Journal: Frontiers in Immunology

Article Title: Serum RNASE1 as a biomarker for disease activity and lupus nephritis in systemic lupus erythematosus

doi: 10.3389/fimmu.2026.1790455

Figure Lengend Snippet: Serum RNASE1 levels are elevated in patients with SLE and correlate with disease activity and renal dysfunction. (A) Serum RNASE1 concentrations measured by ELISA in healthy controls (HC) and patients with SLE. (B) Comparison of serum RNASE1 levels among HC, patients with SLE without nephritis, and patients with LN. (C) Serum RNASE1 levels in patients with SLE stratified according to disease activity based on SLEDAI scores (inactive, mild, moderate, and severe). (D, E) Correlation analyses between serum RNASE1 levels and SLEDAI scores (D) and complement C3 levels (E) in patients with SLE. (F–H) Correlation analyses between serum RNASE1 levels and renal function parameters in patients with LN, including urinary protein excretion (F) , serum creatinine (G) , and blood urea nitrogen (H) . (I) ROC curve evaluating the performance of serum RNASE1 levels in discriminating patients with SLE from healthy controls. (J–L) ROC curve analyses assessing the discriminative ability of serum RNASE1 levels for identifying patients with ≥mild disease activity (J) , ≥moderate disease activity (K) , and ≥severe disease activity (L) . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Serum levels of RNASE1 were quantified using a commercially available ELISA kit (catalog no. SEK13468 ; Sino Biological Inc., Beijing, China) according to the manufacturer’s instructions.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) CD5L-KO mice were generated by CRISPR-Cas9 engineering. Schematic representation of the genome editing strategy to silence Cd5l expression by inserting three in-frame stop codons and a frame shift. (B) Quantification by ELISA of CD5L in the sera of C57BL/6 WT and CD5L-KO mice. DL: detection limit (6.25 pg/mL). Data from at least 3 independent experiments (Mann-Whitney test). (C) Intracellular CD5L was detected, after permeabilization of the cells with Triton X100, by staining with goat anti-CD5L polyclonal antibody followed by Alexa 488-coupled donkey anti-goat secondary antibody. Peritoneal macrophages from WT or CD5L-KO healthy mice were identified through staining with anti-mouse F4/80 mAb followed by Alexa 594-coupled anti-rat secondary antibody. DAPI was used as a nuclear counterstaining. Scale bar: 10 μm. (D) WT or CD5L-KO mice were subjected to surgery to expose and ligate the cecum at approximately half the distance between the distal pole and the base of the cecum, and through-and-through puncture with a 21G needle, to induce a mid-grade sepsis condition. N = 15 (each group). Kaplan–Meier survival curves were generated to compare mortality between the two groups and significance was determined by log-rank (Mantel-Cox) test. (E) Body weight loss quantification. ***, p < 0.005.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Generated, CRISPR, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: WT or CD5L-KO mice were subjected to CLP to induce mid-grade sepsis. For that, animals were subjected to surgery, exposure and ligation of the cecum (corresponding to approximately half the distance between the distal pole and the base of the cecum) and through-and-through puncture with a 21G needle. Control mice (sham) were subjected to the same surgical procedure but without cecum ligation and puncture. Mice were euthanized 6 or 24 h post-surgery. (A) CFU counts from the peritoneal cavity, blood, lung, liver and kidney. (B) Absolute counts assessed by flow cytometry of cellular populations in the peritoneal cavity. Subsets analyzed: leukocytes, CD45 + ; neutrophils, CD45 + CD11b + Ly6G + ; macrophages, CD45 + CD11b + CD11c - F4/80 + . Ly6C and CD206 markers were used to distinguish between M1 and M2 within the macrophage population. (C) Ly6G and CD11b mean fluorescence intensity (MFI) in expressing neutrophils obtained by geometric mean statistics. (D) Lung, liver and kidney tissue sections obtained 24 h post CLP were stained with hematoxylin and eosin. Scale: 100 μm (E) Pathology score of lung, liver and kidney 24 h post CLP in a double blinded analysis. 0, no signs of inflammation; 1, minimal inflammatory signs; 2, mild inflammation; 3, moderate to severe inflammation. (F) The indicated cytokines were quantified by a multiplex bead-based immunoassay in samples from the peritoneal cavity (left panel) and blood serum (right panel). Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05; **, p < 0.01; ns, not statistically significant.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Ligation, Flow Cytometry, Fluorescence, Expressing, Staining, Multiplex Assay, Bead-based Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) Quantification, by ELISA, of CD5L in the peritoneal cavity (left panel) and serum (right panel) of C57BL/6 mice at 0, 6 or 24 h after mid-grade sepsis (CLP) or in sham operated (sham) animals. (B) WT and CD5L-KO naïve healthy mice were subjected to mid-grade CLP and 3 h later were IV administered with recombinant CD5L (rCD5L) at 0 (PBS) or 2.5 mg/Kg (CD5L). Mice were euthanized 1 h after injection to recover the fluids. Amount of CD5L in the peritoneal cavity (left panel) and serum (right panel), quantified by ELISA. (C) Scatter graphs with detailed illustration of the relation between peritoneal and blood CD5L with the indication of Spearman’s rank correlation in both WT and CD5L-KO mice upon CD5L IV injection as detailed above. Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05; **, p < 0.01; ns, not statistically significant.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Injection, IV Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) Percentage of thioglycolate-elicited Ly6G + CD11b + neutrophils among all cells collected from the peritoneal cavity of WT and CD5L-KO mice, determined by flow cytometry. (B) pHrodo™ Red E. coli BioParticles™ were incubated with 0 or 10 μg of rCD5L for 1 h on ice in TBS containing Ca 2+ . After washing, samples were resuspended in Laemmli buffer and heated at 95 °C for 10 min. Western blot detection of CD5L bound to the particles, using mouse anti-His mAb followed by a secondary goat anti-mouse HRP antibody. (C-D) Phagocytosis of pHrodo particles. Neutrophils were cultured in the presence of pHrodo particles pre-coated with 0 or 10 μg of rCD5L. (C) Percentage of Ly6G + CD11b + neutrophils that internalized pHrodo particles, quantified by flow cytometry. (D) MFI values (geometric mean) of pHrodo channel within the pHrodo + neutrophil population. (E) Cecal bacteria were incubated with 0, 2 and 10 μg of rCD5L for 1 h on ice in TBS containing Ca 2+ . Detection of bacteria-bound rCD5L as in (B). (F) Neutrophils from WT or CD5L-KO mice were cultured in the presence of cecal bacteria pre-coated with 0, 2 or 10 μg of rCD5L. After 3 h, cells were washed, lysed and plated for CFU enumeration. (G) Intracellular staining of CD5L in WT or CD5L-KO naïve mouse blood neutrophils (Ly6G + CD11b + ) and monocytes (F4/80 + CD11b + ) was performed after fixation and permeabilization, with goat anti-mCD5L polyclonal antibody followed by Alexa 488-coupled donkey anti-goat secondary antibody and analyzed by flow cytometry. Data from at least 2 independent experiments. Mann-Whitney test did not reveal any statistical difference between relevant groups.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Incubation, Western Blot, Cell Culture, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) CD5L-KO mice were IV injected with 2.5 mg/Kg of rCD5L, and the circulating amount of rCD5L was quantified by ELISA at the indicated time-points. (B) Protocol for analysis of the immune response after CLP and rCD5L IV treatment. Top, analysis at 6 h, mice had been injected IV with 2.5 mg/Kg rCD5L at 3 h post-surgery, or PBS (untreated), and euthanized 3 h later. Bottom, analysis at 24 h, mice were IV injected with 2 doses of 2.5 mg/Kg rCD5L at 3 and 6 h after surgery, or PBS (untreated), followed by euthanasia at 24 h. (C) Absolute counts and frequencies of cellular populations in the peritoneal cavity, measured by flow cytometry. Subsets analyzed: leukocytes, CD45 + ; neutrophils, CD45 + CD11b + Ly6G + ; macrophages, CD45 + CD11b + CD11c - F4/80 + . Ly6C and CD206 markers were used to distinguish between M1 and M2 within the macrophage population. (D) Ly6G and CD11b MFI in expressing neutrophils, obtained by geometric mean statistics. (E) CFU counts of bacteria obtained from the peritoneal cavity and blood, grown in aerobic or anaerobic conditions, or from lung, liver and kidney, grown in aerobiosis. (F) The indicated cytokines were quantified by a multiplex bead-based immunoassay in samples from the peritoneal cavity (upper panels) and blood serum (lower panels). Data from at least 2 independent experiments (Mann-Whitney test). (G) C57BL/6 mice were subjected to CLP to induce highgrade sepsis. At 3 and 6 h after the surgery, mice were injected IV with doses of 2.5 mg/Kg of rCD5L. Kaplan–Meier survival curves were generated to compare mortality between the two groups and significance was determined by log-rank (Mantel-Cox) test. The number of mice per group is indicated (n). *, p < 0.05; ****, p < 0.0001.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Multiplex Assay, Bead-based Assay, MANN-WHITNEY, Generated

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) WT and CD5L-KO mice were subjected to CLP to induce mid-grade sepsis, and CXCL1 was quantified in the peritoneal cavity or blood serum 3 h or 6 h after surgery. (B) WT C57BL/6 mice were subjected to CLP to induce high grade sepsis and IV injected with 2.5 mg/Kg of rCD5L, or with PBS (untreated) 3 h after surgery. CXCL1 was quantified by a multiplex bead-based immunoassay in the peritoneal cavity and blood serum 6 h after CLP. Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Injection, Multiplex Assay, Bead-based Assay, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Isolation and characterization of human Abs directed against IL-4Rα. ( a ) Binding activity of the isolated anti-IL-4Rα Abs to plate-coated human IL-4Rα or GST, as determined by ELISA. Data represented as mean ± SD (n = 3). ( b ) Schematic diagram of the reporter HEK-Blue TM IL-4/IL-13 cell line to monitor the biological activity of anti-IL-4Rα Abs. The details are described in the text. ( c ) IL-4Rα-blocking activity of the indicated Abs, as determined by SEAP secretion levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) in the presence of the Abs (40 and 200 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to PBS-treated samples. ( d ) Binding isotherms of the immobilized anti-IL-4Rα Abs to soluble antigen IL-4Rα, measured by bio‐layer interferometry on OctetRED96 (Fortebio). The concentrations of IL-4Rα analysed are indicated (colored).

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Isolation, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Affinity maturation of 4 R34 and characterization of the isolated clones. ( a ) Scheme of library construction and screening of 4R34 in the format of scFab using yeast surface display technology. The indicated residues in VL-CDR3, VH-CDR2, and VH-CDR3, highlighted by “X,” were randomly mutated, while maintaining the original amino acids at each residue of 4R34 at a frequency of approximately 50%, using designed spiked oligonucleotides. Numbering is according to the Kabat definition. ( b ) Flow cytometric analysis of antigen binding and expression levels of 4R34-based scFab yeast library in each round screening by FACS. The screening conditions of antigen and sorting gate used in each round are indicated. ( c ) Comparison of association and dissociation of soluble IL-4Rα antigen at 10 nM to immobilized anti-IL-4Rα antibodies (Abs), as measured by bio‐layer interferometry. ( d ) IL-4Rα-blocking activity of the indicated Abs, as determined by SEAP secretion levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) in the presence of the Abs (20 and 100 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to PBS-treated samples.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Isolation, Clone Assay, Binding Assay, Expressing, Blocking Assay, Activity Assay

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Engineering and characterization of 4 R34.1.19. ( a ) Library construction scheme of 4 R34.1, where the indicated residues in the VL-CDR1 and VH-CDR1 were randomized with NNK degenerate codon that encodes all 20 amino acids. ( b ) Flow cytometric analysis of antigen binding and expression levels of 4 R34.1-based scFab yeast library in each round screening by FACS. The screening conditions of antigen and sorting gate used in each round are indicated. ( c ) Binding isotherms of the immobilized anti-IL-4Rα Ab 4R34.1.19 to soluble antigen IL-4Rα, measured by bio‐layer interferometry. The concentrations of IL-4Rα analysed are indicated (colored). ( d , e ) IL-4Rα blocking activity of the indicated Abs, as determined by SEAP levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) ( d ) or rhIL-13 (1 nM) ( e ) in the presence of the Abs (20 and 100 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to phosphate buffer saline (PBS)-treated samples. Statistical analysis was performed using a two-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus dupilumab analogue. ( f ) Binding specificity of the indicated Abs (20 and 100 nM) for cell surface expressed IL-4Rα, as analysed in IL-4Rα-expressing THP-1 cells and IL-4Rα-deficient Molt-4 cells by flow cytometry. Representative histograms from three independent experiments are shown.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Binding Assay, Expressing, Blocking Assay, Activity Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Inhibitory effects of anti-IL-4Rα Abs on IL-4-stimulated T cell proliferation and T H 2 differentiation. ( a ) Dose-dependent blocking effects of anti-IL-4Rα Abs on the proliferation of T cells among PHA-activated PBMCs in response to rhIL-4 (500 pM), determined by CTG assay after 72 h culture. Data are represented as mean ± SD (n = 3). ( b,c ) Inhibitory effects of anti-IL-4Rα Abs (100 nM) on the T H 2 differentiation of naïve CD4 + CD45RO − T cells from healthy donors or asthmatic patients after 7 days culture in T H 2-skewing conditions in the presence of rhIL-4 (500 pM) and anti-IL-4Rα Abs (100 nM). The number of IL-4-producing T H 2 cells were determined by ELISPOT. Quantification of spot forming T cells ( b ) and representative image both healthy donor and asthmatic patient ( c ) are shown. In ( b ), error bars, ± SD (n = 3). Statistical analyses were performed using a one-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus PBS-treated group.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Blocking Assay, CTG Assay, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Epitope mapping of anti-IL-4Rα Abs by alanine scanning mutagenesis. ( a ) Competitive ELISA showing the percentage of bound IL-4Rα (10 and 50 nM) to plate-coated hIL-4-mFc in the presence of the indicated Abs (20, 100, and 500 nM) compared to that without the Ab competitor. Data are represented as mean ± SD (n = 3). Statistical analyses were performed using a two-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus dupilumab analogue. ( b ) Overall structure of the human IL-4Rα:IL-4 complex (PDB: 1IAR). Magnified section shows the residues of IL-4Rα putatively involved in IL-4 binding. ( c ) The percent relative binding of the indicated hIL-4-mFc (5 nM) and anti-IL-4Rα Abs (2.5 nM of 4 R34 and 100 pM of 4 R34.1, 4 R34.1.19 and dupilumab analogue) to IL-4Rα alanine mutants compared to that of wild-type IL-4Rα. Data are represented as mean ± SD (n = 3). Statistical analyses were performed using a one-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001 versus binding to wild-type IL-4Rα.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Mutagenesis, Competitive ELISA, Binding Assay