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1.
Saline extracts from the roots of the pokeweed species. Phytolacca octandra were separated by ion-exchange chromatography into three fractions, Po-1, Po-2 and Po-3. Po-1 contained two monomeric proteins with molecular weights of 36,000 and 29,000 and these were partially purified by gel filtration. Po-2 was purified as a single polymeric protein composed of approximately ten 14,000 mol. wt polypeptides and is a new pokeweed mitogen. Po-3 was purified as a single polymeric protein composed of approximately four 31,000 mol. wt subunits, and apart from its polymeric structure closely resembles commercial pokeweed mitogen (PWM). Po-2 and Po-3 were mitogenic for unseparated human peripheral blood lymphocytes but the degree of mitogenic activity in Po-2 preparations was dependent on storage following purification. Purified B cells were not stimulated by either mitogen. Po-3 was a potent mitogen for T cells but preparations of Po-2 required storage before they stimulated T cells. Higher responses were observed in co-cultures of B and T cells than in separated B and T cell cultures. It is suggested that human B and T lymphocytes show synergy in their responses to Po-2 and Po-3.  相似文献   

2.
In pokeweed (Pa-1)-stimulated human lymphocyte cultures, T cells are essential for the survival, proliferation, plasma-cell development, and high-rate Ig secretion of B cells. Their effects are T-cell-specific in the sense that B-cell stimulation does not take place in the absence of T cells even when fibroblasts or monocytes are provided. The experimental system is the most effective model for activation of human B lymphocytes so far described. Plasmablast development requires approximately 7 days in culture. In T + B-cell cultures established at 1 X 10(6)/ml (1 X 10(4)/mm2) initial cell density, plasma cells can secrete, on the average, as much as 40-70 pg IgM or IgG per cell per day. When the initial T-cell density is increased well above 1.0 X 10(6)/ml, a T-cell-mediated depression of Ig synthesis becomes predominant. Thus, in the pokeweed model T-cell effects represent a balance of helper and suppressor influences. The study also shows that B cells are heterogeneous. A non-adherent IgG-committed (smIg-?) TONSIL B-cell population seems to be less susceptible to T suppressor effects than normal tonsil B cells. This subset proliferates particularly well and synthesizes large quantities of IgG in the presence of large initial proportions of T cells.  相似文献   

3.
A study of the events regulating human IgE biosynthesis in vitro was undertaken with tonsillar lymphocytes. IgG synthesis was also studied to evaluate the specificity of our observations. T-cell irradiation significantly enhanced synthesis of IgE by pokeweed mitogen (PWM)-stimulated B cells from 12 of 18 donors and IgG in all 18 donors. This enhancement was the result of de novo immunoglobulin synthesis, since the amount of IgE and IgG spontaneously released from lysed and lysed-and-cultured mononuclear cells was significantly less than that detected in the cell cultures, and the augmentation was completely ablated by the treatment of the cells with cycloheximide or mitomycin C. Enhancement was also dependent on the presence of PWM; T-cell irradiation did not enhance IgE synthesis in unstimulated cultures. Moreover, this enhancement was also observed in the co-cultures of B cells and allogeneic irradiated T cells. These observations suggest that radiosensitive T cells exert a suppressive activity that contributes to regulation of human IgE and IgG synthesis and that the suppressor function as well as the helper function can overcome allogeneic disparities.  相似文献   

4.
Deoxyadenosine (dAdo) levels above 2 microM inhibit plasma cell (PC) differentiation by human blood lymphocytes in pokeweed mitogen (PWM) stimulated cultures containing deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase (ADA). ADA inhibition by dCF alone did not suppress PC differentiation. Thymidine uptake by T cell blasts continuously cultured in conditioned medium was inhibited by dAdo and dCF; two of five EBV-infected B cell lines were also inhibited while three were resistant. Inhibition of PWM-induced PC differentiation of B cells by dCF and dAdo was reversed when conditioned medium (a source of T cell helper factors) was added to the cultures, and dAdo and dCF added to PWM-stimulated cultures 48 hr after their initiation did not inhibit PC differentiation, though thymidine uptake and the total number of cells recovered from the cultures were reduced. Removal of T cells after 48 hr of culture slightly reduced the numbers of PC in PWM-stimulated lymphocyte cultures but no further inhibition was obtained when dCF and dAdo were added to these T-depleted cultures, nor was their thymidine uptake further reduced. These results suggest that the in vitro suppression of B cell differentiation by dAdo in PWM-stimulated cultures is not due to direct toxicity of purine nucleosides to B cells but may be due to interference with T cell help. This is consistent with the view that a relative lack of helper activity by T cells contributes to the antibody deficiency of patients with ADA deficiency.  相似文献   

5.
An extract from the pathogenic actinomycete Nocardia brasiliensis was mitogenic for murine lymphocytes. This deoxyribonucleic acid-synthetic response of whole spleen cells peaked after 48 h in culture at concentrations of Nocardia extract ranging from 10 to 200 micrograms/ml. The extract appeared to be a mitogen for B lymphocytes since cultures of spleen cells from congenitally athymic nude (nu/nu) mice and of antithymocyte serum plus complement-treated spleen cells from conventional (+/+) mice responded as well as untreated spleen cells from normal +/+ mice. Furthermore, thymocytes did not respond mitogenically to the extract. Mitogenic responses were stimulated in spleen cells from H-2(a), H-2(b), H-2(d), and H-2(k) mice, including lipopolysaccharide-nonresponder C3H/HeJ mice. This Nocardia extract also stimulated polyclonal B-cell activation to the hapten trinitrophenyl, serum protein human gamma globulin, and several mammalian erythrocytes in cultures of cells from both euthymic and nude mice. Additionally, the requirement for helper T cells in the primary in vitro immune response to sheep erythrocytes could be circumvented by the addition of this Nocardia extract. These results indicate that an extract from the pathogen N. brasiliensis can nonspecifically activate murine B lymphocytes and raise the possibility that polyclonal activation of B lymphocytes may contribute to the pathogenesis of nocardiosis.  相似文献   

6.
OKT4+ (T helper/inducer) and OKT8+ (T cytotoxic/suppressor) subsets were depleted from peripheral blood lymphocytes (PBL) by complement-mediated lysis and residual cells examined for responsiveness to pokeweed mitogen (PWM) using a protein A haemolytic plaque assay for immunoglobulin secreting B cells. It was shown that: (1) three cycles of cell killing were required to totally abolish T helper function; (2) OKT4- PBL did not respond to PWM, but in a co-culture system, an equal number of unfractionated normal PBL could entirely reconstitute responsiveness of the residual B cells; (3) OKT8- PBL gave enhanced numbers of PWM-induced plaque forming cells (PFC); (4) addition of 4 micrograms/ml concanavalin A (con A) to PWM stimulated OKT8- PBL failed to suppress PFC generation, but suppression was induced by 12.5 and 25 micrograms/ml con A and (5) kinetics of PWM-induced PFC development were similar in the presence or absence of OKT8+ cells.  相似文献   

7.
PHA-induced colony formation and interleukin 2 (IL-2) production were studied in four patients with T cell leukaemia (three cases OKT4+/T helper and one case OKT8+/T cytotoxic suppressor). Cases of T helper cell leukaemia showed colony formation that was comparable to normal purified blood T cells and was not dependent on the addition of conditioned medium, containing IL-2 activity, to cultures. In contrast the T suppressor cell leukaemia formed colonies only when cultures were supplemented with IL-2 containing medium. When IL-2 production by PHA stimulated cells was measured culture supernatants from the three T helper cell leukaemias all showed normal or high levels of activity, when compared to normal blood mononuclear cells, whereas the T suppressor cell leukaemia showed no activity.  相似文献   

8.
In vitro production of IgG and IgM from peripheral blood lymphocytes and B-cell enriched fractions was assessed in a group of Hodgkin's disease (HD) patients and normal controls using pokeweed mitogen (PWM) stimulation. Our studies demonstrated a significant (P less than 0.01) reduction in the absolute number of helper (OKT4 positive) T cells and a significant alteration in the helper/suppressor T-cell ratio (0.89 +/- 0.15) compared to normal (1.83 +/- 0.31). Results from PWM stimulation experiments demonstrated that HD patients produced significantly lower IgG (P less than 0.01) and IgM (P less than 0.01) levels than controls. Synthesis of IgM but not IgG induced by PWM was subnormal after addition to patient B-cell cultures of autologous irradiated T cells or allogeneic irradiated normal T lymphocytes. Irradiated T cells from HD patients were as effective as normal T cells in helping PWM induced IgG and IgM synthesis by normal B cells. Our results suggest that in HD impaired circulating B-cell function is partly due to T-suppressor cell activity and furthermore that B-cell subpopulations producing different immunoglobulin isotypes may either be defective or vary in their susceptibility to T-cell suppression.  相似文献   

9.
H Duclos  M C Maillot    P Galanaud 《Immunology》1982,46(3):595-601
We describe selective effects of azathioprine (Az) on T-cell subpopulations regulating the primary in vitro antibody response of mouse spleen cells to the T-independent antigen TNP-polyacrylamide. This response is susceptible to the effect of two kinds of non-specific suppressor cells: (i) spontaneously-induced suppressor, generated after 4-5 days culture in the presence of 2 micrograms/ml of concanavalin A (Con A). Indeed, both these precultured cells lead to a cell dose-dependent suppression of the anti-TNP response when transferred at the initiation of antigen-stimulated fresh cell cultures. T cells are the effectors of both these suppressions and seem to directly suppress the B-cell response. We tested the in vitro effect of Az (10(-1) micrograms/ml) on the generation of these two sets of suppressors. Whereas that of Con-A-induced suppressors proves to be resistant, that of spontaneously-induced T suppressors is totally prevented by the addition of Az in the preculture medium. Instead, Az treatment allows the manifestation of a spontaneously-induced helper T cell, simultaneously generated, which is able to increase a T-independent antibody response and quite resistant to the in vitro effect of Az. Thus, this study demonstrates that different subpopulations of T lymphocytes regulating the B-cell antibody response exhibit a selectivity to Az, implying different cell proliferation requirements and/or different cellular origin.  相似文献   

10.
As human B lymphocytes and macrophages carry surface receptors for Factor H (B1H), we investigated the possibility that this complement component regulates their function. Factor H inhibits immunoglobulin secretion by peripheral mononuclear cells (MNC) stimulated with pokeweed mitogen if present at the initiation of the cultures and at concentrations greater than 50 micrograms/ml. Factor H also inhibited stimulation and differentiation of purified B cells into immunoglobulin-secreting cells by Epstein-Barr virus (EBV). The inhibitory effect of Factor H was abrogated if anti-Factor H antibody was present in the cultures. EBV-transformed B-cell lines secreted less immunoglobulin if Factor H was present in the culture for at least 4 days. Culture of MNC with Factor H did not lead to the generation of suppressor T cells or macrophages. In contrast, Factor H did not cause proliferation of human peripheral total MNC or enriched T-cell or B-cell subpopulations. Also, Factor H did not inhibit the proliferation of MNC in response to several mitogens and antigens. Our results strongly indicate that Factor H is able to block human B-cell differentiation in vitro without blocking the proliferative ability of the cells. Factor H seems to act directly on the B cells through its receptor on their surface, since it inhibited T-dependent and T-independent B-cell differentiation but generated no suppressor cells.  相似文献   

11.
L Rsnen 《Immunology》1979,37(4):715-721
The ability of antigens and allogeneic cells to induce lymphokine synthesis and cellular co-operation in lymphokine production was investigated. Human peripheral blood lymphocytes were separated into T- and B-cell populations by sheep red blood cell rosette formation and centrifugation on Ficoll--Isopaque. The cells were then stimulated with PPD, SK-SD, candida and with allogeneic cells. The presence of leucocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose migration method. The results indicated that T lymphocytes produced LIF after stimulation with antigens and allogeneic cells. In addition, B cells responded to PPD. Except for PPD the stimulants did not induce significant T-cell LIF production in the absence of monocytes. Only autologous monocytes enhanced LIF synthesis after antigenic stimulation, whereas in mixed lymphocyte cultures allogeneic monocytes were as effective as autologous ones. The monocyte helper effect was mediated by soluble factors in mixed lymphocyte cultures and by soluble factors and direct cell--cell contact in antigen-stimulated cultures. No co-operation between T and B lymphocytes was found. B cells did not enhance LIF production by T cells, nor could T cells induce a B-cell response to antigens or allogeneic cells.  相似文献   

12.
Mitogenesis of human peripheral blood lymphocytes as measured by the uptake of [3H]thymidine was stimulated in vitro by pure orosomucoid glycoprotein when used at concentrations that are considerably lower than the physiological plasma level. The lymphocyte cultures stimulated with PHA or PWM were not affected by low concentration (67 micrograms/ml), but they were mildly suppressed by high concentration (1 mg/ml) of this glycoprotein. The stimulatory response was relatively greater with fractionated T cells than the non-T cells (B cells and monocytes). At 50 micrograms/ml concentration of orosomucoid, the lymphocyte activation was found in randomly selected blood donors which included normal healthy volunteers and patients with T cell immunodeficiency or Alzheimer's disease, demonstrating a consistent immunostimulatory action of this glycoprotein.  相似文献   

13.
Colony Formation by Subpopulations of Human T Lymphocytes   总被引:1,自引:0,他引:1  
Twelve human T-cell lines were derived from cultures stimulated with phytohaemagglutinin (PHA) or with the recall antigen purified protein derivative (PPD), using the one-step agar colony method, followed by further expansion of individual T-cell colonies for at least 50 days. As judged by monoclonal antibodies, two of the PHA-derived T-cell lines carried the helper-inducer (H-I) phenotype and two the suppressor-cytotoxic (S-C) phenotype. Four PPD-derived cell lines displayed the H-I phenotype, and three of these proliferated specifically when challenged with the recall antigen, whereas the fourth did not. Four PPD-induced T-cell lines carried the S-C phenotype, and only one of these was antigen-specific. All cell lines with H-I phenotype displayed helper cell activity as determined by Ig secretion of B lymphocytes stimulated with a T-cell-dependent polyclonal B-cell activator. Cell lines with the S-C phenotype had no such helper activity and suppressed Ig responses in the presence of freshly isolated T cells. Thus, a good correlation between phenotypic markers and functions was found. T helper cells and T suppressor cells can be differentiated by means of both polyclonal and antigenic stimuli.  相似文献   

14.
The effect of anti-immunoglobulin (anti-Ig) on cultures of established human B-cell lines was studied to develop models for the initiation and regulation of immune responses. DNA synthesis in one line, BM, measured by [125I]iododeoxyuridine (IUdR) incorporation, was stimulated by anti-Ig when the continuously proliferating cultures approached high cell density. One of four lines, BJAB, was very sensitive to inhibition of DNA synthesis by anti-Ig at all cell concentrations. Of four human T-cell lines tested for suppressor activity, the CEM line was found to be activated by concanavalin A (Con A) to inhibit the anti-Ig stimulation of BM cells. No cytotoxic activity was detected in Con A-treated CEM cells. Both the anti-Ig stimulation of BM and Con A-induced suppression by CEM occurred with cloned cell lines in long-term culture, in the absence of helper or accessory cells.  相似文献   

15.
The effect of azathioprine (Az) on the primary in vitro antibody response of mouse spleen cell cultures has been studied. The response towards T cell-dependent antigens is suppressed by low Az concentrations (50% inhibition by 10(-2) mug/ml and 100% suppression by 10(-1) mug/ml). The same pattern is observed when Az addition is delayed until day 2, but the suppression is absent or partial when Az is added on day 3. In the early period (day 0 to day 1) the effect of Az is reversible upon addition of an excess of purine nucleosides. In contrast, the response to a T cell-independent antigen (TNP-T4) is relatively insensitive to Az, since 100-fold higher drug concentrations are required to obtain an inhibition. With the assumption that T helper cells are likely to be highly sensitive to Az, the effect of low Az concentrations on the other two cell populations involved in T cell-dependent responses has been evaluated. Adherent cells appear unaffected. In contrast, the B-cell response is markedly sensitive to Az, as shown by the effect of Az on the response of nude mouse cells to a T cell-dependent antigen in the presence of T-cell products, either specific or non-specific. On the other hand, the B-cell response to mitogens is resistant to az. Thus, Az has a differential effect on B-cell response according to the thymus dependency of the antigen. This may suggest the existence of two pathways for B-cell activation or two different B-cell subpopulations.  相似文献   

16.
J Pryjma  H D Flad  M Ernst  E Brandt    A J Ulmer 《Immunology》1986,59(4):485-489
The number of immunoglobulin-secreting cells (ISC) upon stimulation with pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I (SAC) in recombinant interleukin-2 (rIL-2)-supplemented cultures of human peripheral blood mononuclear cells (PBMC) and co-cultures of B and T cells was studied. It has been shown that the addition of rIL-2 to culture can enhance or depress the number of ISC depending on the polyclonal B-cell activator used for culture stimulation. The SAC-induced response was enhanced in the presence of rIL-2, while the number of ISC in PWM-stimulated cultures was depressed. Moreover, in cultures stimulated simultaneously by both activators, the suppressive effect of rIL-2 prevailed, indicating that the reported direct effect of the lymphokine on SAC-activated B cells cannot overcome the suppressive activity of PWM-induced suppressor T cells. rIL-2 could not activate suppressor T cells in the absence of PWM, and has no effect on the number of helper or suppressor cells in the culture.  相似文献   

17.
Tetanus toxoid immunization of humans generates circulating B cells which secrete IgG anti-tetanus toxoid antibodies (IgG-Tet) when stimulated in vitro with T cells and pokeweed mitogen (PWM). A unique property of these cells is the inhibition of maturation into antibody-secreting plasma cells following a 1-hr in vitro pulse with tetanus toxoid. Studies were undertaken to determine if different T-cell subsets could modulate the in vitro generated B-cell unresponsive state. The addition of OKT4+/OKT8- cells to antigen-treated B cells resulted in a partial reversal of the antigen-induced inhibition of IgG-Tet synthesis. The addition of OKT4-/OKT8+ cells to the treated B cells caused a suppression of IgG-Tet synthesis comparable to that seen in cultures containing unfractionated T cells. These results indicate that (1) the B-cell unresponsive state generated by antigen treatment is not absolute, (2) the degree of B-cell unresponsiveness results from a balance of suppressor and helper signals, and (3) T-suppressor cells need to be present to induce and maintain the B-cell unresponsive state.  相似文献   

18.
The responses of resting human B lymphocytes to a variety of activation signals were studied. Human tonsillar B lymphocytes were separated according to size by countercurrent elutriation. The small B lymphocytes were then stimulated in vitro with various concentrations of anti-mu antibody in the presence or absence of B cell growth factor (BCGF) or with Staphylococcus aureus Cowan strain I (SAC). Cellular volume changes and RNA synthesis were measured over the first 24 h of stimulation and were similar with either 15 micrograms/ml of anti-mu, 100 micrograms/ml of anti-mu, or SAC. In the subsequent 24 h, however, substantial increases occurred in the amount of RNA synthesis and cell enlargement only in those cultures stimulated with 100 micrograms/ml of anti-mu or SAC, but not in the cultures stimulated with 15 micrograms/ml of anti-mu. The addition of BCGF to those cultures stimulated with 15 micrograms/ml of anti-mu did not alter the increases in cellular volume and RNA synthesis found 24 h after stimulation with anti-mu alone. However, over the subsequent 24 h, the presence of BCGF in culture enhanced both B cell volume changes and RNA synthesis, when compared to cultures stimulated with 15 micrograms/ml of anti-mu alone. In addition, BCGF enhanced DNA synthesis in cultures stimulated with low and high concentrations of anti-mu. DNA content changes following stimulation with anti-mu, anti-mu plus BCGF, and SAC were also measured using propidium iodide staining and flow cytometry. Optimal concentrations of anti-mu induced 20% of the resting B cells to enter S phase, while optimal concentrations of anti-mu plus BCGF or SAC induced approximately 40%. Finally, prestimulation of resting B cells for 24 h with a low concentration of anti-mu, sufficient for cell enlargement but not S phase progression, allowed for rapid entrance of the prestimulated B cells into S phase when a high concentration of anti-mu or SAC was added. These findings suggest the existence of a control point in the progression of human B cells through the cell cycle. This control point is located in the G1 phase of the cycle and is reached 24 to 36 h after a surface immunoglobulin-mediated stimulus.  相似文献   

19.
The in vitro immunopharmacological effects of phenytoin (PHT) and its major metabolite 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) were investigated by flow cytometric analysis of the DNA content of mitogen stimulated mouse spleen cells. The qualitative effects of PHT and HPPH were similar in concanavalin A stimulated mouse spleen cells with both compounds causing an increase in the percentage of S phase cells. The data suggests that this effect is due to an augmentation of cell cycling as demonstrated by the significant increase in 4N cells in PHT treated cultures relative to control cultures following colcemid treatment. A PHT time course study revealed an increase in S phase cells and a subsequent increase in 4N cells. PHT had no significant effect on congenitally athymic nude mouse spleen cells stimulated with lipopolysaccharide (LPS) except at the highest concentration tested (80 micrograms/ml) where a depression of cell cycling was observed. HPPH caused a colcemid-like accumulation of 4N cells in the LPS stimulated nude mouse spleen cell cultures. PHT and HPPH were found to be effective in enhancing cell cycling in cultures containing a significant population of T-cells stimulated with a T-cell mitogen whereas an inhibitory effect was observed in cultures without T-cells stimulated with a B-cell mitogen. The capacity of PHT to enhance the mitogenic action of concanavalin A may relate to its capacity to induce immunologic abnormalities and lymphadenopathy in humans.  相似文献   

20.
The effects of delta-9-tetrahydrocannabinol (THC) on the growth, DNA synthesis and phagocytic activity of P388D1, a murine macrophage cell line, were investigated. Incubating cell cultures with THC resulted in a dose-dependent inhibition of cell propagation and DNA synthesis. The magnitude of these effects was dependent upon the number of cells in the culture as well as the protein content in the culture medium. As the cell number increased, the THC effect decreased. Using cell cultures in which the cell concentration was standardized, THC (greater than or equal to 5 micrograms/ml) produced pronounced inhibitions of cell growth and DNA synthesis, while lower THC concentrations (less than or equal to 3 micrograms/ml) were less effective. Studies examining the phagocytic activity of the P388D1 cells indicating exposure to THC (5 micrograms/ml for 2 h) only marginally affected the association of latex beads with the external surface of the plasma membrane. However, the ability of these THC-treated cells to internalize the latex particles was severely depressed. Thus, the data indicate that THC inhibited the growth and functional activity of murine macrophages in vitro and suggests that the P388D1 cell line is a useful model to study the effects of cannabinoids on the phagocytic process.  相似文献   

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