芳芝多糖在小鼠体内的抗病毒和免疫促进作用

芳芝多糖是从担系菌杂色芳芝菌系体提出的一种葡聚糖,小鼠口服或腹腔注射芳芝胞肉多糖,可保护流感病毒静脉感染所致死亡和肝脏病理损伤,显著减少死亡和肝病变。但对疱疹病毒Ⅰ型静脉攻击,无明显效果。小鼠腹腔注射可诱出干扰素,促进肝植各细胞吞噬功     

小鼠腹腔巨噬细胞Fc受体的观察

本文对不同免疫水平的小鼠腹腔巨噬细胞作了 EA-花环试验。发现Fc受体功能在昆明杂交系小鼠比近交系C_(57)BL 的同年龄小鼠活跃,而在C_(57)BL系小鼠中,年青动物的Fc受体功能又较老年鼠强,从而证明小鼠腹腔巨噬细胞的Fc受体功能状态可随机体的免疫水平变化而发生改变,因此,对巨噬细胞 Fc受体的观察可作为衡量机体免疫水平的实验指标之一。 当小鼠腹腔巨噬细胞被厌氧棒状杆菌菌苗激活后,发现其 EA-花环形成百分率明显上升,而且吞噬活性也明显增强,尤其是在抗体调理吞噬反应中,还表现出单位细胞吞噬能力的显著提高,这说明菌苗激活的巨噬细胞可促进其表面 Fc 受体的功能,进而使巨噬细胞在调理吞噬反应中变得更为活跃。由此可见,提高巨噬细胞 Fc 受体的功能,对其功能的发挥具有积极意义。     

厌氧棒菌苗对小鼠腹腔巨噬细胞激活的观察

厌氧棒菌苗（ＣｏｒｙｎｅｂａｃｔｅｒｉｕｍＰａｒｖｕｍ，ＣＰ）是抗肿瘤制剂，其抗肿瘤作用的重要机制之一是激活巨噬细胞（Ｍ），用ＭＴＴ法动态观察了ＣＰ对小鼠腹腔Ｍ激活效应，实验用体重２０ｇ±２ｇ的ＮＩＨ雌性小鼠，腹腔注射ＣＰ０．５ｍｌ（ＣＰ每毫升含福尔马林灭活菌苗２ｍｇ，河南医科大学微生物学教研室惠赠），对照组腹腔注射０．５ｍｌ生理盐水，注射后不同时间杀鼠，常规制备腹腔细胞，按３×１０６／ｍｌ的细胞浓度０．１ｍｌ／孔加入９６孔培养板，贴壁２小时后用ＭＴＴ法测Ｍ的活化情况。ＭＴＴ法所测Ａ（吸光度，原…     

厌氧短棒菌苗对环磷酰胺免疫抑制作用的影响

本文研究了厌氧短棒菌苗(CP)对环磷酰胺(Cy)免疫抑制作用的影响。研究结果表明:CP对小鼠肝脾单核巨噬细胞功能和体液免疫具有显著促进作用,并能对抗由Cy造成的小鼠脾重减轻,脾有核细胞数减少和抗体生成抑制等作用。腹腔注射CP可对抗Cy产生的腹腔巨噬细胞(Mφ)吞噬功能抑制,但CP可加剧Cy减轻胸腺重量。     

S-O_2-1菌苗诱生干扰素的研究 Ⅰ小鼠体内诱生

本文研究了免疫增强剂——S-O_2-1菌苗在小鼠体内诱生干扰素的能力。结果表明,给(57BL/6小鼠注射菌苗1.5×1O~9菌/只后,血清中干扰素活性明显升高,于尾静脉注射后2小时,干扰素滴度达高峰(9.06±0.34log_2U/ml)。该干扰素具有小鼠I-型干扰素相同的理化特性。实验还表明,由S-O_2-1菌体提取的核糖体和脂多糖(LPS)亦均具有诱生干扰素的活性。因此我们推测,s-O_2-1菌苗诱生干扰素的能力可能对其抗肿瘤活性的发挥起一定的怍用。     

腹腔巨噬细胞功能平衡对小鼠异位子宫内膜细胞清除的影响

目的探讨腹腔巨噬细胞功能平衡对小鼠腹腔微环境和腹腔异位子宫内膜细胞清除的影响。方法给小鼠、裸鼠腹腔注射子宫内膜上皮和间质细胞,不同时间观察腹腔募集巨噬细胞数,D iI-Ac-LDL鉴定分化巨噬细胞吞噬功能的清道夫受体(SR-A1)。同步反转录-聚合酶链技术(Real-Tim e RT-PCR)检测巨噬细胞的MCP-1/JE和IL-1αmRNA表达。结果腹腔注射子宫内膜细胞后的小鼠、裸鼠募集巨噬细胞增多,上皮细胞的刺激作用强于间质细胞。24 h点为腹腔巨噬细胞募集高峰、MCP-1/JE和IL-1α的基因表达高峰,免疫正常小鼠表达反应吞噬功能的巨噬细胞清道夫受体(SR-A1)强于免疫缺陷裸鼠。结论子宫内膜细胞募集腹腔巨噬细胞为一独立免疫防御反应,募集巨噬细胞的清道夫功能和细胞因子分泌功能的平衡可能对维持正常腹腔微环境和有效清除异位子宫内膜细胞起重要作用,这在内异症的发病机制中具有重要意义。     

分娩后小鼠腹腔巨噬细胞的吞噬功能

本文证明分娩后4天小鼠腹腔巨噬细胞和注射葡聚糖后的小鼠腹腔巨噬细胞,以及正常未妊娠小鼠腹腔巨噬细胞吞噬鸡红细胞和酵母菌的功力明显不同。其中。分娩后4天和注射葡聚糖腹腔巨噬细胞,其吞噬率和吞噬指数都明显高于对照组。该结果表明分娩后小鼠体内单核——吞噬细胞系统活性增强,吞噬活性增高。     

用酶解法制备壳寡糖及其对机体免疫功能的调节作用

目的:研究本课题组酶解制备的主要含3-7聚合度壳寡糖(COS)对机体免疫功能的调节作用。方法:利用本课题组分离的高活性壳聚糖酶通过酶水解法制备壳寡糖,HPLC法对壳寡糖的成分进行鉴定,并用异硫氰酸荧光素(FITC)将壳寡糖进行荧光标记得到FITC-COS,研究小鼠腹腔巨噬细胞对壳寡糖的吞噬作用及其与Toll样受体4(TLR4)的关系,进一步研究了不同浓度壳寡糖对小鼠腹腔巨噬细胞的增殖,吞噬中性红能力及分泌TNF-α能力的影响,并对小鼠灌胃不同剂量的壳寡糖,研究了壳寡糖对小鼠血清IgG和IgM含量及小鼠胸腺、脾脏指数的影响。结果:HPLC分析结果显示壳聚糖酶水解法制备的COS主要为3-7单糖聚合度的寡糖。将FITC-COS作用于小鼠腹腔巨噬细胞不同时间后,荧光显微镜观察结果表明巨噬细胞能够吞噬COS,随着时间的延长吞噬COS的量增加,TLR4单克隆抗体预处理巨噬细胞1小时后再加入FITC-COS,巨噬细胞对COS的吞噬作用几乎完全被抑制。COS被巨噬细胞吞噬后可显著增强巨噬细胞的吞噬功能,刺激巨噬细胞分泌TNF-α。体内研究结果表明小鼠灌胃COS能够显著增加小鼠的脾脏指数,增加血清中IgG的含量,对胸腺指数和血清中IgM的含量没有显著影响。结论:壳寡糖(3-7聚合度)能够被巨噬细胞吞噬,进而激活巨噬细胞,具有较好的体外、体内免疫调节功能,壳寡糖对巨噬细胞的激活是通过细胞表面TLR4受体介导的。     

巨噬细胞在激活过程中形态和功能的改变

实验用石腊油,糖元及淀粉溶液给小鼠腹腔注射,经过16小时,腹腔巨噬细胞(Macrophae-Mφ)的数目开始增多,体积变大。细胞内乳酸脱氢酶、酸性磷酸酶及酸性醋酸萘酚酯酶的活性显著增强;三磷酸腺苷酶、琥珀酸脱氢酶及氨基肽酶的活性稍增强。巨噬细菌吞噬鸡红细胞的能力增强。至注后72小时,上述改变更加明显。表明糖元、淀粉、石腊油可以激活小鼠腹腔巨噬细胞。     

免疫小鼠及正常小鼠巨噬细胞对利什曼无鞭毛期的吞噬作用

内脏利什曼原虫主要寄生在巨噬细胞系统的单核吞噬细胞内,在一般情况下其无鞭毛期能抵抗巨噬细胞的杀灭作用。 为了观察经杜氏利什曼原虫免疫后的小鼠其巨噬细胞的作用,我们采用了CFW纯系小鼠,经不同免疫方法于免疫后不同时间观察了体外培养中巨噬细胞的吞噬功能。实验采用的巨噬细胞与杜氏利什曼原虫前鞭毛期的比例为1:4。从每24小时吞噬功能的结果表明,经利什曼鞭毛体纯抗原免疫及福氏佐剂加利什曼抗原免疫的两组小鼠,均以免疫后3周的吞噬率最高,分别为72％及96％;两组吞噬指数的均值±SD(4.46±1.72,6.99±4.36)亦较正常组小鼠(1.68±1.25,1.72±1.15)为高,并具有显著差异(P<0.05)。提示了特异性抗原以及与佐剂合并具有对吞噬功能的激活作用。实验并观察了巨噬细胞内利什曼原虫无鞭毛期的活力作用,从吞噬原虫后20小时开始至 144小时,正常小鼠巨噬细胞内的无鞭毛期再经三恩氏培养基培养后均能恢复为前鞭毛期,而经免疫小鼠巨噬细胞内的利什曼原虫无鞭毛期在72小时后即消失活力。 另外,对小鼠腹腔巨噬细胞吞噬利什曼原虫的动态亦作了仔细观察。 实验结果说明了经过免疫的小鼠,由于被淋巴细胞激活后的巨噬细胞能杀死利什曼原虫,巨噬细胞在宿主对感染应答中是一个重要部分,对于探索黑热病的免疫机理具有一     

Protection of mice against infection with mouse hepatitis virus type 3 by injection of silica

Injection of silica did not brake the resistance against MHV3 conferred to C57BL/6 mice by injection of C. parvum. However, silica itself had a marked protective effect against MHV3 infection that was maximal when injecting 1 mg 2 hrs before virus infection. The protective effect of silica was observed in a number of inbred mouse strains that differ in their relative resistance to MHV3 infection. No viral titers were observed in the spleen and liver of mice which had received MHV3 plus silica, whereas high titers were observed in the virus-infected controls. Injection of silica caused a marked decrease in the number of esterase-positive macrophages in the peritoneal wash-out population, that may be compatible with the possibility that the cause of the protection is the depletion of target cells for the viral infection. This latter effect, however, was short-lived and 24-48 hrs after injection of silica, high numbers of esterase-positive cells were again observed. This may explain why only little protection was observed when silica was administered 2 days before virus infection.     

The effect of Corynebacterium parvum in influenza infected mice

This paper is the continuation of earlier studies on the effect of the killed suspension of Corynebacterium parvum in influenza virus infected mice. Our investigation showed the normalized effect of these drugs on disturbed function of cell mediated immunity during experimental influenza infection especially in phagocytic and bactericidal activity of granulocytes. The present experiments concern the explanation of these infection mechanisms. Intraperitoneal injection of Corynebacterium parvum stimulated spleen index. Foot pad test is higher than in comparatively treated BCG group. The pathomorphological analysis of the spleen, thymus and peritoneal lymph nodes points out to the multiplication of multiple lymph nodes sinus cells. Generally, C. parvum possessed protective effect in experimental influenza infection. We tested the following parameters: phagocytic and bactericidal activity of granulocytes, liberation of leukocytes migration inhibition factor (LIF).     

Protection of mice against mouse hepatitis virus by Corynebacterium parvum.

C57BL/6 mice that are highly susceptible to infection with mouse hepatitis virus type 3 were protected against intraperitoneal viral infection by simultaneous intraperitoneal injection of Corynebacterium parvum. No protection was observed when C. parvum was given intravenously or when it was injected intraperitoneally 3 days before viral infection. Protective effects were, however, consistently found when C. parvum was given 2 h before or 2 h after viral infection. Activity was seen only against 10 50% lethal doses and not against 100 50% lethal doses. C. parvum also caused a significant decrease of virus type 3. These data suggest a direct effect of C. parvum on virus-susceptible cells. Injection of C. parvum in mice caused activation of natural killer (NK) cells and of interferon production. However, these two effects were equally demonstrable at high and low doses of C. parvum, whereas protection against mouse hepatitis virus type 3 was not demonstrable at low doses of C. parvum. Thus, antiviral protection may be dissociated from activation of NK cells and induction of interferon.     

Induction of resistance to ectromelia virus infection by corynebacterium parvum in murine peritoneal macrophages

An in vitro model has been developed to study the replication of ectromelia virus in murine macrophages (M phi). Infection of mineral oil-elicited peritoneal M phi cultures with either the virulent (Moscow) or attenuated (Hampstead) strain of ectromelia virus led to productive infections. The kinetics of virus synthesis was similar to those seen following infection of murine fibroblasts. In contrast, peritoneal M phi s activated by intraperitoneal injection of Corynebacterium parvum vaccine were found to be totally refractory to infection by the attenuated strain and significantly more resistant to the virulent strain of ectromelia virus. Administration of C. parvum doses as small as 7 micrograms were sufficient to induce antiviral activity. M phi resistance became maximal at 5-9 days after C. parvum administration; however, M phi resistance was unstable during in vitro culture. Decay of antiviral activity was detected within the first 24 hr of culture and complete virus susceptibility returned after 5 days in culture. Peritoneal exudate cells (PEC) from C. parvum-immunized mice could induce resistance in susceptible M phi cultures during overnight cocultivation. In addition, cell-free culture supernatants from C. parvum-immune PEC could also induce resistance in susceptible M phi cultures, suggesting that a soluble factor, induced by C. parvum immunization and possessing interferon activity, may account for the intrinsic resistance to ectromelia virus by activated M phi s.     

Protection against influenza virus infection by intranasal vaccine with surf clam microparticles (SMP) as an adjuvant

A safe and effective adjuvant is necessary to enhance mucosal immune responses for the development of an inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of surf clam microparticles (SMP) derived from natural surf clams as an adjuvant for an intranasal influenza vaccine. The adjuvant effect of SMP was examined when co-administered intranasally with inactivated A/PR8 (H1N1) influenza virus hemagglutinin vaccine in BALB/c mice. Administration of the vaccine with SMP induced a high anti-PR8 haemagglutinin (HA)-specific immunoglobulin A (IgA) response in the nasal wash and immunoglobulin G (IgG) response in the serum, resulting in protection against both nasal-restricted infection and lethal lung infection by A/PR8 virus. In addition, administration of SMP with A/Yamagata (H1N1), A/Beijing (H1N1), or A/Guizhou (H3N2) vaccine conferred complete protection against A/PR8 virus challenge in the nasal infection model, suggesting that SMP adjuvanted vaccine can confer cross-protection against variant influenza viruses. The use of SMP is suggested as a new safe and effective mucosal adjuvant for nasal vaccination against influenza virus infection.     

Protection against influenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant

Chitin in the form of microparticles (chitin microparticles, CMP) has been demonstrated to be a potent stimulator of macrophages, promoting T-helper-1 (Th1) activation and cytokine response. In order to examine the mucosal adjuvant effect of CMP co-administered with influenza hemagglutinin (HA) vaccine against influenza infection, CMP were intranasally co-administered with influenza HA vaccine prepared from PR8 (H1N1) virus. Inoculation of the vaccine with CMP induced primary and secondary anti-HA IgA responses in the nasal wash and anti-HA IgG responses in the serum, which were significantly higher than those of nasal vaccination without CMP, and provided a complete protection against a homologous influenza virus challenge in the nasal infection influenza model. In addition, CMP-based immunization using A/Yamagata (H1N1) and A/Guizhou (H3N2) induced PR8 HA-reactive IgA in the nasal washes and specific-IgG in the serum. The immunization with A/Yamagata and CMP resulted in complete protection against a PR8 (H1N1) challenge in A/Yamagata (H1N1)-vaccinated mice, while that with A/Guizhou (H3N2) and CMP exhibited a 100-fold reduction of nasal virus titer, demonstrating the cross-protective effect of CMP and influenza vaccine. It is suggested that CMP provide a safe and effective adjuvant for nasal vaccination with inactivated influenza vaccine.     

Comparison of protection against H5N1 influenza virus in mouse offspring provided by maternal vaccination with HA DNA and inactivated vaccine

H5N1 influenza virus is one of the viruses that can potentially cause an influenza pandemic. Protection of newborns against influenza virus infection could be effectively provided by maternal immunization. In this study, female mice were immunized with H5N1 HA DNA vaccine or inactivated whole-virion vaccine, and the protection provided by maternal antibodies in their offspring against a lethal homologous influenza virus challenge was compared. The results showed that maternal antibodies, whether induced by a DNA vaccine or an inactivated vaccine, could completely protect offspring aged 1-4 weeks from a lethal influenza virus challenge. Breast-feeding was the major route of transfer for maternal antibodies. Milk-derived antibodies were able to effectively protect the offspring aged 1-4 weeks from lethal influenza virus infection, whereas maternal antibodies transferred through the placenta only partially protected the offspring 1-2 weeks of age. The milk- and placenta-transferred IgG2a antibody levels in offspring from their mothers, whether vaccinated with DNA vaccine or inactivated vaccine, were higher than the IgG1 levels. Our results indicated that maternal vaccination with HA DNA, as well as with whole-virion inactivated vaccine, could offer effective protection to offspring against H5N1 influenza virus infection.     

Long-Term Protective Immunity from an Influenza Virus-Like Particle Vaccine Administered with a Microneedle Patch

Skin vaccination with influenza virus-like particles (VLPs) using microneedles has been shown to induce protection similar to or better than that induced by intramuscular immunization. In this study, we examined the long-term protective efficacy of influenza (H1N1 A/PR/8/34) VLPs after skin vaccination using microneedle patches coated with the vaccine. Microneedle vaccination of mice in the skin induced 100% protection against lethal challenge infection with influenza A/PR/8/34 virus 14 months after a single vaccine dose. Influenza virus-specific total IgG response and hemagglutination inhibition (HAI) titers were maintained at high levels for over 1 year after microneedle vaccination. Microneedle vaccination also induced substantial levels of lung IgG and IgA antibody responses, and antibody-secreting plasma cells from spleen and bone marrow, as well as conferring effective control of lung viral loads, resulting in complete protection 14 months after vaccination. These strong and long-lasting immune responses were enabled in part by stabilization of the vaccine by formulation with trehalose during microneedle patch fabrication. Administration of the stabilized vaccine using microneedles was especially effective at enabling strong recall responses measured 4 days after lethal virus challenge, including increased HAI and antibody-secreting cells in the spleen and reduced viral titer and inflammatory response in the lung. The results in this study indicate that skin vaccination with VLP vaccine using a microneedle patch provides long-term protection against influenza in mice.     

Effects of stimulated or immunologically activated macrophages on the induction of immune responses to Listeria monocytogenes

The influences of peritoneal macrophages induced by proteose peptone, Corynebacterium parvum (C. parvum) or Bacillus Calmette Guérin (BCG) on the initiation and development of immune responses and protection against Listeria monocytogenes infection were studied in mice. Mice treated intraperitoneally (i.p.) with proteose peptone 4 days previously showed much the same level of protection against an intraperitoneal infection with Listeria as untreated mice. Mice treated i.p. with C. parvum 4 days previously, of which peritoneal macrophages had increased abilities for intracellular killing of Listeria and O2- generation as compared with peptone-elicited macrophages, exhibited an enhanced resistance against the listerial infection. The degree of immune responses, as assessed by delayed footpad reaction (DFR), was rather depressed in these mice because C. parvum-activated macrophages acting as scavenger cells reduced the amount of effective antigenic stimulation. BCG-activated peritoneal macrophages from mice treated i.p. with BCG 14 days previously showed a strong ability for antigen presentation in correlation with increases in the number of Ia-bearing macrophages and in the level of interleukin 1 (IL 1) production. These mice showed an early appearance of DFR response and a markedly enhanced resistance against the listerial infection. These results suggested that the differences in macrophage activities as scavenger cells, cytokine-secreting cells and antigen presenting cells may account for the differences in the responsiveness against listerial infection in peptone-, C. parvum- and BCG-treated mice.     

Matrix-M™ adjuvant enhances immunogenicity of both protein- and modified vaccinia virus Ankara-based influenza vaccines in mice

Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M? adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines.