Journal: Kidney International Reports

Article Title: Trial of Amiloride in Type 2 Diabetes With Proteinuria

doi: 10.1016/j.ekir.2017.05.008

Figure Lengend Snippet: Quantitation of urinary plasminogen plus plasmin. Aliquots of urine from 9 patients were subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis and immunoblotting with anti-plasminogen/plasmin antibodies ( n = 3–6). Urine volumes analyzed were optimized for detection of plasminogen and plasmin by immunoblotting (10–300 μl) and are listed at the bottom of the figure. Varying amounts of pure plasminogen (4–43 ng) were included on the same blot to create a standard curve and establish levels of plasminogen and plasmin for each patient. A representative immunoblot (a) and corresponding standard curve (b) are shown for 7 patients. (Two patients consistently lacked a signal.) Line between samples 5 and 6 indicates where the blot was cut. Arrowheads indicate plasminogen (PG) and plasmin (P). Data from 3 to 6 analyses were normalized to urine creatinine (Cr), and values are presented in table format (c) and as a bar graph (d). (e) Correlation between urinary plasminogen plus plasmin per gram of creatinine and urinary albumin (Alb)/Cr. std, standard.

Article Snippet: Proteins were electrophoretically transferred to nitrocellulose (Merck-Millipore, Billerica, MA) and incubated with a mouse anti-plasminogen antibody (MAB2596, R&D Systems, Inc, Minneapolis, MN) overnight and horseradish peroxidase–tagged secondary antibody (Jackson Labs, Bar Harbor, ME) for 90 minutes before incubation with PerkinElmer Western Lightning Plus ECL (PerkinElmer, Billerica, MA) and collection of the signal with a Bio-Rad Versadoc, as previously described.

Techniques: Quantitation Assay, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Frontiers in Immunology

Article Title: The influence of 4G/5G polymorphism in the plasminogen-activator-inhibitor-1 promoter on COVID-19 severity and endothelial dysfunction

doi: 10.3389/fimmu.2024.1445294

Figure Lengend Snippet: The 4G allele of the PAI-1 promoter is associated with higher PAI-1 transcript and lower plasmin activity in COVID-19 patients. (A) PAI-1 levels as measured by ELISA in the study cohort (n=43). (B) Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses of all patient samples showed allele-specific fragments after restriction enzyme treatment. (C) Examples of Sanger Sequencing for confirmation of the identified PAI-1 4G/5G rs1799889 and +43G>A (rs6092) polymorphisms. In the study group, we did not find the 43AA genotype. Therefore, no data were available (not found, N/F) for this genotype in panels (D–G) . (D) Fold change in PAI-1 expression in peripheral mononuclear cells (PBMCs) of COVID-19 patients sorted by genotype (n=42). The expression of indicated genes is normalized to the endogenous reference β-actin and presented as a relative fold change to the expression in 4G/4G groups according to the comparative Ct method (2−ΔΔCt). Experiments were repeated twice with samples run in triplicates each. (E) Circulating total PAI-1 protein sorted according to genotypes (data set is the same as in panel (A) . (F) PAI-1 activity after sorting in genotypes as determined by ELISA [reanalysis of original data published ]. (G) Serum plasmin/a2-antiplasmin complex levels by ELISA. Dots in each panel represent data from one patient sample. N.F., not found, N/A, not available. Data represent mean ± SEM with p values from unpaired Student’s t-test or the Mann-Whitney test. # p<0.05; * p<0.05; ** p<0.01; *** <0.005; n/s, not significant.

Article Snippet: Plasmin/antiplasmin complex (Human PAP ELISA kit; Elabscience #E-EL-H2101), Active TGFβ (BioLegend, #437707), total uPA (American Diagnostics/BioMedica Diagnostics, #897), and uPA/PAI1 complexes (Molecular Innovations, huuPA/PAI-1 KTCX) in sera or EC-supernatants were measured according to the manufacturer’s recommendations.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction, Sequencing, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: The influence of 4G/5G polymorphism in the plasminogen-activator-inhibitor-1 promoter on COVID-19 severity and endothelial dysfunction

doi: 10.3389/fimmu.2024.1445294

Figure Lengend Snippet: Circulating proTGFβ and IL-1β protein but not cytokine transcripts in PBMC of COVID-19 patients were higher in 5G5G patients. NFkB (A) and KLF2 (B) expression in PBMCs of COVID-19 patients were sorted for genotypes of the 4G5G PAI-1 promoter polymorphism. (C) Heat map of Spearman’s correlation coefficient between 4G5G PAI-1 polymorphism coefficient and the transcription factors NFκB and KLF2 in COVID-19 patients. The correlation coefficients are represented in the white-brown color intensity change (left panel), as shown in the color bar. P-values of the parameter correlations are given using white-green color coding (right panel). P values were statistically significant (p<0.05) with 95% CIs for each correlation coefficient. (D–G) Fold change in IFNg (D) , IL-6 (E) , IL-1b (F) , and TGFβ (G) , expression in PBMCs of COVID-19 patients sorted for genotypes of 4G5G PAI-1 promoter polymorphism. The expression of the indicated genes is normalized to the endogenous reference β-actin and presented as a relative fold change to expression in 4G/4G groups according to the comparative Ct method (2−ΔΔCt). Gene expression data: Experiments were repeated twice using at least 2-3 samples per test group. (H, I) Analysis of proTGFβ (H) by western blotting, active TGFβ by ELISA (I) , and IL-1β by western blotting (J) in sera of COVID-19 patients. (H, I) Western data are quantified by band intensity data normalized to a sample from a healthy donor and are the reanalysis of original data published in . Data represent mean ± SEM. Depending on the sample normality, p values were determined using the unpaired Student’s t-test or the Mann-Whitney test. * p<0.05; ** p<0.01; *** <0.005; n/s, not significant.

Article Snippet: Plasmin/antiplasmin complex (Human PAP ELISA kit; Elabscience #E-EL-H2101), Active TGFβ (BioLegend, #437707), total uPA (American Diagnostics/BioMedica Diagnostics, #897), and uPA/PAI1 complexes (Molecular Innovations, huuPA/PAI-1 KTCX) in sera or EC-supernatants were measured according to the manufacturer’s recommendations.

Techniques: Expressing, Gene Expression, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: The influence of 4G/5G polymorphism in the plasminogen-activator-inhibitor-1 promoter on COVID-19 severity and endothelial dysfunction

doi: 10.3389/fimmu.2024.1445294

Figure Lengend Snippet: The 4G4G genotype associates with high tPA/PAI-1 complexes and lower proteolytic fragments angiostatin 38kDa, s-uPAR, and sVACM1 in COVID-19 patient sera and rec IL-1β stimulated ECs. (A, B) ECs with known 4G5G PAI-1 promoter genotypes were cultured overnight with IL-1β. A representative immunoblot of uPA (A) and uPA/PAI-1 complex (B) and band intensity quantification after loading an equal volume of supernatants from EC cultures. (C) Free/uncomplexed uPA [ (C) ; a reanalysis of original data published in ] after band quantification of an immunoblot in COVID-19 sera. Serum uPA/PAI-1 complex (D) levels measured by ELISA in COVID-19 patients with indicated genotypes. (E, F) Free/uncomplexed tPA (E) and tPA/PAI-1 complex (F) levels were determined by western blotting [data were analyzed according to genotypes: original data in ]. (G) Supernatants collected from rec IL-1β-and carrier/control-treated EC cultures were analyzed by western blot analysis for the D2D3 fragment of s-uPAR. Band intensity was quantified by normalizing each rec IL-1β sample to its control sample. (H) Total s-uPAR (D1-D3) in COVID-19 sera as determined by ELISA. (I, J) Western blot analysis for s-uPAR/D2D3 (I) and sVCAM (J) in sera of COVID-19 patients sorted according to their genotypes for the 4G5G PAI-1 Promoter polymorphism [reanalysis of original data in ]. (K) Proposed model for the influence of the 4G5G polymorphism during SARS-CoV-2 infection with a robust inflammatory response: Higher IL-1β was found in the circulation of COVID-19 patients with the 5G5G genotype. IL-1β upregulates PAI-1 and NFκB expression in ECs with the 4G4G but not the 5G5G genotype. Cytokine (IL-1β)-induced PAI-1 forms complexes with its activators tPA and uPA, especially in the 4G4G genotype, resulting in impaired uPA and tPA-driven plasmin generation in the 4G4G genotype, ultimately generating a proteolytic shutdown. In ECs, IL-1β downregulates KLF2 expression, possibly to counterbalance PAI-1 upregulation. This process is the opposite in 5G5G EC. Low PAI-1 levels after IL-1β stimulation contribute to high active plasmin that further fuels IL-1β and TGFβ generation and establishes a proteolytic niche. In vitro data were presented as box plots to discriminate in vivo data from the following in vitro data. All experiments were done in triplicate, and two cell lines for each genotype were used. NFκB, Nuclear factor kappa-light-chain-enhancer of activated B cells; KLF2, Krüppel-like factor 2; PBMC, peripheral blood mononuclear cells; interleukin-1 β, IL-1β, TGF β, transforming growth factor-β; MMP, matrix metalloproteinase. Data represent mean ± SEM. Depending on the sample normality, p values were determined using the unpaired Student’s t-test or the Mann-Whitney test. * p<0.05; **p<0.01; *** p<0.001; n/s, not significant.

Article Snippet: Plasmin/antiplasmin complex (Human PAP ELISA kit; Elabscience #E-EL-H2101), Active TGFβ (BioLegend, #437707), total uPA (American Diagnostics/BioMedica Diagnostics, #897), and uPA/PAI1 complexes (Molecular Innovations, huuPA/PAI-1 KTCX) in sera or EC-supernatants were measured according to the manufacturer’s recommendations.

Techniques: Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Infection, Expressing, In Vitro, In Vivo, MANN-WHITNEY

Journal: Emerging Microbes & Infections

Article Title: Preventing Candida albicans from subverting host plasminogen for invasive infection treatment

doi: 10.1080/22221751.2020.1840927

Figure Lengend Snippet: C. albicans activates host plasminogen to promote invasive infection. (A) Representative confocal microscope analysis of human plasminogen binding with C. albicans SC5314. Scale bar represents 10 μm. (B, C) Assay for yeast or hyphal form of C. albicans SC5314-induced plasminogen activation and the effect of plasminogen activation inhibitor ϵ-ACA (20 mM). (D) Plasminogen promoted the damage effects of C. albicans on HUVECs and the effect of ϵ-ACA (20 mM). HUVECs damage was determined by assaying LDH release after 12 h co-cultured with C. albicans SC5314 (MOI = 0.1). (E-F) C57BL/6 mice were treated ϵ-ACA (30 mg/kg) or vehicle control twice daily for 5 days after intravenously infection with C. albicans SC5314 (1 × 10 6 CFU per mouse, E-H). (E) Survival of the mice was monitored for 30 days. (F) The kidney fungal burden of the mice was determined at day 2 post-infection. (G, H) Blood urea nitrogen (BUN) and creatinine (CRE) levels in mice were determined at day 2 post-infection. UI, uninfected mice; PLG, Plasminogen. Data are represented as means ± SD from triplicates of one representative experiment of three. * P, < 0.05; ** P , < 0.01; *** P , < 0.001 [One-way ANOVA (B, C, D, G, H); Log-rank test (E); Nonparametric t -test (F)].

Article Snippet: Then human plasminogen (0.5 μg per well) or negative control PBS buffer was added and incubated at 37°C for 1 h. Plasminogen binding was detected using a rabbit anti-plasminogen antibody (Acris Antibodies) and HRP-conjugated anti-rabbit IgG (Abcam).

Techniques: Infection, Microscopy, Binding Assay, Activation Assay, Cell Culture, Control

Journal: Emerging Microbes & Infections

Article Title: Preventing Candida albicans from subverting host plasminogen for invasive infection treatment

doi: 10.1080/22221751.2020.1840927

Figure Lengend Snippet: Eno1 plays a central role in C. albicans activating human plasminogen. (A) ELISA assays for recombinant C. albicans cell wall proteins binding to human plasminogen. Recombinant C. albicans Eno1 binding to human plasminogen was regarded as 100 percent. (B) Assays for the ability of recombinant C. albicans cell wall proteins to activate human plasminogen. Recombinant C. albicans Eno1 activating human plasminogen was regarded as 100 percent. (C) ELISA assays for recombinant C. albicans Eno1 binding to human plasminogen. Recombinant C. albicans Eno1 (2.5 μg) binding to human plasminogen was regarded as 100 percent. (D) Assays for recombinant C. albicans Eno1-induced activation of human plasminogen. Recombinant C. albicans Eno1 (2.5 μg) activating plasminogen was regarded as 100 percent. (E-J) Eno1 expression at protein level and mRNA level during C. albicans SC5314 invading human endothelial or epithelial cells in vitro . HUVECs (E, G, I) and Caco-2 intestinal epithelial cells (F, H, J) were co-cultured with C. albicans SC5314 for the indicated time (MOI = 1). C. albicans cell wall-localized (E, F) or cytoplasmic (G, H) Eno1 protein were determined by western blot and Eno1 mRNA expression levels were determined by Q-PCR (I, J). PLG, Plasminogen. Data shown in (A-D), (I) and (J) are means ± SD of triplicates from one representative experiment of three. The immunoblotting analysis shown in (E-H) are representative of three independent experiments. ** P , < 0.01 *** P , < 0.001 (One-way ANOVA).

Article Snippet: Then human plasminogen (0.5 μg per well) or negative control PBS buffer was added and incubated at 37°C for 1 h. Plasminogen binding was detected using a rabbit anti-plasminogen antibody (Acris Antibodies) and HRP-conjugated anti-rabbit IgG (Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Activation Assay, Expressing, In Vitro, Cell Culture, Western Blot

Journal: Emerging Microbes & Infections

Article Title: Preventing Candida albicans from subverting host plasminogen for invasive infection treatment

doi: 10.1080/22221751.2020.1840927

Figure Lengend Snippet: mAb 12D9 could block C. albicans capturing and activating human plasminogen. The assay for C. albicans Eno1 binding to (A) and activating (B) human plasminogen in the presence of the indicated concentration of mAb 12D9. Recombinant C. albicans Eno1 binding to and activating plasminogen in the absence of mAb 12D9 were regarded as 100 percent. (C) The assays for C. albicans SC5314 activating human plasminogen in the presence of mAb 12D9 (10μg). PLG, Plasminogen. Bla, Blank. ** P , < 0.01[One-way ANOVA (A, C)].

Article Snippet: Then human plasminogen (0.5 μg per well) or negative control PBS buffer was added and incubated at 37°C for 1 h. Plasminogen binding was detected using a rabbit anti-plasminogen antibody (Acris Antibodies) and HRP-conjugated anti-rabbit IgG (Abcam).

Techniques: Blocking Assay, Binding Assay, Concentration Assay, Recombinant

Journal: Emerging Microbes & Infections

Article Title: Preventing Candida albicans from subverting host plasminogen for invasive infection treatment

doi: 10.1080/22221751.2020.1840927

Figure Lengend Snippet: mAb 12D9 blocks C. albicans developing invasive infection. (A, B) HUVECs (A) and Caco-2 intestinal epithelial cells (B) damage induced by C. albicans in the presence of plasminogen and mAb 12D9 (10 μg/mL) was determined by assaying LDH release. Relative levels of LDH release from HUVECs or Caco-2 intestinal epithelial cells were measured after 6 h of co-culture with C. albicans (MOI = 0.1). (C) Phagocytosis of C. albicans by thioglycollate-elicited peritoneal macrophages. C. albicans SC5314 was co-cultured with peritoneal macrophages and the indicated concentration of mAb 12D9 at 37°C for 1 h (MOI = 0.4). The suspension was then plated on SDA agar for 48 h, after which C. albicans colonies were counted and the phagocytosis percent was calculated. (D) Killing of C. albicans by thioglycollate-elicited peritoneal neutrophil. C. albicans SC5314 cells were co-cultured with peritoneal neutrophils and the indicated concentration of mAb 12D9 at 37°C for 1 h. The suspension was then plated on SDA agar for 48 h, after which C. albicans colonies were counted and the killing percent was calculated. Data in (A-D) are represented as means ± SD from triplicates of one representative experiment of three. (E-G) C57BL/6 mice were infected with 1 × 10 6 CFU of C. albicans SC5314 and treated with mAb 12D9 (30 mg/kg) and/or anidulafungin (AN) (0.1 mg/kg) via the lateral tail vein. (E) Survival of mice was monitored for 30 days ( n = 10 per group). (F) Quantification of the fungal burden in the kidneys of mice ( n = 6 per group) at day 2 post-infection. (G) Representative H&E (for determining inflammatory cell influx and the extent of tissue necrosis) and PAS (for C. albicans ) staining of kidneys from infected mice with the indicated treatment at day 2 post-infection. Arrows indicate inflammatory cells influx and tissue necrosis (H&E staining) and C. albicans filaments in the tissues (PAS staining). Magnification 200×. Data in (G-J) are representative of three independent experiments. * , P < 0.05; *** , P < 0.001[One-way ANOVA (A-D); Log-rank test (E); Nonparametric One-way ANOVA (F)].

Article Snippet: Then human plasminogen (0.5 μg per well) or negative control PBS buffer was added and incubated at 37°C for 1 h. Plasminogen binding was detected using a rabbit anti-plasminogen antibody (Acris Antibodies) and HRP-conjugated anti-rabbit IgG (Abcam).

Techniques: Infection, Co-Culture Assay, Cell Culture, Concentration Assay, Suspension, Staining

Journal: Emerging Microbes & Infections

Article Title: Preventing Candida albicans from subverting host plasminogen for invasive infection treatment

doi: 10.1080/22221751.2020.1840927

Figure Lengend Snippet: Maximal activity of mAb 12D9 against C. albicans infection depends on host plasminogen. (A) HUVECs damage was determined by assaying LDH release. Relative levels of LDH release from HUVECs were measured after 6 h of co-culture with C. albicans (MOI = 0.1) in the presence of indicated plasminogen, mAb 12D9 (4μg/mL) and ϵ-ACA (20 mM). (B-F) C57BL/6 mice were treated with ϵ-ACA (30 mg/kg) twice daily for 5 days to abolish plasminogen system after intravenously infection with C. albicans SC5314 (1 × 10 6 CFU). (B) Survival of mice with the indicated mAb 12D9 (30 mg/kg) and/or anidulafungin (AN) (0.1 mg/kg) treatment was monitored for 30 days ( n = 10 per group). (C, D) Quantification of fungal burden in kidneys (C) and liver (D) of mice treated with mAb 12D9 (30 mg/kg) and/or anidulafungin (0.1 mg/kg) ( n = 6 per group) at day 2 post-infection. (E, F) Blood urea nitrogen (BUN) (E) and creatinine (CRE) (F) in mice were determined at day 2 post-infection. UI, uninfected mice. Data in (A-F) are representative of three independent experiments. ** , P < 0.01; *** , P < 0.001; [Nonparametric One-way ANOVA (A, C-F); Log-rank test (B)].

Article Snippet: Then human plasminogen (0.5 μg per well) or negative control PBS buffer was added and incubated at 37°C for 1 h. Plasminogen binding was detected using a rabbit anti-plasminogen antibody (Acris Antibodies) and HRP-conjugated anti-rabbit IgG (Abcam).

Techniques: Activity Assay, Infection, Co-Culture Assay

Journal: Emerging Microbes & Infections

Article Title: Preventing Candida albicans from subverting host plasminogen for invasive infection treatment

doi: 10.1080/22221751.2020.1840927

Figure Lengend Snippet: mAb 12D9 binds motif 254 FYKDGKYDL 262 in H6S6 loop of C. albicans Eno1 to prevent it activating plasminogen. (A) Alignment of the H6S6 loop amino acid sequences of T. solium enolase A with enolase of fungal species including C. albicans , C. tropicalis , C. parapsilosis , C. glabrata and A. fumigatus. (B) Schematic diagram of different domains of recombinant C. albicans Eno1. (C, D) ELISA assay for mAb 12D9 binding to C. albicans Eno1 and its different domains (Eno1 254-440aa and Eno1 1-262aa , C; Eno1 263-440aa and Eno1 1-253aa , D). (E) Plasminogen-binding assay with different domains of recombinant C. albicans Eno1. (F) Plasminogen activation induced by different domains of recombinant C. albicans Eno1. (G) Structural model of C. albicans Eno1 (homo-dimer) generation using Swiss-Model. Putative Eno1-plasminogen interaction motif was marked in red, and the key plasminogen-binding residues (F254G, amaranth; K259L, yellow; Y260H, cyan) were calculated by Swiss-Model and marked with “ * ”. A blue box stands for α-helices and a purple arrow represents a β-strand. (H) ELISA assay for mAb 12D9 binding to recombinant C. albicans Eno1 and mutations (F254G, K259L and Y260H). (I) Plasminogen-binding assay for recombinant C. albicans Eno1 and mutations (F254G, K259L and Y260H). (J) Plasminogen activation induced by recombinant C. albicans Eno1 and mutations (F254G, K259L and Y260H). Recombinant C. albicans Eno1 binding to and activating plasminogen in the absence of mAb 12D9 were regarded as 100 percent. PLG, Plasminogen. Data in (C-F and H-J) are representative of three independent experiments. *** P , < 0.001 (One-way ANOVA).

Article Snippet: Then human plasminogen (0.5 μg per well) or negative control PBS buffer was added and incubated at 37°C for 1 h. Plasminogen binding was detected using a rabbit anti-plasminogen antibody (Acris Antibodies) and HRP-conjugated anti-rabbit IgG (Abcam).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Activation Assay