Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: IDIOPATHIC NEPHROTIC SYNDROME. Baseline characteristics of patients affected by idiopathic nephrotic syndrome who underwent evaluation of serum levels of sCD40L.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques:

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: (A-C) Micrographs representative of immunofluorescence staining for nephrin in non-permeabilized GECs: (A) GECs incubated with vehicle alone for 30 min; (B) GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min; (C) Effect of pretreatment with an inhibitor of CD40-CD40L interaction, CD40-muIg fusion protein (20 ng/ml), on loss of nephrin induced by hr-sCD40L. Original magnification: ×400 (F-H). Bars = 10 μm. Images are representative of at least 5 separate experiments with similar results. (D) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with various concentrations of hr-sCD40L for 30 min (upper graph), and of the time-course effect of incubation of GECs with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer; dashed bars) or vehicle alone (open bars) on nephrin expression, as detected by immunofluorescence staining (lower graph). Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (E-G) Micrographs representative of fluorescein isothiocyanate phalloidin staining of actin microfilaments in permeabilized GECs. (E) GECs incubated with vehicle alone for 30 min; (F) GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min; (G) Effect of pretreatment with an inhibitor of CD40-CD40L interaction, CD40-muIg (20 ng/ml), on reorganization of cytoskeleton induced by hr-sCD40L. Original magnification: ×400 (D-I). Bars = 10 μm. Images are representative of at least 5 separate experiments with similar results. (H) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) for 30 min in the absence (black bar) or presence of different inhibitors of CD40-CD40L interaction (dashed bars). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to GEC stimulation, whereas the CD40L-muCD8 fusion protein (50 ng/ml) was added to cultured GECs 30 minutes before adding hr-sCD40L (see for details). Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (I) Immunoblot of a representative experiment on the effect of hr-sCD40L and PF on nephrin expression as detected by Western blot analysis. GEC lysates were immunoblotted with antibodies anti-nephrin or Beta-actin after incubation with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer) or PF (500 ng/ml), in the presence or absence of CD40-muIg fusion protein (20 ng/ml) to inhibit CD40-CD40L interaction. Blots are representative of three independent experiments with similar results.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Immunofluorescence, Staining, Incubation, Expressing, Derivative Assay, Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: P alb was determined after the incubation of rat glomeruli for 30 min with hr-sCD40L (100 ng/ml + 1 μg/ml enhancer). A significant increase in glomerular permeability is expressed by values of P alb greater than 0.5 (black bar). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to glomerulus stimulation, whereas the CD40L-muCD8 fusion protein (50 ng/ml) was added to the glomeruli 30 minutes before adding hr-sCD40L (dashed bars) (see for details). At least five animals were studied per each experimental group. * P < 0.05 and ** P < 0.01 versus unstimulated controls; # P < 0.01 versus rh-sCD40L.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Incubation, Permeability

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: (A-F) Effect of in vivo injection of sCD40L on the glomerular expression of nephrin and podocin. (A-B) Micrographs representative of immunofluorescence staining for nephrin in glomeruli from female C57BL/6 mice injected in vivo with 200 ng sCD40L (C) or, as negative control, 200 ng heat-inactivated sCD40L (B) (x63). Bars = 10 μm. (C) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in glomeruli from female C57BL/6 mice injected in vivo with sCD40L or, as negative control, heat-inactivated sCD40L (Ctrl). (D-E) Micrographs representative of immunofluorescence staining for podocin in glomeruli from female C57BL/6 mice injected in vivo with 200 ng sCD40L (F) or, as negative control, 200 ng heat-inactivated sCD40L (E) (x63). Bars = 10 μm. (F) Semiquantitative analysis of podocin expression as detected by immunofluorescence staining in glomeruli from female C57BL/6 mice injected in vivo with 200 ng sCD40L or, as negative control, 200 ng heat-inactivated sCD40L (Ctrl). Glomerular expression of nephrin and podocin was evaluated by indirect immunofluorescence by confocal microscopy on frozen kidney sections obtained twenty-four hours after injection, as detailed in the Methods section. Six animals were studied per experimental group. (G) Urine protein/creatinine ratio 24 hours after a single injection of 5% bovine serum albumin (BSA–white bars), sCD40L (black bars), or heat-inactivated sCD40L (dashed bars) in female C57BL/6 mice. Each bar is representative of a single experiment.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: In Vivo, Injection, Expressing, Immunofluorescence, Staining, Negative Control, Confocal Microscopy

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: Effect of the partially purified Permeability Factor (PF), prepared from plasmapheresis eluates from patients who presented post-transplant recurrence of FSGS, on nephrin expression in cultured GECs (A) and on Permeability activity of albumin (P alb ) in isolated rat glomeruli (B). (A) Semiquantitative analysis of nephrin expression as detected by immunofluorescence staining in GECs incubated with PF (500 ng/ml) for 30 min in the absence (black bar) or presence of different inhibitors of CD40-CD40L interaction. Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to GEC stimulation, whereas the CD40L-muCD8 fusion protein (10 μg/ml) was added to cultured GECs 30 minutes before adding hr-sCD40L (see for details). ** P < 0.01 versus unstimulated control and PF + inhibitors of CD40-CD40L interaction. Values are derived from 5 or more experiments for each experimental condition and expressed as percent variations from baseline value. (B) Permeability activity of albumin (P alb ) induced by PF on isolated rat glomeruli: P alb was determined after the incubation of rat glomeruli with PF (500 ng/ml), as detailed in the Methods section. A significant increase in glomerular permeability is expressed by values of P alb greater than 0.5 (black bar). Using CD40-muIg fusion protein (20 ng/ml) and the neutralizing antibody against CD40L (5 μg/ml), hr-sCD40L was pre-treated for 10 minutes prior to glomerulus stimulation, whereas the CD40L-muCD8 fusion protein (10 μg/ml) was added to the glomeruli 10 minutes before adding PF. At least five animals were studied per each experimental group.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Purification, Permeability, Expressing, Cell Culture, Activity Assay, Isolation, Immunofluorescence, Staining, Incubation, Derivative Assay

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: Western blot analysis of sCD40L in PF preparations obtained from plasmapheresis eluates of different FSGS patients with elevated P alb (lanes A-F), and from a normal patient with negative P alb (lane G). In the former case, PF was prepared utilizing plasma from FSGS patients undergoing plasmapheresis and separated on Protein A at different conditions; in the second case, normal plasma without P alb activity was obtained as unbound to Protein A material. PF preparations (5μg) were immunoblotted with anti-sCD40L antibody, which identified two bands, at 17 and 34 kDa, corresponding to the monomeric and dimeric forms of the molecule, respectively. hr-sCD40L (500 ng) was used as positive control. Each lane corresponds to an individual patient.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Western Blot, Activity Assay, Positive Control

Journal: PLoS ONE

Article Title: Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS

doi: 10.1371/journal.pone.0188045

Figure Lengend Snippet: (A) Serum sCD40L levels in healthy controls and in patients with idiopathic nephrotic syndrome (NS), subdivided according to clinical features ( i . e . age at onset and response to steroids). Baseline clinical characteristic of the patients with idiopathic nephrotic syndrome are reported in . Children who presented proteinuria under the 1 st year of age underwent biopsy and had a diagnostic molecular approach for several genes implicated in NS; they were classified as congenital NS (cNS). (B) Serum sCD40L levels in healthy controls and in patients with steroid-resistant idiopathic nephrotic syndrome according to the levels of proteinuria (< or > 0.5 g/day). (C) Serum sCD40L levels in patients with nephrotic syndrome and a biopsy-proven diagnosis of FSGS or of iMN. Patients with FSGS were further subdivided according to their age in < or > 18 years. Patients with iMN were always older than 40 years.All patients had eGFR >60 ml/min at the time of serum sampling for sCD40L measurement.

Article Snippet: Human recombinant soluble CD40L (hr-sCD40L) trimeric protein plus enhancer (cross-linking Ab), mouse recombinant soluble CD40L (mr-sCD40L) set, recombinant CD40-murine Ig (muIg) fusion protein, consisting of the extracellular domain of human CD40 fused to mouse IgG2a, recombinant human CD40L-muCD8 fusion protein, constituted by murine CD8 fused to human CD40L, and anti-human CD40L mAbs (MK13A4 and 24–31) were obtained from Alexis Biochemicals (San Diego, CA).

Techniques: Diagnostic Assay, Sampling

Journal: bioRxiv

Article Title: B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

doi: 10.1101/2021.09.09.459588

Figure Lengend Snippet: (A) Irradiated RAG1-deficient mice were reconstituted with bone marrow from either Wnk1 fl/+ RCE or Wnk1 fl/fl RCE mice, or from Wnk1 fl/+ RCE or Wnk1 fl/D368A RCE mice. At least 56 d later, mice were treated with tamoxifen on 3 consecutive days and analyzed 7 d after start of tamoxifen treatment. (B) Mean±SEM levels of Wnk1 mRNA measured across the junction of exons 1 and 2 in control or WNK1-deficient splenic mature B cells, normalized to Hprt expression and to Wnk1 mRNA levels in control B cells (set to 1). (C, D) Mean±SEM numbers of mature B cells in the spleen (B220 + CD19 + CD93 - ) and lymph nodes (TCRβ - B220 + IgM + IgD + ) of RAG1-deficient mice reconstituted with Wnk1 fl/+ RCE or Wnk1 fl/fl RCE marrow (C) or with Wnk1 fl/+ RCE or Wnk1 fl/D368A RCE marrow (D), as described in A. (E) Mean±SEM binding of soluble VCAM-1 complexes to control or WNK1-deficient B cells in response to treatment with anti-IgM or CXCL13 for the indicated times. (F) Mean±SEM surface levels of CD11a, CXCR5, IgM and CD40 on control or WNK1-deficient B cells normalized to expression on control B cells (set to 100). (G) Irradiated RAG1-deficient mice were reconstituted with bone marrow from either Oxsr1 +/+ RCE or Oxsr1 fl/fl RCE mice, or from Oxsr1 +/+ Stk39 +/+ RCE or Oxsr1 fl/fl Stk39 T243A/T243A RCE mice. At least 56 d later, mice were treated with tamoxifen on 5 consecutive days and analyzed 21 d after start of tamoxifen. (H, J) Immunoblot analysis (top) of total cell lysates from splenic B cells from RAG1-deficient mice reconstituted with Oxsr1 +/+ RCE or Oxsr1 fl/fl RCE marrow (H) or with Oxsr1 +/+ Stk39 +/+ RCE or Oxsr1 fl/fl Stk39 T243A/T243A RCE marrow (J), probed with antibodies to OXSR1 and α-TUBULIN. Graph (bottom) shows mean±SEM amount of OXSR1 in the lanes above, normalized to the abundance of α-TUBULIN in each lane. (I, K) Mean±SEM numbers of mature B cells in the spleen (B220 + CD19 + CD93 - ) of RAG1-deficient mice reconstituted with Oxsr1 +/+ RCE or Oxsr1 fl/fl RCE marrow (I), or with Oxsr1 +/+ Stk39 +/+ RCE or Oxsr1 fl/fl Stk39 T243A/T243A RCE marrow (K), as described in G. Mann-Whitney test (B, C, F, H-K); Two-way ANOVA (E), Mann-Whitney test (F, H-K); * 0.01 < P < 0.05, ** 0.001 < P < 0.01; *** 0.0001 < P < 0.001, **** P < 0.0001. Sample sizes: 6 (B, D, E), 5 (C, H), 7 (F, K), 3-4 (I), 12 (J). Data are pooled from 2 independent experiments.

Article Snippet: Where indicated, the cells were stimulated with either 1 µg/ml recombinant murine CXCL13 (CXCL13, R&D Systems; Biotechne), 10 µg/ml AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 10 µg/ml biotin-SP AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (biotinylated anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml recombinant murine CD40L (CD40L, R&D Systems; Biotechne) or 10 µg/ml LPS from Salmonella minnesota R595 (LPS, Enzo Life Sciences, Inc.).

Techniques: Irradiation, Expressing, Binding Assay, Western Blot, MANN-WHITNEY

Journal: bioRxiv

Article Title: B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

doi: 10.1101/2021.09.09.459588

Figure Lengend Snippet: (A-H) Top; immunoblots of cell lysates from mouse B cells stimulated for the indicated times with anti-IgM (A-C, G) or CXCL13 (D-F, H) using WNK1-deficient or control B cells (A, D), B cells expressing kinase-inactive WNK1-D368A or control B cells (B, E), or wild-type B cells treated with vehicle (DMSO), an inhibitor of WNK-family kinases (WNK463) (C, F), a PI3K inhibitor (PI-103) or an AKT inhibitor (MK2206) (G, H), probed with antibodies to phosphorylated OXSR1 (p-OXSR1), α-TUBULIN, phosphorylated AKT (p-AKT), phosphorylated PRAS40 (p-PRAS40) or ERK2. Below; mean±SEM abundance of p-OXSR1, p-AKT and p-PRAS40 in the lanes above, normalized to the α-TUBULIN or ERK2. Mann-Whitney test; * 0.01 < P < 0.05, ** 0.001 < P < 0.01, *** 0.0001 < P < 0.001. Sample sizes: 4 (A, H), 5 (B, D, E, G), 7 (C, F). Data are pooled from two (A, B, D, E, G, H) or three (C, F) independent experiments.

Article Snippet: Where indicated, the cells were stimulated with either 1 µg/ml recombinant murine CXCL13 (CXCL13, R&D Systems; Biotechne), 10 µg/ml AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 10 µg/ml biotin-SP AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (biotinylated anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml recombinant murine CD40L (CD40L, R&D Systems; Biotechne) or 10 µg/ml LPS from Salmonella minnesota R595 (LPS, Enzo Life Sciences, Inc.).

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: B cell-intrinsic requirement for WNK1 kinase in T cell-dependent antibody responses

doi: 10.1101/2021.09.09.459588

Figure Lengend Snippet: (A-D) Mean±SEM binding of soluble ICAM-1 complexes to mouse B cells from control B cells and either WNK1-deficient B cells (A), B cells expressing kinase-inactive WNK1-D368A (B), OXSR1-deficient B cells (C) or OXSR1-deficient B cells expressing a non-phosphorylatable mutant of STK39-T243A (D), stimulated with anti-IgM or CXCL13 or treated with MnCl 2 for the indicated times. (E) Migration of control or WNK1-deficient mouse B cells from the top to the bottom chamber of a Transwell plate in response to CXCL13. (F, G) Violin plots showing mean speed (F) and displacement (G) of control and WNK1-deficient mouse B cells migrating in response to CXCL13. Dashed lines indicate median (red) and 25 th and 75 th percentiles (grey). Two-Way ANOVA (A-D), Mann-Whitney test (E-G); * 0.01 < P < 0.05, ** 0.001 < P < 0.01, *** 0.0001 < P < 0.001, **** P < 0.0001. Sample sizes: 11 (A), 6 (B), 5 (C), 9 (D), 4 mutant and 5 control (E), 5245 mutant cells and 7289 control cells (F, G), Data pooled from two independent experiments.

Article Snippet: Where indicated, the cells were stimulated with either 1 µg/ml recombinant murine CXCL13 (CXCL13, R&D Systems; Biotechne), 10 µg/ml AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 10 µg/ml biotin-SP AffiniPure F(ab’) 2 fragment goat anti-mouse IgM (biotinylated anti-IgM, Jackson ImmunoResearch Laboratories, Inc.), 1 µg/ml recombinant murine CD40L (CD40L, R&D Systems; Biotechne) or 10 µg/ml LPS from Salmonella minnesota R595 (LPS, Enzo Life Sciences, Inc.).

Techniques: Binding Assay, Expressing, Mutagenesis, Migration, MANN-WHITNEY