Cell walls, peptidoglycans, and teichoic acids of gram-positive bacteria as polyclonal inducers and immunomodulators of proliferative and lymphokine responses of human B and T lymphocytes.

In this study, the mitogenic and immunomodulating effects of bacterial cell wall preparations were investigated. Cell walls, peptidoglycans, and teichoic acids from Bacillus subtilis and Staphylococcus aureus Wood 46 activated both human T cells (supplemented with 10% monocytes) and B cells to proliferative and to produce leukocyte migration inhibitory factor. Similar results were obtained with adult and umbilical cord blood cells, suggesting that these bacterial preparations acted as mitogens. Cell walls and peptidoglycans had a modulating effect on purified protein derivative-induced and protein A-induced proliferation. In the presence of suboptimal concentrations of these stimulants, bacterial components enhanced the proliferative response. However, at optimal concentrations of purified protein derivative or protein A, bacterial components suppressed lymphocyte proliferation. Peptidoglycans solubilized by lysozyme activated B lymphocytes but not T cells. Solubilization had no effect on immunomodulating capacity.     

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.     

Agonistic properties of anti-B cell antibodies purified on staphylococcal protein A may be due to contaminating protein A.

Some antibodies directed to cell surface receptors may mimic the physiological ligands by inducing the transmission of activation or growth signals. Such agonistic antibodies have proven very useful when studying functional properties of various receptor molecules on, e.g., lymphoid cells. However, while investigating the agonistic effects on tonsillar B cells of the anti-CD43 monoclonal antibody (mAb) D4B11 and the anti-CD40 mAb S2C6, we made some observations which emphasize the need for caution when using antibodies purified by protein A affinity chromatography. Both antibody preparations were found to elicit changes in the intracellular free calcium concentration ([Ca2+]i) as well as promoting proliferation of phorbol ester activated cells. However, a closer analysis showed that the increase in [Ca2+]i could be attributed to soluble staphylococcal protein A (SpA) desorbed during antibody purification. By using pure soluble SpA, we were able to show that nanogram amounts were sufficient to increase [Ca2+]i by a mechanism that involved both a mobilization from intracellular stores and an influx across the B cell membrane. A similar effect on cytosolic Ca2+ in B cells was also noted for streptococcal protein G (protein G), another bacterial component used for antibody purification. However, in contrast to SpA, protein G had little effect on cell proliferation. These observations suggest that the presence of trace amounts of SpA or protein G in antibodies purified on these bacterial components may lead to incorrect interpretations of the agonistic properties of such antibodies. When the above findings were taken into account, it was found that the CD43 mAb D4B11, like the CD40 mAb S2C6, stimulated growth of B cells without causing any measurable increase in [Ca2+]i. Both CD40 and CD43 may thus be coupled to signalling pathways which do not involve breakdown of membrane phosphoinositides.     

Compartmentalization of T cell responses following respiratory infection with Bordetella pertussis: hyporesponsiveness of lung T cells is associated with modulated expression of the co-stimulatory molecule CD28

We have used a murine respiratory challenge model to examine the local T cell responses in the lung during infection with Bordetella pertussis. T cells from lung parenchyma and airways of naive and infected mice were refractory to both antigen and mitogen stimulation in the presence of lung macrophages. Furthermore irradiated mononuclear cells from the lungs suppressed antigen and mitogen-induced proliferation, but not IFN-γ production, by splenic T cells. Removal of macrophages and stimulation of purified lung T cells in the presence of irradiated splenic antigen-presenting cells fully restored the response to mitogen. However, T cells purified from the lung during the acute phase of infection with B. pertussisfailed to proliferate or produce detectable levels of IL-2, IL-4, IL-5 or IFN-γ in response to purified bacterial antigens. In contrast, splenic T cells from these animals produced high levels of IL- 2 and IFN-γ and proliferated strongly to a range of bacterial components. Phenotypic analy sis of bronchoalveolar lavage cells during the course of infection revealed transient infiltra tion of neutrophils, followed by macrophages, CD4+ T cells and smaller numbers of CD8+ T cells and γ δ+ T cells. Cell surface expression of B7 on infiltrating macrophages and CTLA-4 on T cells did not change significantly during infection. However, expression of the CD28 co- stimulatory molecule was profoundly reduced on lung T cells during the acute phase of infection. In contrast, lung T cells from mice primed by B. pertussisinfection or vaccination were resistant to CD28 down-regulation. These results suggest compartmentalization of T cell responses between the lung and the periphery during B. pertussisinfection and that B. pertussismay have immunomodulatory properties on local T cell populations in the lungs of naive mice.     

Characterisation of cell-wall-derived polypeptide antigens from different species of Mycobacterium

Cell walls from different species of Mycobacterium were purified on a sucrose step gradient. The components derived from these preparations were characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by staining or by Western blotting. Surface-exposed polypeptide molecules were also identified by biotinylation. Many protein and glycoprotein molecules were identified in the cell walls. Some of these molecules were immunogenic in man and experimental animals and showed wide variability from species to species. The data suggest that these molecules could be of significance in the diagnosis and pathophysiology of mycobacterial diseases.     

The roles of interleukin 2 and interferon-gamma in human B cell activation, growth and differentiation

The roles of interleukin 2 (IL 2) and interferon-gamma (IFN-gamma) in human peripheral blood B cell activation, growth and differentiation were examined. Highly purified B cells stimulated with Cowan I Staphylococcus aureus (SA) proliferated minimally and generated no immunoglobulin-secreting cells (ISC) without the addition of T cell supernatants (T sup) produced by mitogen-activated T cells. Recombinant IL 2 (rIL 2) alone was able to promote maximum proliferation and generation of ISC in cultures of highly purified SA-stimulated B cells when present from the initiation of the incubation. IFN-gamma, by contrast, could not support either response alone. When a two-step culture system was employed to determine the effect(s) of T cell influences during both initial activation and in propagating the response following activation, it was found that B cells activated by SA alone subsequently responded maximally to T sup but only minimally to IL 2 and not at all to IFN-gamma. However, the presence of T sup, rIL 2, or rIFN-gamma during initial activation with SA was found to facilitate greatly the subsequent capacity of the activated B cells to proliferate and differentiate in response to either T sup or IL 2. These data suggest two distinct pathways of human B cell responsiveness. Activities in T sup other than IL 2 or IFN-gamma can support the growth and differentiation of B cells initially activated with SA alone, whereas rIL 2 is capable of promoting these responses maximally only when B cells have been initially activated by SA in the presence of T cell lymphokines, such as IL 2 or IFN-gamma. The results emphasize the role of specific T cell factors in determining the outcome of humoral immune responses.     

Inhibition of B cell growth factor (BCGF) by monoclonal antibodies directed against the C3d receptor (CR2)

Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). Human BCGF provides the final proliferative signal to normal, activated B cells. We herein show that anti-CR2 monoclonal antibodies inhibit human B cell responsiveness to purified BCGF. Addition of anti-CR2 antibody, AB5, was capable of completely inhibiting BCGF-mediated enhancement of either anti-mu or staphylococcal protein A-activated human B cells (191 +/- 21 cpm vs. 3942 +/- 622 cpm, mean +/- SEM). Inhibition of B cell response to BCGF by AB5 occurred in a dose-dependent manner. Monoclonal antibody anti-B2, which recognizes the same 140-kDa glycoprotein as AB5, in comparable concentrations also inhibited B cell responsiveness to BCGF. Monoclonal antibodies of the same subclass (IgG1) showed no inhibitory effect on BCGF enhancement of B cell proliferation. The F(ab')2 fragment of AB5 generated by pepsin digestion was similarly inhibitory as was the intact Ig. AB5-mediated inhibition was independent of the target B cell state of activation. Both resting and activated B cells (anti-mu or staphylococcal protein A activated) incubated with similar concentrations of AB5 were unresponsive to BCGF. The ability of anti-CR2 antibodies to block BCGF-dependent B cell proliferation suggests that occupancy of C3d membrane receptors may result in modulation of B cell proliferation in physiologic or clinical disease states.     

Enhancement of human B cell proliferation by an antibody to the C3d receptor, the gp 140 molecule

The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.     

Neurotoxicity of glia activated by gram-positive bacterial products depends on nitric oxide production.

The present study examined the mechanism by which bacterial cell walls from two gram-positive meningeal pathogens, Streptococcus pneumoniae and the group B streptococcus, induced neuronal injury in primary cultures of rat brain cells. Cell walls from both organisms produced cellular injury to similar degrees in pure astrocyte cultures but not in pure neuronal cultures. Cell walls also induced nitric oxide production in cultures of astrocytes or microglia. When neurons were cultured together with astrocytes or microglia, the cell walls of both organisms became toxic to neurons. L-NAME, a nitric oxide synthase inhibitor, protected neurons from cell wall-induced toxicity in mixed cultures with glia, as did dexamethasone. In contrast, an excitatory amino acid antagonist (MK801) had no effect. Low concentrations of cell walls from either gram-positive pathogen added together with the excitatory amino acid glutamate resulted in synergistic neurotoxicity that was inhibited by L-NAME. The induction of nitric oxide production and neurotoxicity by cell walls was independent of the presence of serum, whereas endotoxin exhibited these effects only in the presence of serum. We conclude that gram-positive cell walls can cause toxicity in neurons by inducing the production of nitric oxide in astrocytes and microglia.     

Study of induction of activation of human peripheral blood mononuclear cells with a non-activating form of anti-CD3 MoAb in autoimmune thyroid disease (AITD).

Anti-CD3 (OKT3) MoAb is a mitogenic agent which activates lymphocytes. We have studied the effects of murine anti-human OKT3 MoAb (IgG1) alone or in combination with IL-2, human thyroglobulin (Tg) and thyroperoxidase (TPO) antigens on the proliferation of whole peripheral blood mononuclear cells (PBMC) (including monocytes) or subtypes (T, CD4+, CD8+, B) as measured by tritiated thymidine (3H-TdR) incorporation. B cell differentiation was studied by measuring numbers of IgG-secreting cells and specific anti-TPO/anti-Tg-secreting cells by SPOT ELISA. PBMC or lymphocyte subtypes, obtained from 45 patients with Hashimoto's thyroiditis (HT), 40 Graves' disease (GD) and 51 normal controls were cultured in 96 microtitre plates for 6 days in the presence of OKT3 MoAb at final concentrations 25-250 ng/ml, IL-2 15 U/ml, Tg and TPO (1 micrograms/ml). Then cultures were pulsed with 0.2 microCi 3H-TdR/well and incorporation was measured after 18 h. IgG and anti-TPO/Tg-secreting cells were detected at 7 days. Higher proliferative responses from whole PBMC preparations in response to any of the combinations including OKT3 MoAb were observed in the HT preparations, while the basal values were the lowest. IL-2 alone increased these responses markedly, but equally in all groups. IL-2 in combination with OKT3 had an additive effect on proliferation, with higher responses in HT. Tg and TPO antigens did not change these responses. Most HT preparations responded with their maximum proliferation to the lowest concentration of OKT3 MoAb (25 ng/ml), whereas in GD and control preparations of PBMC these responses were shifted to higher concentrations (250 ng/ml); even with those, proliferation was not so enhanced in controls when compared with HT and GD preparations. In contrast, the proliferative responses of T cells alone and subpopulations of CD8+ suppressor/cytotoxic cells were decreased in HT preparations compared with controls. Monocytes were necessary for proliferation. In the subpopulation of B cells (> 95% pure) and CD4+ helper/inducer cells, differences did not reach significance. In spite of the effect on proliferation, OKT3 MoAb only mildly but significantly increased the numbers of IgG-secreting cells in HT and GD preparations and did not stimulate synthesis of specific antibodies. Our data suggest that the increased proliferative responses of whole PBMC to OKT3 MoAb in HT preparations might be due to insufficient activation of T suppressor/cytotoxic cells.     

Discrimination of T, B, and Null Lymphocytes by Electronic Sizing1

T, B, and null lymphocytes isolated by sheep red blood cells (SRBC) rosetting or adherence to a nylon wool column had distinct size differences according to Coulter counter size analysis. The diameters in microns were as follows: B = 6.42, T = 6.76, null = 7.16, granulocytes = 8.42, and monocytes = 9.02. Coulter size distributions of cell preparations were compared to the proportions of cells with E, EAC, and surface immunoglobulin (SIg) markers in these preparations as well as their reactivity to HLA--DR antisera and rabbit anti-B-lymphocyte antisera. Cell preparations monitored by Coulter sizing produced the expected results in mixed lymphocyte culture (MLC) testing. That is, T cells responded but did not stimulate whereas B lymphocytes and monocytes stimulated but did not respond. NULL lymphocytes both stimulated and responded. In antibody-dependent cell-mediated cytotoxicity (ADCC), null lymphocytes and monocytes functioned as effectors, whereas T lymphocytes and granulocytes did not. B lymphocyte preparations were intermediate in activity. Coulter sizing is a rapid, simple means of assessing the purity of suspensions enriched for T, B, and null lymphocytes.     

Synergy of B cell growth factor and interleukin 2 in the proliferation of activated human B cells

The activity of purified interleukin 2 (IL2), obtained by the recombinant DNA technology, on the proliferative response of human B cells stimulated with low concentrations of anti-mu antibody was investigated. Recombinant IL2 was capable of augmenting the proliferative response of anti-mu-activated B cells and the T cell activation (Tac) antigen was expressed on a substantial proportion of normal B cells stimulated with anti-mu antibody. However, crude supernatants from protein A-stimulated peripheral blood mononuclear cells, which were found to possess both IL2 and B cell growth factor (BCGF) activities, maintained the ability to promote proliferation of anti-mu-activated B cells after depletion of IL2. In addition, supernatants from some T cell clones, apparently free of IL2 activity, displayed strong BCGF activity in the co-stimulation assay with anti-mu antibody. This BCGF activity was found in 25 kDa fractions by gel filtration and it was unaffected by addition to the cultures of anti-Tac antibody, which consistently inhibited the B cell proliferative response promoted by recombinant IL2. The proliferative response of anti-mu-activated B cells to clonal, IL2-free supernatants containing BCGF and recombinant IL2 present together from the beginning of culture was close to the sum of responses to the two stimulants, separately. In addition, the presence of clonal supernatant containing BCGF from the beginning of culture had a synergistic effect in the response of activated B cells to the subsequent addition of IL2, whereas the initial presence of IL2 had no such an effect on the reactivity of anti-mu-stimulated B cells to the late addition of clonal supernatant containing BCGF. The synergistic effect of BCGF in the IL2-promoted B cell proliferation was probably the result of the recruitment of a greater number of IL2-reactive B cells. In fact, the number of Tac-positive cells was significantly higher in 36-h cultures established in the presence of anti-mu antibody plus clonal supernatant containing BCGF than in cultures stimulated with anti-mu antibody alone. Taken together, these data indicate that anti-mu antibody promotes the expression by normal human B cells of distinct receptors for IL2 and a BCGF distinct from IL2. They also suggest that BCGF can exert a synergistic effect in the IL2-promoted proliferation of activated B cells.     

Blastogenesis and Lymphokine Synthesis by T and B Lymphocytes from Patients with Periodontal Disease

Thymus-derived (T) and bone marrow-derived (B) lymphocytes were isolated from human peripheral blood and cultured with various mitogens and antigens. Purified protein derivative of tuberculin stimulated both purified T and B cells from patients with positive skin reactivity to purified protein derivative but did not stimulate nonimmune lymphocytes. Similarly, both T and B lymphocytes from patients with periodontal disease were stimulated to proliferate when incubated with dental plaque, whereas cells from normal individuals without gingivitis were unresponsive. In contrast, one component of plaque, bacterial endotoxins (lipopolysaccharide), minimally stimulated B lymphocytes from both normal or gingivitis patients. T lymphocytes from patients with periodontal disease were also stimulated by plaque antigen to produce chemotactic lymphokine activity (CTX) for human monocytes. B cells purified by the EAC rosetting method nonspecifically produced CTX without concomitant blastogenesis; however, after dissociation of adherent EAC these immune B cells did not spontaneously produce CTX. Lymphokine synthesis by B cells was not dependent on concomitant blastogenesis. Dissociated B cells from periodontitis patients also produced CTX activity after stimulation with dental plaque antigen. Therefore, both T and B lymphocytes, after stimulation with nonendotoxin antigenic components of plaque, proliferated and produced lymphokines, which are presumed to contribute to the pathogenesis of periodontal disease.     

Response of human dendritic cells to different immunomodulatory polysaccharides derived from mushroom and barley

Polysaccharides derived from fungi and plants have been increasingly used as dietary supplement with therapeutic intention for cancer. However, whether these polysaccharides from different sources and structures can elicit similar immunological effects remain unknown. This study aims to investigate and compare the effects of selected groups of purified and crude polysaccharides on human dendritic cells (DCs), the most potent antigen-presenting cells. The selected polysaccharides were from Ganoderma lucidum [(GL) Lingzhi, Reishi], a medicinal mushroom commonly used by oriental; and barley glucan, a purified polysaccharide with known in vivo immunomodulating effect. We found that purified polysaccharides from GL mycelium could induce human PBMC proliferation and phenotypic and functional maturation of DCs with significant IL-12 and IL-10 production. Polysaccharides of GL spore and barley were both rather weak immunostimulator in vitro. In general, all these polysaccharides did not polarize T cells into either T(h)1 or T(h)2 or regulatory T cells, except for crude spore polysaccharides-treated DCs which could suppress T cell proliferation with IL-10 production. This study revealed the polysaccharides of different sources have different immune potency and effect on human immune cells including DCs. Our study also provides a reproducible biological platform for comparing the potential therapeutic effects of different herbal-derived polysaccharides in the future.     

Effects of human alpha-foetoprotein on human B and T lymphocyte proliferation in vitro

Purified human alpha-foetoprotein (AFP) isolated from extracts of foetal and hepatoma tissues, and from cord serum was evaluated as to its suppressive effects on in vitro lymphocyte responses to stimuli which selectively trigger human B or T cells. The effects of equivalent concentrations of individual AFP preparations were compared on lymphocyte cultures stimulated with the human B cell mitogen Staphylococcus aureus strain Cowan I organisms, with the T cell mitogen phytohaemagglutinin (PHA), and with irradiated allogeneic lymphocytes in the one-way mixed lymphocyte reaction (MLR). PHA responses were significantly inhibited by most purified preparations of AFP in a dose-dependent manner, within the concentration range of 300 to 18 μg/ml. However, individual foetal-derived AFP preparations did vary in suppressive potency on PHA responses, and attempts to reactivate an inactive AFP were unsuccessful. In parallel cultures the mitogenic response to protein A expressing Staph. aureus bacteria was normal or even slightly enhanced by AFP. The one-way MLR was effectively suppressed at higher concentrations of AFP (300–600 μg/ml) than were required for inhibition of PHA responses. The inhibitory effect of AFP on PHA-induced lymphocyte proliferation was not altered by increases in the mitogen dose. No evidence was found that AFP merely inhibits PHA responses by direct interference with mitogen or by competition for cell surface receptors with the mitogen. The results reported here indicate that human AFP effectively suppresses certain T cell-mediated reactions, but not B cell responses in vitro, and these are in line with previously reported findings in the murine AFP system.     

IL-4 and transforming growth factor-beta suppress human immunoglobulin secretion in vitro by surface IgD- B cells.

The effect of IL-4 and transforming growth factor-beta (TGF-beta) on immunoglobulin secretion in vitro by peripheral blood mononuclear cells (PBMC) or purified B cells activated with murine EL4 thymoma cells and phorbol myristate acetate (PMA) was investigated. As previously reported, IL-4 induced IgE and IgG4 secretion by B cells in PBMC preparations and B cells activated with EL4 cells and PMA. However, when B cells, either in PBMC preparations or purified and activated with EL4 cells and PMA, spontaneously secreted large quantities of immunoglobulin, IL-4 suppressed the immunoglobulin secretion of all isotypes. IL-4 also suppressed the IgE secretion by B cells from an atopic dermatitis patient. This suppressive effect was not reversed by adding IL-2 or interferon-gamma (IFN-gamma) to the cultures. We also showed that TGF-beta suppressed the immunoglobulin secretion by purified B cells activated by EL4 cells and PMA. To investigate whether IL-4 or TGF-beta suppressed immunoglobulin secretion by in vivo 'switched' and isotype-committed B cells, sIgD- B cells were isolated, activated with EL4 cells and PMA and cultured with IL-4 or TGF-beta. Such activated B cells secreted large quantities of IgG1, IgG2, IgG3, IgA1, IgA2 and IgM, and IL-4 and TGF-beta suppressed all these isotypes by greater than 80%. The data demonstrated that IL-4 and TGF-beta suppress immunoglobulin secretion in vitro by in vivo isotype-committed sIgD- B cells, suggesting that these lymphokines may play a down-regulatory role on differentiated isotype-committed B cells in an isotype-unrestricted manner. The data also showed that IL-4 and TGF-beta acted directly on isolated B cells.     

Detection of cellular and humoral immunity to hepatitis B surface antigen (HBsAg) in asymptomatic HBsAg carriers.

Cell mediated and humoral immunity to hepatitis B surface antigen (HBsAg) was studied in nine asymptomatic HBsAg carriers, nine patients with natural acquired immunity to HB infection and nine HB-susceptible donors. Peripheral T and B lymphocytes from all asymptomatic HBsAg carriers and all HB-immune donors studied were specifically induced into proliferation and anti-HBs secretion when stimulated with low doses of HBsAg (2-30 ng antigen protein/ml) in vitro. This activation was achieved by mixing purified B and/or T cells with optimal concentrations of autologous monocytic cells. T and B cells from the HB-susceptible donors were non-responsive under identical culture conditions. These data do neither substantiate the existence of a qualitative defect in T cell function, nor the absence of circulatory B cells capable of synthesizing anti-HBs in vitro in asymptomatic HBsAg carriers. Thus, the inability to mount a satisfactory antibody response to HBsAg in vivo might be a consequence of altered immune responsiveness to this antigen, which may be a relevant factor in the pathogenesis of asymptomatic HBsAg carriership.     

Immunomodulating activity of seaweed extract on human lymphocytes in vitro.

Effect of eight kinds of seaweed extract (SWE) on human lymphocytes was studied in vitro. The extracts of Hizikiafusiformis and Meristotheca papulosa (green) markedly stimulated human lymphocytes to proliferate, whereas Eucheuma muricatum and Meristotheca papulosa (red) weakly stimulated proliferation. The responder cells are T cells, because T cells purified by sheep red blood cell (SRBC) rosette-formation were significantly stimulated with SWE, but B cells were not. These extracts enhanced the induction of cytotoxic T lymphocyte (CTL) activity, but failed to enhance natural killer (NK) cell activity. These extracts had a stimulatory effect on immunoglobulin (Ig) production by B cells and tumor necrosis factor (TNF) production by monocytes. The activity of Hizikia fusiformis associated with polysaccharides which were extracted with ethanol and purified by ion-exchange and gel filtration chromatography, whose molecular weight was about 100 kDa. These results suggest that SWE has an immunomodulating activity on human lymphocytes and this ability might be useful for clinical application to treat several diseases such as tumors.     

In vitro immunomodulatory activity of ruthenium complexes

OBJECTIVE AND DESIGN: We have explored the in vitro immunomodulatory effects of pure ruthenium red and a series of pyridine and imidazole substituted ruthenium complexes (RCs). MATERIAL: Human peripheral blood lymphocytes and purified T cells were used in these studies along with various cell lines. METHODS: Cells were treated with dilutions of RCs and assessed in various assays of immune function, cytotoxicity and cell cycle progression. RESULTS: RCs efficiently blocked T cell receptor (TCR)-mediated stimulation (IC(50)'s in the low nM range) of human peripheral blood lymphocytes (hPBL) by various agents, including tetanus toxoid, alloantigens, superantigens, and receptor-specific antibodies. RCs are not cytotoxic to T cells. Antiproliferative activity was also observed for B cells. Some non-lymphoid cell lines or primary cultures showed sensitivity to the RCs, but only at higher concentrations. The inhibitory effect on human T cells was assessed and demonstrated at the level of proliferation (DNA synthesis), IL-2 secretion, and IL-2 receptor (CD25) upregulation. RCs also inhibited IL-2-mediated proliferation of antigen-induced T-cell blasts and the IL-2-dependent T cell line Kit-225. Cell cycle analysis indicates that RCs inhibit the progression of activated T cells from G(0)/G(1) to S phase. CONCLUSIONS: Since the mechanism of T cell inhibition by RCs appears to be different than that of rapamycin (RAP) or cyclosporin A (CsA), they may provide a new tool to investigate intracellular signaling in T cells, and may present novel opportunities for immunosuppression