抗腺病毒免疫核糖核酸活性的实验研究

腺病毒(7型)经培养和纯化后免疫绵羊,从免疫羊肝脾抽提抗腺病毒免疫核糖核酸(AdV—i—RNA)。应用ELISA和细胞毒试验研究AdV—i—RNA的免疫活性,实验结果证明:AdV—i—RNA可以在小鼠体内诱生抗腺病毒抗体及增强小鼠脾细胞的细胞毒活性。     

泛素与EB病毒核抗原1融合基因的DNA免疫研究

构建了EB病毒核抗原1(EBNA1)基因的表达质粒pCI-EBNA1和泛素(ubiquitin,Ub)与EBNA1融合基因的表达质粒pCI-Ub-EBNA1.间接免疫荧光和Western blot分析表明,两重组质粒转染HeLa细胞后均能瞬时表达.两种质粒DNA肌肉注射免疫Balb/C小鼠后,分别检测小鼠血清抗EBNA1的抗体和特异性的细胞毒性T淋巴细胞(CTL)反应,比较单基因与融合基因DNA免疫所诱生免疫应答的强度.结果显示:二者诱生抗体的效率无明显差别,但泛素的融合使得针对EBNA1的特异性CTL反应明显增强.     

针对呼吸道合胞病毒的免疫应答

人体染呼吸道合胞病毒（ＲＳＶ）反仅部分人产生保护性免疫,部分人仍可重复感染。在保护性免疫应答中发挥主要作用提针对该病毒Ｆ蛋白和Ｇ蛋白的中和抗体。     

抗菌免疫核糖核酸组份的分离及其活性测定

<正> 抗菌免疫核糖核酸(iRNA)已用于条件致病菌感染的临床治疗,并对其免疫活性做了全面的研究。现已证实iRNA能够诱导特异性抗体的产生和传递特异性的细胞免疫,并能诱生干扰素和白细胞间素Ⅰ、Ⅱ等淋巴因子和单核因子。但制备的iRNA是含有多种RNA种类的混合物,为明确各组份的免疫学功能,我们对iRNA进行了分离,并测定不同组份的免疫活性。     

柯萨奇病毒A组16型灭活抗原对小鼠免疫保护作用的研究

为了研究柯萨奇病毒A组16型(Coxsackievirus A16,CVA16)灭活抗原在小鼠体内所产生免疫保护作用效果,我们选用CVA16临床分离株521-01T,在Vero细胞中进行大量培养,并对培养产物进行甲醛灭活及超速离心纯化。SDS-PAGE和Western blot对纯化的灭活病毒纯度及性质进行初步分析。Al(OH)3+CVA16及单独CVA16抗原,分别经皮下注射免疫雌性ICR小鼠;相同免疫途径、剂量于第14和28d加强免疫2次。ELISA法检测CVA16特异性血清IgG抗体滴度;微量中和试验法鉴定血清中和抗体滴度;酶联免疫斑点试验(ELISPOT)检测特异性T淋巴细胞的活化。对Al(OH)3+CVA16抗原免疫组母鼠所产仔鼠进行脑腔攻毒,检测母传抗体对新生乳鼠的保护作用。结果显示,Al(OH)3+CVA16灭活抗原在小鼠体内能诱生高滴度的特异性抗体,3次免疫后产生的特异性血清IgG抗体滴度最高可达1∶1×105(P=0.000),中和滴度高于1∶256。同时,该抗原还可以诱导特异性T淋巴细胞的活化。以1 000LD50的病毒量脑腔接种48h内新生乳鼠的病毒攻击实验显示,该母传抗体对新生乳鼠具有100%的保护。这一结果表明该灭活CVA16病毒抗原具有较好的免疫原性及保护性,为CVA16灭活疫苗的研究及评价体系提供了参考。     

连续注射抗菌i-RNA诱生内源性细胞毒因子的研究

应用抗菌免疫核糖核酸(i-RNA)对家兔进行连续致敏注射和诱生注射,结果证明在家兔血清中有抑制和杀伤靶细胞(L929)作用较强的细胞毒因子出现。实验对i-RNA诱生内源性细胞毒因子的动态、不同i-RNA的诱生效果,对人和鼠的某些瘤细胞毒作用等做了初步研究,对内源性细胞毒因子的作用主要来自TNF的活性也做了讨论。     

ELISA间接法检测呼吸道合胞病毒和副流感病毒血清IgM和IgA抗体

建立了检测呼吸道合胞病毒(RSV)和副流感病毒(PFV)血清特异性IgM和IgA抗体的间接ELISA方法。在方法统一的基础上比较了检测IgG、IgM和IgA抗体的结果,证明检测血清IgM和IgA可以作为RSV和PFV感染的早期诊断指标。检测了120份临床急性下呼吸道感染患儿的血清,RSV-IgM检出率为33.3％,RSV-IgA为36.7％;PFV-IgM为27.5％,PFV-IgA为31.6％。提出了对RSV和PFV感染以检测特异性IgA替代IgM或两者互补的设想。     

抗菌i－RNA没有传递特异性体液免疫活性能力的确定及其对机体免疫功能影响的研究

本实验用抗绿脓杆菌ｉ－ＲＮＡ致敏小鼠,用酶联免疫吸附试验直接检测鼠体内绿脓杆菌特异性抗体的产生,并通过电镜技术观察补体参与下的溶菌杀菌现象来间接反映体内是否有特异性抗体的产生。结果证实ｉ－ＲＮＡ没有传递特异性体液兔疫活性的能力。但小鼠体内脾细胞抗体产生能力,巨噬细胞吞噬活性,脾细胞ＮＫ活性和ＩＬ－２活性均明显高于正常鼠,提示抗菌ｉ－ＲＮＡ有佐剂活性,可以增强机体的体液免疫功能和细胞免疫功能。     

人呼吸道合胞病毒F-Fc蛋白不同免疫方案对小鼠免疫效果的比较

人呼吸道合胞病毒（Human respiratory syncytial virus,hRSV）是全球婴幼儿和老年人严重呼吸道疾病的主要原因。hRSV感染主要局限于呼吸道，当鼻黏膜中特异性IgA抗体滴度较低时容易引起hRSV反复感染，理想的hRSV疫苗应诱导全身免疫应答，尤其是黏膜免疫。本研究应用CHO细胞表达融合蛋白F-Fc（含有hRSV F蛋白和人IgG1抗体的Fc片段），F-Fc蛋白结合CpG佐剂两次免疫小鼠，比较滴鼻免疫（Intranasal,in）和肌肉注射（Intramuscular,im）免疫安全性和有效性的差异。与佐剂对照组（CpG）相比，四种免疫方式（CpG+F-Fc/in+im,CpG+F-Fc/im+in,CpG+F-Fc/im+im和CpG+F-Fc/in+in）均能诱导高滴度中和抗体，高水平及Th1偏向的细胞免疫应答，减少肺脏病毒的滴度，但是两次滴鼻免疫组小鼠效果是最好的。同时，两次滴鼻免疫组小鼠诱导的IgA抗体最多，小鼠体重恢复速度最快，并且可以显著降低肺脏病理损伤。综上所述，在以上四种免疫方案中，两次滴鼻免疫诱导产生的免疫效果最优。     

栝楼核糖核酸酶的性质研究

关于栝楼核糖核酸酶的性质已经研究.它对核糖核酸和多聚尿嘧喧核苷酸所显示的酶活性,有很大的相似性:(1)栝楼核糖核酸酶的最适度应pH在5左右;(2)稳定的pH范围在5-12之间;(3)最佳反应温度为55℃左右;(4) 热稳定性:在15分钟内,60℃以下酶活性基本稳定,在65℃左右时大约有50%的活性丧失;(5)金属离子中,除CU~(2+)和Ag~+有明显的抑制作用外、一般作用不明显.另外,酸对其它合成的多聚核苷酸的水解作用,显示有碱基特异性;对双链RNA(CPV RNA)也显有活性.实验中未发现该酶有磷酸单酯酶的活性及降解DNA的活性.因此,该酶可能是一种具有碱基特异性的核糖核酸酶.     

Detection of nonspecific cytotoxicity in graft-versus-host reaction as a function of target cell type

The cytotoxic activity of spleen cells from mice undergoing graft-versus-host (GVH) reaction on ⁵¹Cr-labeled target cells was studied under in vitro conditions. Among normal tissues used as target cells, skin fibroblasts proved to be most sensitive to the nonspecific cytotoxic effects of spleen cells from mice undergoing GVH reaction, whereas kidney cells or macrophages were insensitive to these nonspecific cytotoxic effects. Of the two murine neoplastic target cells used, Sarcoma 1 cells were susceptible to these nonspecific cytotoxic effects whereas mastocyoma cells were resistant. However, the target cells which were insensitive to the nonspecific cytolytic effects, were lysed specifically by the spleen cells from animals specifically sensitized. Therefore, both specific and nonspecific cytotoxic effects of spleen cells from mice undergoing GVH reaction could be detected with appropriate targets. These results provide a basis for reconciliation of several apparently contradictory results, reported in the literature, concerning the specificity of the cytotoxic effects of specifically sensitized lymphocytes.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.     

The cryopreservation of immunocompetent cells

Lymphoid cells sensitized against tumor cells in vivo were removed from sheep and mice, frozen and thawed, and then assayed for retention of cytotoxic function. Sheep lymphocytes efferent from immunized nodes have been shown previously to be cytotoxic owing to the production of antibody and these cells retained approximately 50% of their activity after freezing. The specific cytotoxicity of sensitized mouse spleen cells has been shown to be due to thymus-derived lymphocytes and such cells totally retained specific cytotoxic action after freezing. However, spleen cells also generally exhibited a degree of nonspecific cytotoxic action which disappeared on freezing and was attributed to the selective elimination of glass-adherent cells by the freezing process.     

Generation of suppressor lymphocytes during sensitization in culture against a syngeneic tumor: affinity chromatography on insolubilized histamine

We investigated the effect of depletion of histamine-binding lymphoid cells on immunological properties of lymphocytes sensitized in culture against tumor cells. C57BL/6 spleen cells that were sensitized in vitro on monolayers of the syngeneic Lewis lung carcinoma (3LL) became cytotoxic to the tumor cells in vitro after 3 to 5 days of sensitization. Sensitized cells harvested after 4 days of sensitization occasionally enhanced tumor growth in vivo. Fractionation of the sensitized lymphocytes over insolubilized histamine-rabbit serum albumin-Sepharose (HRS) columns decreased or abolished the enhancing activity in vivo and specifically increased the in vitro cytotoxic activity of the depleted lymphocytes. A similar increase in the cytotoxic activity of HRS-fractionated cells was observed in an allogeneic combination of C57BL spleen cells sensitized against C3H fibroblasts. The effect of HRS chromatography on the in vitro cytotoxic activity increased with prolonged incubation of the depleted effector cells with the target cells.     

Augmentation of killer activity of murine spleen cells against allogeneic and syngeneic tumor cells by inoculation of alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.     

Purine metabolic enzymes in lymphocytes. II. Adenosine deaminase and purine nucleoside phosphorylase activities in the immune response

ICR mice were immunized with sheep red blood cells (sRBC). Both adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in spleen lymphocytes increased faster than the serum antibody titer and reached a peak one week after the immunization. ADA activity increased significantly in T lymphocytes but not in B lymphocytes collected from the spleens of the immunized mice. A statistically significant increase in PNP activity was found in both T and B lymphocytes from the spleens of the immunized mice. Spleen lymphocytes collected from ICR mice which had been immunized with mitomycin C-treated sarcoma 180 (S180) cells one week earlier showed cytotoxic activity against viable S180 cells. Both ADA and PNP activities in spleen lymphocytes of S180-immunized mice increased significantly, and both activities increased in T lymphocytes prepared from spleen of immunized mice. In contrast, an increase was found in PNP activity but not in ADA activity in B lymphocytes. These results suggest that an increase in both ADA and PNP activities may by necessary for the T-cell response in both humoral and cellular immune responses, and that an increase in PNP activity may be necessary for the B-cell response.     

Generation of cytotoxic T lymphocytes during coxsackievirus tb-3 infection. II. Characterization of effector cells and demonstration cytotoxicity against viral-infected myofibers1.

This report describes studies characterizing the virus-specific cytotoxic effector cells which are present in the spleens of mice 7 days after infection with Coxsackievirus B-3. An in vitro 51Cr assay employing eyngeneic virus-infected neonatal fibroblasts was used to measure cytotoxic activity. Treatment of immune cells with (anti-thy-1.2) and complement abolished dtheir cytotoxic activity, but no reduction occurred when B cells were removed by incubation with anti-Ig and complement or macrophages eliminated by adherence depletion. The findings therefore imply that the cytotoxic reaction was mediated by sensitized T cells and that B cells and macrophages did not play an important role. Reciprocal assays performed with BALB/c and CBA/J cells showed that Coxsackievirus-immune spleen cells lysed infected syngeneic targets but not allogeneic targets, providing further evidence that cytotoxicity was mediated by effector T cells. In addition and in vitro assay system employing neonatal myocardial cells was developed and used to demonstrate that Coxsackievirus-infected myofibers were susceptible to destruction by immune spleen cells. The evidence suggests that mice infected with Coxsackie B viruses are able to mount a cell-mediated immune response with production of cytotoxic T cells which have the capacity to damage tissues infected with these agents.     

Transfer in vivo of cytotoxic immune lymphocytes obtained in vitro in primary culture against a murine lymphoma antigenically altered by DTIC]

Specific cytotoxic T cells were obtained by coculturing "in vitro" normal spleen cells with inactivated histocompatible DTIC-altered lymphoma cells. The "in vivo" antitumor activity of such sensitized lymphocytes was evaluated by injecting a mixture of lymphocytes + tumor into the brains of lethally irradiated syngenic mice. The results indicated that such lymphocytes demonstrate antitumor activity against the same tumor but not against unrelated tumors.     

Production of cytotoxic antibody to a benz(a)pyrene-induced sarcoma in mice receiving xenogeneic antitumor immune RNA

Summary Groups of normal C3Hf mice were injected subcutaneously and intraperitoneally over a 2-week period with various xenogeneic immune RNA (I-RNA) preparations. I-RNAs were extracted from the lymphoid organs of guinea pigs following immunization with normal C3Hf tissue cells, or tumor cells from a benz(a)pyrene-induced sarcoma (BP-1), a methylcholanthrene-induced sarcoma MC-1, or a spontaneous mammary carcinoma (SMT). In sera from mice injected with anti-BP-1 I-RNA, antibodies were detected which were specifically cytotoxic to BP-1 target cells, in vitro, but not to MC-1 target cells. Absorption with BP-1 sarcoma cells removed this cytotoxic activity while absorption with syngeneic spleen cells, or MC-1 sarcoma cells did not. Sera from mice receiving any of the other I-RNAs were not cytotoxic for either BP-1 or MC-1 target cells. Treatment of mice with BP-1 I-RNA may have induced the production of tumor-specific cytotoxic antibody, in vivo.Dr. Fritze was the recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft     

Generation of cytotoxic T lymphocytes during coxsackievirus B-3 infection. I. Model and viral specificity1.

The production of cytotoxic cells in the spleen of adult male BALB/c mice infected with Coxsackievirus B-3 has been examined.An in vitro 51Cr release assay was used to measure cytotoxic activity against virus-infected and uninfected neonatal sygeneic fibroblasts. Cytotoxicity of immune spleen cells against virus-infected targets was detected on the 3rd day after infection, reached a peak on day 7, and then declined to low levels by days 12 and 14. Spleen cells obtained 3 and 5 days after infection also exerted cytotoxicity against uninfected fibroblasts, but by the 7th day there was little or no reactivity against uninfected target cells, although activity against infected fibroblasts was maximal at this time. Reciprocal assays performed by using Coxsackie and vaccinia viruses provided evidence of virus specificity of the cytotoxic reaction. When spleen cells were obtained 7 days after infection, the Coxsackievirus-immune population was not cytotoxic for vaccinia-infected fibroblasts, and the vaccinia-immune population was not cytotoxic for Coxsackievirus-infected targets, although each immune cell preparation caused significant lysis of fibroblasts infected with the homologous virus. Additional studies showed that primary mouse or hyperimmune rabbit anti-Coxsackieviral serum could not block immune spleen cell cytotoxicity or induce complement-mediated lysis of infected targets. The findings indicate that Coxsackievirus infection results in surface membrane alterations, but no evidence was obtained that antiviral antibody could react with the infected cells.