PHA-Induced Activation of Suppressor Cells in Normal Human Peripheral Blood Lymphocytes

Normal human peripheral blood and tonsil lymphocytes can be stimulated to proliferate by phytohemagglutinin (PHA). When cells cultured with this mitogen for 3 days were transferred fo fresh autologous lymphocytes in fresh medium with PHA, the mitogen response of the fresh lymphocytes was suppressed. The suppression required the presence of viable cells, in that culture supernatants alone were not inhibitory and cell extracts showed only marginal inhibition. Approximately equivalent numbers of previously stimulated cells were required to produce optimal suppression of the PHA response of fresh cells. Cells irradiated after PHA stimulation were as effective as nonirradiated cells in causing suppression. PHA-stimulated cells also inhibited concanavalin A-induced proliferation and a mixed lymphocyte reaction. However, PHA-stimulated cells only partially inhibited the response to pokeweed mitogen. The suppressive effects were fully retained by a nylon-wool-enriched T-cell fraction but not by a B-cell-enriched fraction.     

Multiparametric Flow Cytometric Analysis of the Kinetics of Surface Molecule Expression after Polyclonal Activation of Human Peripheral Blood T Lymphocytes

In this report we have analysed the kinetics of modulation of human peripheral blood T lymphocyte membrane molecules upon activation with optimal amounts of phytohaemagglutinin (PHA) and concanavalin A (ConA). The following activation-related and differentiation/adhesion molecules were selectively and concomitantly investigated on CD4+ and CD8+ subsets by dual colour flow cytometry: CD69, CD25 and CD71; CD2, CD45RA and L-selectin. Cultures were assayed after 24, 48, 72, 120 and 168 h of incubation with PHA and ConA. This approach allowed a comprehensive evaluation of membrane phenomena occurring during activation of normal resting human T lymphocytes. Data show that the kinetics of expression of these molecules follows a precise and consistent time-course with no major differences between CD4 and CD8 subsets. CD69 expression peaked at 24 h, whereas CD25 and CD71 expression peaked at 48/72 h with some differences between PHA and ConA activation. L-selectin expression started an evident decrease in step with culture time whose magnitude was dependent on the lectin used, being higher with PHA than with ConA. Conversely, the expression of CD45RA remained stable for 72 h and then briskly decreased with no major differences between PHA and ConA activation. CD2 molecules increased with time in number and density, although the percentage of positive cells remained essentially constant (greater than 85%). After 48/72 h of stimulation about 10% of cells co-expressed CD4 and CD8 molecules. To ascertain whether the phenomenon was restricted to cells in a particular activation state, the phenotype of cells in the diverse phases of the cell cycle was established. Results obtained show that only actively proliferating cells, that is cells in S and G2-M phases, co-expressed the two molecules, suggesting that such a phenomenon reflects a momentary dysregulation of the normal sequence of gene expression. The present data are also discussed in the light of the dynamic role of T lymphocyte activation and adhesion molecules in regulating cell-cell interactions, tissue localization and eventual immunological function.     

螺旋藻对人外周血淋巴细胞增殖的作用

目的:探讨螺旋藻水提物对人外周血淋巴细胞体外增殖的影响和机制。方法:利用MTT法检测不同剂量组螺旋藻水提膜内、膜外物对人外周血淋巴细胞体外增殖的影响,同时设刀豆蛋白A(ConA)和植物血凝素(PHA)进行对照分析。结果:螺旋藻水提物对人外周血淋巴细胞具有增殖作用,并且高浓度螺旋藻水提膜内物增殖作用优于PHA。结论:螺旋藻水提物对人外周血淋巴细胞具有明显的增殖作用。     

从人外周血B淋巴细胞中应用PCR扩增人抗体基因

本文用常规ＰＣＲ法和半套式ＰＣＲ法，以一组人抗体重链和轻链引物，直接从人外周血淋巴细胞中扩增出抗体重链Ｆｄ基因和轻链基因。一些常规ＰＣＲ法不能直接扩增的人抗体基因，用半套式ＰＣＲ法扩增却得到了阳性结果。扩增的抗体基因的分子量与国内外同类报道一致。本文结果提示，在建立抗体基因文库时，半套式ＰＣＲ法能进一步丰富扩增的抗体基因的多样性     

外周血B淋巴细胞半套式PCR法扩增人抗体基因

目的用半套式PCR法,以一组人抗体重链和轻链引物直接从人外周血淋巴细胞中扩增人全套抗体基因片段.方法从不同人群外周血淋巴细胞中提取总RNA,经反转录后,以免疫球蛋白信号肽序列引物和家族特异性免疫球蛋白可变区基因引物,进行半套式PCR扩增人全套抗体基因片段.结果采用不同的引物进行重链、轻链Kappa和Lambda链的半套式PCR扩增,均能获得相应大小的PCR产物,其结果扩增率达到100%.结论在建立抗体基因文库时,半套式PCR法能进一步丰富扩增的抗体基因的多样性,可弥补由于转化效率不高而降低抗体库多样性的不足.     

Carbohydrate Metabolism in PHA Stimulated Human Peripheral Blood Lymphocytes

Human peripheral blood lymphocytes (PBL) stimulated in vitro by phytohemoagglutinin (PHA) manifest augmented glycolysis and oxidation of glucose-1-¹⁴C, indicating an increased utilization of the pentose pathway. Lactic acid production, as index of increased glycolysis, follows the same kinetic of thymidine incorporation and can he easily quantitated by an enzymatic assay.     

外周血B淋巴细胞半套式PCR法扩增人抗体基因

目的用半套式PCR法，以一组人抗体重链和轻链引物直接从人外周血淋巴细胞中扩增人全套抗体基因片段．方法从不同人群外周血淋巴细胞中提取总RNA，经反转录后，以免疫球蛋白信号肽序列引物和家族特异性免疫球蛋白可变区基因引物，进行半套式PCR扩增人全套抗体基因片段．结果采用不同的引物进行重链、轻链Kappa和Lambda链的半套式PCR扩增，均能获得相应大小的PCR产物，其结果扩增率达到100％．结论在建立抗体基因文库时，半套式PCR法能进一步丰富扩增的抗体基因的多样性，可弥补由于转化效率不高而降低抗体库多样性的不足．     

Reactivity of Antiserum to Human Brain with Peripheral Blood Lymphocytes

Antisera to human brain (AHBS) and human thymocytes (AHTS) were produced in rabbits and selectively absorbed to render them specific for T cells. After absorption AHBS, but not AHTS, lost most of its cytotoxic activity against T cells. Absorbed AHBS bound up to 95% of peripheral blood T lymphocytes as detected by indirect immunofluorescence and inhibited up to 46% of the lytic activity of AHTS; however, it was incapable of inhibiting the E-rosette formation of T lymphocytes. All 10 samples of human peripheral blood lymphocytes, pretreated with AHBS, were significantly suppressed in their response to antigens, but fewer samples were affected in their response to mitogens and to allogeneic stimulation, indicating diversity in the nature of the receptors involved in the cellular responses.     

Modulation of Mitogen-Induced Proliferation of Autologous Peripheral Blood Lymphocytes by Human Alveolar Macrophages

Experiments were carried out to determine the effect of cocultivation of T-cell-enriched human peripheral blood lymphocytes with autologous alveolar macrophages on mitogen-induced proliferation as determined by [³H]thymidine uptake. Cells obtained by fiberoptic bronchoscopy and saline bronchial lavage from 14 normal volunteers were enriched for macrophages by adherence in plastic dishes for 1 h in RPMI 1640 medium supplemented with 10% fetal calf serum. Nonadherent mononuclear cells were prepared from heparinized venous blood after Ficoll-Hypaque sedimentation by passage over nylon wool columns. T-cell-enriched populations were incubated with and without alveolar macrophages, either in the presence or absence of phytohemagglutinin. In these experiments, the number of lymphocytes was held constant (10⁵ per well), while the number of alveolar macrophages was varied (0.1 × 10⁵ to 4.0 × 10⁵ per well). Alveolar macrophages generally tended to stimulate phytohemagglutinin-induced lymphoproliferation at lymphocyte/macrophage ratios of 10:1 but consistently and significantly suppressed proliferation at ratios which approach those usually observed in recovered human bronchial lavage fluid, namely, 1:4. The suppressive effect of alveolar macrophages was observed as early as 48 h after culture initiation, while the magnitude of suppression increased with time. Suppression did not appear to be due to alteration in lymphocyte viability, nor was it sensitive to indomethacin. These results indicate that human alveolar macrophages can modulate the in vitro proliferative response of autologous peripheral blood lymphocytes. This observation may have relevance to interactions between alveolar macrophages and bronchial lymphocytes in the human lung in vivo.     

Triazolobenzodiazepines Exert Immunopotentiating Activities on Normal Human Peripheral Blood Lymphocytes

Previous studies have demonstrated that benzodiazepines (BDZ) (e.g. diazepam) inhibit immune responsiveness. Since these drugs are largely used in psychiatric patients it is of great importance to verify the existence of different types of BDZ, which are not suppressive for the immune system. In this framework, our results indicate that alprazolam and triazolam, two triazolo-BDZ, do not modify in vitro phagocytosis and killing exerted by normal human polimorphonuclear cells and monocytes. On the contrary, they significantly enhance T lymphocyte-dependent antibacterial activity in normal donors. These data support the concept that triazolo-BDZ and, in particular, alprazolam may represent more appropriate drugs for the treatment of psychiatric patients (e.g. patients with phobic disorders and/or migraine) who display immunodeficits.     

Triazolobenzodiazepines Exert Immunopotentiating Activities on Normal Human Peripheral Blood Lymphocytes

Abstract

Previous studies have demonstrated that benzodiazepines (BDZ) (e.g. diazepam) inhibit immune responsiveness. Since these drugs are largely used in psychiatric patients it is of great importance to verify the existence of different types of BDZ, which are not suppressive for the immune system. In this framework, our results indicate that alprazolam and triazolam, two triazolo-BDZ, do not modify in vitro phagocytosis and killing exerted by normal human polimorphonuclear cells and monocytes. On the contrary, they significantly enhance T lymphocyte-dependent antibacterial activity in normal donors. These data support the concept that triazolo-BDZ and, in particular, alprazolam may represent more appropriate drugs for the treatment of psychiatric patients (e.g. patients with phobic disorders and/or migraine) who display immunodeficits.     

In Vitro Humoral Immune Response of Human Peripheral Blood Lymphocytes to Tetanus Toxoid Sepharose 4B

The in vitro immune response of unfractionated human peripheral blood lymphocytes (PBL) from immune donors who had not been re-immunized with tetanus toxoid (TT) prior to donation was investigated. In this study we were able to stimulate PBL with tetanus toxoid coupled to Sepharose 4B (STT) for production of anti-tetanus toxoid antibody (Ab). Soluble tetanus toxoid or STT alone did not stimulate production of specific Ab. Pokeweed mitogen (PWM) and STT were required for optimal production of IgG and IgM antibodies specific to tetanus toxoid. Specific Ab responses were reduced in low and high concentrations of STT. Depletion of monocytes had no effect on either total IgG or specific IgG synthesis, but decreased the synthesis of both total and specific IgM. Depletion of E-rosette-forming cells decreased the production of specific Ab, suggesting T-dependency of the immune response to STT. Simultaneous production of total immunoglobulin and specific Ab by Sepharose 4B was negligible in the absence of PWM. In the presence of PWM, total immunoglobulin production was optimal, and specific anti-TT Ab production was undetectable. The specificity of the antiTT Ab was studied by absorption of the culture supernates with an STT column which removed all measurable specific Ab.     

Synthesis and Fragmentation of DNA in Phytohaemagglutinin-Stimulated Human Peripheral Blood Lymphocytes

In phytohaemagglutinin-stimulated lymphocytes, pulse labelled with tritiated thymidine for one minute, the acid-precipitable radioactivity was in the form of fragments that banded at the top of an alkaline sucrose gradient. When the radioactivity was chased with unlabelled thymidine for 2 hrs, most of the acid-precipitable radioactivity banded with the bulk of the DNA in the lower half of the gradient. On further chasing from 5 to 24 hrs, the radioactive DNA was fragmented and could be located on the lighter side of the gradients. This later fragmentation of radioactive DNA was associated with a loss of acid-precipi table radioactivity from the cells and appearance of DNA in the medium. After 24 hrs, approximately 67% of the acid-precipitable radioactivity was present in the medium. 60 to 70% of the radioactivity put out into the medium was acid precipitable, alkali resistant and DNase sensitive.     

Characterization of NKT Cells in Human Peripheral Blood and Decidual Lymphocytes

PROBLEM: To examine whether natural killer (NKT) cells are present in human pregnancy decidua. METHOD OF STUDY: We calculated the percentage of CD3+CD161+Valpha 24+-NKT cells in peripheral blood and early pregnancy decidua, and analyzed intracellular cytokines, interleukin (IL)-4 and interferon (IFN)gamma in NKT cells using flow cytometry. RESULTS: A distinct subset of CD3+ CD161+ lymphocytes expressing an invariant antigen receptor encoded by the Valpha24 and Vbeta11 segment was accumulated in the decidua. In pregnant subjects the percentages of NKT cells were significantly increased in the decidua compared with peripheral blood. Both NKT cells in the decidua and the peripheral blood had an ability to rapidly produce cytokine associated with Th1 (IFNgamma) and Th2 (IL-4). Interestingly, the percentages of IL-4 and IFNgamma producing NKT cells were significantly higher in the decidua compared with the peripheral blood. CONCLUSIONS: These findings suggest that NKT cells might control the Th1/Th2 balance by producing IL-4 and IFNgamma at the feto-maternal interface.     

Use of Plaque Assays to Study Thyroglobulin Autoantibody Synthesis by Human Peripheral Blood Lymphocytes

Plaque assays have been used to study thyroglobulin autoantibody synthesis and total immunoglobulin production by cultures of peripheral blood lymphocytes from patients with Hashimoto's thyroiditis. Freshly isolated Hashimoto or normal lymphocytes contained small numbers (7-1300) of total IgG plaque-forming cells (PFC) and total IgM PFC, but specific thyroglobulin antibody PFC was undetectable. After 5-8 days' culture with pokeweek mitogen (PWM), total IgG and IgM PFC were markedly increased to a geometric mean of 10,140 (8414-12,220) IgG PFC and 3450 (163-7534) IgM PFC per 10(6) cultured lymphocytes (95% confidence limits in parentheses). Furthermore, a mean of 63 (22-287) specific IgG thyroglobulin antibody PFC per 10(6) lymphocytes were detectable in cultures of Hashimoto lymphocytes. The IgG thyroglobulin antibody PFC were stimulated by Epstein-Barr virus (EBV) infection, suggesting that EBV infection may be useful in obtaining monoclonal thyroid autoantibodies.     

A Micromethod for the Activation of Human Blood Lymphocytes In Vitro

A micro culture system is described in which 2.5 × 10⁴ human blood lymphocytes in aliquots of 100 μL are stimulated by PHA, Pokeweed, “Varidase” antigen, allogeneic small lymphocytes or mitomycin-C-treated allogeneic LCL cells. Careful regulation of the pH by a combination of bicarbonate and MOPS buffers seems to be important for detecting a response to weak stimuli. High and reproducible levels of activation by powerful stimuli (PHA and LCL cells) can be recorded from even smaller cultures (10⁴ responding cells in 40 μL aliquots). The technique allows large numbers of replicate cultures to be set up from a single blood sample so that the time course and/or dose-response relationships can be examined for a range of differen mitogens.     

A Micromethod for the Activation of Human Blood Lymphocytes In Vitro

A micro culture system is described in which 2.5 × 10⁴ human blood lymphocytes in aliquots of 100 μL are stimulated by PHA, Pokeweed, “Varidase” antigen, allogeneic small lymphocytes or mitomycin-C-treated allogeneic LCL cells. Careful regulation of the pH by a combination of bicarbonate and MOPS buffers seems to be important for detecting a response to weak stimuli. High and reproducible levels of activation by powerful stimuli (PHA and LCL cells) can be recorded from even smaller cultures (10⁴ responding cells in 40 μL aliquots). The technique allows large numbers of replicate cultures to be set up from a single blood sample so that the time course and/or dose-response relationships can be examined for a range of differen mitogens.