ANCAASSOCIATED VASCULITIS IN

1
ANCA-ASSOCIATED VASCULITIS IN ISCHEMIC STROKE
AND HEPATITIS C VIRUS INFECTION Manole
Cojocaru Bucharest, Romania
2
Serum autoantibodies are a characteristic
feature of autoimmune diseases Hepatitis C
virus (HCV) infection is a important infection
worldwide Flaviviridae family with extensive
genomic variability associated with different
autoimmune manifestations
3
Several studies have suggested that chronic HCV
may act as a trigger for the development of
autoimmune diseases    FU H. L., HSU T. C.,
CHANG C. C., TSAY G. J. Antigenic specificity
of antineutrophil cytoplasmic antibody. J
Formos Med Assoc 2001 1 35-9.
4
To assess whether HCV infection is associated
with cerebral vasculitis, we studied the
prevalence of ANCA in patients with ischemic
stroke and HCV infection We also evaluated the
clinical significance of positive autoantibodies
in patients with HCV infection
5
MATERIAL AND METHODS
Serum samples were obtained from 36 patients
with ischemic stroke and HCV infection (18
females, 18 males, mean age 7510 years)
6
Assays employed were indirect immunofluorescence
for detection of ANCA and ELISA for anti-PR3-ANCA
These were consecutive patients referred to
the Clinic of Neurology Diagnosis of chronic
HBV or HCV infection is based on the presence of
hepatitis B surface antigen (HBsAg) or anti-HCV
antibodies (anti-HCV), respectively
7
Anti-HCV antibody was tested by a third
generation anti-HCV enzyme-linked immunosorbent
assay (ELISA)
8
HBsAg was also tested by ELISA. Quantitative
measurements of serum HCV-RNA were performed by
the AmpliSensor Assay The diagnosis of ischemic
stroke was based on accepted clinical and
neuroimaging criteria Forty-four normal
subjects were used as a control group
9
Patients with chronic HBV or HCV infection were
evidence for the diagnosis of ischemic stroke
No one of the patients studied received IFN-?
treatment before blood collection
10
Indirect immunofluorescence was used to test for
ANCA on ethanol- or formalin fixed human
neutrophils according to the method described by
Wiik For ANCA tests, titres gt140 were
considered positive Fluorescence patterns were
classified as classic cytoplasmic (cANCA) or
perinuclear (pANCA) patterns
11
We used direct antigen-specific ELISA kits to
detect anti-PR3 and anti-MPO      FU H. L.,
HSU T. C., CHANG C. C., TSAY G. J. Antigenic
specificity of antineutrophil cytoplasmic
antibody. J Formos Med Assoc 2001 1 35-9.
All sera were assayed at a dilution of 1100
12
Three positive and three negative control sera
were included in each plate Normal value of
anti-PR3 and anti-MPO antibodies were 50 IU/L  
13
RESULTS
ANCA were detected in in 21 out of 36 (58 )
patients with ischemic stroke and HCV infection
by indirect immunofluorescence For ANCA test,
titers gt140 were considered positive. All sera
with ANCA showed cANCA pattern None of the sera
from patients with ischemic stroke and HCV
infection had pANCA
14
ANCA was not detected in 44 sera from normal
subjects and 26 sera from patients with hepatitis
B infection Therefore, sera from patients with
HBV infection were not analysed further for
anti-MPO or anti-PR3 antibodies Sera with ANCA
were tested for anti-PR3 and anti-MPO antibodies
by ELISA
15
Anti-PR3 antibodies were detected in all sera
with ANCA Anti-MPO antibodies were detected in
only 3 of 36 sera from patients with HCV
infection, and most anti-MPO antibodies were at
low titers
16
pANCA staining pattern by IIF
17

cANCA staining pattern by IIF
18
The results of anti-PR3 antibodies in HCV
patients grouped according to PCR positivity and
PCR negativity Anti-PR3 antibodies were
detected in 21 of the 36 (58 ) sera from HCV
patients with PCR positivity The frequence of
anti-PR3 antibody in HCV patients with PCR
positivity was higher than those with PCR
negativity (lt0.01)
19
The mean titers of anti-PR3 in HCV patients with
PCR positivity and PCR negativity were 757.6
ELISA units per mL (EU/mL) and 164.3 EU/mL,
respectively The results indicate that the
major antigen of ANCA in HCV infection is PR3
20
We have reported that there is a group of
patients who have HCV infection and who later
develop ischemic stroke The data of this study
indicate that vasculitis becomes more common with
increasing age
21
The results suggest an additional link between
ANCA and cerebral vasculitis Anti-PR3
antibody is a characteristic antibody for
vasculitis in patients with ischemic stroke and
HCV infection
22
Immunofluorescence studies showed cANCA pattern
Therefore, we concluded that ischemic stroke
should be considered the cerebral vasculitis in
hepatitis C virus infection
23
DISCUSSION  
Ischemic stroke is one of the life-threatening
complications of vasculitis in the nervous system
in hepatitis C virus infection       JENNETTE
J.C., FALK R. J. Small-vessel vasculitis. N
Engl J Med 1997 337 1512-23.       MOORE P.
M., RICHARDSON B. Neurology of the vasculitides
and connective tissue diseases. J Neurol
Neurosurg Psychiatry 1998 65 10-22.
24
These autoantobodies were chosen for the study
because they are common antibodies in vasculitis.
Due to its lymphotropism, HCV has been reported
to be associated with a broad spectrum of
autoimmune manifestations, including
cryoglobulinaemia, vasculitis, but there remains
some controversy as to whether it plays a role in
the pathogenesis of autoimmune diseases NASH
M. C., DILLON M. J. Antineutrophil cytoplasm
antibodies and vasculitis. Arch Dis Child
1997 77 261-4.
25
In the present study we investigated the
presence of ANCA In this study, we found that
autoantibodies resulting from HCV infection were
common, and a high prevalence of ANCA with
anti-proteinase 3 (anti-PR3) specificity We
first reported that HCV patients with ANCA had a
higher prevalence of ischemic stroke
26
This is the first report identifying PR3 as an
ANCA major antigen in patients with HCV infection
Using PCR to detect HCV-RNA, anti-PR3 were
found more frequently in HCV patients with PCR
positivity than who were PCR-negative
27
In patients with PCR positivity, titers of
anti-PR3 were also higher WITKO-SARSAT V.,
RIEU P., DESCAMPS-LATSCHA B., et al.
Neutrophils molecules, functions and
pathophysiological aspects. Lab Invest 2000
80 617-53. VASSILOPOULOS D., HOFFMAN G. S.
Clinical utility of testing for antineutrophil
cytoplasmic antibodies. Clin Diagn Lab
Immunol 1999 6 645- 65.
28
ANCA are present in a high percentage of
patients with systemic vasculitis, who often have
associated with ischemic stroke WIIK A.
Rational use of ANCA in the diagnosis of
vasculitis. Rheumatology 2002 41(5)
481-3. NILES M. D. Antineutrophil cytoplasmic
antibodies in the classification of
vasculitis. Ann Rev Medicine 1996 47
303-13.    
29
WIIK A. Delineation of a standard procedure
for indirect immunofluorescence detection of
ANCA. APMIS 1989 97 12-3. STEGEMAN C. A.
Anti-neutrophil cytoplasmic antibody (ANCA)
levels directed against proteinase-3 and
myeloperoxidase are helpful in predicting
disease relapse in ANCA- associated
small-vessel vasculitis. Nephrol Dial
Transplant 2002 17 2077-80. POLLOCK W.,
CLARKE K., GALLAGHER K., et al.
Immunofluorescent patterns produced by
antineutrophil cytoplasmic antibodies (ANCA)
vary depending on neutrophil substrate and
conjugate. J Clin Pathol 2002 55 680-3.
30
The cause of ANCA-related vasculitis in the
nervous system in hepatitis C virus infection is
still not clear HCV may be regarded as a
possible causative factor in ANCA-associated
vasculitis in the nervous system ANCA mediated
directly or indirectly a neutrophil-induced
endothelial injury leading to vasculitis
31
ANCA targets like PR3 and MPO are regularly
expressed on the cell surface of neutrophils
following apoptosis or cytokine-induced priming
of neutrophils Binding of ANCA to their
antigenic targets on the cell surface leads to
neutrophil oxidative burst, degranulation, and
cytokine release
32
Activated neutrophils can then bind to activated
endothelium, leading to endothelial-cell
detachment and lysis Whether ANCA actively
participate in this process by directly binding
to endothelial cells expressing PR3 or MPO
remains a controversial issue Furthermore,
ANCA induce the release of monocyte
chemoattractant protein 1 (MCP-1) and IL-8 from
monocytes in vitro
33
Both molecules are potent chemokines that
recruit monocytes and neutrophils to inflammatory
sites and thus could intensify the initial
endothelial injury Proteinase 3 is a major
target antigen in HCV infection
34
HCV-induced ANCA production may provide a clue
towards understanding the pathogenesis of
autoimmune disease      HARPER L., REN Y.,
SAVILL J., et al. Antineutrophil cytoplasmic
antibodies induce reactive oxygen- dependent
dysregulation of primed neutrophil apoptosis and
clearance by macrophages. Am J Pathol 2000 157
211-20.   ANCA testing should be considered in
patients with HCV infection
35
Ischemic stroke is one of the life-threatening
complications of HCV infection There are a
group of patients with ischemic stroke with HCV
infection, who should be suspected of cerebral
vasculitis if they have PR3-ANCA
36
CONCLUSION
ELISA is a highly sensitive assay for detecting
of PR3-ANCA and should be used in conjunction
with clinical picture Here, we first reported
cases of HCV infection, ischemic stroke and
ANCA-related cerebral vasculitis Anti-PR3 ANCA
constitutes a useful diagnostic tool and a
sensitive marker of disease activity in this
group of patients with cerebral vasculitis
37

• M. Cojocaru
• C. Burcin
• A. Atanasiu
• ?Carol Davila University of Medicine and
  Pharmacy, Clinic of Neurology,
• Colentina Clinical Hospital
• Bucharest, Romania