1,HBeAg Can Up Regulate The Frequency Of Regulatory T Cell In Cord Blood 2,Factors Of Elevated ALT In CHB Patients Treated With Nucleos(t)ide Analogues For 48 Weeks

Objective:To detect the frequency of regulatory t cell(Treg) in cord blood and to analyse relationship between HBeAg and Treg.Methods: According to mother's HBeAg status, 63 cord blood of neonates delivered by HBsAg positive mothers were divided into the positive HBeAg group(n=33) and the negative HBeAg group(n=30), 22 cord blood of neonates come from HBsAg negative mothers as normal control. The cord blood was immediately acquired with disposable sterilized syringe after the umbilical cord was separated from neonates and the placental was striped. Cord blood monocytes(CBMCs) were got with the red blood cells lysis method. Tregs were captured as CD4⁺CD25+CD127low/- cells , myeloid dendritic cells (mDCs) were captured as lineage-HLADR+ CD11c+ cells, plasmacytoid dendritic cells(pDCs) were captured as lineage-HLADR+ CD123+ cells, CD4⁺ effector T cells were captured as CD3⁺CD4⁺ cells and CD8+ effector T cells were captured as CD3⁺CD8+ cells. CBMCs were run and analyzed with three-color immunofluorescence by flow cytometry.HBV serum makers and the level of HBV-DNA both in cord blood and mother's peripheral blood were detected using Electro-Chemiluminescence Immunoassay and Real-time quantitative Polymerase Chain Reaction, respectively.Results: In the HBeAg positive group ,72.7%(24/33) cases of cord blood were HBeAg positive, and none of them were positive in the HBeAg negative group.All of the cord blood HBVDNA were negative. The frequency of Treg in HBeAg positive group, HBeAg negative group and normal control were (7.02±1.46)%, (6.24±1.36)% and(6.01±0.82)%, respectively. Compared with the HBeAg negative group and the health controls , The percentage of Treg increased significantly in HBeAg positive group(P<0.05), but it was not dramatically different between in the HBeAg negative group and the health controls(P>0.05). The percentage of mDC in the three groups were (2.24±0.22)%, (2.45±0.32)% and (1.94±0.24)%,respectively. The percentage of pDC in the three groups were (1.67±0.99)%, (1.64±0.63)% and (1.22±0.67)%, respectively. The percentage of CD4⁺T cell in the three group were (71.37±9.63)%, (74.57±9.67)% and (71.38±10.9)%,respectively. The percentage of CD8⁺T cell in the three group were (24.04±9.47)%, (21.5±6.83)% and (22.62±11.5)%, repectively. The ratio of CD4⁺ and CD8⁺ T cells were (3.68±0.22), (4.05±0.2) and (4.22±0.34), respectively. Among these three groups the percentage of mDC, pDC, CD4⁺/CD8⁺ effector T cell and the ratio of CD4⁺ T cell and CD8⁺ T cell were not remarkably different(P>0.05). There was no relationship between the percentage of Treg in cord blood and mother's serum HBVDNA level(r=-0.171, P>0.05).Conclusion: HBeAg can pass through the placenta and cause HBV persistent infection after the children were born by up regulating the frequency of Tregs in cord blood. Aims: To study the factors of alanine aminotransferase(ALT) level elevation in chronic hepatitis B(CHB)patients treated with nucleos(t)ide analogues for 48 weeks.Methods: CHB patients who were treated with nucleos(t)ide analogues at least 48 weeks in our department between January 2006 to August 2008 were included in this study. Part of them had liver biopsy either at baseline or at week 48. According to their serum ALT level at week 48 patients were divided into ALT normal group and ALT abnormal group. ALT normal was difined as all values within the normal range,with intervals of more than two months over a period of 6 months, and ALT abnormal was difined as all values above the upper limit of normal, with intervals of more than two months over a period of 6 months. Virology breakthrough was defined as increase in serum HBV DNA by> 1 log₁₀(10-fold)above nadir after achieving virologic response during continued treatment at least two determinations more than 4 weeks apart. In addition, patients with negative HBVDNA(PCR) level at week 48 were further divided into HBVDNA negative ALT normal group and HBVDNA negative ALT abnormal group. HBVDNA negative was difined as HBVDNA value was sustained lower than 1000 copies/ml for at least 24 weeks after treatment. A comparison of the demographical, virological and histological features between those groups were performed. Binary logistic regression analysis were used to find predictors of significant elevated ALT at week 48.Results:1,Factors of ALT level elevation for all patients371 CHB patients were enrolled in our study. 17 of them were treated with telbivudine(LDT), 226 with adefovir dipivoxil(ADV), 128 with entecavir(ETV). Males rate was 78.7%. HBeAg positive rate was 75.2%. The mean age of the patients was (31.3±8.3) years old and their body mass index(BMI)was (21.8±2.8)kg/m². Mean ALT levels were (136.7±86.3)U/L and HBVDNA levels were (7.4±2.1)log₁₀copies/ml. The range of liver inflammation grade and fibrosis stage was from 0 to 4, and the medium value was 2.Of the total patients 264 were in ALT normal group and 107 were in ALT abnormal group. In these two groups, the rates of male were 73.1% and 92.5%. The ages were (32.1±8.1)and( 31.4±8.2)years old. At baseline BMI were (21.8±2.9)and (23.2±3.4) kg/m², ALT levels were (147.3±85.1)and(129.6±82.1)U/L,HBVDNA levels were (7.6±1.4)and(7.7±1.4)log₁₀copies/ml, the rates of HBeAg positive were 74.6% and 76.6%. A total of 177 patients received liver biopsy at baseline . The medium inflammation grade and fibrosis stage were 2(range from 0 to 4) and the distribution were as G0-2 (74.8% vs 76.3%),G3-4(25.2% vs 23.7%),S0-2(69.8% vs 76.3%) and S3-4(30.2% vs 23.7%). The rates of liver steatosis were 16.5% and 18.4%.At week 48, the rates of detectable serum HBVDNA level were 35.2% and 58.9%, virology breakthrough were 0.76% and 11.2%, HBeAg seroconversion or loss were 21.8% and 11%. 62 of 177 patients received second liver biopsy at week 48. The medium inflammation grade and fibrosis stage were 2(range from 0 to 4) and the distribution were as G0-2 (88.7% vs 88.9%),G3-4(11.3% vs 11.1%),S0-2(83% vs 77.8%) and S3-4(17% vs 22.2%). The rates of liver steatosis were 17% and 55.6%.For HBeAg positive patients, the rates of ALT normalization in patients treated with ADV and ETV were 66% and 79.4%, respectively. For HBeAg negative patients, they were 78.1% and 61.5%, respectively.According to univariate analysis, the rate of male, BMI at baseline and the rate of dectectable HBVDNA, virology breakthrough and liver steatosis at week 48 in ALT normal group were less than those of in ALT abnormal group(P<0.05). However, the rate of HBeAg seroconversion and the degree of liver inflammation improvement were markedly higher in the ALT normal group(P<0.05). The baseline characteristics as for mean age, serum ALT levels, HBV viral load, the rate of HBeAg positive, the distribution of liver inflammation grade and fibrosis stage and the rate of liver steatosis were compatible in these two groups(P>0.05). For HBeAg positive patients, the rate of ALT normalization was higher in patients treated with ETV than those patients treated with ADV(P<0.05). However, for HBeAg negative patients, it was not dramatically different in patients using those two kind of durgs(P>0.05).The logistic regression analysis revealed that male, BMI≥23 kg/m² at baseline, HBVDNA≥105copies/ml, virology breakthrough, liver steatosis at week 48 were independent predictors of elevated ALT, but HBeAg seroconversion or loss during the treatment and HBVDNA decreased higher than 4 log₁₀ copies/ml were protective factors of normalized ALT for all the patients. For the HBeAg positive patients, compared to ETV using ADV was a risk factor associated with elevated ALT.2,Factors of ALT level elevation for HBVDNA negative patients at week 48A total of 215 patients were HBVDNA negative at week 48, 8 of them were treated with LDT, 98 with ADV, 109 with ETV. Males rate was 77.2%. HBeAg positive rate was 58.6%. The mean age of the patients was (33.5±8.4) years old and their BMI was(22.8±3)kg/m². Mean ALT levels were (156±90.3)U/L and HBVDNA levels were (7.4±1.3)log₁₀copies/ml. The range of liver inflammation grade and fibrosis stage was from 0 to 4, and the medium value was 2. Of the 215 patients 171 were in HBVDNA negative ALT normal group and 44 were in ALT abnormal group. In these two groups, the rates of male were 72.5% and 95.5%. The ages were (34±8.6)and (33±8.5)years old. At baseline BMI were (22±2.9)and (23.6±3.1) kg/m², ALT levels were (152.2±88.2)and(163±102)U/L, HBVDNA levels were (7.4±1.5)and(7.4±1.2)log₁₀copies/ml, the rate of HBeAg positive were 58.8% and 59.1%. 110 of 215 patients received liver biopsy at baseline . The medium inflammation grade and fibrosis stage were 2(range from 0 to 4) and the distribution were as G0-2 (71.8% vs 80%),G3-4(28.2% vs 20%),S0-2(65.9% vs 76%) and S3-4(34.1% vs 24%). The rates of liver steatosis were 17.6% and 28%.At week 48, rates of HBeAg seroconversion or loss were 31% and 26.9%. 39 of 110 patients received second liver biopsy at week 48. The medium inflammation grade and fibrosis stage were 2(range from 0 to 4) and the distribution were as G0-2 (82.4% vs 80%),G3-4(17.6% vs 20%),S0-2(79.4% vs 80%) and S3-4(20.6% vs 20%). The rates of liver steatosis were 14.7% and 60%.For HBeAg positive patients, the rates of ALT normalization in patients treated with ADV and ETV were 82.5% and 78.3%, respectively. For HBeAg negative patients, they were 89.7% and 61.5%, respectively.According to univariate analysis, the rate of male, BMI at baseline and liver steatosis at week 48 in the HBVDNA negative ALT normal group were less than those of ALT abnormal group(P<0.05). However, the baseline characteristics as for mean age, serum ALT levels, HBV viral load, the rate of HBeAg, the distribution of liver inflammation grade and fibrosis stage, the rate of liver steatosis, the rate of HBeAg seroconversion and the degree of liver inflammation improvement at week 48 were compatible in these two groups(P>0.05). For HBeAg negative patients, the rate of ALT normalization was higher in patients treated with ADV than those patients treated with ETV(P<0.05). However, for HBeAg positive patients, it was not dramatically different in patients using those two kind of durgs(P>0.05).The logistic regression analysis revealed that for the HBVDNA negative patients, only male, BMI≥23 kg/m² at baseline and liver steatosis at week 48 were independent predictors of elevated ALT , and for those HBeAg negative patients compared to ETV using ADV was a protective factor associated with normal ALT.Conclusions: Our data indicate that male, overweight at baseline and HBVDNA≥10⁵copies/ml, virology breakthrough, the reduction of HBVDNA<4 log₁₀copies/ml, sustained HBeAg positive and liver steatosis at week 48 might be factors associated with elevated ALT levels in CHB patients treated with nucleos(t)ide analogues at least 48 weeks.