Macrophage heterogeneity role in NAFLD and NASH disease progression / 中华肝脏病杂志

Nonalcoholic fatty liver disease (NAFLD) is a type of metabolic stress liver injury that is closely associated with insulin resistance and genetic susceptibility. The continuum of liver injury in NAFLD can range from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and even lead to cirrhosis and liver cancer. The pathogenesis of NAFLD is complicated. Pro-inflammatory cytokines, lipotoxicity, and gut bacterial metabolites play a key role in activating liver-resident macrophages (Kupffer cells, KCs) and recruiting circulating monocyte-derived macrophages (MoDMacs) to deposit fat in the liver. With the application of single-cell RNA-sequencing, significant heterogeneity in hepatic macrophages has been revealed, suggesting that KCs and MoDMacs located in the liver exert distinct functions in regulating liver inflammation and NASH progression. This study focuses on the role of macrophage heterogeneity in the development and occurrence of NAFLD and NASH, in view of the fact that innate immunity plays a key role in the development of NAFLD.

A survey of loss function of medical image segmentation algorithms / 生物医学工程学杂志

Medical image segmentation based on deep learning has become a powerful tool in the field of medical image processing. Due to the special nature of medical images, image segmentation algorithms based on deep learning face problems such as sample imbalance, edge blur, false positive, false negative, etc. In view of these problems, researchers mostly improve the network structure, but rarely improve from the unstructured aspect. The loss function is an important part of the segmentation method based on deep learning. The improvement of the loss function can improve the segmentation effect of the network from the root, and the loss function is independent of the network structure, which can be used in various network models and segmentation tasks in plug and play. Starting from the difficulties in medical image segmentation, this paper first introduces the loss function and improvement strategies to solve the problems of sample imbalance, edge blur, false positive and false negative. Then the difficulties encountered in the improvement of the current loss function are analyzed. Finally, the future research directions are prospected. This paper provides a reference for the reasonable selection, improvement or innovation of loss function, and guides the direction for the follow-up research of loss function.

Research progress on medical image dataset expansion methods / 生物医学工程学杂志

Computer-aided diagnosis (CAD) systems play a very important role in modern medical diagnosis and treatment systems, but their performance is limited by training samples. However, the training samples are affected by factors such as imaging cost, labeling cost and involving patient privacy, resulting in insufficient diversity of training images and difficulty in data obtaining. Therefore, how to efficiently and cost-effectively augment existing medical image datasets has become a research hotspot. In this paper, the research progress on medical image dataset expansion methods is reviewed based on relevant literatures at home and abroad. First, the expansion methods based on geometric transformation and generative adversarial networks are compared and analyzed, and then improvement of the augmentation methods based on generative adversarial networks are emphasized. Finally, some urgent problems in the field of medical image dataset expansion are discussed and the future development trend is prospected.

Hepatitis B e antigen from chronic hepatitis B patients induces Th1/Th2 cytokine imbalance in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs).</p><p><b>METHODS</b>PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis.</p><p><b>RESULTS</b>Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01).</p><p><b>CONCLUSION</b>HBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.</p>

Effects of antihistamine treatment on immune function in rats with experimental hepatitis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of antihistamine treatment on immune function in rats with experimental hepatitis.</p><p><b>METHODS</b>Thirty Wistar rats were randomly allocated into three groups:experimental hepatitis group (EH group), antihistamine treatment group (AH group) and normal control group (NC group). Rats in the EH group received the subcutaneous injection of 40% carbon tetrachloride oil solution and were fed on diet with low-protein, low-choline, high-fat and high-alcohol,while rats in the AH group received antihistamine treatment(ketotifen + vitamin C) additionally.They were sacrificed after 4 weeks, and the levels of serum alanine aminotransferase(ALT), total bilirubin (TBil), histamine(HA), IFNgamma, IL-12, IL-4 and IL-10 were determined. The levels of IL-12 mRNA and IFN-gamma mRNA in liver tissue were determined via real-time reverse transcriptional polymerase chain reaction(RT-PCR).</p><p><b>RESULTS</b>(1) Compared to the NC group, in the EH group, the levels of ALT, TBil, and circulating and intrahepatic HA were significantly increased(P less than 0.05); intrahepatic HA were significantly decreased(P less than 0.05) after antihistamine treatment. (2) Compared to the NC group, in the EH group, the levels of IL-4, IL-10 were significantly increased((0.504+/-0.202)ng/ml and (29.025+/-1.478) pg/ml vs (0.811+/-0.244)ng/ml and (33.72+/-4.293)pg/ml respectively, P less than 0.05), and the levels of IL-12 were decreased ((6.515+/-2.893)pg/ml vs (3.519+/-1.113)pg/ml, P less than 0.05); and after antihistamine treatment the levels of IL-4 and IL-10 were significantly decreased (were (0.423+/-0.168)ng/ml and (30.412+/-3.275)pg/ml, P less than 0.05), the levels of IL-12 were significantly increased (P less than 0.05), but the level of IFNgamma had no significance (P more than 0.05). The levels of intrahepatic IL-12 mRNA and IFNgamma mRNA had similar results.</p><p><b>CONCLUSION</b>Antihistamine treatment may improve liver function and correct Th1/Th2 unbalance.</p>

Changes in mast cells and hepatic expression of c-kit and stem cell factor in the rat model of chronic hepatitis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the potential role of mast cells and the related molecular mechanism in chronic hepatitis (CH) using a rat model system.</p><p><b>METHODS</b>Thirty Wistar rats (15 males, 15 females; weight range: 230-290 g) were randomly divided into the normal contrast (NC) group and experimental CH group. The CH group received subcutaneous injection of CCl4 and a diet high in cholesterol and alcohol content and low in protein and choline content. Throughout the 4-week modeling period, aseptic blood samples were taken to test plasma tryptase (TS) and hyaluronic acid (HA) levels. The rats were euthanized to assess the changes in liver mast cells by histology and morphology analyses and the changes in liver expression of c-kit and stem cell factor (SCF) proteins by immunohistochemistry and mRNAs by RT-PCR.</p><p><b>RESULTS</b>Compared to the NC group, the CH group had higher plasma and liver concentration of HA (78.09 +/- 38.55 vs. 145.14 +/- 52.54 ng/ml, 51.58 +/- 20.45 vs. 106.59 +/- 43.15 ng/100 mg; t = 2.457 and 2.825 respectively, both P less than 0.05) and TS (0.416 +/- 0.143 vs 0.753 +/- 0.210 mg/ml; t = 4.165, P less than 0.05). The CH group also showed fatty degeneration and fibrosis with many degranulating and degranulated mast cells filled with purple granula located around the liver blood vessels and in fiber-intervals. The CH livers also showed a significantly higher number of mast cells (2.167 +/- 0.924 vs. NC: 10.92 +/- 1.575; t = 7.633, P less than 0.05) and stronger intensity of c-kit staining (2.783 +/- 0.577 vs. 12.86 +/- 3.126; t = 9.511, P less than 0.05) and SCF staining (3.383 +/- 1.583 vs. 15.58 +/- 6.431; t = 9.625, P less than 0.05). The expressions of c-kit and SCF were positively correlated with HA level (r = 0.478 and 0.556 respectively, both P less than 0.05). The c-kit and SCF mRNA expression levels were also significantly higher in the CH liver tissues.</p><p><b>CONCLUSION</b>Mast cell degranulation and histamine release is significantly increased under conditions of chronic hepatitis, and the related mechanism may involve up-regulation of the membrane receptor c-kit and its ligand SCF.</p>

Sequence analysis of varicella-zoster virus gE gene in varicella-zoster virus strains with different clades / 病毒学报

To analyze the gE gene sequence of varicella-zoster virus (VZV) strains of different clades and subclades currently circulating in China. Eighteen skin lesion fluid swabs or skin scab pieces from patients with chickenpox or shingles were obtained from Beijing, Changchun, Lhasa and Urumqi between December 2010 and June 2011. The genotype of the virus strains was determined by a group of single nucleotide polymorphism (SNP) located in 15 ORFs, and the full-length gE genes of 18 strains representing all the clades in the study was amplified by PCR and sequenced. In addition to the synonymous mutations and non-synonymous mutations that were reported in the literature, there were 3 novel non-synonymous mutations (C56T, C1109T, C917A) and 4 new synonymous mutations (C54T, T1075C, T816C, G279A) found in the 8 strains analyzed. We found the VZV strains of clade 5 in Xinjiang for the first time,and the genotypes of some VZV strains circulating in Chagnchun could not be determined by the present methods. The analysis of gE gene sequences,revealed a novel non-synonymous mutations in the e1 and c1 epitopes, corresponding to the amino acid change of serine to tyrosine.

Effects of PPAR-alpha activation on oleic acid-induced steatosis and expression of heme oxygenase-1 in HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of peroxisome proliferator activated receptor-alpha (PPAR-a) activation on oleic acid (OA)-induced steatosis and hepatic expression of heme oxygenase-1 (HO-1) using an in vitro cell model system.</p><p><b>METHODS</b>A steatosis human hepatocyte in vitro model system was established by treating HepG2 cells with 0.2 mmol/L of oleic acid for 24 hours. The steatosis cells were then divided into four groups for an additional 24 hours of treatment with 0.2 mmol/L of oleic acid alone (model control group) or with 5, 10 or 50 pnol/L of fenofibrate (FF, a selective PPAR-a agonist; experimental groups). Untreated HepG2 cells served as non-steatosis controls. Effect of PPAR-a activation on fat accumulation was detected by Oil Red O staining and on intracellular triglyceride (TG) levels by enzymatic assay. mRNA and protein expression of PPAR-alpha and HO-1 were quantified by real-time PCR and immunocytochemistry, respectively. One-way ANOVA and the LSD t-test were used for between-group comparisons, and correlation analysis was performed with the Pearson's correlation coefficient.</p><p><b>RESULTS</b>The steatosis model control cells showed significantly increased TG deposition (379.98 +/- 23.19 mg/g protein, vs. non-steatosis controls F = 148.56, P< 0.01), significantly decreased mRNA and protein expression of PPAR-alpha (0.42 +/- 0.38,F= 177.64,P< 0.01 and 0.47 +/- 0.14, F= 120.76,P< 0.01) and HO-1 (0.36 +/- 0.66, F= 74.77,P< 0.01 and 0.26 +/- 0.10,F= 119.90,P<0.01). FF (5, 10 and 50 micromol/L) inhibited the steatosis induced by OA in a concentration-dependent manner (294.00 +/- 19.80, 250.33 +/- 9.96, and 196.99 +/- 9.14, F = 148.56, P <0.01) and increased the mRNA and protein expression of PPAR-alpha (0.55 +/- 0.65, 0.85 +/- 0.61, and 1.31 +/- 0.36,F= 177.64,P< 0.01; 0.82 + 0.11, 1.31 +/- 0.16, and 1.75 +/- 0.13, F= 120.76,P <0.01) and HO-1 (0.62 +/- 0.05, 0.84 +/- 0.07, and 1.30 +/- 0.11,F= 74.77,P <0.01; 0.44 +/- 0.08, 0.81 +/- 0.08, 1.20 +/- 0.10,F= 119.90,P< 0.01).</p><p><b>CONCLUSION</b>Activation of PPAR-a prevents OA-induced steatosis in HepG2 cells, and HO-1 may function as a downstream effector of this mechanism.</p>

Comparative analysis of the efficacies of entecavir capsules and lamivudine in chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the efficacy profile of entecavir capsule (ETV) as a chronic hepatitis B therapy, as compared to lamivudine (LAM).</p><p><b>METHODS</b>In this multicenter, randomized, double-blind, parallel group evaluation of ETV, 232 subjects were administered a 96-week course of 0.5 mg/day ETV or 100 mg/day LAM. PCR measurement of hepatitis B virus (HBV) was conducted throughout the treatment course to determine achievement of complete virologic response (CVR; defined as less than 500 copies/ml of HBV DNA) or experience of virology rebound ( more than 500 copies/ml of HBV DNA after achievement of CVR).</p><p><b>RESULTS</b>After week-48 of treatment, the ETV group showed a higher CVR rate (90.3% vs. LAM: 59.4%) and lower virology rebound rate (1.9% vs. LAM: 13.9%). After week-96 of treatment, the ETV group continued to have a higher CVR rate (86.0% vs. LAM: 71.4%), and virology rebound was experienced by significantly less subjects in the ETV group (1.2% vs. LAM: 11.9%, P = 0.005).</p><p><b>CONCLUSION</b>ETV therapy can quickly and continuously suppress HBV replication in chronic hepatitis B patients, and has a lower resistance rate than LAM. Compared to LAM, ETV may be a superior long-term treatment choice for chronic hepatitis B.</p>

The genotyping and molecular evolution of varicella-zoster virus / 病毒学报

Varicella-zoster virus (VZV, Human herpesvirus 3) is a member of the family Herpesviridae, and is classified as alpha-subfamily along with HSV-1 and HSV-2. VZV is the causative agent of chicken pox (varicella) mostly in children, after which it establishes latency in the sensory ganglia with the potential to reactivate at a later time to cause shingles (zoster). Increasing molecular epidemiological studies in recent years have been performed to monitor the mutations in VZV genome, discriminate vaccine virus from wild type virus, study the phylogeny of VZV strains throughout the world, and understand the evolution of the different clades of VZV. The progress has great impact on the fields of epidemiology, virology and bioinformatics. In this review, the currently available data concerning the geographic distribution and molecular evolution of VZV clades are discussed.

Effect of reduced glutathione on proliferation of rat hepatic stellate cells and the Nrf2/HO-1 signaling pathway / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the effects of reduced glutathione (GSH) on the proliferation of hepatic stellate cells and the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway.</p><p><b>METHODS</b>The rat HSC-T6 cell line was activated by lipopolysaccharide (LPS; 0.1 mug/ml) and incubated with various concentrations of GSH (0, 1, 2, 5 and 10 mmol/L) for 24 h. Cell proliferation was evaluated by the MTT colorimetric assay. Collagen IV and hyaluronic acid (HA) contents were measured by chemiluminescence immunoassay of cell supernatants. Nrf2 and HO-1 protein expression was observed by immunohistochemistry. Nrf2 and HO-1 mRNA expression was assessed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). HO-1 activity was analyzed by spectrophotometer.</p><p><b>RESULTS</b>GSH treatment inhibited HSC-T6 proliferation and decreased the secretion of HA and collagen IV (P less than 0.05); GSH treatment of HSC-T6 cells also led to increased expression of Nrf2 and HO-1, and increased activity of HO-1 (P less than 0.05).</p><p><b>CONCLUSION</b>GSH can inhibit the proliferation of hepatic stellate cells in vitro and reduce secretion of hyaluronic acid and collagen IV. The underlying mechanism in HSC-T6 cells may involve regulation of the Nrf2/HO-1 signaling pathway.</p>

Dynamic changes and clinical significance of HBcAg18-27 specific cytotoxic T lymphocytes in acute hepatitis B patients / 中华肝脏病杂志

This report aims to investigate the dynamical changes of HBcAg18-27 epitope specific cytotoxic T lymphocytes(CTL), alanine aminotransferase (ALT), HBV DNA and HBsAg in peripheral blood of acute hepatitis B patients, and to explore the roles of HBcAg18-27-specific CTLs in virus clearance and liver injury. Acute hepatitis B (AHB) and chronic hepatitis B (CHB) patients were divided into two groups according to results of HLA-A0201. Patients with positive HLA-A0201 were classified into HBcAg-specific CTL group and those with negative HLA-A0201 were referred as control group. The specific CTLs were stained with HLA-A0201 limited HBcAg18-27 epitope MHC-Pentamer and the frequencies of CTLs, T, B, NK and NKT cells were detected by flow cytometry (FCM). The serum ALT, HBV DNA and HBsAg were examined using speed analysis, quantitative PCR and abbott chemiluminescent technology. The frequencies of HBcAg18-27-specific CTLs in AHB patients were higher in the early three weeks as compared to the late three weeks. The apex time of HBV-specific CTL frequencies lagged behind those of HBV DNA, HBsAg and ALT. The loss of HBsAg in patients with high frequencies of HBV-specific CTL was earlier than that in patients with low frequencies (t = 2.018, P value is less than 0.05). In the second week the peak frequencies of CD3+CD8+ cells overlapped with that of HBcAg18-27-specific CTLs and with a positive correlation between (r = 0.420, P value is less than 0.05). During the early stages of AHB, the frequencies of NK and NKT cells were found significantly lower than that of control group and CHB group and the levels were back to normal after recovery. Moreover, a negative correlation existed between the frequencies of NK cells and the dynamic changes of HBcAg18-27-specific CTLs (r = -0.435, P value is less than 0.01) in AHB group. The frequencies of HBcAg18-27-specific CTLs were significantly higher as compared to CHB group in the first three weeks (z = -3.258, -4.04, and -3.259, P value is less than 0.01). The early loss of HBsAg was closely related to the high frequencies of HBcAg18-27 specific CTLs in AHB patients. HBcAg-specific CTL frequencies in peripheral blood could be used to predict clinical outcome after HBV infection. The frequencies of CD8+ T cells can reflect the changes of frequencies of HBcAg-specific CTL during acute HBV infection.

Effects of lipopolysaccharide on the maturation and secretion of human peripheral dendritic cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of Lipopolysaccharide (LPS) on the maturation and secretion of human peripheral dendritic cells (DCs).</p><p><b>METHODS</b>DCs from healthy human peripheral monocytes (PBMCs) were induced in vitro with rhGM-CSF, rhIL-4, Flt3-L and TNFalpha. The subjects were divided into 3 groups: the long-term group stimulated with LPS 1 microg/ml at day 1, 4, 7, 9 post culture; the short-term group stimulated with LPS 1 microg/ml at day 7 and 8 post culture, and the DCs without LPS stimulation was control group. After 10 days of culture, the morphologic features of DCs were observed by light and electron microscopes, the phenotypic patterns were characterized by flow cytometry, the proliferation of T cell were evaluated with mixed leukocytes reaction (MLR) and the levels of IL-12 and IFNgamma produced by DCs were analyzed with ELISA.</p><p><b>RESULTS</b>Compared with the short-term group, the expressions of HLA-DR (65.81%+/-10.96%), CD86 (48.81%+/-18.13%), CD80 (13.56%+/-5.48%), CD83 (11.52%+/-5.09%), the secretions of IFNgamma(15.60+/-5.83 pg/ml) and IL-12 (51.77+/-11.02 pg/ml) by the DCs in long-term group were decreased obviously (P is less than 0.05) and the proliferation of homogenic lymphocyte cells (1.548+/-0.365) stimulated by DCs was also impaired (P < 0.05).</p><p><b>CONCLUSION</b>Long-term LPS stimulation can suppress the maturation and secretion of DCs, which might be the reason of poor immunity in the patients with intestinal endotoxemia.</p>

A novel missense mutation resulting in X-linked adrenoleukodystrophy in female heterozygotes of a Chinese family / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To identify ABCD1 gene mutation in a Chinese family with three heterozygous female patients.</p><p><b>METHODS</b>Four fragments covering the entire coding sequence of the ABCD1 gene from one of the female patients were amplified by reverse transcription-PCR. The PCR products were directly sequenced. The result of sequencing was confirmed by restriction enzyme digestion of PCR products from genomic DNA. Human ABCD1 gene and ALD protein were aligned with those of rat, monkey, mouse and cattle by Clustal X 1.83. Softwares of Motif Scan, TMpred and ESYpred3D were used to predict the effect of the mutation on the structure of the ALD protein.</p><p><b>RESULTS</b>A novel missense mutation, CAC to CGC, was found at codon 283 of the ABCD1 gene from the patient, resulting in the replacement of histidine by arginine. This mutation abolished an Msl I site in the gene. Her son was free from this mutation. The mutated amino acid residue (283H) was highly conservative in evolution, and the mutation caused a dramatic change in the structure of the ALD protein.</p><p><b>CONCLUSION</b>Three female patients heterozygous for ABCD1 gene mutation were first reported in China, and a novel mutation, p.H283R, was identified in this X-ALD family.</p>

Molecular genetic evolution analysis of new A(H1N1) influenza virus / 病毒学报

In order to analyze the molecular epidemiology of A (H1N1) influenza virus in 2009, the complete genome sequences of influenza strains from different host sources downloaded from the NCBI were analyzed on genetic evolution by DNAstar software in this research. The results showed that 79 mutation sites of new A (H1N1) influenza virus were observed compared to previous human A (H1N1) influenza strain, including 14 mutation sites new in all A (H1N1) influenza sources and 37 mutation sites only observed in swine strain. A significant difference was represented in antigenic sites between new A (H1N1) influenza strain and the previous human A (H1N1) strain. This phenomenon shows the new A (H1N1) influenza strain is either originated from the recombination of human and swine strain or from the infection in pig populations and gradual mutation to human tansmission, which remains to be further studied.

A new method avoids interference of ABCD1 pseudogenes in molecular diagnosis of X-linked adrenoleukodystrophy / 第二军医大学学报

Objective: To introduce a new method which can avoid the interference of ABCD1 pseudogenes in the molecular diagnosis of X-linked adrenoleukodystrophy. Methods: The coding regions of ABCD1 gene of 3 unrelated Chinese patients with X-linked adrenoleukodystrophy were amplified from the total RNA of peripheral blood by long distance RT-PCR; the product was further amplified in 4 segments in a second round PCR; and the PCR products were purified and directly sequenced. To confirm the mutations, the genomic DNA from peripheral blood cells of the patients was analyzed by direct sequencing after amplification of the ABCD1 genes by nested PCR, in which the product of the first round PCR covered the fragment starting from exon 6 and ending at 3′UTR of the ABCD1 gene. Results: The 3 Chinese patients with X-linked adrenoleukodystrophy had 3 different base substitutions(2235C>T,2065C>T and 2190A>T)in the ABCD1 genes of the 3 probands and their mothers, which resulted in 2 missense mutations (R617C and P560L) and one nonsense mutation (K602X). Conclusion: Nested PCR can rapidly and efficiently avoid the interference of ABCD1 pseudogenes in the molecular diagnosis of X-ALD.

Early prediction of lamivudine response in e antigen-negative chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the effects of ALT, HBsAg and HBV DNA at the baseline, 4 and 12 weeks after lamivudine treatment on the long term (104 weeks) response in e antigen-negative chronic hepatitis B patients.</p><p><b>METHODS</b>127 adult e antigen-negative chronic hepatitis B patients were enrolled in this study. All patients received treatment on LAM 100 mg/d for at least 104 weeks. The liver function, serum HBV markers and HBV viral load were regularly checked during the treatment. The effects of ALT, HBsAg and HBV DNA at the baseline, 4 and 12 weeks after lamivudine treatment on the response at 104 weeks were statistically analyzed.</p><p><b>RESULTS</b>The proportion of patients with serum HBV DNA less than 1000 copies / ml at 104 weeks after LAM treatment was 50.0% and 86.8% in patients with baseline ALT less than 5 ULN and ALT is more than or equal to 5 ULN, respectively (P less than 0.01). In patients with baseline HBsAg less than 2000 COI and HBsAg is more than or equal to 2000 COI, the proportion of patients with serum HBsAg less than 500 COI at 104 weeks after LAM treatment was 19.1% and 17.5%, respectively (P more than 0.05). the HBsAg serological conversion rates were respectively 2.1% and 2.5% , respectively (P more than 0.05), the proportion of patients with serum HBV DNA less than 1000 copies/ml was 61.7% and 67.5%, respectively (P more than 0.05). In patients with baseline HBV DNA less than 10(6) copies/ml and HBV DNA is more than or equal to 10(6) copies/ml, the proportion of patients with HBV DNA less than 1000 copies/ml were statistically different at 4 weeks and 12 weeks after treatment, however, the proportion of patients with HBV DNA less than 1000 copies/ml at 104 weeks after LAM treatment was 62.7% and 67.1%, respectively (P more than 0.05). In patients with HBV DNA less than 1000 copies/ml and HBV DNA is more than or equal to 1000 copies/ml at 4 weeks after treatment, the proportion of patients with HBV DNA less than 1000 copies/ml at 104 weeks after LAM treatment was 70.7% and 60.9%, respectively (P more than 0.05). In patients with HBV DNA less than 1000 copies/ml and HBV DNA is more than or equal to 1000 copies/ml at 12 weeks after treatment, the proportion of patients with HBV DNA less than 1000 copies/ml at 104 weeks after treatment was 78.8% and 38.1%, respectively (P less than 0.01).</p><p><b>CONCLUSION</b>e antigen negative chronic hepatitis B patients with baseline ALT is more than or equal to 5 ULN and HBV DNA less than 1000 copies/ml at 12 weeks after treatment have better virological response at 104 weeks after LAM treatment. The baseline HBsAg and HBV DNA load are associated with the virological response at 104 weeks after LAM treatment.</p>

Molecular diagnosis of a Chinese pedigree with osteogenesis imperfecta type I / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To perform molecular diagnosis for a Chinese pedigree with osteogenesis imperfecta type I.</p><p><b>METHODS</b>Thirty pairs of primers were designed to amplify all the 52 exons, exon boundaries and promoter region of the COL1A1 gene from genomic DNA of peripheral blood cells of the family members. The PCR products were purified and directly sequenced. To check the mutation in normal controls, the genomic DNA from peripheral blood cells of the index patient, his mother and 60 normal controls were analyzed by amplification refractory mutation system.</p><p><b>RESULTS</b>A missense mutation of GAT>CAT was identified at codon 1441 of the COL1A1 gene from the family, which resulted in the replacement of aspartic acid by histidine (D1441H). This mutation was not found in a group of 60 normal controls.</p><p><b>CONCLUSION</b>The method for molecular diagnosis of osteogenesis imperfecta was established and a novel COL1A1 gene mutation, D1441H, was identified in the Chinese pedigree with osteogenesis imperfecta type I.</p>

Mutation analysis of SMN gene in a patient and his family with spinal muscular atrophy / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To perform mutation analysis and describe the genotype of the SMN gene in a patient with spinal muscular atrophy (SMA) and his family.</p><p><b>METHODS</b>Deletion analysis of the SMN1 exon 7 by conventional PCR-restriction fragment length polymorphism (RFLP) and allele-specific PCR, and gene dosage of SMN1 and SMN2 by multiplex ligation-dependent probe amplification (MLPA) were performed for the patient and his parents; reverse transcriptase (RT)-PCR and sequencing were performed for the patient. To determine whether the SMN variant was exclusive to transcripts derived from SMN1, the RT-PCR product of the patient was subcloned and multiple clones were sequenced directly; PCR of SMN exon 5 from the genomic DNA of the parents and direct sequencing were performed to confirm the mutation.</p><p><b>RESULTS</b>In SMN1 exon 7 deletion analysis, no homozygous deletion of the SMN1 was observed in the family; the gene dosage analysis by MLPA showed that the patient had 1 copy of SMN1 and 1 copy of SMN2 his father had 2 copies of SMN1 and 2 copies of SMN2, and his mother had 1 copy of SMN1 and no SMN2. A previously unreported missense mutation of S230L was identified from the patient and this mutation was also found in his father.</p><p><b>CONCLUSION</b>A novel missense mutation of S230L was identified in the SMA family and the genotype of the family members were investigated.</p>

Protein expression and function of gene 2 transregulated by hepatitis B virus pre-s1 protein and its cloning / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen proteins in leukocytes interacting with PS1TP2 by yeast-two hybrid and to view their subcellular localization in HepG2 cells.</p><p><b>METHODS</b>The function and structure of PS1TP2 were studied by bioinformatic analysis. PS1TP2 gene was amplified and cloned into plasmid pET32a (+) and pGBKT7 to construct recombinant expression vectors pET32a (+)-PS1TP2 and pGBKT7-PS1TP2. They were transduced into E. coli Rosetta strain and yeast AH109. The transformed yeast mated with yeast Y187 containing leukocyte cDNA library plasmid in a 2xYPDA medium. Diploid yeast cells were plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) for selecting twice and then screening. Then a green fluorescent protein (GFP) expression vector pEGFP-C1-PS1TP2 was established, transduced into HepG2, and its subcellular localization was studied by fluorescence microscopy and confocal microscopy.</p><p><b>RESULTS</b>Bioinformatic analysis showed that the PS1TP2 gene was located at 6q24.1, the protein was unstable and the aliphatic index was very high. After transformation of the E. coli and yeast AH109, the expression protein showed: (1) the molecular weight of the expressed product was about 41000 Da, and (2) PS1TP2 existed within the cells. Diploid yeast cells were plated on the synthetic dropout nutrient medium containing X-a-gal for selecting twice and then screening. Twenty-six colonies from blue colonies were sequenced, pEGFP-C1-PS1TP2 was successfully expressed in the HepG2 cells, and PS1TP2 was located in the cell plasma.</p><p><b>CONCLUSION</b>A prokaryotic expression vector pET32a(+)-PS1TP2 was constructed successfully and the PS1TP2 was successfully expressed in the yeast system. Genes of PS1TP2 interact with leukocyte proteins. These results bring some new clues for studying the biological functions of HBV.</p>