Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies

Downes, Frances Pouch; Barrett, Timothy J.; Green, J H; Aloisio, Carol H.; Spika, John S.; Strockbine, N A; Wachsmuth, I K

doi:10.1128/iai.56.8.1926-1933.1988

Cited by 87 publications

(28 citation statements)

References 34 publications

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“…Stx1 was purified by receptor analog affinity chromatography [26] on globotriose-Fractogel (IsoSep AB, Lund, Sweden). Stx2 was obtained according to the method described by Downes et al [27]. Stx preparations contained low amounts of LPS (2-3 ng/mg), as assayed by using the Limulus amoebocyte lysate Pyrogent plus (Cambrex, Walkersville, MD, USA).…”

Section: Stx Purificationmentioning

confidence: 99%

Interactions between Shiga toxins and human polymorphonuclear leukocytes

Brigotti

Carnicelli

Ravanelli

et al. 2008

Journal of Leukocyte Biology

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Human intestinal infections by Shiga toxin (Stx)-producing Escherichia coli cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), which represents the main cause of acute renal failure in early childhood. In HUS, Stx released in the gut enter the bloodstream and are targeted to renal endothelium. The mechanism of toxin delivery is still a matter of debate, although the role of polymorphonuclear leukocytes (PMN) as a Stx carrier has been indicated. The aim of this paper was to better define the interactions between Stx and human PMN. Direct and indirect flow cytometric analysis and binding experiments with radiolabeled toxins demonstrated that Stx bind to the surface of human mature PMN but not to immature PMN from G-CSF-treated donors. The use of the human myeloid leukemia cell (HL-60) model for inducible cell differentiation confirmed that the toxin binding occurs only after granulocytic differentiation. Stx binding caused a delay of the spontaneous apoptosis of PMN, as shown by the delayed appearance of apoptotic nuclei and activation of caspase 3 and by the higher number of cells negative to the annexin V-binding assay after 48 h. Moreover, flow cytometric analysis of mixed Stx-positive and Stx-negative PMN populations showed that the toxins were transferred from positive to negative PMN. The delayed, spontaneous apoptosis and the passage of the toxic ligand from older PMN to new, mature cells entering the circulation from the bone marrow may explain the previously reported persistence of Stx in the blood of children with HUS.

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Section: Stx Purificationmentioning

confidence: 99%

Interactions between Shiga toxins and human polymorphonuclear leukocytes

Brigotti

Carnicelli

Ravanelli

et al. 2008

Journal of Leukocyte Biology

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“…However, passive antibody therapy may hold promise. Murine monoclonal antibodies (mAbs) against Stx have been reported to neutralize the activity of Stx1 and ⁄ or Stx2 in vitro [16][17][18][19][20][21]. Stx1-neutralizing mAbs will have potential clinical utility for the prevention and treatment of HUS caused by Stx1-producing EHEC.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Monoclonal Immunoglobulin A and G Against Shiga Toxin Binding Subunits Produced by Intranasal Immunization

et al. 2008

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Immunoglobulin A (IgA) is considered to play a major role in protection of the mucosal surface. However, its immunological and biological properties have not been extensively studied because the production of IgA class monoclonal antibodies (mAbs) is difficult. We compared the properties of IgA and IgG mAbs against Shiga toxin B subunits (Stx1B). These mAbs were secreted from hybridomas that had been produced from mice after intranasal immunization with recombinant Stx1B and cholera toxin. The dose response curves for the binding of the IgA (clone G2G7) and IgG (clone D11C6) mAbs to immobilized Stx1B were similar, as revealed on ELISA. The majority of the IgA mAb formed dimers while the IgG mAb was monomeric, as judged by immunoblot analysis. The IgG mAb completely inhibited the binding of Stx1B to Burkitt’s lymphoma cell line Ramos, while the inhibition by the IgA mAb was only partial. The IgG mAb was able to neutralize the cytotoxicity of Stx1 holotoxin towards Vero cells, whereas the IgA mAb was not. The binding affinity of each binding site was compared by means of surface plasmon resonance analysis involving a capture method, with which the binding of soluble Stx1B to immobilized mAb was detected. The association rate was similar but the dissociation rate was twofold faster in the case of the IgA mAb, resulting in twofold higher affinity of the IgG mAb. These results suggest that one can obtain high affinity IgA mAb but toxin neutralization is another challenge as to therapeutic antibodies of the IgA class.

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“…The LD 50 of crude Stx-2 (corresponding to c. 1AE0-1AE5 · 10 )6 lg of purified Stx-2; Downes et al 1988;Yoshimura et al 2000) isolated from E. coli O157:H7 43889 was placed into flat-bottomed Immunopure Polysorp 96-well plates (Nunc, Roskilde, Denmark). These plates were incubated at 37°C for 1 h, and then overnight at 4°C, washed once with PBST (PBS containing 0AE05% Tween-20), and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at 37°C.…”

Section: Shiga-like-toxin-binding Assaysmentioning

confidence: 99%

Inhibitory effects ofLactobacillus acidophiluslysates on the cytotoxic activity of shiga-like toxin 2 produced fromEscherichia coliO157:H7

Kim

Han

Imm

et al. 2006

Letters in Applied Microbiology

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Aims: The purpose of this study was to characterize the degree to which four cell lysates obtained from Lactobacillus acidophilus strains affected the cytotoxic activity of Escherichia coli O157:H7 in vitro and in vivo. Methods and Results: In a cytotoxic inhibition test that used 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide and toxin‐binding ELISA assays, the activity of shiga‐like toxin 2 (Stx‐2) was inhibited profoundly by the cell lysates (10 mg ml−1) from two strains of L. acidophilus A4 and 30SC (>85% of survival rates compared with the control) among the five strains tested. In particular, a significant decline in the virulence level of E. coli O157:H7, under the presence of the cell lysates of L. acidophilus A4, was observed by killing assay of Caenorhabditis elegans in vivo model. Conclusions: According to our results, L. acidophilus strains might be capable of attenuating the virulence of Stx‐2 produced from E. coli O157:H7. Significance and Impact of the Study: The cell lysates of L. acidophilus can be applied to a variety of foods, and can be used as adjuncts for the inhibition of Stx‐2‐mediated cytotoxicity.

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Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies

Cited by 87 publications

References 34 publications

Interactions between Shiga toxins and human polymorphonuclear leukocytes

Interactions between Shiga toxins and human polymorphonuclear leukocytes

Characterization of Monoclonal Immunoglobulin A and G Against Shiga Toxin Binding Subunits Produced by Intranasal Immunization

Inhibitory effects ofLactobacillus acidophiluslysates on the cytotoxic activity of shiga-like toxin 2 produced fromEscherichia coliO157:H7

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