家蝇蛹血淋巴及其提取物的免疫调节作用

目的:观察家蝇蛹血淋巴及其提取物对小鼠脾淋巴细胞增殖的影响.方法:以不同浓度血淋巴及其提取物与脾淋巴细胞、T细胞和B细胞共孵育72小时,采用MTT法测定淋巴细胞的吸光度值,观察它们对淋巴细胞增殖的促进作用,采用免疫印迹法研究提取物对B淋巴细胞增殖的信号转导通路.结果:与对照组相比,血淋巴冻干粉、分子量在50 kD以上的冻干粉及其提取物对混合淋巴细胞和B细胞增殖有显著促进作用,然而对于T细胞的增殖却没有明显的促进作用.MTT实验和免疫印迹结果表明血淋巴提取物激活了B淋巴细胞的ERK通路,促进了B淋巴细胞的增殖.结论:家蝇蛹血淋巴及其提取物具有免疫调节作用,为天然免疫增强剂的开发提供了一定的参考依据.     

胚芽乳杆菌胞壁肽聚糖对细胞免疫的作用

用体外刺激、~8H-TdR掺入测定淋巴细胞增殖的方法观察到胚芽乳杆菌的胞壁肽聚糖(PG)可以直接诱导小鼠脾淋巴细胞的增殖反应,在所采用剂量范围内细胞增殖的倍数与PG剂量呈线性关系;但对胸腺细胞和分纯的脾T淋巴细胞则无直接作用。还观察到PG有协同ConA诱导小鼠脾细胞和胸腺细胞增殖作用。可见PG具有B细胞的丝裂原性,并有增强T细胞丝裂原的作用。     

丙型肝炎病毒高变区1模拟表位7肽刺激小鼠脾淋巴细胞增殖

目的:探讨丙型肝炎病毒(HCV)高变区1(HVR1)模拟表位7肽在体外刺激小鼠经脾内直接免疫后脾淋巴细胞增殖的变化。方法:选用经7肽脾内直接注射免疫的小鼠,分离脾细胞后,脾细胞再经7肽刺激,应用单溶液细胞增殖检测方法,体外检测7肽在不同浓度和不同培养时间点,对小鼠脾淋巴细胞增殖的影响。结果:7肽刺激免疫小鼠脾淋巴细胞后,实验组的脾淋巴细胞增殖反应与对照组相比有明显促进作用。结论:HCV高变区1模拟表位7肽在体外对脾内直接免疫小鼠脾淋巴细胞具有一定的促增殖作用。     

TLSFJM在体内对同种异体抗原诱导的小鼠T细胞增殖和杀伤细胞分化的影响

目的：探讨JM急性T淋巴细胞白血病细胞分泌的抑制因子(TLSJM)对BALB／C小鼠胸腺发育的影响及其在体内对同种异体抗原诱导的小鼠辅助性T细胞(Th)增殖及杀伤性T细胞(CTL)分化的影响。方法：建立小鼠同种异基因型肿瘤排斥模型，实验小鼠腹腔注射含TLSFJM的JM细胞培养上清，对照小鼠注射RPMI1640。无菌取小鼠脾脏细胞，分别以^3H-TdR掺入法和^51Cr释放法检测T细胞增殖和CTL的分化。结果：①TLSFJM明显抑制多克隆刺激剂PHA联合IL-2诱导的小鼠胸腺细胞增殖；②体内注射TLSFJM明显抑制多克隆刺激剂PHA联合IL-2诱导的小鼠T细胞活化、增殖；③TLSFJM在体内能有效抑制同种异体抗原诱导的小鼠T细胞增殖；④TLSFJM在体内能有效抑制同种异体抗原诱导的小鼠CTL分化。结论：TLSFJM在体内能有效抑制同种异体抗原诱导的小鼠T细胞增殖和小鼠CTL分化。     

北豆根提取物对小鼠和人淋巴细胞及巨噬细胞作用的体外实验研究

目的：探讨中药北豆根提取物的免疫学调节作用及其作用机制；比较北豆根水提物、醇提物和多糖成分的生物学活性。方法：采用水提取、醇提取及水回流等方法对北豆根进行成分分离。采用MTT法、中性红法检测了北豆根提取物对淋巴细胞增殖的作用以及对小鼠巨噬细胞代谢和吞噬功能的影响。结果：北豆根多糖成分(RMP)具有丝裂原样作用，可刺激小鼠脾细胞及人淋巴细胞增殖，对小鼠巨噬细胞的吞噬功能有一定的促进作用。而北豆根水提物(RMW)和醇提物(RME)对小鼠脾细胞、人淋巴细胞的增殖具有抑制作用，对小鼠巨噬细胞的代谢及吞噬功能也有抑制作用。结论：不同的北豆根成分对免疫细胞有不同的作用，水提物和醇提物对免疫细胞的增殖、代谢和功能具有一定的抑制作用；北豆根多糖成分对淋巴细胞具有丝裂原样作用，能增强小鼠巨噬细胞的吞噬功能，该活性可用于临床辅助治疗肿瘤。     

小鼠脾淋巴细胞可T能存在5HT3受体

本文用3H-TdR掺人法和MTT法观察5HT3受体激动剂1-phenylbiguanide(PBG)、5HT3受体拮抗剂格拉司琼(granisetron)和托烷司琼(tropisetron)对体外培养ICR小鼠脾淋巴细胞ConA,LPS刺激的增殖和NK细胞活性的影响.结果表明PBG(10-6～10-4mol/L)抑制ConA刺激的脾细胞增殖反应和脾细胞IL-2的生成(P＜0.05);增强LPS剌激的脾细胞增殖反应(P＜0 05)格拉司琼和托烷司琼双相影响,即在低浓度(10～～10-6mol/L)促进、在较高浓度(10-5～10-4mol/L)抑制ConA刺激的增殖反应(P＜0.05);二药均浓度依赖抑制LPS刺激的脾细胞增殖效应.PBG对脾淋巴细胞增殖的影响被同时加入格拉司琼或托烷司琼拮抗,格拉司琼和托烷司琼减弱PBG 10-5mol/L对脾细胞增殖反应的影响.本实验5HT3受体激动剂或拮抗剂浓度不明显影响脾NK细胞活性和无丝裂原刺激的增殖反应.结果提示小鼠脾T细胞和B细胞表面可能存在对淋巴细胞增殖有不同影响的5HT3受体.     

TLSF_(JM)对T细胞IL-2R表达和蛋白酪氨酸磷酸化的调节作用

应用免疫组化染色技术,研究了TLSF_(JM)对T细胞IL-2R表达和蛋白酪氨酸磷酸化的调节作用。结果表明:TLSF_(JM)对PHA或PA活化的人PBMC以及小鼠CTLL-2 IL-2R表达和蛋白酪氨酸磷酸化具有明显的抑制作用,但对在T淋巴细胞增殖、分化过程中起重要作用的细胞因子IL-1、IL-2和IL-6的产生无明显影响。     

驱虫斑鸠菊对正常小鼠免疫功能的影响

目的探讨驱虫斑鸠菊对小鼠免疫功能的影响,揭示其免疫作用机理.方法利用[3H]-TdR掺入法分别测定了驱虫斑鸠菊对环胞菌素A(Con A)诱导的小鼠脾脏T淋巴细胞和细菌脂多糖(LPS)诱导的B淋巴细胞增殖活性以及脾脏T淋巴细胞分泌白细胞介素-2(IL-2)活性的影响,运用酶联免疫吸附试验测定了驱虫斑鸠菊对小鼠血清中抗体活性的影响;采用流式细胞仪测定脾脏B淋巴细胞表面抗原CD19的表达水平.结果驱虫斑鸠菊的低、中、高3个剂量体内对脾脏T、B淋巴细胞的增殖活性、血清总抗体和针对人A375黑素瘤细胞抗原特异性抗体含量、B细胞表面抗原CD19的表达以及对脾脏T淋巴细胞分泌IL-2活性都具有明显抑制作用.结论驱虫斑鸠菊对机体体液免疫和细胞免疫功能都具有明显抑制作用.     

淫羊藿苷(ICA)对化疗后免疫抑制小鼠的免疫促进作用

目的:通过观察淫羊藿苷(ICA)对化疗药环磷酰胺(Cy)所致免疫抑制小鼠免疫功能的影响,探讨ICA促进免疫功能的作用及机理.方法:除正常组小鼠外,所有小鼠经腹腔注射Cy(300 mg/kg);第二天开始给予实验组小鼠灌胃不同剂量的ICA[150、80、40 mg/(kg·d)],阳性对照组小鼠尾静脉注射参芪扶正注射液(1 ml/d),模型组给予等量生理盐水,连续干预10天.所有小鼠均于末次给药12小时后处死,计算胸腺指数(TI)和脾指数(SI),光镜下计数外周血和脾淋巴细胞数量.MTT法检测小鼠脾淋巴细胞增殖反应.ELISA法检测TNF-α的含量.乳酸脱氢酶实验(LDH)检测小鼠脾脏NK和CTL细胞的活性.流式细胞术(FACS)检测脾淋巴细胞中NKT和CD3+T细胞的比例.结果:模型组小鼠以上各项免疫指标均有所下降.ICA处理组小鼠脾脏指数和胸腺指数均升高(P<0.01),脾淋巴细胞数量显著增加(P<0.01),但未达到正常对照组水平;ICA明显提高小鼠脾淋巴细胞的增殖反应,促进了小鼠脾NK和CTL细胞对肿瘤细胞的杀伤活性(P<0.01),提高了小鼠脾细胞TNF-α的产生(P<0.01).ICA处理组小鼠脾细胞中CD3+T、NKT细胞比例明显增加(P<0.01).结论:ICA能促进小鼠免疫功能,具有逆转化疗后小鼠免疫抑制状态的作用.     

海洋无脊椎动物提取物免疫调节活性的初步研究

目的：为了从海洋无脊椎动物中寻找生物活性物质，对山东青岛及其附近海域常见的21种海洋无脊椎动物的粗提物进行了免疫调节活性测定。方法：测定了它们的乙醇提取物以及不同极性部位对T、B淋巴细胞增殖的调节作用。在小鼠脾细胞体外培养中用T细胞丝裂原ConA(伴刀豆蛋白A)和B细胞丝裂原IPS(脂多糖)分别刺激T、B细胞增殖，以DNA合成前体^3H-TdR(胸腺嘧啶核苷)掺入法检测T、B淋巴细胞的增殖强度。结果：海洋无脊椎动物对T、B淋巴细胞具有ConA或IPS协同刺激作用或者抑制作用，其中10种有明显的剂量依赖性，6种呈现小剂量增殖、大剂量抑制的双向调节作用。结论：海洋无脊椎动物粗提物具有一定的免疫调节功能，这些数据为从海洋无脊椎动物中寻找天然药物活性前体奠定了基础。     

B cell growth factor activity of immunoaffinity-purified and recombinant human interleukin 2

We investigated the effect of recombinant and affinity-purified human interleukin 2 (IL2) on human B cell proliferation. Five X 10(4) nonadherent spleen cells that had been depleted twice of T cells were activated by 3-day culture with formaldehyde-killed Staphylococcus aureus Cowan strain I (SAC) prior to addition of tested growth factors. Cultures were harvested 72 h later. It was found that both IL2 preparations led to optimal cell proliferation compared with a control supernatant obtained by 36 h phytohemagglutinin stimulation of spleen mononuclear cells. Moreover, the effect of such spleen supernatant on B cell proliferation correlated with the IL2 activity since its B cell growth factor activity (BCGF) was not greater than that of purified IL2 and no residual BCGF activity could be detected after absorption of all IL2 activity by the IL2-dependent cytotoxic T lymphocyte line cells. T cells, enumerated as E-rosetting cells as well as T3+ cells, represented 0.2 to 2% of the cells recovered at termination of the cultures (day 6) and there were less than 1% E-rosetting cells in freshly purified or SAC-activated (day 3) B cell populations. Therefore, we conclude that IL2 is a growth factor not only for activated T cells but also for activated human B cells.     

Low responsiveness of hepatitis B virus-transgenic mice in antibody response to T-cell-dependent antigen: defect in antigen-presenting activity of dendritic cells.

The experiments presented here were performed to evaluate immune responsiveness of hepatitis B virus (HBV)-transgenic mice (transgenic mice), as a model of HBV-carrier state to a T-cell-dependent antigen, keyhole limpet haemocyanin (KLH). The transgenic mice which were completely unresponsive to hepatitis B surface antigen (HBsAg), responded poorly to KLH. The levels of anti-KLH antibodies (Ab) produced in vivo were significantly lower in transgenic mice compared with the normal control mice at respective immunizing doses of KLH. In addition, a little or no anti-KLH Ab production was detected in culture supernatants of KLH-primed transgenic mice spleen cells. KLH-primed T cells from normal and transgenic mice induced anti-KLH Ab production from transgenic B cells in the presence of antigen-presenting spleen adherent cells (SAC) from normal mice, but not those from transgenic mice. Depletion of dendritic cells from normal mice-derived SAC completely abrogated the anti-KLH Ab response in transgenic spleen cell culture and their addition to the culture restored the response. Low efficiency of transgenic dendritic cells was demonstrated in sodium periodate (NaIO4)-induced non-specific and allogenic antigen-induced T-cell proliferation. Finally, cytofluorometric analyses showed a reduced Ia antigen expression on transgenic dendritic cells. These results indicate that low responsiveness of transgenic mice in specific-antibody response is not due to functional defects in T cells or B cells but rather to a defect of antigen-presenting activity of dendritic cells.     

T cell clones interacting with accessory and T helper cells for a proliferative response

The requirement for cell interactions in T cell activation has been studied with two continuously in vitro growing T cell clones. These clones are specific for minor histocompatibility antigens, are H-2K restricted, and one clone is functionally a cytolytic T lymphocyte. Both can proliferate when interleukin 2 is added to the cultures, but for continuous growth they require irradiated spleen cells carrying the specific minor histocompatibility antigen and the restricting H-2. In this study we show that for proliferation the clones require at least two cell populations in the stimulator spleen, one is a splenic-adherent cell (SAC), the other a T cell. The SAC are plastic adherent, Thy-1-, Ia+. The T cells are nylon wool nonadherent, Thy-1+, Lyt-1+2- and Ia-. Cell mixing experiments of stimulator cells (all were done with H-2-syngeneic cells), depleted of either SAC or T cells confirm the requirement for a specific interaction between these two cell types and the T clone. Neither SAC, syngeneic with the T clone when mixed with T cells of the stimulator type, nor T cells syngeneic with the clone added to stimulator SAC, can induce an optimal proliferative response. Such a response is obtained only if both cell types, SAC and T cells, are of the stimulating genotype. This suggests that, in addition to an interaction of clonal T cells with SAC, a specific recognition at the T cell level between T stimulator and T clone is necessary. The interaction of the T clones with stimulator SAC and T cells leads to an activation, mediated by antigen recognition, of all three cell populations. Since we also show that each of the stimulator cell types are impaired by ultraviolet light irradiation, we conclude that factor production by SAC and T helpers is the final prerequisite for clonal expansion.     

Diminished proliferation of B blast cell in response to cytokines in ethanol-consuming mice

We and others have demonstrated that ethanol suppresses the antibody response in humans and animals. The purpose of this study was to determine whether ethanol affects cytokine-induced proliferative responses of splenic B blast cells, and whether the decreased response was due to an imbalance of the cytokine activity. Thus, the ability of spleen cells from individual ethanol-diet-fed C57BL/6 mice to proliferate and produce cytokines was determined. The ability of anti-IgM monoclonal antibodies (mAb)-activated splenic B blast cells in response to mouse recombinant IL-2 (rIL-2) or rIL-4 was also assessed. A thymidine incorporation assay was used to determine cell proliferation, and the conventional bioassays for cytokine-dependent cell proliferation were used for determining the bioactivity of cytokines. Data were analyzed with general linear model procedure. Our results showed that ethanol weakened the proliferative response of B cells in response to mitogen as well as to mouse rIL-2 and rIL-4. The decreased B cell responses may result from an increase in the production of IL-4 by helper T cells. Finally, in the presence of excessive dose of rIL-4, the proliferative responses of B blast cells from all three groups of mice were diminished (p<0.01). Thus, our data clearly indicated that the diminished B cell proliferation in ethanol-consuming mice was due in part to an excessive amount of IL-4 produced and an inability of the B cell to interact properly with IL-4 that was secreted by helper T cells. The results should extend our basic understanding of the mechanism by which chronic alcoholism impairs the interactions and interdependence of T- and B-cell immune functions.     

Study of the thymic-derived or -independent nature of mouse spleen cells induced to proliferate in culture by various mitogens and antigens

The nature of mouse lymphoid cells induced to proliferate in vitro by a number of stimulating agents has been explored by 1) karyotypic analysis of the mitoses observed in stimulated spleen cell cultures (arrested on sequential days) of chimaeras bearing chromosomally marked T cells; 2) comparison between the response in DNA synthesis of total spleen cells and of spleen cells depleted of thymus-dependent lymphocytes by treatment with anti-MSLA (anti-mouse-specific lymphocyte antigen) + C, and 3) study of the response of cortisone-resistant thymocytes (CRT) to some of these agents. Pokeweed mitogen stimulated both B and T cells equally and simultaneously. Rabbit anti-mouse Ig induced moderate proliferation of B cells exclusively. The response to lipopolysaccharide was entirely B cell-dependent, but evidence was found of some T cell proliferation following or accompanying the stimulation of B cells. A similar observation was made in the case of “primary” or “secondary” in vitro response to bacteriophage T4. Good stimulation with sheep red blood cell stromas or mitogen-treated xenogenic spleen cells were observed only in cultures from primed animals, and consisted of a mixed B and T cell proliferation with a majority of B mitoses, even at the earlier stages of the response. That this “secondary” response was not totally T cell-dependent was suggested by the observation of a stimulatory effect of these antigens on T-depleted spleen cell cultures which were found in parallel experiments to be totally unresponsive to PHA. Finally, using chimeric mice treated in vivo with vinblastine prior to establishment of the spleen cell cultures, evidence was obtained that part of the B cells stimulated to divide in vitro by several of these mitogens belong to a rapidly dividing cell population in vivo.     

Subpopulations of mouse spleen lymphocytes. III. Cellular interactions in the response to concanavalin A.

The profile of response to concanavalin A (con A) of purified mouse T cells was found to differ appreciably from that of non-fractionated spleen cells, in agreement with results previously published by other investigators. Experiments designed to elucidate the reasons underlying these differences have revealed that the response of the spleen cells to con A is determined by a complex interplay between several cell types. (a) B cells contribute to the overall incorporation of thymidine in the presence of con A-stimulated T cells. However, the B cells participate in the response only if the T cells are dividing. (b) A population of 'adherent cells' is present in the spleen, which enhances the stimulation of the spleen cells by low doses of con A but suppresses the response to high doses of mitogen. These adherent cells include most likely the conventional macrophages, but probably also a population of 'suppressor T cells'. (c) Such 'suppressor T' cells can be readily detected among the peritoneal exudate cells. Addition of the exudate cells to cultures of purified T cells enhances the response to low doses of con A. This effect can be further increased by treating the peritoneal cells with a cell T-specific antiserum and complement, i.e. by eliminating the T cells.     

Partial characterization of chicken spleen cell culture supernatants stimulated with Staphylococcus aureus

The role of accessory cells in the proliferative response of chicken spleen cells to Staphylococcus aureus Cowan I (SAC) was examined. It was found that chicken spleen cells cultured with SAC produced a soluble molecule capable of causing proliferation when culture supernatants were added to spleen cells. The molecules responsible for this activity were stable in terms of exposure to extremes of heat and pH. Gel filtration of culture supernatants revealed biological activity, over a broad range of molecular weights, as measured by spleen cell proliferation. Similar findings were obtained when SAC was sonicated and evaluated following gel filtration. Exposure of culture supernatants to trypsin abrogated biological activity. The pivotal role of adherent cells in the generation of biologically active molecules is suggested by the ability of peritoneal exudate cells incubated with SAC to produce biologically active supernatants. In addition, the proliferative response of spleen cells to SAC was sensitive to chloroquine.     

Study on the Effect of Calf Thymic Peptides Preparation (Tp) on Human B' Lymphocytes

Extensive experimental evidence has shown that thymic hormones (or factors) affect and regulate the differentiation and function of T lymphocytes. However, little is known about the action, if any, of thymic hormones on B lymphocytes. This paper reports the results of an investigation of the effect of a calf thymic peptide preparation (TP) prepared in our laboratory, on the proliferation and differentiation of human B lymphocytes.

TP at concentrations higher than 1 μg (protein)/ml inhibited the proliferative responses of human B lymphocytes to the stimulation by Staphylococcus aureus Cowen strain I (SAC) and F(ab')₂ fragments of goat anti-human IgM μ chain specific antibody (anti-μ). TP itself had neither toxic nor mitogenic effect on B cells. TP at concentrations of 60 and 100 μg/ml did not affect the differentiation of B cells driven by SAC and PWM in a reverse PFC assay, but appeared to suppress the production in some individuals of total IgG and IgM in culture supernatants in a PWM system. Preculture of B cells with 60 μg/ml of TP for 40 hrs showed a suppression of the proliferative response to SAC and anti-μ stimulation, suggesting that TP might act on cells directly and persistently for some time. When TP was added to the culture on day 0 or on day 1, a similar decrease of inhibition of B cell proliferation was observed. A decrease in monocytes from 15-17% to 5% did not appreciably influence the suppression of SAC-or anti-μ-induced proliferation of B cells by TP. These preliminary results suggest that a calf thymic peptides preparation might have some direct effect on B lymphocytes.     

In vitro IgM and IgM rheumatoid factor production in response to Staphylococcus aureus Cowan I: evidence for the role of Leu-2-positive T suppressor cells and radiosensitive T helper cells.

Staphylococcus aureus Cowan I (SAC) is a potent stimulus of B cell proliferation and differentiation, the latter being T cell-dependent. It has been suggested that immunoglobulin and IgM rheumatoid factor (RF) production in response to SAC involves radiosensitive T helper cells. We studied normal peripheral blood mononuclear cell (PBMC) cultures to assess the roles of radiosensitive T cells and Leu-2 positive suppressor cells in the cellular control of SAC-stimulated IgM and IgM RF responses. Depletion of Leu-2 positive T cells from reconstituted autologous PBMC cultures resulted in an increase in SAC-stimulated IgM production in the majority of individuals, implying the involvement of Leu-2 positive suppressor T cells in this system. Suppression by Leu-2 positive cells is less evident in the SAC-induced IgM RF response, suggesting qualitative differences between IgM and IgM RF SAC-stimulated responses in PBMC cultures from the same normal individuals. Irradiation (1000 rads) of the T cell-enriched subpopulation, either with or without Leu-2 positive cell depletion, resulted in statistically significant decreases in IgM and IgM RF production in response to SAC in reconstituted autologous cultures, providing further evidence of a Leu-2 negative radiosensitive sheep-cell rosetting cell active in in vitro SAC responses. In contrast, PWM-stimulated PBMC cultures showed almost exclusively increases in total IgM and IgM RF production when T cells were irradiated (1000 rads) before culture, consistent with the radiosensitive T suppressor cell involved in the in vitro immunoglobulin responses to PWM. The same five out of nine individuals produced IgM RF, under different culture conditions, in response to PWM and SAC, suggesting that the ability of an individual to produce IgM RF lies intrinsically within the B cell.     

Sendai virus glycoproteins are T cell-dependent B cell mitogens

UV-inactivated Sendai virus is mitogenic for murine splenocytes, whereas infectious Sendai virus kills spleen cells in vitro. The isolated hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of Sendai virus are also mitogenic for cultured mouse spleen cells. A mixture of these glycoproteins (1 microgram/well) gives maximum stimulation 96 h after culture initiation. Viral proteins remaining insoluble after Triton X-100 extraction are also mitogenic for mouse spleen cells, with maximum stimulation occurring at 72 h after culture initiation with 1 to 5 microgram/well. On the basis of protein concentration, the HN and F glycoproteins are approximately three times more mitogenic than the Triton X-100-insoluble material. The mitogenic response of the HN and F glycoproteins has two components, a T cell-independent B cell proliferation, which is less than one-half of the total stimulation observed, and a T cell-dependent B cell proliferation. In contrast, the Triton X-100-insoluble material is a T cell-dependent B cell mitogen. Purified T lymphocytes do not respond to the mitogenic signal of either HN-F or Triton X-100-insoluble material.