elevated phosphorylated neurofilament heavy chains in serum and cerebrospinal fluid of patients with amyotrophic lateral sclerosis." Journal of Neurology, Neurosurgery & Psychiatry 89, no. 4 (October 20, 2017): 367–73. http://dx.doi.org/10.1136/jnnp-2017-316605.
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ObjectivePhosphorylated neurofilament heavy chain (pNfH) levels are elevated in cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). Instead of CSF, we explored blood as an alternative source to measure pNfH in patients with ALS.MethodsIn this single centre retrospective study, 85 patients with ALS, 215 disease controls (DC) and 31 ALS mimics were included. Individual serum pNfH concentrations were correlated with concentrations in CSF and with several clinical parameters. The performance characteristics of pNfH in CSF and serum of patients with ALS and controls were calculated and compared using receiver operating characteristic (ROC) curves.ResultsCSF and serum pNfH concentrations in patients with ALS correlated well (r=0.652, p<0.0001) and were significantly increased compared with DC (p<0.0001) and ALS mimics (p<0.0001). CSF pNfH outperformed serum pNfH in discriminating patients with ALS from DC and ALS mimics (difference between area under the ROC curves: p=0.0001 and p=0.0005; respectively). Serum pNfH correlated inversely with symptom duration (r=−0.315, p=0.0033). CSF and serum pNfH were lower when the disease progression rate was slower (r=0.279, p<0.01 and r=0.289, p<0.01; respectively). Unlike CSF, serum pNfH did not correlate with the burden of clinical and electromyographic motor neuron dysfunction.ConclusionsCSF and serum pNfH concentrations are elevated in patients with ALS and correlate with the disease progression rate. Moreover, CSF pNfH correlates with the burden of motor neuron dysfunction. Our findings encourage further pursuit of CSF and serum pNfH concentrations in the diagnostic pathway of patients suspected to have ALS.
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Abu-Rumeileh, Samir, Simone Baiardi, Anna Ladogana, Corrado Zenesini, Anna Bartoletti-Stella, Anna Poleggi, Angela Mammana, et al. "Comparison between plasma and cerebrospinal fluid biomarkers for the early diagnosis and association with survival in prion disease." Journal of Neurology, Neurosurgery & Psychiatry 91, no. 11 (September 14, 2020): 1181–88. http://dx.doi.org/10.1136/jnnp-2020-323826.
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ObjectiveTo compare the diagnostic accuracy and the prognostic value of blood and cerebrospinal fluid (CSF) tests across prion disease subtypes.MethodsWe used a single-molecule immunoassay to measure tau and neurofilament light chain (NfL) protein levels in the plasma and assessed CSF total(t)-tau, NfL and protein 14-3-3 levels in patients with prion disease (n=336), non-prion rapidly progressive dementias (n=106) and non-neurodegenerative controls (n=37). We then evaluated each plasma and CSF marker for diagnosis and their association with survival, taking into account the disease subtype, which is a strong independent prognostic factor in prion disease.ResultsPlasma tau and NfL concentrations were higher in patients with prion disease than in non-neurodegenerative controls and non-prion rapidly progressive dementias. Plasma tau showed higher diagnostic value than plasma NfL, but a lower accuracy than the CSF proteins t-tau and 14-3-3. In the whole prion cohort, both plasma (tau and NfL) and CSF (t-tau, 14-3-3 and NfL) markers were significantly associated with survival and showed similar prognostic values. However, the intrasubtype analysis revealed that only CSF t-tau in sporadic Creutzfeldt-Jakob disease (sCJD) MM(V)1, plasma tau and CSF t-tau in sCJD VV2, and plasma NfL in slowly progressive prion diseases were significantly associated with survival after accounting for covariates.ConclusionsPlasma markers have lower diagnostic accuracy than CSF biomarkers. Plasma tau and NfL and CSF t-tau are significantly associated with survival in prion disease in a subtype-specific manner and can be used to improve clinical trial stratification and clinical care.
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Ding, Yan-Li, Mei-Yun Liu, Wei Han, Sheng-Li Yang, Hui Liu, and Yi Gong. "Application of Phage-displayed Single Chain Antibodies in Western Blot." Acta Biochimica et Biophysica Sinica 37, no. 3 (March 1, 2005): 205–9. http://dx.doi.org/10.1093/abbs/37.3.205.
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Abstract A phage display single chain fragment variable library constructed on pIII protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.
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Stals, Patrick J. M., Chi-Yuan Cheng, Lotte van Beek, Annelies C. Wauters, Anja R. A. Palmans, Songi Han, and E. W. Meijer. "Surface water retardation around single-chain polymeric nanoparticles: critical for catalytic function?" Chemical Science 7, no. 3 (2016): 2011–15. http://dx.doi.org/10.1039/c5sc02319j.
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A library of water-soluble dynamic single-chain polymeric nanoparticles (SCPN) was prepared using a controlled radical polymerisation technique followed by the introduction of functional groups, including probes at targeted positions.