奶牛γ干扰素基因的高效表达及活性测定

经刀豆素(conA)刺激诱导奶牛外周血淋巴细胞,应用RT-PCR方法从其总RNA中对奶牛γ干扰素基因cDNA进行扩增,然后将特异性片段连接到pMD18-T载体,测序结果表明,与已知序列同源性为100%.然后将特异性片段连在pRLC载体上进行表达,经SDS-PAGE分析,原核表达产物为16kDa的重组蛋白,占菌体总蛋白的42%,表达产物以包涵体形式存在.经7mol/L盐酸胍的变性液溶解及0.5mol/L盐酸胍复性液处理,表达产物进行脱盐、凝胶层析纯化,细胞病变抑制法结果表明,重组牛IFN-γ具有较高的干扰素活性,约为6.0×105U/mg.     

菌群失调小鼠感染鼠伤寒沙门菌后Th1和Th2细胞因子的动态研究

目的研究鼠伤寒沙门菌感染小鼠在菌群失调下Th细胞因子的动态变化,以探讨菌群失调对沙门菌感染的免疫机制。方法分别建立菌群失调、感染和空白对照的小鼠模型。各组动物在感染不同时点处死,观察小肠、肝脏和脾脏病理改变。采用流式细胞仪检测脾脏细胞中IFN-γ和IL-4表达,以此代表Th1和Th2细胞。结果菌群失调组病理损害最重,Th1明显增加,表达水平高,Th2变化不大,Th1/Th2比值升高。结论菌群失调后,能够加重小鼠感染鼠伤寒沙门菌引起的的Th1型反应,产生炎症性损伤。     

Microglia and macrophages as innate producers of interferon-gamma in the brain following infection with Toxoplasma gondii

We previously reported the requirement of interferon-gamma (IFN-gamma) expression by cells other than T and natural killer (NK) cells in the brain, in addition to T cells, for prevention of toxoplasmic encephalitis following infection with Toxoplasma gondii. In the present study, we analysed the identity of the IFN-gamma-producing non-T, non-NK cells in the brain using infected athymic nude and SCID mice that lack T cells but express IFN-gamma in their brains. Intracellular staining for IFN-gamma followed by flow cytometry revealed that approximately 45-60% of the cells expressing IFN-gamma in their brains were positive for CD11b or F4/80 on their surfaces. Smaller portions of the cells were positive for pan-NK marker. Further smaller portions were positive for CD11c, and these cells were less than 5% of the IFN-gamma-expressing cells in brains of infected SCID mice. In addition to IFN-gamma proteins, large amounts of mRNA for IFN-gamma were detected in CD11b+ cells purified from brains of infected mice, but it was not the case in the cells obtained from uninfected animals. In infected SCID mice depleted of NK cells by treatment with anti-asialo-GM1 antibody, cells expressing IFN-gamma in their brains were all positive for CD11b, and the IFN-gamma-producing cells were detected in both CD45low and CD45high populations. These results suggest that CD11b+ CD45low microglia and CD11b+ CD45high blood-derived macrophages are the major non-T, non-NK cells which express IFN-gamma in the brain of mice infected with T. gondii.     

Biological activity of hamster interferon-gamma is modulated by the carboxyl-terminal tail

The Syrian golden hamster (Mesocricetus auratus) is highly susceptible to a number of intracellular pathogens. Interferon-gamma (IFN-γ), the primary macrophage-activating cytokine, plays a key role in the host defense against intracellular pathogens. The hamster IFN-γ cDNA encodes a 174 amino acid protein that has an additional 17 amino acids at the carboxyl-terminus compared to IFN-γ of mice and rats. A homologous C-terminal tail is also found in other non-murine rodents. The biological activity of hamster IFN-γ had not been investigated previously so we first demonstrated the activity of native IFN-γ in assays of IFN-γ-induced receptor signaling and antiviral activity against vesicular stomatitis virus. We then tested the hypothesis that the C-terminal tail of hamster IFN-γ could influence its biological activity. A truncated hamster IFN-γ, in which the C-terminal 17 aa were removed by insertion of a stop codon at the position corresponding to the stop codon in the mouse sequence, had approximately 10-fold greater activity than the full length protein when measured in the two bioassays. Polyclonal and monoclonal anti-hamster IFN-γ antibodies specifically inhibited this biological activity. Collectively, these data indicate that this unique structural feature influences the biological activity of hamster IFN-γ.     

Mice lacking the IFN-γ receptor or fyn develop severe experimental autoimmune uveoretinitis characterized by different immune responses

Endogenous interferon (IFN)- negatively regulates experimental autoimmune uveoretinitis (EAU), a Th1-mediated disease. Although it is well known that IFN- exerts its effects by binding to the IFN- receptor (IFN-R), the role that IFN-R plays in the development of EAU has not been investigated. Fyn has been reported to inhibit Th2 differentiation. We aimed to investigate how endogenous IFN-R and fyn, which influence Th1/Th2 differentiation, participate in the development of EAU. Sex-matched 6- to 10-week-old C57BL/6 wild-type (WT), IFN-R knockout (GRKO) and fyn knockout (fyn KO) mice were compared. Mice were immunized subcutaneously with human interphotoreceptor retinoid-binding protein peptide 1–20 emulsified in Freunds complete adjuvant together with an intraperitoneal injection of Bordetella pertussis toxin. Three weeks later, mice were sacrificed, and their eyes and spleens were harvested for histopathologic analyses and examination of cellular immune responses, respectively. Cellular immune responses were evaluated by measuring the proliferative responses and cytokine production [interleukin (IL)-4, IL-5, IL-6, IL-13, IFN- and tumor necrosis factor (TNF)-] of splenocytes. The incidence of EAU was 40.0% in WT mice, 59.3% in GRKO mice and 78.6% in fyn KO mice. The average EAU score was 0.294 in WT mice, 0.917 in GRKO mice and 1.063 in fyn KO mice. Upon EAU induction, significant infiltration of eosinophils into the eyes was observed in GRKO and fyn KO mice compared to WT mice. Splenocytes from GRKO mice proliferated against the antigen and a mitogen more vigorously than those from WT and fyn KO mice. Stimulation of splenocytes with the antigen induced a higher production of IL-4, IL-6, IL-13 and IFN- in GRKO mice compared to WT and fyn KO mice. In contrast, IL-5 and TNF- were most abundantly produced by splenocytes from fyn KO mice compared to WT and GRKO mice. The incidence and mean severity of EAU were significantly higher in GRKO and fyn KO mice than in WT mice, suggesting that endogenous IFN-R and fyn negatively regulate the development of EAU. The different cytokine production patterns by the GRKO and fyn KO mice indicate that the negative regulatory mechanism mediated by IFN-R and fyn may differ.     

慢加急性重型乙型病毒性肝炎患者血清干扰素γ水平的检测及临床意义

目的：探讨血清干扰素-γ（IFN-γ）在慢加急性重型乙型病毒性肝炎（acute．on．chronihepatitisBloverfailure,ACHBLF）发病中的作用,旨在为ACHBLF的诊治提供理论参考。方法：采用ELSIA法测定30例ACHBLF患者和30例CHB患者血清IFN-γ水平,采用全自动生化仪检测肝功能常规指标。结果：AcHBLF组IFN-γ水平显著高于CHB组,相比较有显著性差异（P〈0．05）;ACHBLF组TBIL、ALT水平显著高于CHB组,相比较有显著性差异（P〈0．05）;ACHBLF组PTA水平显著低于CHB组,相比较有显著性差异（P〈0．05）;ACHBLF患者的IFN．叮水平与TBIL呈显著正相关（r=0．818,P〈0．01）,与PTA呈显著负相关（r=0．529,P〈0．05）,而与ALT无明显相关性（r=0．172,P〉0．05）。结论：IFN-γ参与ACHBLF的发病,并且是ACHBLF病情严重程度的重要指标之一。     

重组人γ-干扰素辅助治疗脊柱结核手术的临床研究

目的:观察重组人γ-干扰素辅助治疗脊柱结核手术的临床疗效。方法:选择2009年6月至2012年6月在我院治疗脊柱结核患者110例,随机分成实验组和对照组,每组55例。对照组患者行手术治疗,手术前后予抗结核治疗,观察组在对照组治疗的基础上加用γ-干扰素治疗。观察和比较两者患者症状改善情况、Frankel分级、并发症及复发率。结果:治疗组优良率为(96.4%)高于对照组(83.6%),两组患者功能评价比较差异均有统计学意义(X2=8.0,p=0.005);治疗组并发症发生率为5.5%,对照组为18.2%,治疗组并发症发生率比对照组低(X2=8.3,p=0.004),两组相互比较差异均有统计学意义;治疗组患者神经功能Frankel分级E级比例(98.2%)较对照组为(81.8%)高,差异有统计学意义(X2=5.1,p=0.02)。结论:人重组r-干扰素有改善脊柱结核患者的临床症状,促进伤口愈合,减少并发症及复发率等特点,值得在临床推广。     

奶牛γ干扰素基因的高效表达及活性测定

经刀豆素(conA)刺激诱导奶牛外周血淋巴细胞,应用RT-PCR方法从其总RNA中对奶牛γ干扰素基因cDNA进行扩增,然后将特异性片段连接到pMD18-T载体,测序结果表明,与已知序列同源性为100%。然后将特异性片段连在pRLC载体上进行表达,经SDS-PAGE分析,原核表达产物为16kDa的重组蛋白,占菌体总蛋白的42%,表达产物以包涵体形式存在。经7mol/L盐酸胍的变性液溶解及0.5mol/L盐酸胍复性液处理,表达产物进行脱盐、凝胶层析纯化,细胞病变抑制法结果表明,重组牛IFN-γ具有较高的干扰素活性,约为6.0×105U/mg。     

Tumor sensitivity to IFN-γ is required for successful antigen-specific immunotherapy of a transplantable mouse tumor model for HPV-transformed tumors

Purpose: Many human tumors lose responsiveness to IFN-, providing a possible mechanism for the tumor to avoid immune recognition and destruction. Here we investigate the importance of tumor responsiveness to IFN- in the successful immunotherapy of TC1 tumors that were immortalized with human papillomavirus proteins E6 and E7. Methods: To investigate the role of IFN- in vivo, we constructed a variant of TC1, TC1.mugR, that is unresponsive to IFN- due to overexpression of a dominant negative IFN- receptor. Results: Using recombinant Listeria monocytogenes that express HPV-16 E7 (Lm-LLO-E7) to stimulate an antitumor response, we demonstrate that sensitivity to IFN- is required for therapeutic efficacy in that Lm-LLO-E7 induces regression of TC1 tumors but not TC1.mugR. In addition, we show that tumor sensitivity to IFN- is not required for inhibition of tumor angiogenesis by Lm-LLO-E7 or for trafficking of CD4⁺ and CD8⁺ T cells to the tumor. However, it is required for penetration of lymphocytes into the tumor mass in vivo. Conclusions: Our findings identify a role for IFN- in immunity to TC1 tumors and show that loss of tumor responsiveness to IFN- poses a challenge to antigen-based immunotherapy.Mary E. Dominiecki and Gregory L. Beatty contributed equally to this work.