Journal: Scientific Reports

Article Title: Bovine antibodies targeting primary and recurrent Clostridium difficile disease are a potent antibiotic alternative

doi: 10.1038/s41598-017-03982-5

Figure Lengend Snippet: ELISA analysis of C . difficile -specific antibodies in colostrum from cows immunised with C. difficile antigens. ELISA plates were coated with C . difficile spore, exosporium, vegetative cell, SLP or recombinant TcdB antigens to determine the specific colostrum antibody titres of Spore-HBC ( a ), Exo-HBC ( b ), Veg-HBC ( c ), SLP-HBC ( d ) or TcdB-HBC ( e ), respectively, compared with colostrum from non-immune cows (NI-BC).

Article Snippet: Strains used in this study were: DLL3109 (TcdA + TcdB + ) , M7404 (TcdA + TcdB + ) , R20291 (TcdA + TcdB + ) , VPI10463 (TcdA + TcdB + ) , 630∆ERM (TcdA + TcdB + ) , DLL3111 (TcdA + TcdB + ), DLL7518 (TcdA − TcdB + ), JGS6133 (TcdA + TcdB + ) , AI35 (TcdA − TcdB + ) , 1470 (ATCC 43598; TcdA − TcdB + ) , and CD37 (TcdA − TcdB − ) .

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Scientific Reports

Article Title: Bovine antibodies targeting primary and recurrent Clostridium difficile disease are a potent antibiotic alternative

doi: 10.1038/s41598-017-03982-5

Figure Lengend Snippet: C. difficile -specific antibodies in colostrum are cross-reactive and able to neutralise the cytotoxicity of TcdB. C. difficile whole cell lysates from a variety of strains were used to detect vegetative cell-specific IgG purified from Veg-HBC ( a ) or SLP-HBC ( b ), respectively. Crude toxin preparations from the panel of strains indicated were used to detect TcdB-specific IgG purified from TcdB-HBC; control lanes contained purified TcdB from an 027 strain (tgcBIOMICS; Lane 11) while a non-toxigenic strain (CD37) was used to show that binding was toxin-specific (Lane 10) ( c ). Exosporium proteins isolated from a variety of clinical and animal isolates of C. difficile were used to detect spore-specific IgG purified from Spore-HBC ( d ) or Exo-HBC ( e ), respectively. C. difficile strains used are indicated alongside each panel ( a – e ). To determine if TcdB-HBC contained IgG capable of neutralising TcdB, purified TcdB from strain VPI10463 (Abcam) was incubated for 60 minutes with either PBS, NI-BC IgG or TcdB-HBC IgG before being added to Vero cell monolayers. After a 24 hour incubation, the percentage of cell death was determined by directly visualising cells for cell rounding ( f ). The experiment was performed in triplicate. Data represent the mean ± SEM and statistical significance was assessed using a two-way ANOVA with a post hoc Bonferroni multiple comparison test. ****Indicates P < 0.0001.

Article Snippet: Strains used in this study were: DLL3109 (TcdA + TcdB + ) , M7404 (TcdA + TcdB + ) , R20291 (TcdA + TcdB + ) , VPI10463 (TcdA + TcdB + ) , 630∆ERM (TcdA + TcdB + ) , DLL3111 (TcdA + TcdB + ), DLL7518 (TcdA − TcdB + ), JGS6133 (TcdA + TcdB + ) , AI35 (TcdA − TcdB + ) , 1470 (ATCC 43598; TcdA − TcdB + ) , and CD37 (TcdA − TcdB − ) .

Techniques: Purification, Control, Binding Assay, Isolation, Incubation, Comparison

Journal: Scientific Reports

Article Title: Bovine antibodies targeting primary and recurrent Clostridium difficile disease are a potent antibiotic alternative

doi: 10.1038/s41598-017-03982-5

Figure Lengend Snippet: HBC-mediated treatment of C. difficile -infected mice. Mice were uninfected or infected with C. difficile spores and treated six hours post-infection with NI-BC, TcdB-HBC, vancomycin or were untreated. Mice were monitored daily for survival ( a ) and weight loss ( b ). Weight loss is presented as the % weight relative to the day of infection (day 0 or D0). Numbers in brackets indicate the number of surviving mice at the completion of the experiment compared to (/) the total number of mice at the beginning of the experiment. Faecal spore load was determined 24 hours post-infection and is presented as CFU/gram faeces (log 10 ), with each point representing a single mouse ( c ). The limit of detection is represented as a dotted line. Error bars represent the mean ± SEM of n = 10–15 mice. ****Indicates P < 0.0001. Representative images of PAS/Alcian blue stained colonic ( d ) tissue from mice. Square brackets ([) indicate crypt hyperplasia, arrow heads (▲) represent epithelial damage and asterisks ( * ) represent oedema and inflammation. Scale bars represent 200 µm.

Article Snippet: Strains used in this study were: DLL3109 (TcdA + TcdB + ) , M7404 (TcdA + TcdB + ) , R20291 (TcdA + TcdB + ) , VPI10463 (TcdA + TcdB + ) , 630∆ERM (TcdA + TcdB + ) , DLL3111 (TcdA + TcdB + ), DLL7518 (TcdA − TcdB + ), JGS6133 (TcdA + TcdB + ) , AI35 (TcdA − TcdB + ) , 1470 (ATCC 43598; TcdA − TcdB + ) , and CD37 (TcdA − TcdB − ) .

Techniques: Infection, Staining

Journal: Scientific Reports

Article Title: Bovine antibodies targeting primary and recurrent Clostridium difficile disease are a potent antibiotic alternative

doi: 10.1038/s41598-017-03982-5

Figure Lengend Snippet: HBC-mediated prevention of C. difficile disease in mice. Mice were untreated or were pre-treated for two days with NI-BC, Spore-HBC, Exo-HBC, Veg-HBC, SLP-HBC, TcdB-HBC or vancomycin prior to infection with C. difficile spores. Colostrum or vancomycin was administered daily for the duration of the experiment. Uninfected, untreated mice served as controls. Mice were monitored daily for survival ( a ) and weight loss ( b ). Weight loss is presented as the % weight relative to the day of infection (day 0 or D0). Numbers in brackets indicate the number of surviving mice at the completion of the experiment compared to (/) the total number of mice at the beginning of the experiment. Faecal spore load was determined 24 hours post-infection and is presented as CFU/gram faeces (log 10 ), with each point representing a single mouse. The limit of detection is represented as a dotted line ( c ). Error bars represent the mean ± SEM of n = 10–24 mice. **Indicates P < 0.01, ****Indicates P < 0.0001. Representative images of PAS/Alcian blue stained colonic (d) tissue from mice. Square brackets ([) indicate crypt hyperplasia, arrow heads (▲) represent epithelial damage and asterisks ( * ) represent oedema and inflammation. Scale bars represent 200 µm.

Article Snippet: Strains used in this study were: DLL3109 (TcdA + TcdB + ) , M7404 (TcdA + TcdB + ) , R20291 (TcdA + TcdB + ) , VPI10463 (TcdA + TcdB + ) , 630∆ERM (TcdA + TcdB + ) , DLL3111 (TcdA + TcdB + ), DLL7518 (TcdA − TcdB + ), JGS6133 (TcdA + TcdB + ) , AI35 (TcdA − TcdB + ) , 1470 (ATCC 43598; TcdA − TcdB + ) , and CD37 (TcdA − TcdB − ) .

Techniques: Infection, Staining

Journal: Scientific Reports

Article Title: Bovine antibodies targeting primary and recurrent Clostridium difficile disease are a potent antibiotic alternative

doi: 10.1038/s41598-017-03982-5

Figure Lengend Snippet: Prevention of primary CDI by HBC mixtures. Mice were untreated (no colostrum) or were pre-treated with NI-BC, TcdB-HBC, Mix1-HBC, Mix2-HBC, Mix3-HBC or vancomycin prior to infection. Uninfected, untreated mice were controls. Mice were monitored daily for survival ( a ) and weight loss ( b ). Weight loss is presented as the % weight relative to the day of infection (day 0 or D0). Numbers in brackets indicate the number of surviving mice at the completion of the experiment compared to (/) the total number of mice at the beginning of the experiment. Faecal spore load is presented as CFU/gram faeces (log 10 ), with each point representing a single mouse. The limit of detection is represented as a dotted line ( c ). Error bars represent the mean ± SEM of n = 5–10 mice.

Article Snippet: Strains used in this study were: DLL3109 (TcdA + TcdB + ) , M7404 (TcdA + TcdB + ) , R20291 (TcdA + TcdB + ) , VPI10463 (TcdA + TcdB + ) , 630∆ERM (TcdA + TcdB + ) , DLL3111 (TcdA + TcdB + ), DLL7518 (TcdA − TcdB + ), JGS6133 (TcdA + TcdB + ) , AI35 (TcdA − TcdB + ) , 1470 (ATCC 43598; TcdA − TcdB + ) , and CD37 (TcdA − TcdB − ) .

Techniques: Infection

Journal: Scientific Reports

Article Title: Bovine antibodies targeting primary and recurrent Clostridium difficile disease are a potent antibiotic alternative

doi: 10.1038/s41598-017-03982-5

Figure Lengend Snippet: Prevention of recurrent CDI by HBC mixtures. Infected mice were orally treated with vancomycin prior to receiving either vancomycin or vancomycin and a mixture of Exo-HBC, SLP-HBC and TcdB-HBC. After vancomycin treatment ceased, mice either received no treatment or the HBC mixture. Mice were monitored daily for survival ( a ) and weight loss ( b ). Weight loss is presented as the % weight relative to the day before, with each point representing a single mouse. Faecal spore load was determined daily after cessation of vancomycin treatment and is presented as CFU/gram faeces (log 10 ) as described above ( c ). Error bars represent the mean ± SEM of n = 9 mice. **Indicates P < 0.01.

Article Snippet: Strains used in this study were: DLL3109 (TcdA + TcdB + ) , M7404 (TcdA + TcdB + ) , R20291 (TcdA + TcdB + ) , VPI10463 (TcdA + TcdB + ) , 630∆ERM (TcdA + TcdB + ) , DLL3111 (TcdA + TcdB + ), DLL7518 (TcdA − TcdB + ), JGS6133 (TcdA + TcdB + ) , AI35 (TcdA − TcdB + ) , 1470 (ATCC 43598; TcdA − TcdB + ) , and CD37 (TcdA − TcdB − ) .

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Soluble P2X7 Receptor Is Elevated in the Plasma of COVID-19 Patients and Correlates With Disease Severity

doi: 10.3389/fimmu.2022.894470

Figure Lengend Snippet: Treatment with ATP or Clostridium difficile toxin B induces the release of the P2X7 receptor. P2X7 receptor (left) or IL-1β (right) from human peripheral blood mononuclear cells from healthy individuals treated for 3 h with LPS (1.6 μg/ml) and then treated with ATP (5 mM) for 45 min or with Clostridium difficile toxin B (TcdB, 1 μg/ml) for 1 h. Each dot represents data from an independent individual.

Article Snippet: The different reagents used in this assay and their sources were ultrapure Escherichia coli lipopolysaccharide (LPS) serotype 0111:B4 (InvivoGen, SanDiego, CA, USA), adenosine 5′-triphosphate (ATP) from Sigma-Aldrich (St. Louis, MO, USA) and Clostridium difficile toxin B (TcdB) from Enzo Life Sciences (Farmingdale, NY, USA).

Techniques: