Journal: BMC Immunology

Article Title: The effect of pneumococcal immunization on total and antigen-specific B cells in patients with severe chronic kidney disease

doi: 10.1186/s12865-019-0325-9

Figure Lengend Snippet: a . Numbers of total IgG antibody secreting cells (ASC) per 200,000 peripheral blood mononuclear cells (PBMC) pneumococcal vaccine naïve ( n = 18) and previously immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23) ( n = 29) patients with severe chronic kidney disease on day 7 post-immunization with PCV13. The median is displayed counts above the upper limit of detection (200) were assigned a value of 210 (8 pneumococcal vaccine naïve and 13 previously immunized with PPV23). If no spots were detected, a value of 0.5 was assigned for statistical purposes. PPV23 naïve and PPV23 immunized each had 4 values below the limit of detection. Control wells coated with methylated human serum albumin were not displayed (all values were below the limit of detection). b . Numbers of IgG antibody secreting cells (ASC) specific for pneumococcal capsular polysaccharides of serotypes 6B or 14 per 200,000 peripheral blood mononuclear cell (PBMC) in pneumococcal vaccine naïve (n = 18) and previously immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23) ( n = 31) patients with severe chronic kidney disease 7 days post-immunization with PCV13 (median is shown). If no spots were detected a value of 0.5 was assigned for statistical purposes. For serotype 6B, 9 pneumococcal vaccine naïve and 16 previously immunized with PPV23 patients had ASC below the lower limit of detection. For serotype 14, 7 pneumococcal vaccine naïve and 13 previously immunized with PPV23 patients had ASC below the lower limit of detection. Control wells coated with methylated human serum albumin were not displayed (all below the limit of detection)

Article Snippet: Multi-screen IP 96-well PVDF membrane filter plates (Millipore Canada Ltd., Etobicoke, ON, CAN) were coated with either goat anti-human IgG (20 μg/ mL) (Cedarlane, Burlington, ON, CAN), or pneumococcal capsular polysaccharides of serotypes 6B, or 14 (10 μg/ mL) (Cedarlane) conjugated with methylated human serum albumin (mHSA, 10 μg/ mL) (National Institute for Biological Standards and Control, Hertfordshire, UK).

Techniques: Control, Methylation

Journal: The Journal of Biological Chemistry

Article Title: Meningococcal Group W-135 and Y Capsular Polysaccharides Paradoxically Enhance Activation of the Alternative Pathway of Complement *

doi: 10.1074/jbc.M110.184838

Figure Lengend Snippet: Only group W-135 and Y capsular polysaccharides bound to bacteria, but not free soluble polysaccharide, enhance alternative pathway activation. A, purified capsular polysaccharides in solution do not generate C3a. C2-depleted serum (25% (v/v)) was incubated with soluble purified capsular polysaccharides (each at a concentration of 125 μg/ml) for 10 min at 37 °C and C3a generated was measured by ELISA. Each bar represents the mean ± S.D. of 2 separate experiments. B, binding of purified capsular polysaccharides to Cap− N. meningitidis. W171 Cap−/fHbp−/NspA− (referred to as W Cap−) was incubated separately with purified polysaccharides (125 μg/ml) representing each of the 5 major meningococcal serogroups. Bound polysaccharide was detected using a mAb specific for each of the serogroups (gray shaded histograms). Binding of the mAb to the corresponding Cap+ mutant strain used in this study (i.e. A2594, H44/76, C2120, W171, and Y2220) is shown by the solid black histogram. Controls where the Cap− W171 mutant without any added polysaccharide was incubated with the anti-capsule mAb and secondary conjugate are shown by the broken lines. One of two reproducible experiments is shown. C, group W-135 and Y polysaccharides induce rapid C3a generation when bound to bacteria. Strain W171 Cap− was incubated with soluble purified capsular polysaccharides (each at a concentration of 125 μg/ml), followed by the addition of C2-depleted serum (25% (v/v)) for 10 min at 37 °C; C3a generated in the reaction mixture was measured by ELISA. Encapsulated strains W171 and Y2220 served as positive controls, whereas W171 Cap− plus C2-depleted serum (no added capsular polysaccharide) and C2-depleted serum alone were used as negative controls. Each bar represents the mean ± S.D. of 3 separate experiments. D, deposition of iC3b on W171 Cap− coated with purified capsular polysaccharides. The bacterial strains incubated with C2-depleted serum as described in C were washed, lysed, and Western blotted to analyze iC3b binding.

Article Snippet: Serogroup B meningococcal capsular polysaccharide was the kind gift of Dr. Jo Anne Welsch (Novartis Vaccines).

Techniques: Bacteria, Activation Assay, Purification, Incubation, Concentration Assay, Generated, Enzyme-linked Immunosorbent Assay, Binding Assay, Mutagenesis, Western Blot