用构建的新型BmNPV载体在家蚕高效表达人β-干扰素

家蚕核多角体病毒(Bombyx mori Nuclear Polyhedrosis Virus.BmNPV)和家蚕细胞已成功地用来大量生产具有生物活性的重组蛋白。但是BmNPV的通用载体的类型较少。因此,本实验构建了BmNPV新型载体pBm92,该载体将多角体蛋白基因的起始密码ATG改变为ATT,然后在多角体蛋白基因的+12位外连接有5个外源基因的克隆位点。将HuIFN-β基因克隆在多角体蛋白基因的+12位后,构建了pBmIFN+12;同时构建HuIFN-β克隆在-3位后的转移栽体pBmIFN-3。将两种转移载体DNA分别与BmNPV基因组DNA共转染Bm-N细胞。利用重组病毒不产生多角体蛋白的特征,筛选重组病毒。用HuIFN-β基因探针与重组病毒DNA进行杂交鉴定。重组病毒BmIFN+12感染Bm-N细胞,其上清IFN活性最高时可达2.0×10~6IU/ml,将BmIFN+12注射5龄家蚕虫体,表达水平为50×10~7IU/ml,是HuIFN-β基因克隆在多角体蛋白基因的-3位后获得的重组病毒的表达量的2～4倍。家蚕体生产的rHulFN-β为糖基化蛋白具有天然HuIFN-β的抗原性。

Expression of interferon-alpha/beta receptor protein in liver of patients with hepatitis C virus-related chronic liver disease

ABM: To study the expression of interferon-alpha/beta (IFN-α/β) receptor protein in liver of patients with hepatitis C virus (HCV)-related chronic liver disease and its clinical significance. METHODS: A total of 181 patients with HCV-related chronic liver disease included 56 with HCV-related liver cirrhosis (LC) and 125 with chronic hepatitis C (CHC). CHC patients were treated with five megaunits of interferon-α1b six times weekly for the first 2 weeks and then every other day for 22 wk. The patients were divided into interferon (IFN) treatment-responsive and non-responsive groups, but 36 patients lost follow-up shortly after receiving the treatment. The expression of IFN-α/β receptor (IFN-α/βR) protein in liver of all patients was determined with immunofluorescence. RESULTS: In liver of patients with HCV-related chronic liver disease, the expression of IFN-α/βR protein in liver cell membrane was stronger than that in cytoplasm and more obvious in the surroundings of portal vein than in the surroundings of central vein. Moreover, it was poorly distributed in hepatic lobules. The weak positive, positive and strong positive expression of IFN-α/βR were 40% (50/125), 28% (35/125), 32% (40/125), respectively in CHC group, and 91.1% (51/56), 5.35% (3/56), and 3.56% (2/56), respectively in LC group. The positive and strong positive rates were higher in CHC group than in LC group (P<0.01). In IFN treatment responsive group, 27.8% (10/36) showed weak positive expression; 72.2% (26/36) showed positive or strong positive expression. In the non-responsive group, 71.7% (38/53) showed weak positive expression; 28.3% (15/53) showed positive or strong positive expression. The expression of IFN-α/βR protein in liver was more obvious in IFN treatment responsive group than in non-responsive group. CONCLUSION: Expression of IFN-α/βR protein in liver of patients with HCV-related chronic liver disease is likely involved in the response to IFN treatment.

Effect of in vitro interferon-beta administration on hepatitis C virus in peripheral blood mononuclear cells as a predictive marker of clinical response to interferon treatment for chronic hepatitis C

AIM:To test whether in vitro incubation of peripheral blood mononuclear cells (PBMC) with interferon (IFN) could efficiently decrease hepatitis C virus-RNA (HCV-RNA) amount and to analyze whether this effect was associated with clinical response to IFN.METHODS:Twenty-seven patients with histologically proven chronic hepatitis C were given intravenous administration of 6 million units (MU) IFN-β daily for 6 weeks followed by three times weekly for 20 weeks. PBMC collected before IFN therapy were incubated with IFN-β and HCV-RNA in PMBC was semi-quantitatively determined.RESULTS: Twenty-five patients completed IFN therapy.Eight patients (32%) had sustained loss of serum HCV-RNA with normal serum ALT levels after IFN therapy (complete responders).HCV-RNA in PBMC was detected in all patients,whereas it was not detected in PBMC from healthy subjects.In vitro administration of IFN-β decreased the amount of HCV-RNA in PMBC in 18 patients (72%). Eight of these patients obtained complete response. On the other hand,none of the patients whose HCV-RNA in PBMC did not decrease by IFN-β was complete responders. Multiple logistic regression analysis revealed that the decrease of HCV-RNA amount in PBMC by IFN-β was the only independent predictor for complete response (P<0.05).CONCLUSION:The effect of in vitro IFN-β on HCV in PBMC reflects clinical response and would be taken into account as a predictive marker of IFN therapy for chronic hepatitis C.

Interferon β-1a alone or in combination with ribavirin: A randomized trial to compare efficacy and safety in chronic hepatitis C

AIM： To compare the efficacy and safety of recombinant human IFN β-la alone or in combination with ribavirin in treatment-naive subjects with chronic hepatitis C. METHODS： Open, randomized trial was performed in 6 Italian tertiary centers： 102 of the 108 patients screened were randomized to receive 6 MIU of recombinant human IFN β-la subcutaneously daily for 24 wk, alone （Group 1, n = 51） or in combination with ribavirin 1 000 to 1 200 mg/d （Group 2, n = 51）. RESULTS： The end-of-treatment virologic response rate was 29.4% in Group 1 and 41.2% in Group 2 （nonsignificant）. Twenty-four weeks after stopping therapy, sustained virologic response rate was 21.6% in Group 1 and 27.4% in Group 2 （non-significant）. All subjects in Group 1 completed treatment, while two subjects in Group 2 stopped therapy due to treatment-related adverse events. CONCLUSION： Recombinant human IFN β-la, alone or in combination with ribavirin, has an excellent safety profile and, may represent an alternative for chronic hepatitis C patients who are unable to tolerate pegylated α-interferon.

Is interferon-beta an alternative treatment for chronic hepatitis C?

The treatment of chronic hepatitis C （CHC） is still far from optimal, particularly for those subpopulations that do not respond to the standard combination therapy with Interferon-α （IFNα） plus ribavirin. Although in some cases the use of higher doses or longer treatment periods may be effective, these approaches are generally associated with a higher incidence of adverse events, which may either lead to a reduction in patient compliance or require drug withdrawal. IFNβ could represent an interesting alternative for treating CHC patients. Controversial data about IFNβ efficacy in CHC exist, the main reason being that many results stem from pilot studies with small cohorts of patients. However, promising results have been obtained in some subgroups of patients that fail to respond to IFNα. Additionally, the good tolerability of IFNβ represents an important advantage of the drug. The rates of dropouts in controlled clinical trials, as well as the need for dose reductions or treatment discontinuation are very low. It might be worth assessing the value of IFNβ plus ribavirin in randomized studies with a larger cohort of patients, not eligible or not tolerating standard therapy, and for non-responders.

Effect of expression of TGF-beta and TNF-alpha with Qidan granule treatment in rats by bleomycin-induced pulmonary fibrosis

Objective: To evaluate the therapeutic effects and mechanisms of Qidan granule in blemycinA5-induced pulmonary interstitial fibrosis (PIF)in rats. Methods: PIF models were established by blemycinA5-induced in rats. They were treated by Qidan granule and Hydrocortisone respectively. The pathological changes and collagen protein disposition were observed, and the expression of TGF-β, TNF-α proteins were measured by immunohistochemical technique. Results: The pulmonary alveolitis and fibrosis were alleviated remarkably in Qidan granule group compared with those in the model control group and hydrocortisone group (P<0.01). The expression of TGF-β and TNF-α protein were higher in Qidan granule group than those in normal group ,and were significantly less than those in the model control group and in hydrocortisone group (P<0.01). Conclusion: Qidan granule would ameliorate the pulmonary alveolitis and fibrosis. TGF-β and TNF-α might play an important role in the development of alveolitis and fibrosis in rats.

Interferon-γ inhibits in situ expression of PDGF-β mRNA by smooth muscle cells in injured rabbit arteries after transluminal balloon angioplasty

Objective To elucidate the mechanism of interferon-gamma (IFN-γ) to inhibit the restenosis after successful percutaneous transluminal angioplasty (PTA).Methods A rabbit vascular restenotic model was constructed and the proliferation of intimal smooth muscle cells (SMCs) were observed by monitoring their expression of proliferating cell nuclear antigen (PCNA) and platelet-derived growth factor β chain mRNA (PDGF-β mRNA) at the indicated time points. Results IFN-γ could significantly inhibit the expression of PCNA by intimal SMCs one week after denudation, when counting 200 intimal cells for PCNA-positive reactions with an inhibitory rate of 88.50% (P<0.001). IFN-γ could downregulate in situ expression of PDGF-β mRNA by these cells as we calculated the average number of PDGF-β mRNA positive cells per square millimetre area at ×400 magnification with reduced rates of 86.85% in 1 week group (P<0.001), of 93.66% in 2 week group (P<0.001) and of 52.92% in 4 week group (0.02<P<0.05), respectively. Conclusions The local production of PDGF-β by vascular intimal SMCs via an autocrine mechanism may be responsible for continuous proliferation of these cells and the formation of neointima after injury. This could be inhibited by IFN-γ through downregulating the expression of PDGF-β mRNA. These results provide an in vivo basis for IFN-γ to be used clinically for the management of restenosis after percutaneous transluminal angioplasty.