人类疱疹病毒7型体外生长特点的研究

研究了人类疱疹病毒７型（ＨＨＶ－７）南京株ＹＹ５在脐血单个核细胞（ＣＢＭＣｓ）及ＳＵＰＴ１细胞株上生长特点。观察病毒感染细胞后不同时间病变效应程度,记数死亡细胞百分率,间接免疫荧光染色记数抗原表达阳性细胞数,并用透射电镜观察病毒感染细胞超微结构变化及病毒复制不同时期的特点。结果发现：ＨＨＶ－７在ＣＢＭＣｓ及ＳＵＰＴ１上出现ＣＰＥ时间迟于ＨＨＶ－６,且ＣＰＥ程度也低于ＨＨＶ－６;ＨＨＶ－７在ＳＵＰＴ     

从病人外周血单个核细胞中检测HHV—6：分离培养和基因扩增

收集婴幼儿急疹及淋巴系统增生性疾病患者外周血单个核细胞进行体外培养,从７例婴幼儿急疹及２例淋巴系统增生性疾病患者中分离出一种病毒,此病毒能在ＰＨＡ激活的人脐血单个核细胞中传代生长,产生典型ＣＰＥ：形成气球样巨细胞。电镜下观察,感染细胞中可见直径１８０ｎｍ左右,有包膜,疱疹样病毒颗粒;血清学试验证明分离株与ＨＳＶ－１,２、ＨＣＭＶ、及ＥＢＶ无抗原交叉,而与ＨＨＶ－６ＧＳ株间存在抗原一致性;多聚酶链反     

不同刺激对小鼠B细胞表面IgD表达的影响

本文通过细胞流式计（ＦＡＣＳ）技术,利用单克隆抗体抗－ＩｇＤ［Ｉｇ（５ｇ（５ａ）７．２］研究了膜表面ＩｇＤ（ｓＩｇＤ）在新制备的小鼠Ｂ细胞上的表达。并通过用不同的丝裂原刺激脾脏休止Ｂ细胞,研究了不同刺激对Ｂ细胞ｓＩｇＤ表达的调节,发现脾脏Ｂ细胞形成两个主要细胞群,高浮力密度小Ｂ细胞高度表达ｓＩｇＤ的细胞主要是低浮力密度大Ｂ细胞。无论用细菌脂多糖（ＬＰＳ）或抗－ＩｇＭ和白细胞介素－６（ＩＬ－６）刺激     

乙型肝炎病毒（HBV）体内外对单个核细胞白细胞介素—1和白细胞介素…

本研究用ＰＡＰ法、胸腺细胞增殖法、脾细胞增殖法,分别检测１６例体外ＨＢＶ感染和骨髓单个核细胞与１６例慢性乙型肝炎患者体内感染的骨髓单个核细胞中的ＨＢｃＡｇ和白细胞介素－１、白细胞介素－２的诱生活性。结果显示,体外ＨＢＶ感染组成体内ＨＢＶ感染组骨髓ＭＮＣｓ中ＨＢｃＡｇ检出率分别为５０％和４３．７％。本实验结果表明,ＨＢＶ在体外感染骨髓ＭＮＣｓ,且与体内自然感染相符,但光镜下未观察到致细胞病变效应。体     

EB病毒转化Hu—TLC—SCID小鼠脾细胞的实验研究

利用ＥＢ病毒转化可产生较高水平人ＩｇＧ和特异性抗２型登革病毒人抗体的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞,通过免疫组化、免疫荧光和ＰＣＲ法检测转化细胞的人Ｂ细胞表面标志、ＥＢ病毒抗原和ＥＢ病毒基因。结果表明,被团体的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞能继续产生抗２型登革病毒的特异性人抗体,并具有人Ｂ细胞的ＣＤ２０＾＋、ＳｍＩｇＧ标志及ＥＢ病毒潜伏膜蛋白－１（ＬＭＰ－１）基因,可表达ＬＭＰ－１和ＥＢ病毒核     

灵芝多糖对人脐血LAK细胞活性的影响

本文研究了灵芝多糖（ＧＬＰ）对人脐血ＬＡＫ（ＣＢ—ＬＡＫ）细胞活性的影响,结果发现,单独ＧＬＰ能刺激人脐血单个核细胞（ＣＢＭＣ）增殖,但不能诱导ＬＡＫ活性,当与５０ｕ／ｍｌｒＩＬ—２伍用时,可增殖ＣＢ—ＬＡＫ细胞诱导活性,不同剂量ＧＬＰ（０．５—１００μｇ／ｍｌ）影响作用不同,以１０μｇ／ｍｌ浓度最好．在不同浓度ｒＩＬ—２（１０—１００ｕ／ｍｌ）诱导ＣＢ—ＬＡＫ细胞过程中加入ＧＬＰ（１０μｇ／ｍｌ）,可明显提高细胞增殖能力,减少ｒＩＬ—２用量。ＧＬＰ亦能促进效应阶段ＣＢ—ＬＡＫ细胞对Ｒａｊｉ肿瘤靶细胞的杀伤作用（Ｐ＜０．００１）。由此看出,ＧＬＰ具有增强ＣＢ—ＬＡＫ细胞活性的作用,是一很好的生物反应调节剂（ＢＲＭ）,有必要进行深入的研究。     

EB病毒转化Hu－TLC－SCID小鼠脾细胞的实验研究

利用ＥＢ病毒转化可产生较高水平人ＩｇＧ和特异性抗２型登革病毒人抗体的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞,通过免疫组化、免疫荧光和ＰＣＲ法检测转化细胞的人Ｂ细胞表面标志、ＥＢ病毒抗原和ＥＢ病毒基因。结果表明,被转化的Ｈｕ－ＴＬＣ－ＳＣＩＤ小鼠脾细胞能继续产生抗２型登革病毒的特异性人抗体,并具有人Ｂ细胞的、ＳｍＩｇＧ标志及ＥＢ病毒潜伏膜蛋白－１（ＬＭＰ－１）基因,可表达ＬＭＰ－１和ＥＢ病毒核抗原（ＥＢＮＡ）。     

HHV—6体外感染对外周血单个核细胞IL—6,IL—8的诱生和NK活性的影响

采用生物活性法和／或酶联免疫吸附法和以及乳酸脱氢酶释放法,研究了人类疱疹病毒６型南京地方株ＣＮ８对人体周血单个核细胞的ＩＬ－６,ＩＬ－８的诱生和ＮＫ活性的影响,并与国外的ＧＳ侏作比较。结果发现,ＨＨＶ－６ＣＮ８,ＧＳ两种病毒感染均苛诱导ＰＢＭＣｓ产生ＩＬ－８,４８ｈ达到峰值。     

人类疱疹病毒6型感染细胞免疫学特性研究

采用间接免疫荧光法、ＡＰＡＡＰ法及ＭＴＴ法,研究了人类疱疹病毒６型（ＨＨＶ６）中国南京地方株ＣＮ５感染细胞病毒抗原表达的形态学和动力学特征、ＣＤ抗原表达阳性细胞百分率的变化及ＰＨＡ诱导的细胞增殖反应的改变。结果显示,ＣＮ５感染脐血单个核细胞（ＣＢＭＣｓ）后８～１２ｈ即可在细胞内检出病毒抗原,至接种后４８ｈ,病毒抗原阳性细胞可达３６％;ＣＮ５感染ＣＢＭＣｓ和成人外周血单个核细胞（ＰＢＭＣｓ）后可引起两者ＣＤ３阳性细胞减少、ＣＤ４阳性细胞增多,而对ＣＤ２、ＣＤ８、ＣＤ４５ＲＡ阳性细胞百分率未见明显影响;ＣＮ５感染细胞裂解液对ＰＨＡ诱导的ＰＢＭＣｓ增殖反应具有抑制作用,这种抑制作用与该裂解液的蛋白浓度之间呈一剂量依赖关系,且可被ＨＨＶ６抗血清所逆转。     

从病人外周血单个核细胞中检测HHV－6：分离培养和基因扩增

收集婴幼儿急疹及淋巴系统增生性疾病患者外周血单个核细胞进行体外培养,从７例婴幼儿急疹及２例淋巴系统增生性疾病患者中分离出一种病毒,此病毒能在ＰＨＡ激活的人脐血单个核细胞中传代生长,产生典型ＣＰＥ：形成气球样巨细胞。电镜下观察,感染细胞中可见直径１８０ｎｍ左右,有包膜,疱疹样病毒颗粒;血清学试验证明分离株与ＨＳＶ－１,２、ＨＣＭＶ、及ＥＢＶ无抗原交叉,而与ＨＨＶ－６ＧＳ株间存在抗原一致性;多聚酶链反应表明该分离株ＨＨＶ－６特异性ＤＮＡ阳性;综合以上结果,初步认为该分离株为ＨＨＶ－６。同时还用ｐＣＲ法对所收集的标本直接检测ＨＨＶ－６特异性ＤＮＡ。ＰＣＲ法与病毒分离法相比较,前者ＨＨＶ－６检出率为８８．８％（１６／１８）．后者为３８．９％（７／１８）。     

The role of T cells in immunoglobulin class switching of specific antibody production system in vitro in humans

Only antibodies of the IgM class were produced in vitro by peripheral blood mononuclear cells stimulated with streptococcal carbohydrate. B cells of the peripheral blood mononuclear cells, however, synthesized both IgM and IgG class antibodies when combined with tonsillar T cells, suggesting that T cells inducing immunoglobulin class switching are present in the tonsils. Peripheral blood T cells also became capable of inducing B cells to produce IgG class antibodies when the T cells were incubated with antigen-pulsed macrophages. Surface IgM-positive, IgG-negative high-density B cells produced IgG antibodies for streptococcal carbohydrate in the presence of these T cells or tonsillar T cells. The culture supernatant solutions from these T cells or tonsillar T cells, however, failed to cause the B cells to produce IgG, indicating that class switching is not mediated by factors released from T cells. Lymphokines such as interleukin-2, human B cell growth factor, helper T cell factor, or interferon-gamma were also incapable of inducing IgG production. These results suggest that the cognate interaction between T cells and B cells is necessary for the immunoglobulin class switching.     

Effect of lymphokine-activated killer cells on human retinoblastoma cells (Y-79) in vitro: enhancement of the activity by a polysaccharide preparation, krestin

Peripheral blood mononuclear cells (PBMC) cultured in a medium containing interleukin 2 (IL 2) develop the ability to kill fresh tumor cells. This function has been termed lymphokine activated killing (LAK). Recently, cord LAK cell activity was demonstrated to be equally as cytotoxic against similar in vitro targets as adult (peripheral) LAK cells. We investigated the future therapeutic use of LAK adoptive immunotherapy by examining LAK in vitro cytotoxicity from both cord and peripheral blood mononuclear cells against pediatric malignant tumor cell lines Y-79 (retinoblastoma). Cord LAK cells show higher levels of cytotoxicity toward Y-79 targets than do adult LAK cells. Attempts to enhance the rIL 2-induced LAK activity by addition of rIFN-gamma or PSK (krestin) were successful. Furthermore, we found that PSK has a function to enhance rIL 2-induced IFN-gamma and TNF-alpha production. These findings suggest that combined administration of cord LAK cells and PSK may account for the improvement of advanced retinoblastoma in the neonatal period.     

8-Mercaptoguanosine overcomes unresponsiveness of human neonatal B cells to polysaccharide antigens

We have shown previously that pneumococcal polysaccharides behave as human T cell-independent type 2 Ag. When cultured in vitro with type 4 pneumococcal polysaccharides (PS4), human neonatal B cells do not or only marginally respond. Limiting dilution analysis of neonatal B cells polyclonally activated by a combination of phorbol esters, calcium ionophore, and T cells and T cell factors, however, showed that Ag-reactive B cells are present in cord blood. The frequency of anti-PS4 reactive B cells in cord blood is comparable with that of adult peripheral blood. In order to obtain more insight into the activation requirements of these PS4-reactive neonatal B cells, 8-mercaptoguanosine (8MGuo) was added to in vitro cultures. Addition of 0.5 to 1.0 mM 8MGuo resulted in a 3- to 10-fold amplification of the anti-PS4 response. the effect of 8 MGuo was most prominent when added 3 days after initiation of the culture. Based on kinetic studies, we propose that in vitro activated cells are target for 8MGuo. These data further indicate that neonatal unresponsiveness to polysaccharide Ag is not due to the physical absence of Ag-reactive B cells but more likely to be the consequence of different activation requirements as compared with adult B cells.     

Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

Patterns of isotype commitment in human B cells: limiting dilution analysis of Epstein Barr virus-infected cells

The frequency of Epstein Barr virus- (EBV) inducible IgM, IgG, and IgA-secreting cells in human peripheral blood and tonsil was determined by performing limiting dilution experiments in suspension culture. We devised a method of propagating small numbers of EBV-infected B cells with irradiated umbilical cord blood cells as a feeder layer. Precursor cell frequencies can be derived from these experiments; we have shown by statistical analysis that they conform to the single hit model of the Poisson distribution. By using this technique, a significant percentage of surface immunoglobulin-bearing lymphocytes are activated to secrete immunoglobulin in vitro. On the average, 8 to 38% of B cells in peripheral blood and tonsil can be propagated and the secreted immunoglobulin from the clonal progeny can be analyzed. Neither the EBV immune status of the donor nor the source of the umbilical cord blood feeder layer could account for the variations in cloning efficiency observed among donors. A surprisingly high frequency of B cells secreted IgA in vitro and we have shown that a small proportion of B cell clones in tonsil and peripheral blood secrete both IgM and IgA during the 4-wk culture period. Other B cells, including all those that produce IgG, appear to be committed to the secretion of a single isotype. Thus, these studies establish methodology for the analysis of the secreted products of human B cells at the single cell level and demonstrate that the progeny of at least some clones can secrete more than one isotype in vitro.     

Generation of activated killer (AK) cells by recombinant interleukin 2 (rIL 2) in collaboration with interferon-gamma (IFN-gamma)

Activation of human peripheral blood mononuclear cells (PBMC) by interleukin 2 (IL 2) and the role of interferon-gamma (IFN-gamma) in the IL 2-induced activation were investigated. Activated killer (AK) cells against NK-resistant tumor cell lines were induced in the medium containing recombinant IL 2 (rIL 2) and autologous serum without any other stimulating agents. AK activity was induced by doses of rIL 2 as low as 3 U/ml, and reached a maximum at 10(3) U/ml. Incubation of PBMC with rIL 2 resulted in IFN-gamma production and augmented NK activity after 1 day of culture, and in induction of AK cells and proliferative response after 2 days of culture. These results suggested that endogenous IFN-gamma was required for rIL 2-induction of AK cells and proliferative response. To prove this, PBMC were cultured with rIL 2 and rIFN-gamma or were pretreated with rIFN-gamma before culture with rIL 2. Both rIFN-gamma treatments of PBMC augmented rIL 2-induced AK activity and proliferative response. rIL 2-induced IFN-gamma production was also enhanced by the rIFN-gamma pretreatment of PBMC. The addition of anti-IFN-gamma antibody to rIL 2 cultures abrogated the rIL 2-induced NK augmentation, AK generation, and proliferative response in proportion to the decreased amounts of endogenous IFN-gamma detectable in culture. rIFN-gamma and/or rIL 2 cultures of PBMC increased Tac antigen expression on cell surfaces as measured by flow cytometry. Enhanced Tac expression by rIL 2 was abrogated by adding anti-IFN-gamma antibody. These data indicate that: 1) AK generation and IFN-gamma production are mediated by IL 2, and 2) IFN-gamma production may be required for IL 2 induction of AK cells and proliferative response. These finding are consistent with the hypothesis that AK generation involves a collaboration between IL 2 and IFN-gamma, in which IL 2 stimulates PBMC to produce IFN-gamma, which in turn acts as a differentiation signal that may be involved in the IL 2-initiated AK generation and proliferative response.     

Immunoglobulin M and immunoglobulin G secretion by human B cells exposed to RU 41.740, a glycoprotein extract from Klebsiella pneumoniae

RU 41.740, a glycoprotein extract from Klebsiella pneumoniae, was seen to activate human B cells to immunoglobulin secretion in vitro. The effects of RU 41.740 on human B cells were compared to those induced by pokeweed mitogen, a T-cell-dependent polyclonal B-cell activator, and Epstein-Barr virus, a T-cell-independent polyclonal B-cell activator. Exposure of human B cells to all of these agents resulted in increased immunoglobulin M (IgM) and immunoglobulin G (IgG) secretion. IgM and IgG secretion induced by RU 41.740 appeared to be T cell dependent when B cells were isolated from human peripheral blood. However, this activity may have been T cell independent when B cells were isolated from human spleen. RU 41.740-induced IgM secretion by peripheral blood B cells was seen to peak after 6 days in culture; IgG secretion peaked after 7 days in culture. The optimal concentration of RU 41.740 for the induction of IgM and IgG secretion by human B cells in vitro was seen to be 200 micrograms/ml.     

Fetal immune responses to Plasmodium falciparum antigens in a malaria-endemic region of Cameroon

Plasmodium falciparum infection during pregnancy can lead to the transplacental passage of malarial Ags that are capable of inducing acquired immune responses in the fetus. Studies have identified cytokines produced by malaria-specific cord blood (CB) T cells, but information on fetal B cells is limited. Thus, CB mononuclear cells from 120 Cameroonian newborns were cultured for 7 days in vitro and supernatants were assessed by ELISA for Abs to an extract of malarial schizonts (MA), recombinant apical merozoite Ag 1 (AMA-1), the 42-kDa C-terminal region of merozoite surface protein 1 (MSP-1(42)), a B epitope of ring-infected erythrocyte surface Ag (RESA), and the dominant B epitope of the circumsporozoite protein (CSP). Only 12% of supernatants contained IgM to MA but 78% had IgG to one or more malarial Ags, with 53% having IgG to AMA-1, 38% to MSP-1(42), 3% to RESA, and 0% to CSP. The Abs to AMA-1 and MSP-1(42) were predominantly IgG1 and IgG3. CB mononuclear cells were also tested for the ability to secrete cytokines in response to MA and a pool of conserved MSP-1 T cell epitopes. Among the Ag-reactive samples, 39.3% produced only Th2-type cytokines, whereas 60.6% produced a combination of Th1- and Th2-type cytokines. Although a Th2 bias was observed, the in utero cytokine environment was adequate to support isotype switching to cytophilic IgGs, the isotypes that are protective in adults. Because many infants living in a low transmission area are born with malaria-specific B and T cells, the influence of in utero priming on neonatal immunity merits further investigation.     

Mode of in vitro augmentation of natural killer cell activity by recombinant human interleukin 2: a comparative study of Leu-11+ and Leu-11- cell populations in cord blood and adult peripheral blood

Human newborn natural killer (NK) cell activity against K562 target cells was observed to be low compared with adult controls. Although Leu-7 (HNK-1)+ cells were negligible in cord blood, the proportions of Leu-11+ cells were equal to those of adult peripheral blood. Leu-11+ cells sorted from cord blood lymphocytes, as well as from adult lymphocytes exhibited the morphology of granular lymphocytes. In this study, we have investigated the phenotypic characterization of recombinant interleukin 2 (rIL 2)-induced cytotoxic lymphocytes against K562 cells by using anti-Leu-11 monoclonal antibody. Spontaneous cytotoxicity of lymphocytes was restricted to Leu-11+ cells in cord blood, as well as in adult blood, but this activity was low in cord blood Leu-11+ cells as compared with that of adult ones. NK cell activity of adult Leu-11+ cells could not be additionally enhanced after an 18-hr incubation with rIL 2(25 U/ml), whereas rIL 2 could potentiate the cytotoxicity of cord blood Leu-11+ cells approximately to the adult levels. It should be noted that cytotoxic activity of both Leu-11- cells from cord blood and adult blood that had no basal NK cells activity could be significantly potentiated by rIL 2. On the other hand, lymphokine-activated killer cells cytotoxic for HL-60 cell line could not be generated, and no proliferation of the lymphocytes was detected after an 18-hr incubation with rIL 2. It was shown that rIL 2 could not enhance the ability to bind to target cells in Leu-11+ and Leu-11- cells by means of a single cell conjugate assay, but the rate of target lysis of Leu-11+ cells from cord blood was significantly enhanced by rIL 2. These results suggested that rIL 2-induced cytotoxic effector cells were heterogeneous, and rIL 2 might potentiate the cytotoxicity of functionally immature NK cells or NK precursor cells.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.