JC virus infects the enteric glia of patients with chronic idiopathic intestinal pseudo-obstruction

Abstract

Background and aims: Chronic idiopathic intestinal pseudo-obstruction (CIIP) is characterised by severe impairment of intestinal propulsive motility that mimics bowel obstruction. JC virus (JCV) is a polyomavirus that can infect brain glial cells causing a fatal disease, but may also be found throughout the normal gastrointestinal tract. The hypothesis that JCV infects the myenteric plexuses of patients with CIIP was tested.

Methods: 10 patients with CIIP and 61 normal specimens (30 ascending colon and 31 ileum) from patients with uncomplicated colon cancer were studied. DNA was extracted from the myenteric plexuses, and JCV T antigen (TAg) DNA and the viral regulatory region were detected by PCR and sequencing. Immunohistochemistry was performed to detect JCV viral protein expression, neuronal and glial markers. Fluorescence in situ hybridisation was performed for cellular localisation of the JCV infection.

Results: Clinical studies demonstrated neurogenic impairment, and pathological analyses showed neuropathy in each patient with CIIP. JCV TAg DNA was found in the myenteric plexuses of 8/10 (80%) of the patients with CIIP and 3/31 (9.7%) of the control patients (p<0.001). All samples were JCV Mad-1 strains. Seven of the 10 CIIP specimens expressed both JCV TAg and the JCV viral protein VP1, while none of the controls expressed either. JCV infection co-localised with glial fibrillary acidic protein expression, a marker of enteric glial cells.

Conclusion: JCV infection occurs in the myenteric plexuses of patients with CIIP. The JCV localisation in enteroglial cells suggests a possible pathological role for this virus in enteric neuropathy.

Figure 1

Immunohistochemical staining of the enteric neural network in patients with chronic idiopathic intestinal pseudo-obstruction (CIIP) compared with controls. Representative examples of immunolabelling experiments on cross-sections of patients with CIIP investigated in the present work. Note the marked reduction in the density and intensity of non-specific enolase- (NSE), enteroglial protein S100-β-, substance P/related tachykinins- (SP) and vasoactive intestinal polypeptide (VIP)-immunoreactive perikarya and nerve processes (right panels) compared with controls (left panels).

Figure 2

Upper panel (A–C): microdissection of the myenteric plexus. (A) A myenteric plexus is stained with neuron-specific enolase-specific antibody before DNA extraction. (B) Microdissection of the selected area was performed before DNA extraction. (C) Agarose gel electrophoresis of JC virus (JCV)-positive and -negative samples. Samples are numbered from 1 to 10; C1 and C2 are two samples extracted from control patients. B, water lane; M, the molecular size standard. Lower panel (D–F): fluoresce in situ hybridisation (FISH) analysis for JCV DNA. The left column shows nuclear (4′,6-diamidino-2-phenylindole (DAPI)) staining, the middle column shows the same tissue hybridised with FISH markers for JCV DNA, and the right column shows the merged images. (D) The myenteric plexus from a patient with chronic idiopathic intestinal pseudo-obstruction (CIIP) with positive PCR for JCV DNA shows multiple areas of hybridisation. (E) The myenteric plexus from a control sample does not show hybridisation. (F) A section of hamster brain previously transfected with JCV T antigen was used as positive control. Multiple areas of hybridisation were observed.

Figure 3

Immunohistochemical staining of myenteric plexuses for JC virus (JCV) T antigen (TAg), JCV viral protein 1 (VP1) and glial fibrillary acidic protein (GFAP). Upper panel. Immunohistochemistry shows expression of TAg (Pab2003) (A) and VP1 (B) in a patient affected by CIIP (patient 3). This patient was found to be positive for JCV DNA by both PCR and fluorescene is situ hybridisation. Patient 7, who was found positive for JCV DNA, tested negative for both TAg and VP1 expression. For a negative control we employed myenteric plexuses from patients affected by colon cancer. For a positive control for TAg we employed hamster brain tissues expressing the protein. Lower panel: (C) dual immunofluorescence shows co-localisation (merged panel) between VP1 expression and GFAP, indicating that the virus actively replicates in enteric glial cells. As positive controls for VP1 expression we employed human glial cells expressing the protein.