Intronic (TTTGA)n insertion in SAMD12 also causes familial cortical myoclonic tremor with epilepsy.

BACKGROUND: Intronic (TTTCA)n insertions in the SAMD12, TNRC6A, and RAPGEF2 genes have been identified as causes of familial cortical myoclonic tremor with epilepsy. OBJECTIVE: To identify the cause of familial cortical myoclonic tremor with epilepsy pedigrees without (TTTCA)n insertions in SAMD12, TNRC6A, and RAPGEF2. METHODS: Repeat-primed polymerase chain reaction, long-range polymerase chain reaction, and Sanger sequencing were performed to identify the existence of a novel (TTTGA)n insertion. Targeted long-read sequencing was performed to confirm the accurate structure of the (TTTGA)n insertion. RESULTS: We identified a novel expanded intronic (TTTGA)n insertion at the same site as the previously reported (TTTCA)n insertion in SAMD12. This insertion cosegregated with familial cortical myoclonic tremor with epilepsy in 1 Chinese pedigree with no (TTTCA)n insertion. In the targeted long-read sequencing of 2 patients and 1 asymptomatic carrier in this pedigree, with 1 previously reported (TTTCA)n -insertion-carrying patient as a positive control, a respective total of 302, 159, 207, and 50 on-target subreads (predicated accuracy: ≥90%) spanning the target repeat expansion region were generated. These sequencing data revealed the accurate repeat expansion structures as (TTTTA)114-123 (TTTGA)108-116 in the pedigree and (TTTTA)38 (TTTCA)479 in (TTTCA)n -insertion-carrying patient. CONCLUSION: The targeted long-read sequencing helped us to elucidate the accurate structures of the (TTTGA)n and (TTTCA)n insertions. Our finding offers a novel possible cause for familial cortical myoclonic tremor with epilepsy and might shed light on the identification of genetic causes of this disease in pedigrees with no detected (TTTCA)n insertion in the reported causative genes. © 2019 International Parkinson and Movement Disorder Society.

Competitive binding between miR-122 and p68 onto hepatitis C viral RNA.

BACKGROUND: Liver-specific microRNA (miR)-122 has been shown to be involved in regulating translation of hepatitis C viral (HCV) RNA. This study aimed to explore the molecular mechanism of miR-122 in regulating HCV RNA translation initiation. MATERIAL/METHODS: In human liver hepatocellular carcinoma cell line HepG2, UV cross-link assay was performed on a large scale to identify RNA-binding proteins with gradient concentrations of miR-122. Analytical ultracentrifugation was then used to separate the translation initiation complexes. All RNA-binding proteins were then identified by Western blotting. RESULTS: The binding of 68 kDa protein (p68) to HCV RNA was suppressed by the addition of miR-122 via the competitive binding assay. Such inhibition can be eliminated by the addition of 2'-O-methylated oligonucleotides. This binding suppression was determined to be specific for miR-122, which used the mature single-stranded RNA to suppress the binding of p68 onto HCV RNA. This binding inhibition was further validated by using authentic miR-122 with conserved regions and mutated sequences. CONCLUSIONS: The binding of p68 onto HCV RNA can be specifically inhibited by miR-122 via a competitive binding process.

Role of A20 in interferon-α-mediated functional restoration of myeloid dendritic cells in patients with chronic hepatitis C.

Hepatitis C virus (HCV) infection is a global health problem characterized by a high rate of chronic infection, which may in part be due to a defect in myeloid dendritic cells (mDCs). This defect appears to be remedied by treatment with interferon-α (IFN-α) -based antiviral therapies; however, the molecular mechanisms underlying mDC dysfunction in HCV infection and restoration by IFN-α treatment are unclear. The ubiquitin-editing protein A20 plays a crucial role in controlling the maturation, cytokine production and immunostimulatory function of mDCs. We propose that the expression of A20 correlates with the function of mDCs during HCV infection and IFN-α therapy. In this study, we observed that A20 expression in mDCs isolated from chronically HCV-infected subjects was significantly higher than healthy subjects or subjects achieving sustained virological responses (SVR) following antiviral treatment. Notably, A20 expression in mDCs from HCV patients during IFN-α treatment was significantly lower than for untreated patients, SVR patients, or healthy subjects. Besides, A20 expression in mDCs stimulated by polyI:C differed between HCV patients and healthy subjects, and this difference could be abrogated by the treatment with IFN-α in vitro. Additionally, A20 expression by polyI:C-activated mDCs, with or without IFN-α treatment, negatively correlated with the expression of HLA-DR, CD86 and CCR7, and the secretion of interleukin-12 (IL-12), but positively associated with the production of IL-10. Importantly, silencing A20 expression using small interfering RNAs increased the production of IL-12 in mDCs of chronically HCV-infected individuals. These findings suggest that A20 plays a crucial role in negative regulation of innate immune responses during chronic viral infection.

Green Preparation of Lightweight, High-Strength Cellulose-Based Foam and Evaluation of Its Adsorption Properties.

In recent years, the application scope of most cellulose-based foams is limited due to their low adsorbability and poor recyclability. In this study, a green solvent is used to extract and dissolve cellulose, and the structural stability of the solid foam is enhanced by adding a secondary liquid via the capillary foam technology, and the strength of the solid foam is improved. In addition, the effects of the addition of different gelatin concentrations on the micro-morphology, crystal structure, mechanical properties, adsorption, and recyclability of the cellulose-based foam are investigated. The results show that the cellulose-based foam structure becomes compact, the crystallinity is decreased, the disorder is increased, and the mechanical properties are improved, but its circulation capacity is decreased. When the volume fraction of gelatin is 2.4%, the mechanical properties of foam are the best. The stress of the foam is 55.746 kPa at 60% deformation, and the adsorption capacity reaches 57.061 g/g. The results can serve as a reference for the preparation of highly stable cellulose-based solid foams with excellent adsorption properties.

Fibroblast growth factor 1 (FGFR1) modulation regulates repair capacity of oligodendrocyte progenitor cells following chronic demyelination.

The adult mammalian brain contains multiple populations of endogenous progenitor cell types. However, following CNS trauma or disease, the regenerative capacity of progenitor populations is typically insufficient and may actually be limited by non-permissive or inhibitory signals in the damaged parenchyma. Remyelination is the most effective and simplest regenerative process in the adult CNS yet is still insufficient following repeated or chronic demyelination. Our previous in vitro studies demonstrated that fibroblast growth factor receptor 1 (FGFR1) signaling inhibited oligodendrocyte progenitor (OP) differentiation into mature oligodendrocytes. Therefore, we questioned whether FGFR1 signaling may inhibit the capacity of OP cells to generate oligodendrocytes in a demyelinating disease model and whether genetically reducing FGFR1 signaling in oligodendrocyte lineage cells could enhance the capacity for remyelination. FGFR1 was found to be upregulated in the corpus callosum during cuprizone mediated demyelination and expressed on OP cells just prior to remyelination. Plp/CreER(T):Fgfr1(fl/fl) mice were administered tamoxifen to induce conditional Fgfr1 deletion in oligodendrocyte lineage cells. Tamoxifen administration during chronic demyelination resulted in reduced FGFR1 expression in OP cells. OP proliferation and population size were not altered one week after tamoxifen treatment. Tamoxifen was then administered during chronic demyelination and mice were given a six week recovery period without cuprizone in the chow. After the recovery period, OP numbers were reduced and the number of mature oligodendrocytes was increased, indicating an effect of FGFR1 reduction on OP differentiation. Importantly, tamoxifen administration in Plp/CreER(T):Fgfr1(fl/fl) mice significantly promoted remyelination and axon integrity. These results demonstrate a direct effect of FGFR1 signaling in oligodendrocyte lineage cells as inhibiting the repair capacity of OP cells following chronic demyelination in the adult CNS.

Clinical and genomic analysis of a large Chinese family with familial cortical myoclonic tremor with epilepsy and SAMD12 intronic repeat expansion.

Objective: Our goal was to perform detailed clinical and genomic analysis of a large multigenerational Chinese family with 21 individuals showing symptoms of Familial Cortical Myoclonic Tremor with Epilepsy (FCMTE) that we have followed for over 20 years. Methods: Patients were subjected to clinical evaluation, routine EEG, and structural magnetic resonance imaging. Whole exome sequencing, repeat-primed PCR, long-range PCR, and PacBio sequencing were performed to characterize the disease-causing mutation in this family. Results: All evaluated patients manifested adult-onset seizures and presented with progressive myoclonic postural tremors starting after the third or fourth decade of life. Seizures typically diminished markedly in frequency with implementation of antiseizure medications but did not completely cease. The electroencephalogram of affected individuals showed generalized or multifocal spikes and slow wave complexes. An expansion of TTTTA motifs with addition of TTTCA motifs in intron 4 of SAMD12 was identified to segregate with the disease phenotype in this family. Furthermore, we found that the mutant allele is unstable and can undergo both contraction and expansion by changes in the number of repeat motifs each time it is passed to the next generation. The size of mutant allele varied from 5 to 5.5 kb with 549-603 copies of TTTTA and 287-343 copies of TTTCA repeat motifs in this family. Significance: Our study provides a detailed description of clinical progression of FCMTE symptoms and its management with antiseizure medications. Our method of repeat analysis by PacBio sequencing of long-range PCR products does not require high-quality DNA and hence can be easily applied to other families to elucidate any correlation between the repeat size and phenotypic variables, such as, age of onset, and severity of symptoms.

Fibroblast growth factor receptor-1 is required for long-term potentiation, memory consolidation, and neurogenesis.

BACKGROUND: Although substantial evidence supports the view that adult neurogenesis is involved in learning and memory, how newly generated neurons contribute to the cognitive process remains unknown. Fibroblast growth factor 2 (FGF-2) is known to stimulate the proliferation of neuronal progenitor cells (NPCs) in adult brain. Using conditional knockout mice that lack brain expression of FGFR1, a major receptor for FGF-2, we have investigated the role of adult neurogenesis in hippocampal synaptic plasticity and learning and memory. METHODS: The Fgfr1 conditional knockout mice were generated by crossing the Fgfr1-null line, the Fgfr1-flox line, and the Nestin-Cre transgenic mice. Bromodeoxyuridine (BrdU) labeling, slice electrophysiology, and Morris Water Maze experiments were performed with the Fgfr1 conditional mutant mice. RESULTS: Bromodeoxyuridine labeling experiments demonstrate that FGFR1 is required for the proliferation of NPCs as well as generation of new neurons in the adult dentate gyrus (DG). Moreover, deficits in neurogenesis in Fgfr1 mutant mice are accompanied by a severe impairment of long-term potentiation (LTP) at the medial perforant path (MPP)-granule neuron synapses in the hippocampal dentate. Moreover, the Fgfr1 mutant mice exhibit significant deficits in memory consolidation but not spatial learning. CONCLUSIONS: Our study suggests a critical role of FGFR1 in adult neurogenesis in vivo, provides a potential link between proliferative neurogenesis and dentate LTP, and raises the possibility that adult neurogenesis might contribute to memory consolidation.

Interaction of fibroblast growth factor 2 (FGF2) and notch signaling components in inhibition of oligodendrocyte progenitor (OP) differentiation.

Oligodendrocyte progenitor (OP) cell differentiation is a critical process of developmental myelination, tumor formation, and remyelination in the CNS. Activation of the fibroblast growth factor 2 (FGF2) or notch pathway can inhibit differentiation of OP cells. The current study examines the interaction of FGF2 and notch signaling components in regulating OP differentiation. Cultured neonatal rat brain OP cells were used for transfection-based promoter assays and for infection with retroviruses expressing a GFP reporter to monitor OP differentiation into oligodendrocytes or astrocytes. FGF2 treatment resulted in a four-fold increase of transcriptional activity from the promoter region of Hes5, a notch pathway target gene. FGF2 inhibition of OP differentiation into oligodendrocytes was perturbed by retroviral expression of a dominant negative construct for mastermind-like 1, which is an important co-activator of transcription for notch target genes. OP differentiation into oligodendrocytes was reduced by co-culture with fibroblasts expressing Jagged1, a ligand for notch receptors. This Jagged1 inhibition of OP differentiation was not altered by retroviral expression of a dominant negative FGF receptor construct. Constitutive activation of notch signaling, by retroviral expression of the Notch1 intracellular domain, greatly reduced OP differentiation into either oligodendrocytes or astrocytes and did not require FGF2 signaling. These findings indicate that inhibition of OP differentiation through the Notch1 pathway was not influenced by FGF2 signaling. However, FGF2 signaling may interact with down stream components of the notch signaling pathway, including mastermind-like1 and Hes5, to inhibit OP differentiation into oligodendrocytes.

Endogenous cell repair of chronic demyelination.

In multiple sclerosis lesions, remyelination typically fails with repeated or chronic demyelinating episodes and results in neurologic disability. Acute demyelination models in rodents typically exhibit robust spontaneous remyelination that prevents appropriate evaluation of strategies for improving conditions of insufficient remyelination. In the current study, we used a mouse model of chronic demyelination induced by continuous ingestion of 0.2% cuprizone for 12 weeks. This chronic process depleted the oligodendrocyte progenitor population and impaired oligodendrocyte regeneration. Remyelination remained limited after removal of cuprizone from the diet. Fibroblast growth factor 2 (FGF2) expression was persistently increased in the corpus callosum of chronically demyelinated mice as compared with nonlesioned mice. We used FGF2 mice to determine whether removal of endogenous FGF2 promoted remyelination of chronically demyelinated areas. Wild-type and FGF2 mice exhibited similar demyelination during chronic cuprizone treatment. Importantly, in contrast to wild-type mice, the FGF2 mice spontaneously remyelinated completely during the recovery period after chronic demyelination. Increased remyelination in FGF2 mice correlated with enhanced oligodendroglial regeneration. FGF2 genotype did not alter the density of oligodendrocyte progenitor cells or proliferating cells after chronic demyelination. These findings indicate that attenuating FGF2 created a sufficiently permissive lesion environment for endogenous cells to effectively remyelinate viable axons even after chronic demyelination.

Correlation between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models.

AIM: To evaluate serum TIMP-1 level and the correlation between TIMP-1 expression and liver fibrosis in immune-induced and CCL4-induced liver fibrosis models in rats. METHODS: Immune-induced and CCL4-induced liver fibrosis models were established by dexamethasone (0.01 mg) and CCL4 respectively. Serum TIMP-1 level was detected with ELISA, while histopathological grade of liver biopsy was evaluated. Spearman rank-correlation test was used to analyse the difference of the correlation between the TIMP-1 expression and hepatic fibrosis in the two fibrosis models. Furthermore, in situ hybridization was used to determine the expression difference of TIMP-1 mRNA in the two models. RESULTS: Positive correlation existed between serum TIMP-1 level of immune induced group and the histopathological stages of fibrosis liver of corresponding rats (Spearman rank-correlation test, r(s) = 0.812, P < 0.05), and the positive in situ hybridization signal of TIMP-1 mRNA was strong. In CCL4-induced liver fibrosis model, the correlation between the serum TIMP-1 level and the severity of hepatic fibrosis was not statistically significant(Spearman rank-correlation test, r(s) = 0.229, P > 0.05). And compared with immune-induced model, the positive in situ hybridization signal of TIMP-1 mRNA was weaker, while the expression variation was higher in hepatic fibrosis of the same severity. CONCLUSION: The correlations between TIMP-1 expression and liver fibrosis in two rat liver fibrosis models are different. In immune-induced model, serum TIMP-1 level could reflect the severity of liver fibrosis, while in CCL4-induced model, the correlation between the serum TIMP-1 level and the severity of hepatic fibrosis was not statistically significant.

Lack of huntingtin-associated protein-1 causes neuronal death resembling hypothalamic degeneration in Huntington's disease.

Huntington's disease (HD) is caused by a polyglutamine expansion in the disease protein huntingtin. The polyglutamine expansion causes huntingtin to interact abnormally with a number of proteins. However, it is unclear whether, and how, huntingtin-associated proteins are involved in the neurodegeneration in HD. Here, we show that huntingtin-associated protein-1 (HAP1), which is involved in intracellular trafficking of epidermal growth factor receptor (EGFR), is highly expressed in the hypothalamus. Mice lacking HAP1 die after birth because of depressed feeding activity. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining and electron microscopic examination revealed the degeneration in hypothalamic regions that control feeding behavior. Hypothalamic degeneration was also observed in HD transgenic mice that have a significant loss of body weight. Inhibition of HAP1 expression decreases EGFR signaling and cell viability, whereas overexpression of HAP1 enhances this signaling activity and inhibits mutant huntingtin-mediated cytotoxicity. These results suggest that the effect of mutant huntingtin on HAP1 and EGFR signaling may contribute to the hypothalamic neurodegeneration and loss of body weight in HD.

Induction of hepatitis C virus-specific cytotoxic T and B cell responses by dendritic cells expressing a modified antigen targeting receptor.

AIM: To find a novel antigen (Ag) presentation strategy to improve the immune responses induced by dendritic cell (DC) vaccine expressing hepatitis C virus (HCV) core antigen (pcDNA3HCV C-Fc) in Balb/c mice (H-2d). METHODS: pcDNA3HCV C-Fc plasmid and eukaryotic expression vector pcDNA3 were injected into mice sc. Immune responses to pcDNA3HCV C-Fc were studied. Meanwhile the effect of pcDNA3HCV C-Fc on anti-translated subcutaneous tumor of SP2/0 cells stably expressing HCV C Ag (SP2/0-HCV C-FC) was also studied. Anti-HCV C in serum was detected by enzyme-linked immunoadsordent assay (ELISA) and HCV specific cytotoxic T lymphocyte (CTL) activity was measured by LDH release assay. After 3 wk of DNA immunization, the cells of SP2/0-HCV C-FC were inoculated into mice subcutaneously and tumor growth was measured every 5 d. The survival rate and living time of mice were also calculated. RESULTS: After 4 wk of DC immunization, the A(450 nm) values of sera in mice immunized with pcDNA3HCV C-Fc-DC and pcDNA3-DC were 0.56+/-0.17 and 0.12+/-0.03 respectively. The antibody titres in mice codeliveried with pcDNA3HCV C-Fc with DC were significantly higher than those of mice injected with pcDNA3-DC. The HCV specific CTL activities in mice coinjected with DC and pcDNA3HCV C-Fc or empty expression vectors were (73.2+/-3.1)% and (24.4+/-8.8)%, which were significantly higher than those of mice injected with water. The DC vaccine could evidently inhibit tumor growth, prolong the survival time of mice and improve the survival rate of mice and these effects could be improved by HCV C-Fc (pcDNA3HCV C-Fc) gene codelivered. CONCLUSION: DC vaccine has a strong antigenicity in humoral and cellular immunities, which can be promoted by transduced pcDNA3HCV C-Fc expressing HCV C or Fc. Thus, pcDNA3HCV C-Fc-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.

Artery of Percheron Infarction as an Unusual Cause of Korsakoff's Syndrome.

The Korsakoff syndrome is defined as "an abnormal mental state in which memory and learning are affected out of all proportion to other cognitive functions in an otherwise alert and responsive patient." Confabulation refers to false or erroneous memories arising, not deliberately, in the context of a neurological amnesia and is often thought of as pathognomonic of the Korsakoff syndrome. Although the exact pathophysiology is unknown, various studies have identified brain lesions in the thalami, mammillary bodies, and frontal cortex. We report a case of a 68-year-old male presenting with acute altered mental status on July 16, 2015. The neuropsychological dysfunctions included prominent Korsakoff's syndrome, which became apparent when the altered mental status resolved. Amnesia was accompanied by prominent confabulation, disorientation, and lack of insight into his own disability. Neuroradiological data indicated that the intralaminar and dorsomedial nuclei in bilateral thalami were infarcted by occlusion of the artery of Percheron. We believe that ours is one of few reported cases of Korsakoff syndrome in a patient with infarction involving the territory of the artery of Percheron. We conclude that bilateral thalamic lesions could cause Korsakoff's syndrome and the intralaminar and dorsomedial nuclei might be important structures in the pathogenesis of confabulation.

Inhibitor RNA blocks the protein translation mediated by hepatitis C virus internal ribosome entry site in vivo.

AIM: To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA (IRNA) on gene expression mediated by HCV IRES in vivo. METHODS: By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA (mIRNA) were established and characterized, and HCV replicons containing the 5' untranslated region (5'UTR) were constructed by using the same method. Cotransfection of pCMVNCRluc containing HCV 5'UTR-luc fusion genes and eukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression plasmid of IRNA was transfected transiently into HCV replicons. pCMVNCRluc or pCDNA-luc was cotransfected with pSV40-beta Gal into IRNA expressing hepatoma cells by using lipofectamine 2000 in vitro. Then the reporting gene expression level was examined at 48 h after transfection by using a luminometer and the expressing level of HCV C antigen was analysed with a confocal microscope. RESULTS: Transient expression of IRES specific IRNA could significantly inhibit the expression of reporter gene and viral antigen mediated by HCV IRES by 50% to 90% in vivo, but mIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in the HHCC constitutively expressing IRNA. At 48 h after transfection, the expression level of reporter gene decreased by 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression 24 h after transfection and the highest inhibitory activity was 80% by 72 h, and the inhibitory activity was not increased until 7d after transfection. CONCLUSION: IRNA can inhibit HCV IRES mediated gene expression in vivo.

A small yeast RNA inhibits HCV IRES mediated translation and inhibits replication of poliovirus in vivo.

AIM: To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA). METHODS: IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was transfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccines line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay. RESULTS: Constructive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constructively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus. CONCLUSION: IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication.

Expression of TIMP-1 and TIMP-2 in rats with hepatic fibrosis.

AIM: To investigate the location and expression of TIMP-1 and TIMP-2 in the liver of normal and experimental hepatic fibrosis in rats. METHODS: The rat models of experimental immunity hepatic fibrosis (n=20) were prepared by the means of immunologic attacking with human serum albumin (HSA), and normal rats (n=10) served as control group. Both immunohistochemistry and in situ hybridization methods were respectively used to detect the TIMP-1 and TIMP-2 mRNA and related antigens in liver. The liver tissue was detected to find out the gene expression of TIMP-1 and TIMP-2 with RT-PCR. RESULTS: The TIMP-1 and TIMP-2 related antigens in livers of experimental group were expressed in myofibroblasts and fibroblasts (TIMP-1: 482+/-65 vs 60+/-20; TIMP-2: 336+/-48 vs 50+/-19, P<0.001). This was the most obvious in portal area and fibrous septum. The positive signals were located in cytoplasm, not in nucleus. Such distribution and location were confirmed by situ hybridization (TIMP-1/beta-actin: 1.86+/-0.47 vs 0.36+/-0.08; TIMP-2/beta-actin: 1.06+/-0.22 vs 0.36+/-0.08, P<0.001). The expression of TIMP-1 and TIMP-2 was seen in the liver of normal rats, but the expression level was very low. However, the expression of TIMP-1 and TIMP-2 in the liver of experimental group was obviously high. CONCLUSION: In the process of hepatic fibrosis, fibroblasts and myofibroblasts are the major cells that express TIMPs. The more serious the hepatic fibrosis is in the injured liver, the higher the level of TIMP-1 and TIMP-2 gene expression.

Methodologic research on TIMP-1, TIMP-2 detection as a new diagnostic index for hepatic fibrosis and its significance.

AIM: To set up a new method to detect tissue inhibitors of metalloproteinase-1 and -2(TIMP-1 and TIMP-2) in sera of patients with hepatic cirrhosis, and to investigate the expression and location of TIMP-1 and TIMP-2 in liver tissue of patients with hepatic cirrhosis, and the correlation between TIMPs in liver and those in sera so as to discuss whether TIMPs can be used as a diagnosis index of hepatic fibrosis. METHODS: The monoclonal antibodies (McAbs) of TIMP-1 and TIMP-2 were used to sensitize erythrocytes, and solid-phase absorption to sensitized erythrocytes (SPASE) was used to detect TIMP-1 and TIMP-2 in the sera of patients with hepatic cirrhosis. Meanwhile, with the method of in situ hybridization and immunohistochemistry, we studied the mRNA expression and antigen location of TIMP-1 and TIMP-2 in the livers of 40 hepatic cirrhosis patients with pathologic diagnosis. RESULTS: With SPASE, they were 16.4% higher in the acute hepatitis group, 33.3% higher in the chronic hepatitis group, and the positive rates were 73.6% and 61.2% respectively in sera of hepatic cirrhosis patients, which were remarkably higher than those in chronic hepatitis and acute hepatitis group (P<0.001). In 40 samples of hepatic cirrhosis tissues, all of them showed positive expression of TIMP-1 and TIMP-2 mRNA detected with immunohistochemistry or in situ hybridization (positive rate was 100%). Expression of TIMPs in different degrees could be found in liver tissue with cirrhosis. TIMPs were located in cytoplasm of liver cells of patients with hepatic cirrhosis. There was a significant correlation between serum TIMPs level and liver TIMPs level. CONCLUSION: SPASE is a useful method to detect the TIMP-1 and TIMP-2 in sera of patients with hepatic cirrhosis, and TIMP-1 and TIMP-2 can be considered as a useful diagnostic index of hepatic fibrosis, especially TIMP-1.

DC-SIGN: binding receptors for hepatitis C virus.

OBJECTIVE: To review the recent developments in and research into binding receptors of hepatitis C virus (HCV) and especially the role of dendritic cell-specific adhesion receptor (DC-SIGN) in HCV. DATA SOURCES: Both Chinese- and English-language literature was searched using MEDLINE (2000 - 2003) and the databank of Chinese-language literature (2000 - 2003). STUDY SELECTION: Relevant articles on DC-SIGN and HCV binding receptors in recent domestic and foreign literature were selected. DATA EXTRACTION: Data were mainly extracted from 40 articles which are listed in the references section of this review. RESULTS: DC-SIGN, a dendritic cell-specific adhesion receptor and a type II transmembrane mannose-binding C-type lectin, is very important in the function of dendritic cells (DC), both in mediating naïve T cell interactions through ICAM-3 and as a rolling receptor that mediates the DC-specific ICAM-2-dependent migration processes. It can be used by HCV and other viral and bacterial pathogens including human immunodeficiency virus (HIV), Ebola virus, CMV and Mycobacterium tuberculosis to facilitate infection. Both DC-SIGN and DC-SIGNR can act either in cis, by concentrating virus on target cells, or in trans, by transmission of bound virus to a target cell expressing appropriate entry receptors. Recent report showed that DC-SIGN not only plays a role in entry into DC, HCV E2 interaction with DC-SIGN might also be detrimental to the interaction of DC with T cells during antigen presentation. CONCLUSIONS: DC-SIGNs are high-affinity binding receptors for HCV. The clinical strategies that target DC-SIGN may be successful in restricting HCV dissemination and pathogenesis as well as directing the migration of DCs to manipulate appropriate immune responses in autoimmunity and tumorigenic situations.

Promoting hepatic growth factor in the treatment of heavy type hepatitis and severe chronic hepatitis: a multicenter clinical study.

BACKGROUND: The mortality rate of heavy type hepatitis is high. No special treatment is available except general treatment. This multicenter clinical study was designed to observe the safety and efficacy of promoting hepatic growth factor (PHGF) in the treatment of heavy type hepatitis and severe chronic hepatitis. METHODS: 347 patients with heavy type hepatitis and 324 with severe chronic hepatitis were subjected to administration of 120 microg of PHGF per day for 4 weeks on the basis of general treatment. Those who were being effectively treated would last additional 2 to 4 weeks. Blood routine, urine routine, blood urea nitrogen (BUN), blood creatinine (Cr), blood ammonia, alpha fetoprotein (AFP), electrolyte, alanine transaminase (ALT), aspartate transaminase (AST), serum total bilirubin (TBIL), serum direct bilirubin (DBIL), prothrombin time activity (PTA), total protein (TP) and albumin (ALB) were detected in the patients before treatment, 2 weeks after treatment, and at the end of the treatment. Any side-effect would be recorded. RESULTS: In the patients with severe chronic hepatitis, the total effective rate of the treatment was 88.9%. The levels of ALT, AST and TBIL decreased significantly (P<0.001), whereas those of PTA and ALB increased significantly (P<0.001), and the level of AFP increased slightly. In patients with heavy type hepatitis, the total effective rate of this treatment was 78.4%, and patients at different stage showed different results. The total effective rates of patients with early, medium and terminal stage heavy type hepatitis were 89.9%, 84.8% and 27.5%, respectively. No severe side-effect was shown. CONCLUSION: PHGF is effective and safe in the treatment of patients with heavy type hepatitis and severe chronic hepatitis. But it should be administered early in patients with heavy type hepatitis so as to get better curative effects.

[Effect of inhibitor RNA on intracellular inhibition of viral gene expression in 5'-noncoding region of hepatitis C virus].

OBJECTIVE: To determine the effect of internal ribosome entry site (IRES)specific inhibitor RNA (IRNA) on viral gene expression mediated by HCV 5' noncoding region (NCR) in human hepatic carcinoma cell line. METHODS: Cotransfection of pCMVNCRluc containing 5'NCR-luc fusion genes and eukaryotic vectors of IRNA into human hepatic carcinoma cells (HHCC) was performed and the luciferase activity of reporter gene was measured by luminometer. RESULTS: pCMVNCRluc was efficiently expressed in HHCC. IRES specific IRNA significantly inhibited the expression of luc mediated by HCV IRES by 50% to 90%, but the mutant IRNA lost the activity of inhibition. CONCLUSION: IRNA can inhibit HCV IRES mediated gene expression in vivo.