الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Autoimmune hepatitis is an immune-mediated liver disorder characterized by female preponderance, elevated transaminases and immunoglobulin G levels, seropositivity for autoantibodies and interface hepatitis.
Early and prompt diagnosis is very important as autoimmune AIH is exquisitely responsive to immunosuppressive treatment, which should be instituted promptly to prevent rapid deterioration and promote remission and long-term survival.
Overlap/variant forms of the disease known as autoimmune hepatitis/sclerosing cholangitis overlap syndrome (ASC) has the same prevalence as AIH-1 in childhood and in spite of abnormal cholangiograms, have no histological features that suggest bile duct involvement, and the diagnosis of sclerosing cholangitis is only possible because of the cholangiographic studies.
The pathogenesis of AIH are not understood, but it is assumed that the disease is driven by a dysregulated adaptive immune response to liver antigens.
The aim of this work is to evaluate the intrahepatic status of regulatory T cells in children with autoimmune hepatitis and also the significance of the intrahepatic IgG/IgM plasma cell ratio in the diagnosis of autoimmune hepatitis.
We included 100 pediatric patients in this study (53 girls and 47 boys) and they were classified into 2 groups:
1- Autoimmune hepatitis group: included 50 patients, which were diagnosed as AIH by satisfying the international criteria for diagnosis of AIH Summary and conclusion
2- Non-autoimmune hepatitis group: included also 50 patients who diagnosed to have other chronic liver diseases to be compared with the AIH group.
These patients were selected retrospectively and prospectively from all cases attending the Pediatric Hepatology department and outpatient clinics at the National Liver Institute, Menoufiya University and their data at the time of diagnosis in the initial presentation were included to be studied.
All children in this study were subjected to the following:
1-Clinical assessments: Full history taking and thorough clinical examination.
2-Laboratory and radiological assessments:
- Complete blood count.
- Liver function tests.
- Prothrombin time and concentration.
- Serum autoantibodies: ANA, ASMA and LKM-1.
- Protein electrophoresis.
- Serum viral markers for HBV.
- Hepatitis C virus antibody (anti-HCV) in serum.
- HCV-RNA by qualitative polymerase chain reaction (PCR) in serum.
- Abdominal ultrasonography.
3-Liver biopsy and histopathological assessment:
Liver biopsies were performed for diagnostic purpose before starting the treatment.
Immunohistochemical staining of liver tissue: done by using the following antibodies: FOXP3 monoclonal antibody, plasma cell IgG monoclonal antibody, plasma cell IgM monoclonal antibody, T-cell CD4 monoclonal antibody and T-cell CD8 monoclonal antibody.
Positive immunostaining was identified by brownish discoloration of the cytoplasm and/or cell membrane. All cells morphologically consistent with
plasma cells or T cells showing unequivocal immunohistochemical expression of either FOXP3, IgM, IgG, CD4 or CD8 were counted. The absolute number of positive cells for each studied antibody was assessed. IgM/IgG ratio was analyzed.
Data were analyzed using the SPSS for Windows, version 18.0, SPSS Inc., and Chicago, Illinois, USA. Qualitative data were expressed as frequency and percent and quantitative data are shown as mean, range and SD.
The major findings in this study were:
1- Autoimmune hepatitis group included 17 males and 33 females. Twenty nine patients were type 1 AIH, 5 patients were type 2 AIH, 6 patients were diagnosed to have ASC, 2 patients had both HCV infection and AIH and 8 patients were seronegative. Patients had different clinical presentations and received different lines of treatment. Forty one patients are responder to treatment, while 9 patients didn’t show a response to treatment till the time of writing the results.
2- Non-AIH group included 30 males, 20 females with different etiologies. The most common included etiology was HCV infection (25 cases) followed by glycogen storage disease (12 cases), Wilson disease (5 cases), congenital hepatic fibrosis (2 cases), galactosemia (3 cases) and there was one case of biliary atresia, one case of giant cell hepatitis and a case of mucopolysacharidosis.
3- Female gender was statistically significant higher in the AIH group than non-AIH group (P<0.019).
4- The age of patients in the AIH group is significantly older than the non- AIH group (P=0.019).
5- Patients with AIH were divided according to response to treatment into responder (41 patients) and non-responder (9 patients) groups.
The mean of ALT level at presentation was significantly higher in the responder group; 305 IU/ml versus 160 IU/ml in the non-responder group, P=0. 028.
GGT level was significantly higher in the non-responder group (mean of 182 IU/ml versus 93 IU/ml in responder with P=0.016.
Gamma globulin level was found to be statistically significantly higher in the responder group (6.8± 9.6 gm/dl) than in the non-responder (1.6±0.7 gm/dl) with value of P=0.002.
Fibrosis stage was more significantly advanced at presentation in the non-responder group (4.5±1.2) than in responder (3.4±1.3) with value of P=0.026.
The IgG/IgM ratio was statistically significantly higher in the responder group (mean is 3±3) while in the non-responder the mean is (1.6±0.5) with value of P=0.009.
6- The serum ALT was significantly higher in the AIH patients than in non- AIH group with a mean of ALT 279±329 IU/ml in AIH and 135±173 IU/ml in the other group (P=0.007).
7- Total bilirubin levels were significantly higher in the AIH patients than in non-AIH group with a mean of bilirubin level is 3.7±3.5 mg/dl in AIH and 2±4.1 mg/dl in non-AIH patients (P=0.037).
8- Prothrombin time was found to be significantly more prolonged in AIH than in non-AIH group with a mean of PT 16.9±4.8 Sec in AIH and 13.1±1.8 Sec in non-AIH patients (P<0.001).
9- Hemoglobin level was of a mean of 10.6±1.3 gm/dl in AIH versus 11.4±1.1 gm/dl in non-AIH patients (P=0.003).
10- Platelet count was significantly lower in AIH patients than for non-AIH patients with a mean of (186±94 versus 330 ±156) X 1000/mm3 (P<0.001).
11- Histopathological evaluation of liver biopsy revealed that the histological activity index (HAI) showed a mean of 9.4±3.5 in AIH group versus 4.5±1.8 in non-AIH group with P<0.001. The stage of fibrosis revealed a mean of 3.6±1.4 in AIH group versus 2.2±1.2 in non-AIH group with P<0.001.
12- Immunohistochemistry (IHC) staining of FOXP3 in the liver tissue The mean number of FOXP3+ T cells in patients with AIH (6.3±5) was statistically significantly higher than that in the non-AIH patients (2.1±2.6) with a value of P<0.001.
13- FOXP3+ T cells were detected in liver tissue with marked inflammatory cellular infiltrate. Whereas, weak reaction or negative for FOXP3 cells was noticed in conditions associated with minimal inflammatory infiltrate.
14- The mean percentage of FOXP3+ cells to CD4+ cells was 15.6% in the AIH group while was7 % in non-AIH group.
15- Immunostaining of CD4+ cells and CD8+ cells showed that the mean number of CD4+ T cells and CD8+ T cells infiltrating the liver tissue was significantly higher in AIH patients than in the non-AIH patients with value of P<0.001.
16- IgG or IgM plasma cells were absent in some conditions of chronic liver diseases other than AIH. Weak presence of IgG positive cells was detected in some conditions of non-AIH patients. The mean number of IgG positive cells in AIH was 17.8±7.6 while was 14.8±6.9 in non-AIH patients with value of P=0.046. The mean number of IgM positive cells in AIH was 8.0±4.4 while was 7.8±4.5 in non-AIH patients with no significant difference.
17- The relation of intrahepatic frequencies of FOXP3+, CD4+, CD8+T-cells, IgG and IgM plasma cells to all the biochemical data of both AIH and non-AIH groups and also to all the patients included in this research were studied.
A- AIH group:
1- Relation of FOXP3 Treg cells to IgG plasma cells, IgM plasma cells, CD4, CD8 cells and IgG/IgM ratio and to all studied parameters:
There were statistically significant positive correlation between FOXP3 cells and IgG plasma cells; P=0.029, CD4; P<0.001 and CD8 T cells; P<0.001.
2- Relation between the mean number of IgG plasma cells and all studied parameters:
There were statistically significant positive correlation between the number of IgG plasma cells and the number of IgM plasma cells; P<0.001, FOXP3 cells; P=0.029, CD4; P=0.005 and CD8 T cells; P=0.050.
3- Correlation between the mean number of IgM plasma cells and all studied parameters:
There were statistically significant positive correlation between the number of IgM plasma cells and the number of IgG plasma cells; P<0.001, CD4 T cells; P=0. 013, CD 8 T cells; P=0. 015 and grade of activity in liver biopsy P=0. 017. Also, there was statistically significant negative correlation between the number of infiltrating IgM plasma cells and the IgG/IgM ratio; P <0.001.
4- Correlation between the mean number of CD4 T lymphocytes and the patient parameters:
There were statistically significant positive correlation between the number of CD4 T lymphocytes and the number of FOXP3 cells; P<0.001, CD8 T lymphocytes; P<0.001, IgG plasma cells; P=0.005 and IgM plasma cells; P=0.013.
5- Correlation between the mean number of CD8 T lymphocytes and the patient parameters:
There were statistically significant positive correlation between the number of infiltrating CD8 lymphocytes and FOXP3; P<0.001, CD4 lymphocytes; P<0.001, IgG; P=0.050 and IgM plasma cells; P=0.015.
6- Correlation between the IgG/IgM ratio and all the studied parameters:
There were statistically significant positive correlation between the IgG/ IgM ratio and AIH score; P=0.023 and statistically significant negative correlation between this ratio and number of infiltrating IgM plasma cells; P<0.001.
B- Non-AIH group:
1- Correlation between the mean number of Foxp3 cells and all the patient parameters:
There were statistically significant positive correlation between the FOXP3 cells and the CD4; P <0.001 and CD8 T lymphocytes; P=0.002, IgG; P=0.057 and IgM plasma cells; P=0.023, total bilirubin; P=0.034 and direct bilirubin level; P=0.010.
2- Correlation between the mean number of CD4 T lymphocytes and all the studied parameters:
There were statistically significant positive correlation between the CD4 T lymphocytes and FOXP3 cells; P<0.001, CD8 T lymphocytes; P< 0.001 IgG; P<0.001 and IgM plasma cells; P<0.001. Also, there was
statistically significant negative correlation between the number of infiltrating CD4 lymphocytes and the white blood cell count; P=0. 016.
3- Correlation between the mean number of CD8 T lymphocytes and all patient parameters:
There were statistically significant positive correlation between the CD8 T lymphocytes and FOXP3 cells; P=0.002, CD4 T lymphocytes; P<0.001, IgG; P<0.001 and IgM; P<0.001 plasma cells. Also, there was statistically significant negative correlation between the number of infiltrating CD4 lymphocytes and the white blood cell count; P=0. 007.
4- Correlation between the mean number of IgG cells and all the studied parameters:
There were statistically significant positive correlation between the IgG plasma cells and FOXP3 cells; P=0.057, IgM plasma cells; P=0.01, IgG/ IgM ratio; P<0.001, CD4; P<0.001 and CD8 T lymphocytes; P<0.001.
5- Correlation between the mean number of IgM plasma cells and all the studied parameters:
There were statistically significant positive correlation between the number of IgM cells and FOXP3 cells; P=0.023, IgG plasma cells; P=0.011, CD4; P<0.001 and CD8 T lymphocytes; P<0.001 and statistically significant negative correlation between the IgM plasma cells and IgG/IgM ratio; P< 0.001.
6- Correlation between the IgG/IgM ration and all the studied parameters:
There were statistically significant positive correlation between the IgG/IgM ratio and the IgG plasma cells; P<0.001 and negative correlation between this ratio and the IgM plasma cells; P<0.001.
C -AIH and non-AIH groups:
1- Relation of FOXP3 Treg cells to IgG plasma cells, IgM plasma cells, CD4, CD8 cells and IgG/IgM ratio and to all studied parameters of both AIH and non-AIH groups:
There were statistically significant positive correlation between FOXP3 and IgG (P<0.001), IgM (P=0.013), CD4 (P<0.001), CD8 (P<0.001), ALT (P=0.016), AST (P=0.002), T-BIL (P<0.001), D-BIL (P<0.001), PT (P =0.006). Grade of necroinflammatory activity P<0.001 and stage of fibrosis P=0.024. There were also statistically significant negative correlation between FOXP3, Hb level (P=0.009) and platelet count (P=0.038).
2- Relation of IgG plasma cells to IgM plasma cells, FOXP3, CD4, CD8 cells and IgG/IgM ratio and to all studied parameters of both AIH and non-AIH groups:
The predominance of IgG plasma cells in liver tissue is associated with the histologically detected an increased number of FOXP3 cells (P<0.001), IgM plasma cells (P<0.001), CD4 T cells (P<0.001), CD 8 T cells (P<0.001) and increased ratio of IgG plasma cells to the IgM cell ratio (P =0. 004) and increase the degree of activity in liver biopsy in all patients at initial diagnosis (P=0. 004).
3- Relation of IgM plasma cells to IgG plasma cells, FOXP3, CD4, CD8 T-cells, IgG/IgM ratio and to all the studied parameters of both AIH and non- AIH groups:
The number of IgM plasma cells is significantly correlated positively with the number of FOXP3 cells (P=0.013), IgG plasma cells
(P<0.001), CD4 cells (P<0.001), CD8 cells (P<0.001), the ratio of IgG plasma cells to IgM plasma cells (P<0.001) and to the grade of necroinflammatory activity in liver tissue (P=0.021).
4- Relations of the CD4 T-cells to the CD8 T- cells, FOXP3 cells, IgG plasma cells, IgM plasma cells, IgG/IgM ratio and to all studied parameters of both AIH and non-AIH groups:
The increased number of infiltrating CD4 T-lymphocytes were associated with increase infiltrations of CD8 T-lymphocytes (P<0.001), FOXP3 cells (P<0.001), IgG plasma cells (P<0.001) and IgM plasma cells (P<0.001), increased grade of necroinflammatory activity in liver tissue (P< 0.001), the age of patients at presentation (P=0. 021), T-Bil level (P=0. 046) and PT (P=0. 017). While negative correlation was observed between the number of CD4 T lymphocytes and the number of platelets (P=0. 036).
5- Relations of the CD8 T-cells to the CD4 T- cells, FOXP3 cells, IgG plasma cells, IgM plasma cells, IgG/IgM ratio and to all studied parameters of both AIH and non-AIH groups:
The increased number of infiltrating CD8 T-lymphocytes were associated with increased infiltrations of CD4 T-lymphocytes (P<0.001), FOXP3 cells (P<0.001), IgG plasma cells (P<0.001) and IgM plasma cells (P<0.001), the grade of activity in liver tissue (P<0.001), the age of patients at presentation (P=0.021), T-Bil level (P=0.021) and PT (P=0.024). While negative correlation was observed between the number of CD4T lymphocytes and platelet number (P=0.022).
Cutoff values of the immunological markers between AIH and non-AIH groups:
1- It was found that the number of intrahepatic Foxp3 cells in portal tracts can differentiate between the AIH and other chronic liver diseases with a number more than 2.5/HPF favor for diagnosis of AIH and below in favor with a diagnosis of liver diseases other than AIH (P<0.001) with an accuracy of 73% and a sensitivity of 82% and specificity of 64%.
2- Intrahepatic CD4 T-lymphocytes number more than 26.5/HPF supports the diagnosis of AIH (P<0.001) and number below this is in favor of diagnosis of other chronic liver disease with an accuracy of 71% sensitivity of 84% and specificity of 54%.
3- The number of intrahepatic CD8 T-lymphocyte more than 15.5/HPF support the diagnosis of AIH (P<0.001) and number below this favor of diagnosis of other chronic liver disease with accuracy of 73% with sensitivity of 82% and specificity of 64%.
4- The number of IgG plasma cells in portal tracts more than 11.5/HPF predict the diagnosis of AIH (P=0. 038) while a lesser number suggests the presence of other CLD with an accuracy of 58% with sensitivity of 84% and specificity of 32%.
In conclusion:
1- Intrahepatic regulatory T cells were not decreased in frequency in patients with AIH in the initial presentation at diagnosis, but they were increased in number and their presence is associated with increased activity and inflammation in liver biopsy.
2- Serum level of ALT, GGT, gamma globulin and intrahepatic IgG/IgM ratio and fibrosis stage in liver biopsy can help in early, prediction of the non-responder patients to be considered for early shift to the second line in treatment or referred early for liver transplantation. 3- The IgG/IgM ratio doesn’t differentiate between AIH and other chronic liver diseases, but it may differentiate between cases of AIH and ASC if a larger group of patients with ASC were studied.
4- The cutoff level of the intrahepatic FOXP3, CD4, CD8 and IgG cells may be used for confirmation of some cases difficult to be diagnosed and in the near future they can be included in a pediatric scoring system.