Introduction
Autoimmune liver disease (AILD) is mainly caused by
excessive activation of body immunity against own cells. This
auto-activation of immunity further leads to a series of chronic
and progressive liver diseases (1)
like liver inflammatory response, liver cell and bile duct
necrosis. AILD is mainly classified as primary biliary cirrhosis
(PBC), autoimmune hepatitis (AIH) and primary sclerosing
cholangitis (PSC). All three types of AILD are closely related to
microorganism infection, biological heterologous materials,
malnutrition, vaccination, ultraviolet radiation and environmental
factors (2). Further, evolving
advances in the fields of biochemistry, immunology and
histopathology allowed efficient as well as specific detection of
AILD, thus contributing towards timely management of AILD.
Helicobacter pylori (HP) is a Gram-negative
bacilli planting in stomach and duodenum. HP infection is a major
factor responsible for peptic ulcers (3). Moreover, HP could also exist in liver
or kidneys. Moreover, there is a fair chance of its association
with incidence of AILD (4). This
study aims at analyzing the correlations between HP infection and
AILD and will provide a new idea for clinical diagnosis and
treatment of AILD.
Patients and methods
Study subjects
A total of 60 patients diagnosed with AILD (from
January 2013 to January 2016 in Binzhou Hospital) were enrolled as
study subjects in the present study. PBC diagnosis was performed in
reference to the standards of American Association for the Study of
Liver Diseases (AASLD, 2009). AIH diagnosis was referred to AASLD
standard 2010; and PSC diagnosis was according to Mayer standards.
AILD, liver cancer, viral hepatitis, alcoholic hepatitis, atrophic
gastritis and gastric ulcers and pregnant/lactating women were
excluded from the study. Study subjects included 39 male patients
and 21 female patients, with age range of 45–78 years and mean age
of 58.7±13.6 years. This study was approved by the Ethics Committee
of Binzhou Central Hospital. Signed written informed consents were
obtained from the patients and/or guardians.
Study methods
HP infection was detected by 13C-urea breath test
(13C-UBT). The levels of anti-myeloperoxidase (MPO) were tested by
enzyme-linked immunosorbent assay (ELISA). The positive rate of
rabbit polyclonal anti-nuclear antibody (ANA) (dilution, 1:100;
cat. no. ab151216), rabbit polyclonal anti-mitochondrial antibody
(AMA) (dilution: 1/100; cat. no. ab194396), rabbit monoclonal
anti-smooth muscle anti-body (SMA) (dilution, 1:100; cat. no.
ab108531) and rabbit polyclonal anti-neutrophil cytoplasm antibody
(ANCA) (dilution, 1:500; cat. no. ab178623) were tested by indirect
immunofluorescence (IIF). The positive rates of rabbit polyclonal
anti-mitochondrial antibody (AMA-M2) (dilution, 1:500; cat. no.
ab193668), rabbit monoclonal anti-liver-kidney microsomal antibody
(LKM-1) (dilution, 1:500; cat. no. ab14554), rabbit polyclonal
anti-liver cytoplasm antibody I (LC-1) (dilution, 1:500; cat. no.
ab48394) and rabbit polyclonal anti-soluble liver
antigen/liver-pancreas antigen (SLA/LP) (dilution, 1:500; cat. no.
ab174136) were tested by immunoblotting (IBT). All antibodies were
all purchased from Abcam (Cambridge, MA, USA). Liver function
indexes alanine transaminase (ALT), aspartate transaminase (AST),
alkaline phosphatase (ALP) and glutamyltransferase (GGT), were
analyzed with a fully automatic biochemical analyzer. The levels of
serum cytokine IFN-γ, interleukin-6 (IL-6), IL-10 and tumor
necrosis factor-α (TNF-α) were tested by ELISA.
Statistical analysis
SPSS 20.0 (SPSS, Inc., Chicago, IL, USA) statistical
software was used for statistical analysis. The measurement data
were expressed as mean ± standard deviation (SD) and comparison
among groups adopted the independent sample t-test. Enumeration
data were expressed by case number or (%) and comparison among
groups adopted χ2 test. P<0.05 was considered to
indicate a statistically significant difference.
Results
HP-positive rate analysis
A total of 37 patients (61.67%) were observed to be
HP-positive. HP ratio in positive patients was 4.1–7.6, with an
average of 5.8±1.3.
Comparison of MPO levels
The MPO-positive rate in HP-positive patients was
significantly higher than that of HP-negative patients [35.14%
(13/37) vs. 8.70% (2/23), χ2=5.288, P=0.021].
Comparison of positive rate of ANA,
AMA, SMA and ANCA
The positive rates of ANA, AMA, SMA and ANCA were
observed to be significantly higher in HP-positive patients than
that of HP-negative patients (P<0.05) (Table I).
 | Table I.Comparison of positive rate of ANA,
AMA, SMA and ANCA (case, %). |
Table I.
Comparison of positive rate of ANA,
AMA, SMA and ANCA (case, %).
| Groups | Case no. | ANA | AMA | SMA | ANCA |
|---|
| HP-positive
infection | 37 | 18 (48.65) | 16 (43.24) | 15 (40.54) | 20 (54.05) |
| Negative
infection | 23 | 4 (17.39) | 3 (13.04) | 3 (13.04) | 5 (21.74) |
| χ2
test |
| 5.967 | 5.978 | 5.107 | 6.094 |
| P-value |
| 0.015 | 0.014 | 0.024 | 0.014 |
Comparison of positive rate of AMA-M2,
LKM-1, LC-1 and SLA/LP
The positive rates of AMA-M2, LKM-1, LC-1 and SLA/LP
were significantly higher in HP-positive patients in comparison to
HP-negative patients (P<0.05) (Table
II).
| Table II.Comparison of positive rate of AMA-M2,
LKM-1, LC-1 and SLA/LP (case, %). |
Table II.
Comparison of positive rate of AMA-M2,
LKM-1, LC-1 and SLA/LP (case, %).
| Groups | Case no. | AMA-M2 | LKM-1 | LC-1 | SLA/LP |
|---|
| HP-positive
infection | 37 | 21 (56.76) | 20 (54.05) | 23 (62.16) | 22 (59.46) |
| Negative
infection | 23 | 2 (8.70) | 2 (8.70) | 3 (13.04) | 3 (13.04) |
| χ2
test |
| 13.859 | 12.566 | 13.935 | 12.572 |
| P-value |
| <0.001 | <0.001 | <0.001 | <0.001 |
Comparison of liver function
indexes
The levels of ALT, AST, ALP and GGT in HP-positive
patients did not show any significant difference (Table III).
| Table III.Comparison of liver function indexes
(U/l). |
Table III.
Comparison of liver function indexes
(U/l).
| Groups | ALT | AST | ALP | GGT |
|---|
| HP-positive
infection | 58.73±9.62 | 53.84±14.52 | 123.62±25.47 | 49.68±15.05 |
| Negative
infection | 54.62±12.33 | 51.22±13.65 | 118.47±28.63 | 47.52±14.66 |
| t-test | 0.162 | 0.184 | 0.231 | 0.206 |
| P-value | 0.965 | 0.932 | 0.857 | 0.883 |
Comparison of IFN-γ, IL-6, IL-10 and
TNF-α level
The levels of IFN-γ, IL-6, IL-10 and TNF-α in
HP-positive patients were significantly higher than that of
HP-negative patients (P<0.05) (Table
IV).
| Table IV.Comparison of IFN-γ, IL-6, IL-10 and
TNF-α level (µmol/l). |
Table IV.
Comparison of IFN-γ, IL-6, IL-10 and
TNF-α level (µmol/l).
| Groups | IFN-γ | IL-6 | IL-10 | TNF-α |
|---|
| HP-positive
infection | 17.83±5.46 | 45.62±12.23 | 35.92±7.62 | 13.68±4.25 |
| Negative
infection | 8.25±2.26 | 24.35±10.24 | 17.52±8.34 | 6.56±2.73 |
| t-test | 8.624 | 7.965 | 9.234 | 9.625 |
| P-value | <0.001 | <0.001 | <0.001 | <0.001 |
Discussion
AILD could be preliminarily diagnosed by analyses of
liver function tests. Significant elevation of ALT and AST enzyme
activities confirm serious liver damage. However, conventional
indexes are hard to identify between AILD and viral hepatitis
(5). The mechanism of immune
tolerance is missing in patients with AILD, thereby, inducing T
lymphocyte-mediated damage.
High sensitive and specific antibodies included ANA,
AMA, SMA, ANCA, MPO, AMA-M2, LKM-1, LC-1 and SLA/LP. ANA has been
positively correlated with range of motion of diseases and responds
to autoantibodies leading to inflammation and aggravation of the
liver tissue damage in patients with AILD (6,7). AMA is
an autoantibody without organ specificity and observation of its
higher levels signified necrosis, apoptosis or lymphoid
infiltration into the intra-hepatic bile ducts (8). AMA-M2 is an ingredient of AMA, plays a
leading role in AMA reaction and directly reflects the activeness
of AMA (9). On the other hand, SMA
is classified as one of diagnostic criteria in AILD guideline of
AASLD, with species and organ-specific characteristics. The
detection rate has been observed to be 70% in patients with AILD
(10). When neutrophils receive
antigenic stimulation in patients with AILD, the cytoplasm could
produce ANCA that is associated with AILD activities and damage to
the liver tissues (11). Further,
MPO may inhibit the activation of neutrophils and inactivate the
chemokines. However, in combination with ACNA, its inhibitory
function was limited resulting in a large number of neutrophils
leading to liver cell damage (12).
Although the positive rates of anti-LKM-1, LC-1 and SLA/LP AILD are
relatively low in the antigen spectrum, it is almost 100% in
prediction of specificity. It is rather significant in diagnosis of
AILD (13,14).
The present study observed HP-positive rate of
61.67% in patients with AILD. HP contains some cytotoxin-related
proteins (CagA). When HP is observed to be positive in patients
with AILD, its concentration may be increasing and plenty of CagA
may have transferred to the epithelial cells of gastric mucosa
accordingly. The signaling pathway downstream of cells is thus
activated resulting in aggravation of infection symptoms, thereby,
inducing cellular immune response (15). When an AILD patients is in certain
condition of immune tolerance, his/her defense capability would be
reduced. Therefore, it may easily lead to endotoxemia resulting in
generation of hepatic inflammatory injuries (16). Further in the present study
MPO-positive rate, positive rates of ANA, AMA, SMA and ANCA,
positive rates of AMA-M2, LKM-1, LC-1 and SLA/LP were higher than
in patients with negative HP. After HP infection, ANA has been
reported to react with autoantibodies to form immune complexes
resulting in immune dysfunction. Further, upon release of plasma
inflammatory mediators in quantity, the inflammation has been
reported to further aggravate the damage to liver tissues leading
to AILD as observed in our study (17,18).
Moreover, observed elevation in MPO in combination with AMA and
ANCA could be related to aggravation in the liver injury thereby
causing lymphocytes to infiltrate into intra-hepatic bile duct and
speed up liver cirrhosis (19,20).
Furthermore, the levels of IFN-γ, IL-6, IL-10 and TNF-α were
significantly higher than in patients with negative HP. It
suggested that the positive rate of HP infection in patients with
AILD was rather high and was closely associated with elevation in
various positive immune antibodies and cytokines. The present study
concludes from the above observation that the ratio of correct
diagnosis and clinical treatment outcome for AILD could be enhanced
via early HP detection and intervention. However, the above results
still need further confirmation in clinical setting with large
sample size for a concrete conclusion.
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