Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

AIM： To determine the effect of hammerhead ribozyme targeting connective tissue growth factor （CCN2） on human hepatic stellate cell （HSC） function.METHODS： CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor （TGF）-β1 to the culture medium. Semiquantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell /ysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry.RESULTS： In pTriEx2-transfected LX-2 cells, TGF-β1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels, pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-β1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase.CONCLUSION： Endogenous CCN2 is a mediator of basal or TGF-β1-induced collagen I production in human HSCs and regulates entry of the cells into S phase.

Connective tissue growth factor is overexpressed in human hepatocellular carcinoma and promotes cell invasion and growth

AIM:To determine the expression characteristics of connective tissue growth factor(CTGF/CCN2) in human hepatocellular carcinoma(HCC) in histology and to elucidate the roles of CCN2 on hepatoma cell cycle progression and metastasis in vitro.METHODS:Liver samples from 36 patients(who underwent hepatic resection for the first HCC between 2006 and 2011) and 6 normal individuals were examined for transforming growth factor β1(TGF-β1) or CCN2 mRNA by in situ hybridization.Computer image analysis was performed to measure integrated optimal density of CCN2 mRNA-positive cells in carcinoma foci and the surrounding stroma.Fibroblast-specific protein-1(FSP-1) and E-cadherin were examined to evaluate the process of epithelial to mesenchymal transition,α-smooth muscle actin and FSP-1 were detected to identify hepatic stellate cells,and CD34 was measured to evaluate the extent of vascularization in liver tissues by immunohistochemical staining.CCN2 was assessed for its stimulation of HepG2 cell migration and invasion using commercial kits while flow cytometry was used to determine CCN2 effects on HepG2 cell-cycle.RESULTS:In situ hybridization analysis showed that TGF-β1 mRNA was mainly detected in connective tissues and vasculature around carcinoma foci.In comparison to normal controls,CCN2 mRNA was enhanced 1.9-fold in carcinoma foci(12.36 ± 6.08 vs 6.42 ± 2.35) or 9.4-fold in the surrounding stroma(60.27 ± 28.71 vs 6.42 ± 2.35),with concomitant expression of CCN2 and TGF-β1 mRNA in those areas.Epithelial-mesenchymal transition phenotype related with CCN2 was detected in 12/36(33.3%) of HCC liver samples at the edges between carcinoma foci and vasculature.Incubation of HepG2 cells with CCN2(100 ng/mL) resulted in more of the cells transitioning into S phase(23.85 ± 2.35 vs 10.94 ± 0.23),and induced a significant migratory(4.0-fold) and invasive(5.7-fold) effect.TGF-β1-induced cell invasion was abrogated by a neutralizing CCN2 antibody showing that CCN2 is a downstream mediator of TGF-β1-induced hepatoma cell invasion.CONCLUSION:These data support a role for CCN2 in the growth and metastasis of HCC and highlight CCN2 as a potential novel therapeutic target.

An immortalized rat pancreatic stellate cell line RP-2 as a new cell model for evaluating pancreatic fibrosis,inflammation and immunity

BACKGROUND： Pancreatic stellate cells （PSCs） play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells, immune cells or intermediaries in pancreatic exocrine secretion. Increasing evidence has shown that desmin as an exclusive cytoskeleton marker of PSC is only expressed in part of these cells. This study was to establish a desmin-positive PSC cell line and evaluate its actions on pancreatic fibrosis, inflammation and immunity.

Focal autoimmune pancreatitis and chronic sclerosing sialadenitis mimicking pancreatic cancer and neck metastasis

Type 1 autoimmune pancreatitis (AIP) or chronic sclerosing sialadenitis (Küttner’s tumour) is an uncommon disorder that has recently been confirmed as an IgG4-related disease. Here, we describe a rare case of a 53-year-old male patient who primarily presented with pancreatic body mass, left neck mass and several lumps in his lower lip mimicking pancreatic cancer (PC) and neck metastasis. The patient underwent pancreatic body mass and labial gland lumps resection as well as an ultrasound-guided biopsy of the left neck mass. He was diagnosed with IgG4-related focal type of AIP (f-AIP) and Küttner’s tumour by immunohistochemistry. The patient responded well to corticosteroid therapy and remains healthy with no signs of recurrence at one year follow-up. The differentiation of f-AIP from PC is very important to avoid unnecessary pancreatic resection.

Hepatitis B virus-related liver cirrhosis complicated with dermatomyositis: A case report

BACKGROUND Twenty percent of patients infected with hepatitis B virus(HBV)develop extrahepatic manifestations with HBV detected in the lymph nodes,spleen,bone marrow,kidneys,and skin.HBV infection has been associated with some autoimmune disorders.Dermatomyositis(DM)is an idiopathic inflammatory myopathy,which involves a viral infection,and DM has been identified in patients infected with HBV,but there is no direct histological evidence for an association between HBV and DM.CASE SUMMARY We describe a familial HBV-infected patient admitted with liver function abnormality,rashes,a movement disorder,and an elevated level of creatine kinase(CK).A computed tomography scan of the lung showed pulmonary fibrosis,and a liver biopsy identified nodular cirrhosis.An electromyogram revealed myogenic damage,and a muscle biopsy showed nuclear migration in local sarcolemma and infiltration of chronic inflammatory cells.Immunohistochemical staining showed negative results for HBsAg and HBcAg.Fluorescence in situ hybridization showed a negative result for HBV DNA.The patient was diagnosed with HBV-related liver cirrhosis complicated with DM and was treated with methylprednisolone,mycophenolate mofetil,and lamivudine.Eight months later,the patient was readmitted for anorexia and fatigue.The blood examination showed elevated levels of aminotransferases and HBV DNA,however,the CK level was within the normal range.The patient developed a virological breakthrough and lamivudine was replaced with tenofovir.CONCLUSION DM in chronic HBV-infected patients does not always associate with HBV.Antiviral and immunosuppressive drugs should be taken into consideration.

CD4+Foxp3+CD25+/-Tregs characterize liver tissue specimens of patients suffering from drug-induced autoimmune hepatitis:A clinical-pathological study

Background: The diagnosis of drug-induced autoimmune hepatitis(DIAIH) and its differentiation from idiopathic autoimmune hepatitis(AIH) is challenging. This study aimed to differentiate DIAIH from AIH by comparing the biochemical changes, histological features, and frequencies of CD4~+Foxp3~+CD25+/-regulatory T cells(Tregs) in liver tissues or peripheral blood lymphocytes.Methods: A total of 15 DIAIH patients and 24 AIH patients who underwent liver biopsies at initial presentation were enrolled in this study. The liver histological changes were assessed by HE staining. The phenotypic recognition and distribution of CD4~+Foxp3~+CD25+/-Tregs in liver tissues were evaluated by single/double immunostains in serial sections. The CD4~+Foxp3~+CD25+/-Tregs in peripheral blood were analyzed by flow cytometry.Results: The median values of ALT and AST were 404.50 U/L and 454.10 U/L in DIAIH patients and309.50 U/L and 315.00 U/L in AIH patients, respectively. More importantly, for the first time we found that patients with DIAIH had higher levels of serum ALT and AST, more severe degree of lobular inflammation,higher frequencies of zone 3 necrosis and higher number of lobular CD4~+Foxp3~+CD25~-Tregs compared with AIH(P < 0.05). Furthermore, there were positive correlations in DIAIH between the degree of lobular inflammation and either the AST/ALT level or the number of lobular CD4~+Foxp3~+CD25~-Tregs(P < 0.05).However, the frequency of peripheral blood CD4~+Foxp3~+CD25+/-Tregs were not significantly different between DIAIH and AIH.Conclusions: The differences of ALT, AST and the number of lobular CD4~+Foxp3~+CD25~-Tregs between patients with DIAIH and those with AIH are clinically helpful in differentiating these two diseases in their early stage.

Rare case of Helicobacter pylori-positive multiorgan Ig G4-related disease and gastric cancer

A 61-year-old male from Northeast China presented with a 2-mo history of abdominal distension,pruritus and jaundice.Laboratory testing revealed an elevated serum Ig G4 level.A computed tomography scan showed a typical feature of autoimmune pancreatitis(AIP) and cholecystocholangitis.Early gastric cancer was incidentally discovered when endoscopic untrasound-guided fine needle aspiration(EUSFNA) of the pancreas was carried out.The patient underwent radical subtotal gastrectomy for gastric cancer combined with cholecystectomy.Helicobacter pylori(H.pylori) and Ig G4-positive plasmacytes were detected in gastric cancer tissue,pancreatic EUSFNA sample and resected gallbladder specimen by immunohistochemistry.The patient was diagnosed with H.pylori-positive Ig G4-related AIP and sclerosing cholecystocholangitis as well as H.pylori-positive gastric cancer.He responded well to steroid therapy and remains healthy with no signs of recurrence at one year follow-up.We speculate that H.pylori might act as a trigger via direct or indirect action in the initiation of onset of gastric cancer and multiorgan Ig G4-related disease.

Clinical significance of connective tissue growth factor in hepatitis B virus-induced hepatic fibrosis

AIM:To determine the utility of connective tissue growth factor(CCN2/CTGF) for assessing hepatic fibrosis in hepatitis B virus(HBV)-induced chronic liver diseases(CLD-B).METHODS:Enzyme-linked immunosorbent assay was used to measure CCN2 in sera from 107 patients with chronic hepatitis B(CHB) and 39 patients with HBVinduced active liver cirrhosis and 30 healthy individuals.Liver samples from 31 patients with CHB,8 patients with HBV-induced liver cirrhosis and 8 HBV carriers with normal liver histology were examined for transforming growth factor β-1(TGF-β1) or CCN2 mRNA levels by in situ hybridization,and computer image analysis was performed to measure integrated optimal density(IOD) of CCN2 mRNA-positive cells in liver tissues.Histological inflammation grading and fibrosis staging were evaluated by H and E staining and Van Gieson's method.RESULTS:Serum CCN2 concentrations were,respectively,4.0-or 4.9-fold higher in patients with CHB or active liver cirrhosis as compared to healthy individuals(P < 0.01).There was good consistency between the levels of CCN2 in sera and CCN2 mRNA expression in liver tissues(r = 0.87,P < 0.01).The levels of CCN2 in sera were increased with the enhancement of histological fibrosis staging in patients with CLD-B(r = 0.85,P < 0.01).Serum CCN2 was a reliable marker for the assessment of liver fibrosis,with areas under the receiver operating characteristic(ROC) curves(AUC) of 0.94 or 0.85 for,respectively,distinguishing normal liver controls from patients with F1 stage liver fibrosis or discriminating between mild and significant fibrosis.CONCLUSION:Detection of serum CCN2 in patients with CLD-B may have clinical significance for assessment of severity of hepatic fibrosis.

IgG4-related autoimmune pancreatitis overlapping with Mikulicz's disease and lymphadenitis:A case report

Autoimmune pancreatitis(AIP)is a form of chronic pancreatitis that is categorized as type 1 or type 2according to the clinical profile.Type 1 AIP,which predominantly presents in a few Asian countries,is a hyper-IgG4-related disease.We report a case of IgG4-related AIP overlapping with Mikulicz’s disease and lymphadenitis,which is rare and seldom reported in literature.A 63-year male from Northeast China was admitted for abdominal distension lasting for one year.He presented symmetric swelling of the parotid and submandibular glands with slight dysfunction of salivary secretion for 6 mo.He had a 2-year history of bilateral submandibular lymphadenopathy without pain.He underwent surgical excision of the right submandibular lymph node one year prior to admission.He denied any history of alcohol,tobacco,or illicit drug use.Serological examination revealed high fasting blood sugar level(8.8 mmol/L)and high level of IgG4(15.2 g/L).Anti-SSA or anti-SSB were negative.Computed tomography of the abdomen showed a diffusely enlarged pancreas with loss of lobulation.Immunohistochemical stain for IgG4 demonstrated diffuse infiltration of IgG4-positive plasma cells in labial salivary gland and lymph node biopsy specimens.The patient received a dose of 30 mg/d of prednisone for three weeks.At this three-week follow-up,the patient reported no discomfort and his swollen salivary glands,neck lymph node and pancreas had returned to normal size.The patient received a maintenance dose of 10mg/d of prednisone for 6 mo,after which his illness had not recurred.

IgG4-related sclerosing cholangitis and chronic sclerosing sialadenitis mimicking cholangiocarcinoma and neck malignancy

To the Editor:IgG4-related sclerosing cholangitis（IgG4-SC）has re cently been recognized as a biliary manifestation of IgG4-related disease（IgG4-RD）.Type 3 IgG4-SC pre sented biliary strictures in both the porta hepatis and the distal common bile duct（CBD）.;Its manifestation especially in the absence of autoimmune pancreatitis,is extremely rare and very similar to that of cholangiocarci noma（CC）.