Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Relationship between sLAIR1, sLAIR2, and C1q serum levels in children with malaria. Differences between circulating sLAIR1 (ng/mL), sLAIR2 (ng/mL), and C1q (ng/mL) in children with non-SMA and SMA were determined using Student t -test and are presented as mean ± SEM. Relationships between sLAIR1, sLAIR2, C1q, and haemoglobin levels were determined by Spearman's correlation test. (a) Normalised sLAIR1 levels in children with non-SMA and SMA. Children with SMA had elevated sLAIR1 levels relative to non-SMA. (b) Normalised sLAIR1 vs. haemoglobin concentrations in children with non-SMA and SMA. Circulating sLAIR1 levels were inversely correlated with haemoglobin levels. (c) Normalised sLAIR1 levels in children with non-SMA and SMA. sLAIR2 serum levels were elevated in children with SMA relative to non-SMA. (d) Normalised sLAIR2 vs. haemoglobin concentrations in children with non-SMA and SMA. Normalised sLAIR2 vs. haemoglobin. sLAIR2 levels were inversely correlated with haemoglobin concentrations. (e) Normalised sLAIR1 vs. normalised sLAIR2 in children with non-SMA and SMA. Circulating sLAIR1 and sLAIR2 levels were positively correlated. (f) Circulating C1q levels in children with non-SMA and SMA. Circulating C1q levels were lower in children with SMA.

Article Snippet: C1q was measured in serum using matched antibody pair (Cat No. H00000712-AP21) and human recombinant C1q protein (Cat No. H00000712-P01) (Novus Biologicals, 8100 Southpark Way # A8, Littleton, CO 80120).

Techniques:

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Effect of intraleukocytic Pf Hz on LAIR1 and SHP-1 phosphorylation. Temporal kinetics of the signalling pathway in response to the different treatment conditions was determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). (a) Human Phospho-immunoreceptor array results showing LAIR1 and SHP-1 phosphorylation upon no treatment (baseline), C1q (25 μg/mL), Pf Hz (10 μg/mL), and Pf Hz + C1q. Phosphorylation (spots) in the different conditions with control represented by blue boxes, LAIR1 by red boxes, and SHP-1 by green boxes. (b) Densitometric analysis of human phosphor-immunoreceptor array data. Data presented as (mean ± SEM). Across group comparisons analysed using ANOVA. *indicates significant differences ( P < .05) determined by Student t-test in pLAIR1 and pSHP-1 compared to C1q treatment. Phagocytosis of Pf Hz resulted in a reduction of pLAIR1 relative to C1q (alone) treatment. Similarly, ingestion of Pf Hz also caused a marked decrease in pSHP-1 levels relative to C1q treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: C1q was measured in serum using matched antibody pair (Cat No. H00000712-AP21) and human recombinant C1q protein (Cat No. H00000712-P01) (Novus Biologicals, 8100 Southpark Way # A8, Littleton, CO 80120).

Techniques: Phospho-proteomics, Control

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Effect of haemozoin on NF-κB activation and cytokine production. Measurements ndwere determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). Data presented as mean ± SEM. Across group and pairwise comparisons determined using Student t-test and Anova analyses, respectively. (a) Comparisons of phosphorylated NF-κB immediately after stimulation of PBMCs with no treatment (NT), C1q (25 μg/mL), Pf Hz (10 μg/mL), and Pf Hz + C1q. NF-κB phosphorylation levels were elevated in the Pf Hz and Pf Hz + C1q treatment groups relative to no treatment (denoted by * P < .05). (b) IL-1β levels (pg/mL) were elevated in culture supernatants from PBMCs treated with Pf Hz and Pf Hz + C1q. (c) IL-6 levels (pg/mL) were elevated in culture supernatants from PBMCs treated with Pf Hz and Pf Hz + C1q. (d) TNF-α levels (pg/mL) were elevated in culture supernatants from PBMCs treated with Pf Hz and Pf Hz + C1q.

Article Snippet: C1q was measured in serum using matched antibody pair (Cat No. H00000712-AP21) and human recombinant C1q protein (Cat No. H00000712-P01) (Novus Biologicals, 8100 Southpark Way # A8, Littleton, CO 80120).

Techniques: Activation Assay, Phospho-proteomics

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: LAIR1 signalling pathway in malaria. The LAIR1 pathway is activated by attachment of collagen and collagenous ligands (C1q) to LAIR1 extracellular surface. This results in phosphorylation of intracellular LAIR1 ITIM tyrosine residues by Src family kinases. Phosphorylated ITIMs serve as docking sites for recruitment of SHP-1 and SHP-2 phosphatases. SHP-1 and SHP-2 become localized to phosphorylated ITIMs through their regulatory SH2 domains, subsequently inducing their phosphatase activity. Activated SHP-1 has been shown to block activation and nuclear translocation of nuclear factor nuclear factor-kappa beta (NF-κB) through de-phosphorylation of inhibitor of kappa-beta kinase complex (IKK). SHP-1 phosphatase activity also inhibits activation and translocation of IRFs from the cytoplasm to the nucleus by preventing TANK-binding kinase 1 (TANK-1) phosphorylation of interferon regulatory factors (IRFs). These events subsequently block transcription of inflammatory mediator encoding genes with response elements for IRFs and NF-κB. SHP-2 inhibits activation of IRF 8 and blocks expression of phagocyte NADPH oxidase (gp91 PHOX ). LAIR1 inhibitory signalling is regulated by soluble LAIR1 and LAIR2 (soluble homolog of LAIR1) through competition for collagenous ligands. Children with SMA had increased circulating levels of sLAIR1 and sLAIR2 indicative of enhanced receptor shedding. C1q was reduced in children with SMA, thereby, limiting ligand availability. Phagocytosis of Pf Hz antagonizes LAIR1 signalling through down-regulation of LAIR1 ITIM and SHP-1 phosphorylation, but does not alter SHP-2 phosphorylation. Leukocytic ingestion of Pf Hz also decreases LAIR1 transcripts and protein. These events result in NF-κB activation and the consequent production of pro-inflammatory mediators that enhance the pathogenesis of SMA. Red arrows represent ligand-receptor interaction, black solid arrows represent activation, and dashed black arrows represent blockade. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: C1q was measured in serum using matched antibody pair (Cat No. H00000712-AP21) and human recombinant C1q protein (Cat No. H00000712-P01) (Novus Biologicals, 8100 Southpark Way # A8, Littleton, CO 80120).

Techniques: Phospho-proteomics, Activity Assay, Blocking Assay, Activation Assay, Translocation Assay, De-Phosphorylation Assay, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein

doi: 10.1101/2024.03.27.587088

Figure Lengend Snippet: (A) To produce A1AT with a human-like glycan profile, a CHO parent clone whereby 9 genes involved in protein glycosylation (Mgat4a, Mgat4b, Mgat5, St3gal3, St3gal4, St3gal6, B3gnt2, Fut8 and Sppl3) have been knocked out were made to express human ST6GAL1 and human A1AT via random integration. (B) The molecular size of plasma-derived and recombinant A1AT was also analyzed using SEC-MALS and DLS. The chromatographs of each protein are shown as blue (pdA1AT) or cyan (geCHO-rhA1AT) lines. The MALS scattering sizes between the peak half-maxima are shown as red (pdA1AT) or maroon (geCHO-rhA1AT) points.

Article Snippet: Collected blood extracted from each animal was processed into plasma and levels of rhA1AT or pdA1AT were measured via sandwich ELISA using a matched antibody pair specific for human A1AT (Novus Biologicals NBP2-79565) and following the manufacturer’s instructions.

Techniques: Glycoproteomics, Clinical Proteomics, Derivative Assay, Recombinant

Journal: bioRxiv

Article Title: Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein

doi: 10.1101/2024.03.27.587088

Figure Lengend Snippet: N-glycan analysis of (A)pdA1AT, (B)geCHO-rhA1AT, and (C) wild-type CHO-derived recombinant A1AT was performed using LC-MS.

Article Snippet: Collected blood extracted from each animal was processed into plasma and levels of rhA1AT or pdA1AT were measured via sandwich ELISA using a matched antibody pair specific for human A1AT (Novus Biologicals NBP2-79565) and following the manufacturer’s instructions.

Techniques: Glycoproteomics, Derivative Assay, Recombinant, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein

doi: 10.1101/2024.03.27.587088

Figure Lengend Snippet: (A) In vitro inhibitory activity of purified rhA1AT against elastase was evaluated and compared with that of commercial pd A1AT. Data shown represents at least two experimental replicates (mean ± SEM). (B) Half-life of pdA1AT and rhA1AT was measured in SD rats after a single-dose, intravenous injection at a concentration of 150 µg/kg. Blood was collected at 5mins, 15 mins, 30 mins, 1 hour, 2 hours, 6 hours, 24 hours, 48 hours, and 120 hours post-injection, and levels of either protein remaining in the serum was measured using sandwich ELISA. Figure represents data from 6 animals per group, with each group receiving an injection of either pdA1AT or geCHO-rhA1AT (mean ± SEM).

Article Snippet: Collected blood extracted from each animal was processed into plasma and levels of rhA1AT or pdA1AT were measured via sandwich ELISA using a matched antibody pair specific for human A1AT (Novus Biologicals NBP2-79565) and following the manufacturer’s instructions.

Techniques: In Vitro, Activity Assay, Purification, Injection, Concentration Assay, Sandwich ELISA