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Identification of toxemia in patients with Clostridium difficile infection.

Yu H, Chen K, Wu J, Yang Z, Shi L, Barlow LL, Aronoff DM, Garey KW, Savidge TC, von Rosenvinge EC, Kelly CP, Feng H - PLoS ONE (2015)

Bottom Line: C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay.Toxins were relatively stable in stored sera.Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, United States of America.

ABSTRACT
Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.

No MeSH data available.


Related in: MedlinePlus

Glucosyltransferase activity in the serum samples.Rac1 glucosylation was assessed with serum from Case I on mRG1-1 cell (A) and Case II on Vero cells (B). mRG1-1 cells were co-cultured with serum (Case I) in the presence of A1H3 for 4hr at 37°C. Vero cells were cultured with serum (Case II) alone for 4hr at same condition. Treated cells were lysed and non-glucosylated form Rac1 was detected by WB with specific anti-Rac1 (clone 102, BD Biosciences, San Diego, CA). Serum treatment led to glucosylation of Rac1, which was blocked by specific anti-TcdA or (and) TcdB antibody in Case I; and blocked by anti-TcdB not anti-TcdA in Case II. The data shown was a representative experiment from three independent experiments. The normalized data from 3 experiments (see Materials and Methods) were presented in the lower panels. P value < 0.05 was considered as significance when compared to negative control (cell cultured in medium only). *, p< 0.05; **, p< 0.01
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pone.0124235.g002: Glucosyltransferase activity in the serum samples.Rac1 glucosylation was assessed with serum from Case I on mRG1-1 cell (A) and Case II on Vero cells (B). mRG1-1 cells were co-cultured with serum (Case I) in the presence of A1H3 for 4hr at 37°C. Vero cells were cultured with serum (Case II) alone for 4hr at same condition. Treated cells were lysed and non-glucosylated form Rac1 was detected by WB with specific anti-Rac1 (clone 102, BD Biosciences, San Diego, CA). Serum treatment led to glucosylation of Rac1, which was blocked by specific anti-TcdA or (and) TcdB antibody in Case I; and blocked by anti-TcdB not anti-TcdA in Case II. The data shown was a representative experiment from three independent experiments. The normalized data from 3 experiments (see Materials and Methods) were presented in the lower panels. P value < 0.05 was considered as significance when compared to negative control (cell cultured in medium only). *, p< 0.05; **, p< 0.01

Mentions: To conform that the cytotoxicity of the two toxemia-positive serum samples was due to glucosyltransferase activity of the toxins, we analyzed the glucosylation of host small Rho GTPases Rac1. Cell lysates from recombinant TcdA- and TcdB-exposed cells (positive control) showed significantly decreased levels of non-glucosylated Rac1/CDC42 as compared to untreated cells (negative control). A reduction in the level of the non-glucosylated Rho GTPases was also seen in mRG1-1 cell lysates treated with toxemia-positive serum samples; the addition of anti-TcdA or anti-TcdB antibodies led to a partial recovery of the non-glucosylated Rho GTPases, while combined both antibodies completely prevented the loss of the non-glucosylated protein in case I (Fig 2A). In case II, the reduction of the non-glucosylated Rac1/CDC42 induced by the toxin-positive serum sample was completely blocked by anti-TcdB antibody (Fig 2B). These results correlated with the cytotoxicity data described above (Fig 1).


Identification of toxemia in patients with Clostridium difficile infection.

Yu H, Chen K, Wu J, Yang Z, Shi L, Barlow LL, Aronoff DM, Garey KW, Savidge TC, von Rosenvinge EC, Kelly CP, Feng H - PLoS ONE (2015)

Glucosyltransferase activity in the serum samples.Rac1 glucosylation was assessed with serum from Case I on mRG1-1 cell (A) and Case II on Vero cells (B). mRG1-1 cells were co-cultured with serum (Case I) in the presence of A1H3 for 4hr at 37°C. Vero cells were cultured with serum (Case II) alone for 4hr at same condition. Treated cells were lysed and non-glucosylated form Rac1 was detected by WB with specific anti-Rac1 (clone 102, BD Biosciences, San Diego, CA). Serum treatment led to glucosylation of Rac1, which was blocked by specific anti-TcdA or (and) TcdB antibody in Case I; and blocked by anti-TcdB not anti-TcdA in Case II. The data shown was a representative experiment from three independent experiments. The normalized data from 3 experiments (see Materials and Methods) were presented in the lower panels. P value < 0.05 was considered as significance when compared to negative control (cell cultured in medium only). *, p< 0.05; **, p< 0.01
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401762&req=5

pone.0124235.g002: Glucosyltransferase activity in the serum samples.Rac1 glucosylation was assessed with serum from Case I on mRG1-1 cell (A) and Case II on Vero cells (B). mRG1-1 cells were co-cultured with serum (Case I) in the presence of A1H3 for 4hr at 37°C. Vero cells were cultured with serum (Case II) alone for 4hr at same condition. Treated cells were lysed and non-glucosylated form Rac1 was detected by WB with specific anti-Rac1 (clone 102, BD Biosciences, San Diego, CA). Serum treatment led to glucosylation of Rac1, which was blocked by specific anti-TcdA or (and) TcdB antibody in Case I; and blocked by anti-TcdB not anti-TcdA in Case II. The data shown was a representative experiment from three independent experiments. The normalized data from 3 experiments (see Materials and Methods) were presented in the lower panels. P value < 0.05 was considered as significance when compared to negative control (cell cultured in medium only). *, p< 0.05; **, p< 0.01
Mentions: To conform that the cytotoxicity of the two toxemia-positive serum samples was due to glucosyltransferase activity of the toxins, we analyzed the glucosylation of host small Rho GTPases Rac1. Cell lysates from recombinant TcdA- and TcdB-exposed cells (positive control) showed significantly decreased levels of non-glucosylated Rac1/CDC42 as compared to untreated cells (negative control). A reduction in the level of the non-glucosylated Rho GTPases was also seen in mRG1-1 cell lysates treated with toxemia-positive serum samples; the addition of anti-TcdA or anti-TcdB antibodies led to a partial recovery of the non-glucosylated Rho GTPases, while combined both antibodies completely prevented the loss of the non-glucosylated protein in case I (Fig 2A). In case II, the reduction of the non-glucosylated Rac1/CDC42 induced by the toxin-positive serum sample was completely blocked by anti-TcdB antibody (Fig 2B). These results correlated with the cytotoxicity data described above (Fig 1).

Bottom Line: C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay.Toxins were relatively stable in stored sera.Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, United States of America.

ABSTRACT
Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.

No MeSH data available.


Related in: MedlinePlus