Upregulation of Shiga toxin receptor CD77/Gb3 and interleukin-1β expression in the brain of EHEC patients with hemolytic uremic syndrome and neurologic symptoms - PubMed (original) (raw)

Upregulation of Shiga toxin receptor CD77/Gb3 and interleukin-1β expression in the brain of EHEC patients with hemolytic uremic syndrome and neurologic symptoms

Christian Hagel et al. Brain Pathol. 2015 Mar.

Abstract

In 2011, a large outbreak of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infections occurred in northern Germany, which mainly affected adults. Out of 3842 patients, 104 experienced a complicated course comprising hemolytic uremic syndrome and neurological complications, including cognitive impairment, aphasia, seizures and coma. T2 hyperintensities on magnet resonance imaging (MRI) bilateral in the thalami and in the dorsal pons were found suggestive of a metabolic toxic effect. Five of the 104 patients died because of toxic heart failure. In the present study, the post-mortem neuropathological findings of the five EHEC patients are described. Histological investigation of 13 brain regions (frontal, temporal, occipital cortex, corpora mammillaria, thalamus, frontal operculum, corona radiata, gyrus angularis, pons, medulla oblongata, cerebellar vermis and cerebellar hemisphere) showed no thrombosis, ischemic changes or fresh infarctions. Further, no changes were found in electron microscopy. In comparison with five age-matched controls, slightly increased activation of microglia and a higher neuronal expression of interleukin-1β and of Shiga toxin receptor CD77/globotriaosylceramide 3 was observed. The findings were confirmed by Western blot analyses. It is suggested that CD77/globotriaosylceramide upregulation may be a consequence to Shiga toxin exposure, whereas increased interleukin-1β expression may point to activation of inflammatory cascades.

Keywords: CD77/Gb3; enterohemorrhagic Escherichia coli; hemolytic uremic syndrome; microglia; neuropathology; vasculature.

© 2014 International Society of Neuropathology.

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Figures

Figure 1

Figure 1

Vasculature‐related parameters in adult enterohemorrhagic

E

scherichia coli (

EHEC

) patients and controls. A. Upper row: small vessels in thalamus and brain stem of controls and

EHEC

brains showing some congestion but no thrombotic microangiopathy; second row: no signs of previous microbleeds in Turnbull stain; third row: regular endothelial expression of von Willebrand Factor (

vWF

) without evidence for

vWF

multimers; forth row: zonulo‐occludens‐1 (

ZO

‐1) immunohistochemistry showing regular expression in controls and

EHEC

cases; fifth row: normal glial fibrillary acidic protein (

GFAP

) expression in controls and

EHEC

cases; sixth row: normal vascular endothelial growth factor (

VEGF

) expression in neurons in both, controls and

EHEC

cases; seventh row: normal angiopoietin‐1 expression in neurons of controls and

EHEC

patients; all microphotographs from case #2 and #6, respectively; counter stain for all immunohistochemical samples hemalum. Scale bar: 50 μm. B. Semiquantitative evaluation of vasculature related antigens expressed in controls and

EHEC

patients, median values ± 95% confidence intervals.

Figure 2

Figure 2

Lack of apoptosis and normal ultrastructure in adult enterohemorrhagic

E

scherichia coli (

EHEC

) patients and controls. Upper row: representative samples of thalamus and brain stem of

EHEC

patients and controls depicting neurons with normal morphology,

H

&

E

; middle row: negative labeling with antibodies against activated caspase 3, counter stain hemalum, all microphotographs in the first two rows from case #2 and #6, respectively; scale bar for first two rows, 50 μm; lower row: normal ultrastructure of neurons in post‐mortem brain tissue showing intact nuclei (

N

), mitochondria (

M

) and endoplasmic reticulum (

ER

), scale bar in left picture 5 μm, scale bar in left close up view 1 μm, scale bars apply accordingly to the

EHEC

sample on the right.

Figure 3

Figure 3

Inflammation‐related parameters in the brain of adult enterohemorrhagic

E

scherichia coli (

EHEC

) patients and controls. A. First row: representative samples of thalamus and brain stem of

EHEC

patients and controls showing few scattered lymphocytes in both groups by labeling with antibodies against common leukocyte antigen (

LCA

); second and third row: labeling of major histocompatibility complex

II

(

HLA‐DR

) and

IBA

‐1 demonstrating slightly increased ramified microglia cells in

EHEC

patients; fourth row; no differences in

TNF

‐α expression in both groups; all microphotographs in the first five rows from case #2 and #6, respectively; scale bar 50 μm applies for the first five rows, counterstain hemalum; fifth row: increased

IL

‐1β expression in cortical neurons in

EHEC

patients in immunofluorescence, nuclear staining with

DAPI

. Scale bar: 10 μm. B. Semiquantitative evaluation of expression of inflammation associated antigens in controls and

EHEC

patients, median values ± 95% confidence intervals. C. Quantification of cytoplasmic

IL

‐1β‐immunofluorescence in 50 neurons of the frontal cortex in a control and an

EHEC

brain in relation to the area, arbitrary units, mean values ± 1 standard error of the mean, P < 0.0001.

Figure 4

Figure 4

Neuronal and endothelial

CD

77 expression in the brain of enterohemorrhagic

E

scherichia coli (

EHEC

) patients and controls. A.

CD

77 immunohistochemistry of thalamus samples of all 10 cases demonstrating increased

CD

77 expression in

EHEC

cases in the second row compared with controls in the first row, counter stain hemalum. Scale bar: 50 μm. B. Median expression levels of

CD

77 in thalamus and brain stem of controls and

EHEC

patients, median values of semiquantitative evaluation ± 95% confidence intervals. C. Percent of vessels with endothelial expression of

CD

77 in a randomly chosen area of 9 mm2 ± 1 standard error of the mean. D. Example of a vessel with

CD

77‐positive endothelia, counter stain hemalum. Scale bar: 50 μm.

Figure 5

Figure 5

Immunoblots of

CD

77,

H

sp70, actin, IBA‐1 and

IL

‐1β. Western blot analyses of frontal cortex of

EHEC

patients compared with controls using antibodies against

CD

77/Gb3,

H

sp70 IBA‐1 and the mature (cleaved) and precursor forms of

IL

‐1β. Actin is shown as loading control and sizes of marker is indicated in kDa. Elevated steady‐state levels in homogenates of frontal cortex of

EHEC

patients compared with controls.

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