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Typical Hus: Evidence of Acute Phase Complement Activation from a Daycare Outbreak.

Brady TM, Pruette C, Loeffler LF, Weidemann D, Strouse JJ, Gavriilaki E, Brodsky RA - J Clin Exp Nephrol (2016)

Bottom Line: We present the clinical course of a child who contracted Shiga toxin-positive E. coli (STEC) from a daycare center during an outbreak.Utilizing the modified Ham test which is a rapid, serum-based functional assay used to detect activation of the alternative pathway of complement as observed in atypical HUS, patient sera revealed evidence of increased complement activation in the acute phase of the syndrome but not after resolution.Our report suggests that complement blockade may be effective in the treatment of STEC-HUS when initiated early in the disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pediatric Nephrology, Johns Hopkins University School of Medicine, USA.

ABSTRACT

The clinical manifestations of typical hemolytic uremic syndrome (HUS) encompass a wide spectrum. Despite the potentially severe sequelae from this syndrome, treatment approaches remain supportive. We present the clinical course of a child who contracted Shiga toxin-positive E. coli (STEC) from a daycare center during an outbreak. Utilizing the modified Ham test which is a rapid, serum-based functional assay used to detect activation of the alternative pathway of complement as observed in atypical HUS, patient sera revealed evidence of increased complement activation in the acute phase of the syndrome but not after resolution. Further, this complement activation was attenuated by eculizumab in vitro, an effect that was replicated in vitro utilizing Shiga toxin as a stimulus of complement activation in normal serum. Our report suggests that complement blockade may be effective in the treatment of STEC-HUS when initiated early in the disease. Given the epidemic nature of the disease that limits the feasibility of randomized clinical trials, further studies are needed to determine the value of early eculizumab treatment in STEC-HUS.

No MeSH data available.


Related in: MedlinePlus

A): Complement activation in Shiga-toxin associated HUS- Increased percentage of non-viable cells in the modified Ham test was observed in the acute phase of Shiga-toxin associated HUS (STEC-HUSa) The same patient was tested twice after resolution of the syndrome (4 and 7 days after the first sample, symbolized as STEC-HUSb and c), showing normalization of cell killing. The dotted line symbolizes the cut-off value (21.5% non-viable cells) above which percentage of non-viable cells suggests increased complement activation observed in atypical HUS. Results from two independent experiments are shown.B): Complement activation in Shiga-toxin associated HUS- Eculizumab containing serum (ECU) was collected within 60 minutes of eculizumab infusion from a PNH patient. ECU was mixed with serum from the acute STEC-HUS in different percentages (50–50%, 25–75% and 12.5–87.5% of STEC-HUS and ECU sera respectively). Total amount of serum in the assay remained unchanged (20%). Eculizumab containing serum resulted in a normalization of the modified Ham test results in all ratios. Results from two independent experiments are shown.C): Complement activation in Shiga-toxin associated HUS- Recombinant shiga-toxin 2 from E.coli was added in normal serum (NS) to replicate the effects of STEC-HUS. Addition of shiga-toxin (stx) resulted in increased percentage of non-viable cells compared to normal serum alone and with heat-inactivated shiga-toxin (hi-stx), as well as heat-inactivated normal serum (HNS) with maximum amount of shiga-toxin. Results from two independent experiments are shown.
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Figure 1: A): Complement activation in Shiga-toxin associated HUS- Increased percentage of non-viable cells in the modified Ham test was observed in the acute phase of Shiga-toxin associated HUS (STEC-HUSa) The same patient was tested twice after resolution of the syndrome (4 and 7 days after the first sample, symbolized as STEC-HUSb and c), showing normalization of cell killing. The dotted line symbolizes the cut-off value (21.5% non-viable cells) above which percentage of non-viable cells suggests increased complement activation observed in atypical HUS. Results from two independent experiments are shown.B): Complement activation in Shiga-toxin associated HUS- Eculizumab containing serum (ECU) was collected within 60 minutes of eculizumab infusion from a PNH patient. ECU was mixed with serum from the acute STEC-HUS in different percentages (50–50%, 25–75% and 12.5–87.5% of STEC-HUS and ECU sera respectively). Total amount of serum in the assay remained unchanged (20%). Eculizumab containing serum resulted in a normalization of the modified Ham test results in all ratios. Results from two independent experiments are shown.C): Complement activation in Shiga-toxin associated HUS- Recombinant shiga-toxin 2 from E.coli was added in normal serum (NS) to replicate the effects of STEC-HUS. Addition of shiga-toxin (stx) resulted in increased percentage of non-viable cells compared to normal serum alone and with heat-inactivated shiga-toxin (hi-stx), as well as heat-inactivated normal serum (HNS) with maximum amount of shiga-toxin. Results from two independent experiments are shown.

Mentions: Further laboratory testing was performed after written informed consent (see Supplementary Methods). Three samples was provided: one in the acute phase of the syndrome and two after the resolution of the syndrome. We utilized the modified Ham test that has been recently described as a functional assay able to detect activation of the alternative pathway of complement observed in complement-mediated hemolytic anemias [10,11]. The test was performed as previously described. Briefly, this rapid and simple cell proliferation assay detects complement-mediated cell killing in the patient serum. As shown in Figure 1A, the acute phase sample caused increased cell killing in the modified Ham test, while the two samples during remission showed normalization of cell killing. These findings suggest that increased complement activation is evident in the acute phase of the disease. To further investigate these findings, we first sought to determine whether complement inhibition with eculizumab ifies the effects of acute STEC-HUS serum. Therefore, we incubated STEC-HUS serum with serum containing eculizumab at different ratios and tested its effect on the modified Ham test. Indeed, addition of eculizumab containing serum resulted in a normalization of the modified Ham test results in all ratios (Figure 1B).


Typical Hus: Evidence of Acute Phase Complement Activation from a Daycare Outbreak.

Brady TM, Pruette C, Loeffler LF, Weidemann D, Strouse JJ, Gavriilaki E, Brodsky RA - J Clin Exp Nephrol (2016)

A): Complement activation in Shiga-toxin associated HUS- Increased percentage of non-viable cells in the modified Ham test was observed in the acute phase of Shiga-toxin associated HUS (STEC-HUSa) The same patient was tested twice after resolution of the syndrome (4 and 7 days after the first sample, symbolized as STEC-HUSb and c), showing normalization of cell killing. The dotted line symbolizes the cut-off value (21.5% non-viable cells) above which percentage of non-viable cells suggests increased complement activation observed in atypical HUS. Results from two independent experiments are shown.B): Complement activation in Shiga-toxin associated HUS- Eculizumab containing serum (ECU) was collected within 60 minutes of eculizumab infusion from a PNH patient. ECU was mixed with serum from the acute STEC-HUS in different percentages (50–50%, 25–75% and 12.5–87.5% of STEC-HUS and ECU sera respectively). Total amount of serum in the assay remained unchanged (20%). Eculizumab containing serum resulted in a normalization of the modified Ham test results in all ratios. Results from two independent experiments are shown.C): Complement activation in Shiga-toxin associated HUS- Recombinant shiga-toxin 2 from E.coli was added in normal serum (NS) to replicate the effects of STEC-HUS. Addition of shiga-toxin (stx) resulted in increased percentage of non-viable cells compared to normal serum alone and with heat-inactivated shiga-toxin (hi-stx), as well as heat-inactivated normal serum (HNS) with maximum amount of shiga-toxin. Results from two independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940046&req=5

Figure 1: A): Complement activation in Shiga-toxin associated HUS- Increased percentage of non-viable cells in the modified Ham test was observed in the acute phase of Shiga-toxin associated HUS (STEC-HUSa) The same patient was tested twice after resolution of the syndrome (4 and 7 days after the first sample, symbolized as STEC-HUSb and c), showing normalization of cell killing. The dotted line symbolizes the cut-off value (21.5% non-viable cells) above which percentage of non-viable cells suggests increased complement activation observed in atypical HUS. Results from two independent experiments are shown.B): Complement activation in Shiga-toxin associated HUS- Eculizumab containing serum (ECU) was collected within 60 minutes of eculizumab infusion from a PNH patient. ECU was mixed with serum from the acute STEC-HUS in different percentages (50–50%, 25–75% and 12.5–87.5% of STEC-HUS and ECU sera respectively). Total amount of serum in the assay remained unchanged (20%). Eculizumab containing serum resulted in a normalization of the modified Ham test results in all ratios. Results from two independent experiments are shown.C): Complement activation in Shiga-toxin associated HUS- Recombinant shiga-toxin 2 from E.coli was added in normal serum (NS) to replicate the effects of STEC-HUS. Addition of shiga-toxin (stx) resulted in increased percentage of non-viable cells compared to normal serum alone and with heat-inactivated shiga-toxin (hi-stx), as well as heat-inactivated normal serum (HNS) with maximum amount of shiga-toxin. Results from two independent experiments are shown.
Mentions: Further laboratory testing was performed after written informed consent (see Supplementary Methods). Three samples was provided: one in the acute phase of the syndrome and two after the resolution of the syndrome. We utilized the modified Ham test that has been recently described as a functional assay able to detect activation of the alternative pathway of complement observed in complement-mediated hemolytic anemias [10,11]. The test was performed as previously described. Briefly, this rapid and simple cell proliferation assay detects complement-mediated cell killing in the patient serum. As shown in Figure 1A, the acute phase sample caused increased cell killing in the modified Ham test, while the two samples during remission showed normalization of cell killing. These findings suggest that increased complement activation is evident in the acute phase of the disease. To further investigate these findings, we first sought to determine whether complement inhibition with eculizumab ifies the effects of acute STEC-HUS serum. Therefore, we incubated STEC-HUS serum with serum containing eculizumab at different ratios and tested its effect on the modified Ham test. Indeed, addition of eculizumab containing serum resulted in a normalization of the modified Ham test results in all ratios (Figure 1B).

Bottom Line: We present the clinical course of a child who contracted Shiga toxin-positive E. coli (STEC) from a daycare center during an outbreak.Utilizing the modified Ham test which is a rapid, serum-based functional assay used to detect activation of the alternative pathway of complement as observed in atypical HUS, patient sera revealed evidence of increased complement activation in the acute phase of the syndrome but not after resolution.Our report suggests that complement blockade may be effective in the treatment of STEC-HUS when initiated early in the disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pediatric Nephrology, Johns Hopkins University School of Medicine, USA.

ABSTRACT

The clinical manifestations of typical hemolytic uremic syndrome (HUS) encompass a wide spectrum. Despite the potentially severe sequelae from this syndrome, treatment approaches remain supportive. We present the clinical course of a child who contracted Shiga toxin-positive E. coli (STEC) from a daycare center during an outbreak. Utilizing the modified Ham test which is a rapid, serum-based functional assay used to detect activation of the alternative pathway of complement as observed in atypical HUS, patient sera revealed evidence of increased complement activation in the acute phase of the syndrome but not after resolution. Further, this complement activation was attenuated by eculizumab in vitro, an effect that was replicated in vitro utilizing Shiga toxin as a stimulus of complement activation in normal serum. Our report suggests that complement blockade may be effective in the treatment of STEC-HUS when initiated early in the disease. Given the epidemic nature of the disease that limits the feasibility of randomized clinical trials, further studies are needed to determine the value of early eculizumab treatment in STEC-HUS.

No MeSH data available.


Related in: MedlinePlus