الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Chronic hepatitis C virus (HCV) infection is considered a health problem
worldwide, especially in Egypt. It is associated with systemic complications including
both hepatic and extrahepatic manifestations, so it is described as a syndrome. However,
HCV is not considered a direct cytolytic or cytopathic virus, but it causes these
complications via immunopathogenic mechanisms throughout manipulation and/or
dysregulation of innate and adaptive immune system. Moreover, HCV, as a hepatotropic
and B lymphotropic virus, utilizes B cells as reservoir during extrahepatic replication
resulting in phenotypical and functional alterations of these cells.
Interleukin 7 (IL-7) plays essential roles in precursor (pre-) B cell survival and
development into mature B cells as well as acting as an antiviral cytokine by
downregulating the suppressor of cytokine signaling 3 (SOCS3).
Furthermore, toll-like receptor 7 (TLR7) has an antiviral activity when triggered by
viral single-stranded ribonucleic acid (ssRNA) in both interferon-dependent and -
independent mechanisms. It also induces B cell survival, differentiation and cytokine
secretion. Additionally, TLR7 contributes in activation of B cells during persistent HCV
infection, as well as initiation of HCV-related autoimmune diseases and B cell
malignancies.
The aim of this study was to assess the immunomodulatory effects of treatment with
direct-acting antivirals (sofosbuvir and daclatasvir) with/without ribavirin according to the
European Association for the Study of the Liver) EASL guideline, 2016 on phenotypic
restoration/normalization of peripheral B cell subpopulation among treated patients
(n=20) who achieved SVR12, as well as untreated (treatment naïve) chronically infected
patients (n=20) and healthy volunteer (n=20) as positive and negative controls,
respectively. Also, this study aimed to evaluate the correlation of the frequencies of B cell
subsets with both IL-7 and TLR7 in these groups.
Written informed consent was taken from all recruited subjects and the study
protocol was approved by Ethical Committee of Medical Research Institute, Alexandria
University.
All participants in this study were subjected to thorough history taking, clinical
examinations, laboratory investigations (complete blood count, liver function tests,
hemostatic screening tests), virological seromarkers such as anti-HCV antibody (Ab),
hepatitis B surface antigen (HBsAg) and anti- human immunodeficiency virus (HIV) Ab to
exclude other related viral infections, as well as molecular investigations (HCV RNA).
Peripheral blood samples were collected from all subjects under study. After the separation
of mononuclear cells from the peripheral blood, total RNA was extracted and transcribed
into complementary DNA (cDNA) in order to quantify TLR7 gene expression via
Quantitative PCR (q-PCR). Moreover, the phenotypic frequencies of peripheral B cell
subpopulations were investigated by flow cytometry using appropriate mouse anti-human
monoclonal Abs (anti-CD19, anti-CD10, anti-CD21 and anti-CD27). In addition, serum
IL-7 level was assayed by enzyme-linked immunosorbent assay (ELISA) technique.
Our findings showed that there were no statistically significant differences between
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106
the frequencies of total B cells (p=0.523) as well as naïve B cells (p=0.621) among the
three studied groups.
On the other hand, there was a significant statistical difference in the frequency of
immature transitional (IT) B cells (p=0.041) between the three studied groups, being
increased in untreated patients compared to control (p=0.035).
Statistically, there was a highly significant difference in the frequency of activated
mature (AM) B cells (p=0.006) between the three studied groups, being increased in
treated HCV patients compared to control (p=0.005).
Also, the statistical difference in the frequency of resting memory (RM) B cells was
highly significant (p=0.005) between the three studied groups, being decreased in both
untreated (p=0.007) and treated HCV patients (p=0.038) compared to normal control.
The frequency of tissue-like memory (TLM) B cells also showed a highly
significant difference (p<0.001) between the three studied groups. It was more increased in
untreated HCV patients than both treated HCV patients (p=0.02) and control (p<0.001).
Concerning the total memory (TM) B cell frequency, there was a significant
difference (p=0.043) between the three studied groups, being decreased in untreated HCV
patients versus control group (p=0.034).
Also, the frequency of CD19+CD21low/- B cells showed a highly significant
difference (p<0.001) between the three studied groups. It was increased in both untreated
(p<0.001) and treated HCV patients (p<0.001) compared to normal control, and it was
increased in untreated versus treated HCV patients (p<0.001).
Our study also demonstrated that the gene expression of TLR7 showed a significant
difference (p=0.001) between the three studied groups. It was significantly downregulated
in untreated patients compared to treated patients (p=0.036) and control group (p=0.001).
Our study also showed that the serum IL-7 levels were not significantly different between
untreated HCV patients, treated HCV patients and healthy control (p=0.363).
Finally, no correlations were noted between B cell subpopulations and both serum
IL-7 levels and gene expression of TLR7, whereas a positive correlation was found with
TM B cells (r=0.44, p=0.04) among studied groups. Additionally, TM B cells were
statistically correlated with age (r= -0.345, p=0.025), serum ALT (r= -0.387, p=0.014),
serum total bilirubin (r= -0.395, p=0.012), serum albumin (r= 0.356, p=0.024) and platelet
count (r= 0.383, p=0.015). Also, naive B cells were statistically correlated with age
(r= 0.395, p=0.011), serum total bilirubin (r= 0.331, p=0.037), serum albumin (r= -0.335,
p=0.035) and platelet count (r= -0.321, p=0.043). Moreover, IT B cells, RM B cells and
TLM B cells were showed statistical significant correlations with serum total bilirubin
(r= 0.337, p=0.034), age (r= -0.337, p=0.033) and ALT (r= -0.415, p=0.007), respectively.
We concluded that the frequencies of some B cell subsets (AM B cells, RM B cells
and CD19+CD21low/- B cells) among treated HCV patients compared to both untreated
HCV patients and control were not completely normalized or restored and should be
monitored after SVR12. So, the different B cell subsets among treated patients could be
considered partially recovered after achieving SVR12.
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107
This study was provided a scientific addition about an important local filed due to the
immunomodulatory effects of DAAs on the frequencies of peripheral B cell phenotypes
and TLR7 gene expression among Egyptian HCV patients treated with DAAs. Targeting
TLR7 may be used as a potential prophylactic and/or therapeutic agents during chronic
HCV infection as well as immunopotentiation of total memory B cells. Also, we
recommended to assess TLR7 gene expression as a predictor for response to HCV
treatment and/or disease outcome including extrahepatic manifestations in further studies.
Regarding the findings of the present study, we can conclude the following:
1. Biochemical and hematologic disturbances during chronic HCV infection is
correlated with alterations and abnormalities of the phenotypes/ frequencies of
peripheral B cell compartment in Egyptian HCV patients. These altered B cell
subsets include immature/ transitional, activated mature, resting memory and tissuelike
memory.
2. The frequencies of some peripheral B cell subpopulations were partially restored
among Egyptian HCV patients treated with direct acting antivirals (sofosbuvir plus
daclatasvir) with or without ribavirin who achieved SVR12. These unrecovered B
cell subsets include both activated mature and resting memory cells.
3. Accumulation of peripheral CD19+CD21low/- B cells in both untreated and treated
HCV patients may be responsible for the variation in total memory B cell
frequencies among these groups.
4. The expression of TLR7 gene was downregulated by persistent HCV infection,
while it became normalized in HCV patients treated with DAAs, which confirms
the evasion of the virus from immunosurveillance by impairment of innate immune
receptors.
5. There was an increased level of interleukin 7 among untreated HCV patients without
a statistically significant differences compared to treated HCV patients and healthy
control. It was also not correlated with the frequencies of peripheral B cell
subpopulations, including immature/ transitional B cell subsets in all studied groups.
6. The expression of TLR7 gene didn’t correlate with the frequencies of peripheral B
cell subpopulations, except total memory B cells in all participating groups.
According to the results of current study, we can recommend the following:
1. Egyptian treated HCV patients with partially normalized frequencies of some
peripheral B cell subpopulations as well as accumulation of CD19+CD21low/- B cells
may be at risk to develop B cell-associated extrahepatic manifestations. Monitoring
B cells at several points of time after completion of treatment should be considered.
2. Further studies should be conducted to assess TLR7 gene expression as possible
predictive marker for response to HCV treatment by DAA between responders and
non-responders.
3. Targeting TLR7 by its agonists and antagonists may be used in immunoregulation
of interaction between HCV and immune system in order to limit HCV-related
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108
disorders. It should be considered as a potential prophylactic and/or therapeutic
target during chronic HCV infection, as well as immunopotentiator of total memory
B cell subsets.
4. The present study encompassed a limited number of studied groups. Evaluation of
more samples in further studies would improve the statistical validity of our data.
5. Future works should include HCV patients receiving other DAA regimens with
different stages of HCV-associated liver diseases according to periodically updated
standard HCV treatment guidelines.
6. Treatment-experienced HCV patients who are candidates for DAA treatment
protocols should be included in further studies in parallel to their counterparts of
treatment in naïve HCV patients.
7. More immunomodulatory parameters other than those included in the current study
which may shape the phenotypes and function of B cells should be investigated in
further studies to understand the molecular basis of normal physiological and
pathological B cell behavior among chronically infected HCV patients, particularly
in different anatomical sample sites.