THAI NGUYEN UNIVERSITY
UNIVERSITY OF MEDICINE & PHARMACY
LE QUOC TUAN
STUDY OF CLINICAL AND SUBCLINICAL
CHARACTERISTICS AND CYTOKINE LEVELS IN
PATIENTS WITH ALCOHOLIC CHRONIC LIVER
DISEASE
Major: Internal Medicine
ID: 97.20.107
DISSERTATION ABSTRACT
THAI NGUYEN - 2018
Dissertation was completed at: Thai Nguyen University of
Medicine & Pharmacy
Advisors:
1. Assoc.PhD. Tran Viet Tu
2. Assoc.PhD. Nguyen Ba Vuong
Reviewer 1: Assoc.PhD.
Reviewer 2: Assoc.PhD.
Reviewer 3: Assoc.PhD.
Dissertation will be defensed in front of dissertation’s university
council at: h:
201.
Held in: Thai Nguyen University of Medicine & Pharmacy
The information from this thesis can be found at:
- Vietnam National Library
- Library of Thai Nguyen University
- Library of Thai Nguyen University of Medicine & Pharmacy
1
INTRODUCTION
Alcohol Liver Disease (ALD) is the result of harmful alcohol
abuse and prolonged exposure. The first stage of ALD is
asymptomatic, reversible if alcohol withdrawal occurs, but the later
stages of ALD can not be reversed, usually resulting in death due to
esophageal varices. There is no radical treatment except liver
transplantation. It affect the quality of life of patients, but also have a
great impact on the socio-economic development.
Studies show that altered immunity and inflammation are key
factors contributing to the progression of ALD. Mediators of the
immune system, such as cytokines or inflammatory factors, are
primarily involved in the stages of the disease. In ALD, the use of
chronic alcohol activates Kupffer cells through receptors, increases
interleukin-1 (IL-1) production, and alpha tumor necrosis factor
(TNF-α), contributes to confusion, hepatic dysfunction, necrotizing
fasciitis, hepatic cellular dysfunction, progressive liver fibrosis, and
cirrhosis. Studies of alcoholic hepatitis patients showed TNF-α, IL-1,
IL-8, leukemia, mononuclear activation and macrophage. This further
demonstrates that inflammation is a key factor in the progression of
ALD.
Inflammation is a major factor contributing to the development
of ALD, and therapies that inflammation are a reasonable strategy.
Understanding the role of some cytokines in ALD stages helps to
detect new therapies that inhibit inflammation at an early stage and
fibrosis in the later stages of the disease is actually beneficial to slow
down progression of the disease.
2
In Vietnam, the increasing number of patients with alcoholic
liver disease is a matter of concern and concern due to the increasing
use of alcohol in Vietnam. However, studies on the association of
cytokines with clinical, subclinical and clinical characteristics in
patients with alcoholic liver disease have not been studied by
researchers. For that reasons, we study: "Study of some plasma
cytokines in patients with alcoholic liver disease" with the aim:
1. Describe the clinical, subclinical characteristics and TNF-α,
IL-12, IL-1β, TGF-β in plasma in patients with chronic liver disease
caused by alcohol
2. Analysis of the relationship between TNF-α, IL-12, IL-1β,
TGF-β in plasma with clinical, subclinical and clinical features in
patients with chronic liver disease caused by alcohol.
SCIENTIFIC SIGNIFICANCE
Emphasizing the role of some cytokines in the diagnosis,
prognosis, providing useful evidence is the scientific basis for the
application of cytokine therapy in the treatment of alcoholic liver
disease.
PRACTICAL SIGNIFICANCE
Determination of TNF-α, IL-12, IL-1β, TGF-β levels in plasma
in patients with alcoholic hepatitis and alcoholic cirrhosis.
Evaluate the relationship between these 4 cytokines and some
clinical and laboratory characteristics in patients with ALD.
SUMMARY OF NEW CONTRIBUTIONS OF DISSERTATION
The first thesis studied 4 cytokines in patients with ALD
plasma.
3
Contributing additional information about the cytokine role
in alcoholic liver disease is the scientific basis for the use of
cytokine-based therapies for the treatment of alcoholic liver disease.
Suggestting use of biomarkers for clinical use to detect
inflammation and fibrosis of the liver.
The layout of the dissertation:
+ The dissertation consists of 102 pages including of the following
sections: the introduction (2 pages): chaper 1: overview (37 pages):
chapter 2: objects and methods (14 pages): chapter 3: results (17
pages): chapter 4: discussion (22 pages): conclusions (2 pages):
petition (1 pages). The dissertation consists of 52 tables, 12 pictures.
The theis references include 102 references consist of 7 Vietnamese
document, 95 English document.
+ The three related articles to the dissertation have been published in
the Vietnam Medical Journal.
4
Chapter 1. OVERVIEW
1.1. Alcoholic liver disease
Epidemiology: Alcohol abuse is prevalent throughout the world,
with an estimated 18 percent of adults in the United States. In 2010,
alcoholic cirrhosis caused 493,300 deaths (accounting for 1% of total
deaths). In the United States, the National Institute of Health
estimates that in 2009, there were more than 31,000 deaths from
cirrhosis and in which cirrhosis accounted for 48% of deaths. The
incidence of alcoholic liver disease is higher in areas with high levels
of alcohol consumption per capita. Areas with high rates of alcohol
consumption and alcoholic liver disease include Eastern Europe,
Southern Europe, and the United Kingdom. Countries with large
numbers of Muslims have the lowest rates of alcohol consumption
and alcoholic liver disease. The United States has an average
consumption of 9.4 L/adult/year, compared with 13.4 L/year in
England and 0.6 L/year in Indonesia.
1.1.2. Clinical features, laboratory results of alcohol liver
disease
In most cases, the clinical is less or asymptomatic in the early
stages and in the compensated period. Clinical manifestations of
ALD vary from asymptomatic or mild to fatal cirrhosis. Therefore,
the diagnosis is highly dependent on clinical signs, different tests and
invasive or non-invasive techniques.
Typical ALD:
- Anorexia, nausea, vomiting, discomfort, weight loss, abdominal
pain and jaundice.
Fever sometimes up to 390C, 50% of cases.
5
- Examination: most have large liver, pain, one third of cases
have large spleen.
- More likely: ascites, edema, hemorrhage, hepatic brain
syndrome.
Symptoms of jaundice, ascites, and hepatic brain syndrome can
be reduced by abstaining from alcohol. If the patient continues to
drink alcohol and poor nutrition can lead to repeated episodes with
manifestations of decompensated cirrhosis, resulting in death.
Some patients with early signs of alcohol abuse such as salivary
gland dysfunction, body weakness, malnutrition, may have peripheral
neurological signs, but usually patients without symptoms and
Reluctance to admit alcohol may be the cause of abnormal liver
function.
During clinical examination of patients with cirrhosis, typical
skin manifestations of liver disease include: cardiovascular disease,
palmar edema and glossy tongue. Jaundice, hepatic brachial
syndrome, ascites and edema may also be seen in patients with endstage liver disease.
Consider ALD when patients have a history of excessive
drinking ( > 40-50 g/day) and clinical abnormalities and abnormal
test indices suggest liver damage. However, when drinking history is
often forgotten, it is often necessary to use indirect alcohol screening
tools.
In patients with alcoholic liver disease, alcohol withdrawal
symptoms, delirium, hypertension, mild fever, abdominal pain,
paranoid hallucinations and hallucinations, have been reported after
72-96 hours of withdrawal. .
6
AST/ALT > 2. Increasing AST < 500 U/L, increasing ALT < 300
U/L. Increasing GGT . MCV > 100 fL.
Histopathology:
Mallory
hepatic
degeneration,
Mallory
syndrome, giant mitochondria, inflammatory cell infiltration, liver
fibrosis and fatty liver.
1.4. Definitive diagnosis of alcoholic liver disease
According to the guidelines of the American Liver Disease
Research Association (AASLD - 2010): Diagnosis based on a history
of alcohol use (screening alcohol use of the World Health
Organization, AUDIT - WHO), clinical symptoms of liver disease,
and abnormal liver enzymes. Liver biopsy helps diagnose the cause,
and identifies the stages of liver damage.
Observational studies showed that serum TNF-α levels as well as
those of the liver increased in patients with alcoholic hepatitis, and
were correlated with the severity of the disease. Apply this scientific
basis to use TNF-α inhibitor in the treatment of patients with ALD.
1.5. The role of some cytokines in alcoholic liver disease
Serum IL-12 levels are increased in patients with alcohol
poisoning, alcoholic hepatitis, alcoholic cirrhosis. IL-12 is highest in
patients with alcoholic hepatitis and gradually decreases with alcohol
abstinence.
TGF-β is central in chronic liver disease, which is related to the
progression of the disease, from initial liver damage through
inflammatory and fibrosis reactions leading to cirrhosis and
hepatocellular carcinoma. TGF-β activates the production of collagen
from astrocytes. Damage to the liver causes TGF-β to enhance
astrocytomatic regulation and activate fibroblasts leading to a wound
healing
response,
including
myofibroblast
and
extracellular
7
deposition. Recognizing as a major profibrogenic cytokine, TGF-β
signaling pathways are associated with the inhibition of progression
of liver disease.
Numerous data indicate that the important role of IL-1β in
alcoholic liver damage depends on the formation and activation of
the inflammasome. It relates to the progression of the disease.
Chapter 2. MATERIAL AND METHODS
2.1. Focus group
2.1.1. The study group
- 95 inpatients with ALD who were treated at the Gastrointestinal
Department of Thai Nguyen Nation Hospital and 103 Military
Hospital; 40 healthy students of Military Medical University.
- Time: From January 2015 to December 2015.
Inclusion criteria
* Study group:
95 inpatients had been diagnosed with ALD according to the
guideline of the American Association for the Study of Liver
Diseases (AASLD) 2010: The diagnosis of ALD is based on a
combination of features, including a history of significant alcohol
intake, clinical evidence of liver disease, and supporting laboratory
abnormalities.
- The histological features of alcohol-induced hepatic injury vary,
depending on the extent and stage of injury. These may include
steatosis (fatty change), lobular in-flammation, periportal fibrosis,
Mallory bodies, nuclear vacuolation, bile ductal proliferation, and
fibrosis or cirrhosis.
- Patients who agreed to take part in the research.
8
* Control Group:
40 healthy students of Military Medical University were
interviewed to investigate alcohol abuse based on the AUDIT
questionnaire; comprehensive clinical examination and clinical
examination; Make laboratory tests to detect and eliminate liver
disease. If they are really healthy (without Hepatitis B and Hepatitis
C, perfectly healthy, liver function tests are normal) agree to
participate in research. They will be selected for the study.
2.1.2. Criteria for exclusion of disease group
The patients were excluded from the study as follows: Simple
steatosis and non alcoholic liver disease of any etiology (drug use,
bile clogging, cancer, hepatitis B, hepatitis C), liver disease patients
with unknown etiology, contraindications for liver biopsy. Do not
agree to participate in research.
- Patients are infected, fungal, virus, allergic, autoimmune.
2.2. Method
2.2.1.Design research: prospective, descriptive, cross-sectional,
comparative itself control.
2.2.2. Template size and selection:
- Sample size were calculated by the equation:
In which:
n is the minimum number of participants diagnosed with ALD
Z is confidence level, 95% level of confidence used, Z= 1.96
: Mean cytokine index in patient with ALD taken from a previous
study.
9
:Standard deviation of mean cytokine index taken from a previous
study.
: Relative precision, selection of = 0.1.
According to González-Reimers et al, TNF-α in the ALD group was
7.18 ± 3.51 pg/ml, appling this equation. The minimum sample size in
this study was: n = 57, we estimated that theoretical sample size is 95
patients.
2.2.3. Research steps
2.3.3.1. Select the patient
All patients who are diagnosed with ALD in Gastrointestinal
Department were eligible for case selection and were not included in
the exclusion criteria.
2.3.3.2. Clinical examination
- All objects will be examined and recorded in medical records which
have been designed specifically to gather and identify the following
information:
- Screening for alcohol use: AUDIT questionnaire.
2.3.3.5. The technique of testing TNF-α, IL-12, TGF-β, IL-1β in blood
- TNF-α, IL-12, TGF-β, IL-1β in blood was measured by using
ELISA kit supplied by Wkea-China. The plasma samples determine
the cytokines index were stored at -800C. Use ELISA reader–
Diagnostic Automation, USA at Military Medical University.
2.3.3.6.Liver biopsy
Liver biopsies were performed with ultrasound guidance. It’s
Germany's Pajunk automatic biopsy gun in the study. The liver tissue
sample was evaluated by doctors of the Department of Anapath Military Hospital 103.
2.3. Study index
10
- Identify the association between 4 cytokines and some clinical
features and laboratory results.
- Association with age, sex, history of alcohol abuse and
alcoholism, some clinical symptoms and laboratory results.
- Association with stage of liver fibrosis by Metavir classification.
- Association with the characteristic of ALD histological lesion in
results.
2.4. Evaluation criteria used in the study
- AUDIT questionnaire - WHO was used to detect alcohol dependence
or abuse: To score the AUDIT questionnaire, sum the scores for each of the
10 questions. A total 8 for men up to age 60, or 4 for women, adolescents, or
men over age 60 is considered a positive screening test.
- Stage of liver fibrosis by Metavir classification.
2.5. Data processing
Data was coded and analyzed by SPSS software version 20.0
+ Descriptive variables: mean, median, standard deviation, min
and max value.
+ Comparing 2 proportions: χ2 test, with statistical significant
at p < 0.05.
+ Comparing 2 means: Student t-test, with statistical significant
at p < 0.05.
Chapter 3. RESEARCH RESULT
95 patients were researched in the Gastrointestinal Department of
Thai Nguyen Central Hospital and Military Hospital 103 from 1/2015
to 6/2017, the results are as follows
11
3.1. Clinical, subclinical characteristics and TNF-α, IL-12,
IL-1β, TGF-β in plasma in patients with chronic liver disease
caused by alcohol.
Table 3.1. Age characteristics in patients with ALD
Age group
Number
Ratio (%)
< 45
45-59
60-74
≥75
Tổng
average age
30
48
15
2
95
31,6
50,5
15,8
2,1
100
50,65
The age group of 45-59 was the most common, accounting for
50,5%. The average age of the researched patients: 50,65. There were
95 male patients and no female patients.
Table 3.2. Clinical characteristics in patients with ALD
Clinical characteristics Number
Ratio (%)
(n =95)
61
64,2
Anorexia
18
18,9
Hematemesis
60
63,2
Hepatomegaly
Collateral circulation
Jaundice
Vasodilatation
14
63
29
14,7
66,3
30,5
- The number of patients having hepatomegaly accounts for
63,2%. The number of patients having jaundice accounts for 66,3%.
12
Table 3.5. Evaluation of liver enzyme in patients with ALD
Liver enzyme test
Number (n=95)
Ratio (%)
Nomal
2
2,1
increasing
89
93,7
AST (U/L)
ALT < 500
≥ 500
4
4,2
198,19
±
155,05
X ± SD
Nomal
24
25,3
increasing
63
66,3
ALT (U/L)
ALT < 200
≥ 200
8
8,4
85,86
±
71,06
X ± SD
Nomal
2
2,1
GGT (U/L)
increasing
93
97,9
749,10 ± 716,18
X ± SD
The mean AST was 198,19 ± 155,05 U/L. 77,1% of patients had
increased ALT < 200 U/L. The mean ALT was 85,86 ± 71,06 U/L.
The mean GGT was 749,10 ± 716,18 U/L.
Table 3.6. Histological findings in ALD
Histological Findings in ALD
Number
(n=95)
21
Large-droplet fat
Features
of
8
Small-droplet fat
fatty liver
66
Mixed
Steatosis
grade
Location
fatty liver
5-33%
34-66%
> 66%
of Zone 1
Zone 2
Zone 3
Ratio
(%)
22,1
8,4
69,5
25
40
30
91
26,3
42,1
31,6
95,8
68
66
71,6
69,5
13
F0
F1
Fibrosis stage
F2
(n=95)
F3
F4
Significant fibrosis
Fibrosis grade (≥ F2)
(n=95)
Severe fibrosis (≥ F3)
Cirrhosis (F4)
Lipogranuloma
Foamy degeneration
Hemosederosis
Some
indicators
of Mallory-Denk
histopathology Bodies
(n=95)
Megamitochondria
Eosin hepatocellular
change
14
20
25
24
12
14,7
21,1
26,3
25,3
12,6
61
64,2
36
12
9
80
54
37,9
12,6
9,5
84,2
56,8
61
64,2
59
62,1
60
63,2
The grade of steatosis of 34%-66% accounted for 42,1%, the
fatty liver zone 1 accounted for 95,8%. Stage of fibrosis F2
accounted for 26,3%. Foamy degeneration accounted for 84,2%, the
megamitochondria accounted for 64,2%, the Mallory body accounted
for 60.2%. Significant stage of fibrosis (≥ F2) accounted for 64,2%.
14
Table 3.8. Results of TNF-α, IL-12, IL-1β, TGF-β indicators in
patients with ALD and the controls
Cytokine indicators
TNF-α
(pg/mL)
IL-12
(ng/L)
IL-1β
n
95% CI
Median
Study subjects 95
110,61 - 326,42
173,64
Controls
153,71 - 166,71
158,23
24,45 - 32,10
27,47
40
Study subjects 95
Controls
40
Study subjects 95
-
4,00
13,42 - 15,34
14,36
< 0,001
Controls
(ng/L)
< 0,001
< 0,001
(ng/L)
TGF-β
p
40
Study subjects 95
3,19 - 3,30
3,19
1126,43 - 1320,91
1191,46
< 0,001
Controls
40
79563,90- 666819,53
110829,44
There was a statistically significant decrease in the
plasma TGF-β in patients with alcoholic liver disease as
compared to controls.
There was a statistically significant increase in the plasma
TNF-α, IL-12, IL-1β in patients with alcoholic liver disease as
compared to controls.
15
3.2. The relationship between levels of TNF-α, IL-12, IL-1β,
TGF-β in plasma with clinical, subclinical features in patients
with chronic liver disease caused by alcohol.
Table 3.14. Relationship between
TNF-α
in plasma with
histological findings in ALD
Histological findings in ALD
95% CI
Median
F0
14
146,37 - 251,43
164,17
F1
20
145,81 - 190,02
155,97
F2
25
147,50 - 185,40
166,71
F3
24
149,76 - 158,80
153,72
F4
12
154,28 - 312,77
168,12
Disease
Hepatitis
83
151,74 - 166,71
157,10
stage
Cirrhosis
12
152,59 - 362,41
168,12
Fibrosis
stage
TNF-α
n
(pg/mL)
The median TNF-α decreased in the hepatitis group (157,10
pg/mL) was lower than in the cirrhosis group (168,12 pg/mL), the
difference was significant (p < 0,05).
p
> 0,05
< 0,05
16
Table 3.26. Relationship between IL-1β in plasma with
histological findings in ALD
Histological findings in
n
ALD
Median
F0
14
11,30 - 17,65
15,55
F1
20
10,88 - 19,28
16,11
F2
25
11,42 - 16,60
14,09
F3
24
10,60 - 15,23
13,22
F4
12
11,16 - 38,06
13,81
Disease
Hepatitis
83
13,21 - 15,89
14,36
stage
Cirrhosis
12
11,16 - 27,61
13,81
Fibrosis
stage
IL-1β
95% CI
(ng/L)
p
> 0,05
< 0,05
The median IL-1β in the cirrhosis group (13,81 ng/L) was lower
than in the hepatitis group (14,36 ng/L), the difference was significant
(p < 0,05).
Table 3.31. A Relationship between TGF-β in plasma with
histological findings in ALD
Histological findings in ALD
TGF-β
(ng/L)
Features of fatty
liver
Steatosis
grade
n
Largedroplet
fat
Smalldroplet
fat
95%CI
Median
988,42 - 1542,33
1246,59
1119,54 - 1460,42
1289,92
p
21
8
Mixed
66
1083,58 - 1320,91
1169,89
Mild
25
1119,54 - 1460,42
1246,59
Moderate
40
1050,03 - 1474,32
1221,43
> 0,05
> 0,05
17
Severe
30
1059,61 - 1344,88
1136,33
No
4
-
1089,58
Yes
91
1138,30 - 1320,91
1208,24
No
27
1059,61 - 1459,97
1154,26
Yes
68
1119,54 - 1344,88
1199,85
No
29
1119,54 - 1492,30
1270,57
Yes
66
1083,58 - 1321,09
1169,89
No
86
1119,54 - 1342,48
1181,87
Yes
9
1049,21 - 1474,32
1246,59
Foamy
No
36
1229,59 - 1512,69
1331,70
degeneration
Yes
59
1049,21 - 1191,46
1124,34
No
15
1145,91 - 1491,11
1239,40
Yes
80
1091,11 - 1344,88
1163,27
Mallory-Denk
No
41
1131,53 - 1343,68
1208,24
Bodies
Yes
54
1069,12 - 1370,26
1181,87
No
34
1018,86 - 1460,42
1163,27
Yes
61
1126,43 - 1344,88
1208,24
No
35
1232,21 - 1491,11
1342,48
Yes
60
1041,64
1210,07
1121,94
Zone 1
Zone 2
Zone 3
Lipogranuloma
Hemosederosis
Megamitochondria
Eosin
hepatocellular
change
-
> 0,05
> 0,05
> 0,05
> 0,05
> 0,05
> 0,05
> 0,05
> 0,05
< 0,05
The median TGF-β in the Eosin hepatocellular change group
(1121,94 ng/L) was lower than in the without Eosin hepatocellular change
group (1342,48 ng/L), the difference was significant with p < 0,05.
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