|Year : 2019 | Volume
| Issue : 3 | Page : 124-130
Hepatic expression of integrin β-8 protein as a risk factor in infants with biliary atresia
Mohammed A Khedr1, Nermin M Adawy1, Behairy E Behairy1, Nermine A Ehsan2, Ahmed El-Refaei2, Fatma E.-Z.S Abou-Shaaban1
1 Department of Pediatric Hepatology, Gastroenterology and Nutrition, National Liver Institute, Menoufia University, Menoufia, Egypt
2 Department of Pathology, National Liver Institute, Menoufia University, Menoufia, Egypt
|Date of Submission||13-May-2019|
|Date of Decision||10-Jun-2019|
|Date of Acceptance||10-Jun-2019|
|Date of Web Publication||27-Apr-2020|
Nermin M Adawy
Department of Pediatric Hepatology, Gastroenterology and Nutrition, National Liver Institute, Menoufia University, Menoufia, National Liver Institute 32511
Source of Support: None, Conflict of Interest: None
Introduction Biliary atresia (BA) is a progressive obliteration of the extrahepatic bile ducts that affects as many as 1 in 5000 children in certain areas of the world.
Aim The aim was to study the expression of integrin β-8 in patients with BA and compare it with patients with non-BA cholestasis.
Patients and methods This study was conducted on 30 infants with BA and 30 infants with cholestasis other than BA. Integrin β-8 immunohistochemical (IHC) staining was done to all studied groups.
Results In this study, infants with BA had statistically significant more integrin β-8 expression than non-BA patients (P=0.001) with significantly higher expression at IHC score 3 (P<0.0001). While non-BA infants had significantly higher expression at low IHC score 1 (P<0.0001) integrin β-8 IHC score at a cutoff value greater than 2 could discriminate between BA and non-BA groups that had 0.729 area under the receiver-operating characteristic curve with 83.3% sensitivity, 60% specificity, and 71.6% accuracy.
Conclusion Integrin β-8 expression is significantly more expressed in patients with BA than non-BA patients and can significantly discriminate BA at an IHC score of greater than 2 from other causes of neonatal cholestasis.
Keywords: biliary atresia, cholestasis, integrin
|How to cite this article:|
Khedr MA, Adawy NM, Behairy BE, Ehsan NA, El-Refaei A, Abou-Shaaban FE. Hepatic expression of integrin β-8 protein as a risk factor in infants with biliary atresia. Alex J Pediatr 2019;32:124-30
|How to cite this URL:|
Khedr MA, Adawy NM, Behairy BE, Ehsan NA, El-Refaei A, Abou-Shaaban FE. Hepatic expression of integrin β-8 protein as a risk factor in infants with biliary atresia. Alex J Pediatr [serial online] 2019 [cited 2020 Jun 2];32:124-30. Available from: http://www.ajp.eg.net/text.asp?2019/32/3/124/283325
| Introduction|| |
Biliary atresia (BA) is a progressive obliteration of the extrahepatic bile ducts that affects as many as 1 in 5000 children in certain areas of the world, always within the first 3 months of life . There is no medical treatment for the disease, and treatment options are limited to surgical Kasi portoenterostomy, with nearly 90% of patients requiring eventual liver transplant . Many theories were postulated to explain the etiology of BA, including viral infection, abnormal morphogenesis, and autoimmunity, though none has been proven .
Hepatic fibrosis leading to cirrhosis is the major morbidity in patients with BA. This fibrosis is due to an imbalance in the extracellular matrix breakdown and deposition .
Integrins are a group of cell adhesion molecules, which work through other receptors such as cadherins, the immunoglobulin superfamily cell adhesion molecule, selectins and syndecans to mediate cell–cell and cell–matrix interaction. Ligands for integrins include collagen, fibronectin, vitronectin, and laminin .
Integrins can bind and activate latent, transforming growth factor-β (TGF-β) complexes . TGF-β is the main regulator in chronic liver disease, contributing to nearly all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma .
| Aim|| |
The study aimed to study the expression of integrin β-8 in patients with BA and compare it to patients with cholestasis due to other causes.
| Patients and methods|| |
This diagnostic test accuracy study was conducted on 60 infants with cholestatic liver disease admitted to the Pediatric Hepatology Department, National Liver Institute (NLI), Menoufia University, Egypt. Thirty patients with BA (diagnosed according to the biliary scoring system  and confirmed by intraoperative cholangiogram) and 30 patients with cholestasis due to diseases other than BA were included. The study was conducted from January 2015 to January 2016, as the rate of diagnosis of BA in our department is about 18 patients per year. Kokkat et al.  reported that there is no difference in histopathological findings between fresh and frozen samples; so, we completed our data from inpatient medical files of the previous years. Files with incomplete medical data were excluded.
The parents of each infant signed an informed consent. The study was approved by the Research Ethics Committee of National Liver Institute, Menoufiya University, Egypt, in accordance with the Declaration of Helsinki, 1975, and its updates.
Paraffin blocks and corresponding slides for all liver biopsies from cases included in this study were retrieved from the Archives of the Pathology Department, NLI. The sections were stained in Hematoxylin and Eosin, Masson’s trichrome, Perls’, orcein, and periodic acid-Schiff. Reexaminations of histological sections were performed and only sections that contained more than 10 portal tracts were included in the study.
Histological evaluation of BA cases was performed based on the scoring system proposed by Lee and Looi . Liver fibrosis was categorized into mild for stages 1, 2; moderate for stages 3, 4; and severe for stages 5, 6. Non-BA cases were evaluated histologically for any pathological changes in portal tract and hepatic parenchyma.
Sctions of 4-µm thickness were cut from paraffin-embedded, formalin-fixed liver tissues, and placed on positively charged slides. Each paraffin section was deparaffinized and rehydrated through a graded series of ethanol. Antigen retrieval was performed by steaming the slides in an appropriate buffer at different temperatures. Endogenous peroxidase was blocked using a 3% H2O2 methanol solution. The immunostaining technique applied in this study was the improved **horseradish peroxidase amplified system ,.
The presence of integrin β-8 was identified by immunohistochemical (IHC) expression of the rabbit anti-human polyclonal antibody (clone NBPI-87447; Novusbio, Santa Cruz, California, USA) in a dilution of 1 : 150 in citrate buffer pH 6.0. Visualization of immunoreactive, positive integrin β-8 was performed using the En Vision System (Dako, Cambridge, UK) Integrin B8 (H-160)sc-25714(lot HO414) Rabbit polyclonal IgG with 3, 3′ diaminobenzidine as a chromogen, according to standard protocols. IHC results were evaluated on anonymized tissue sections without knowing the clinical or histological diagnosis .
Interpretation of immunostaining
Immunoreactive positive cells of integrin β-8 were identified by brownish discoloration of the cytoplasm. Endothelial cells lining the liver vasculature (blood sinusoids, portal vein, and hepatic artery) as well as bile duct epithelium were examined for immunoreaction. Ten fields were examined per liver section for each case. Interpretations of immunoreactive cells were graded on a 4-scale IHC score depending on the intensity of the chromogen (3, 3′ diaminobenzidine) brown coloration. Score 0 was assigned for negative staining. Score 1 was assigned for a faint brown color of immunoreaction. Score 3 was assigned for the strongest brown color obtained from the immunoreaction. Score 2 showed brown color of intermediate intensity . As all the cases showed positive immunoreaction for hepatic vasculature, the percentage of positivity was 100%. For the calculation of IHC score, the percentage was considered 1 as it was equal in all cases (100%) and the final results relied on the intensity score (1, 2, or 3). The results had been checked by two histopathologists who agreed perfectly on all 60 specimens.
Data were collected and entered into a computer using the Statistical Package for the Social Sciences program for statistical analysis version 18 (SPSS Inc., Chicago, Illinois, USA). Data were expressed as numerical or categorical, as appropriate. Data were not normally distributed. Quantitative data were shown as median (interquartile range) (25th−75th percentile). Qualitative data were expressed as frequency and percentage. χ2-test (odd’s ratio and Yates’ continuity) correction was used to measure the association between qualitative variables. Comparisons between two studied independent non-normally distributed subgroups were done using the Mann–Whitney U test. The diagnostic performance of IHC scores were measured as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), and all were expressed as percentages. The cutoff for optimal clinical performance was determined from the receiver-operating characteristic curve. Accuracy was assessed mathematically through the following equation: Accuracy=TP+TN/TP+TN+FP+FN, where TP is true positive, FP false positive, TN true negative, and FN refers to false negative. An α level was set to 5% with a significance level of 95% .
| Results|| |
This study included 60 patients: 30 patients diagnosed with BA (the BA group) and 30 patients with cholestasis due to causes other than BA (the non-BA group). This group comprised 25 infants with Progressive Familial Intrahepatic Cholestasis (PFIC), two infants with cytomegalovirus infection, two infants with Alagille syndrome, two infants with resolving hepatitis, one infant with idiopathic neonatal hepatitis, one infant with congenital hepatic fibrosis, one infant with glycogen storage disease type IV, and one infant with inspissated bile syndrome. Both groups were age and sex matched (P>0.05). The median (interquartile range) age of the BA group at diagnosis was 55 (44–67.5) days, and that of the non-BA group was 60 (37.5–90) days. The BA group included 12 males (40%) and 18 females (60%), while the non-BA group included 18 males and 18 females (60%) in the BA group, respectively.
On assessing the clinical data, clay-colored stool was found in 100% of the BA group compared with 20% of the non-BA group and the difference was statistically significant (P<0.05).
The majority of patients (86.6 in BA group, 80% in non-BA) had hepatomegaly, while splenomegaly was detected in 26.6, 20% of patients, respectively. Also, no significant difference was found between alanine aminotransferase and aspartate aminotransferase in the two groups.
However, the median serum γ-glutamyl transferase level was significantly higher in the BA group reaching 827 (449–1276) U/l compared with 94.5 (46.5–160) U/l in the non-BA group (P<0.05) ([Table 1]).
By ultrasonography, this study showed that 87.5% of the BA group had hepatic subcapsular flow compared with 36.8% of the non-BA group, which was significant (P<0.05). In the same context, an abnormal GB sonography (non-contractile or atretic GB) was found in 96.7% of the BA group compared with 20% in the non-BA group (P≤0.05) ([Table 2]).
Histopathological examination in the current study showed that there was a significant difference between BA and non-BA groups in portal ductal proliferation, presence of bile plug in portal ductules, portal–portal bridging, and neutrophil infiltrate (P<0.0001). The odd’s ratio values [confidence interval (CI) 95%] were 18.02 (2.631–123.496), 3.361 (1.791–6.307), and 15.615 (2.286–106.681), respectively.
This study showed higher grades of portal fibrosis in BA cases compared with the non-BA group ([Table 3]).
|Table 3 Histopathological findings in liver biopsy of the studied groups|
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Our results showed that patients with BA had statistically significant higher integrin β-8 IHC score than non-BA patients (P=0.001). Higher expression of low-scale 1 integrin β-8 was predominant in non-BA patients (P<0.0001, OR: 5.091, 95% CI: 1.366–18.872) and higher scale 3 integrin β-8 in BA (P=0.001) and is considered a risk factor for BA (OR: 2.416, 95% CI: 1.445–4.029) ([Table 4] and [Figure 1]).
|Table 4 Comparison of integrin β-8 immunohistochemical score between the studied groups|
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|Figure 1 (a) Immunostaining of integrin β-8 in a case of biliary atresia showing bile duct epithelium immunoreaction (arrows) and endothelial cells lining the portal tract blood vessels (arrowhead) [(a1) magnification ×200 and (a2) magnification ×400]; (b) immunostaining of integrin β-8 in another case of biliary atresia showing focal expression (immunoreaction) of bile duct epithelium (arrows) and endothelial cells lining portal tract blood vessels (arrowhead) (magnification ×200); (c) immunostaining of integrin β-8 in a case of nonbiliary atresia showing negative immunoreaction for bile duct epithelium (arrows) and endothelial cells lining the portal tract blood vessels (arrowhead) (magnification ×200).|
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In this study, a significant statistical difference had been found between the stages of liver fibrosis and integrin β-8 (P<0.005) ([Table 4]). IHC score 3 had significantly more numbers of patients with late fibrosis (P<0.0001) and considered as a risk factor for it. Patients with IHC score 3 are 13.778 times more susceptible to the late stages of fibrosis than other scores; this may suggest the impact of integrin β-8 in fibrosis in the two studied groups.
Integrin β-8 IHC score at a cutoff value greater than 2 could discriminate between BA and non-BA groups. It had area under the receiver-operating characteristic curve of 0.736 with 83.3% sensitivity, 60% specificity, 67.6% PPV, 78.3% NPV, and 71.6% accuracy ([Figure 2]).
|Figure 2 Receiver-operating characteristic curve for integrin β-8 immunohistochemical score for differentiation between biliary atresia and nonbiliary atresia patients.|
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| Discussion|| |
Integrins and other cell adhesion molecules regulate numerous physiological and pathological mechanisms by mediating the interaction between cells and their extracellular environment. Although the significance of integrins in the evolution and progression of certain cancers is well recognized, their involvement in nonmalignant processes, such as organ fibrosis or inflammation, is only beginning to emerge .
In this study, the presence of clay stool, positive hepatic subcapsular flow, abnormal GB sonography (noncontractile or atretic GB), absence of bile in duodenal tube aspirate, and elevated serum γ-glutamyl transferase were the hallmarks in differentiating the BA and non-BA groups.
Portal ductal proliferation, bile plug in portal ductules, portal–portal bridging and neutrophil infiltrate are the main histopathological markers in BA. In accordance with these results, Rastogi et al.  have reported that portal tract changes, including ductular proliferation and bile plugs, were significantly more frequent in BA compared with other disorders causing cholestasis.
Our data has shown that BA patients had more advanced fibrosis grades than the non-BA group. Iordanskaia et al.  have found that patients with BA showed significantly more fibrosis than controls. The overexpression of integrin β8 IHC in BA than non-BA suggests a potential role for integrin ανβ8 in the pathogenesis of human BA. In agreement with the findings of Iordanskaia et al. , who proposed that integrin ανβ8 protein expression is increased in liver specimens of patients with BA, IHC evaluation showed increased integrin ανβ8 protein expression when compared with controls (2.67±0.81 vs 1.72±0.62, P<0.005).
Both integrin ανβ6 and integrin ανβ8 form high-affinity bonds with TGF-β, leading to more efficient activation of TGF-β . Interestingly, integrin ανβ8 activation of TGF-β is influenced by interactions with MMP-14 and likely other MMPs , which are also dysregulated in human BA .
In this study, a significant statistical difference had been found between the stages of liver fibrosis and integrin β8 with a higher score 3 in late fibrosis (P<0.0001) and is considered as a risk factor (OR: 13.778, 95% CI: 2.094–44.053). This implies that integrin overexpression is part of a uniform and coordinated process involved in the progression of liver fibrosis regardless of the underlying etiology. But surprisingly, only a few integrin inhibitors have been tested as potential antifibrotic agents so far .
Integrin β-8 IHC score at a cutoff value greater than 2 could discriminate between BA and non-BA groups with 83.3% sensitivity and 60% specificity.
A limitation of this study is the difficulty of obtaining liver biopsies from healthy infants.
| Conclusion|| |
Integrin β-8 is significantly overexpressed in patients with BA than non-BA patients and can discriminate BA at an IHC score of greater than 2 from other causes of neonatal cholestasis and stages of liver fibrosis. Integrin β-8 is implicated in the evolution of liver fibrosis and, therefore, its targeting represents an attractive concept of therapeutic intervention.
Mohammed A. Khedr, Nermin M. Adawy, Behairy E. Behairy contributed in study concept and design; Mohammed A. Khedr, Nermin M. Adawy, Fatma El-Zahraa S. Abou-Shaaban contributed in recruitment of patients, clinical management and follow-up, and contributed to data acquisition; Nermine A. Ehsan, Ahmed El-Refaei contributed in histopathological examination; Mohammed A. Khedr contributed in statistical analysis and designed the figures; Mohammed A. Khedr, Nermin M. Adawy, Behairy E. Behairy contributed to data interpretation; Mohammed A. Khedr, Nermin M. Adawy, Behairy E. Behairy contributed in writing the manuscript, all the authors reviewed the manuscript and finally approved it for submission; SAM is the guarantor.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]