1-BP inhibits NF-kappaB activity and Bcl-xL expression in astrocytes in vitro and reduces Bcl-xL expression in the brains of rats in vivo.

1-Bromopropane (1-BP) has been widely used as a substitute for chlorofluorocarbon that destroys the ozone layer. Although the central neurotoxicity of 1-BP has been recently reported, a molecular mechanism is not clear. In particular, the effects on cells in brain have not been fully analyzed. Here, we studied the effects of 1-BP on the activation of transcription factors involved in anti-apoptotic function or cell survival in astrocytes. Astrocytoma cell lines, U251, U373 and VM, or murine primary astrocytes were used for in vitro assay. DNA binding activities of NF-kappaB in these cells induced by interleukin (IL)-1 or LPS were inhibited by 1-BP. Consequently, the treatment of U251 cells with 1-BP resulted in suppression of NF-kappaB reporter activity. Furthermore, 1-BP blocked IkappaBalpha degradation, which is important for NF-kappaB activation. In addition, the level of Bcl-xL mRNA, which is known as an anti-apoptotic gene, were reduced in U251 treated with 1-BP or in the brain from rat exposed to 1-BP (400 ppm, 12 weeks). These results suggest that subchronic inhalation exposure to 1-BP vapor may affect the Bcl-xL expression in astrocytes.

Prostaglandin E2 down-regulates viable Bacille Calmette-Guérin-induced macrophage cytotoxicity against murine bladder cancer cell MBT-2 in vitro.

The regulatory effect of prostaglandin (PG) E2 and a cyclooxygenase (COX) inhibitor on Bacille Calmette-Guérin (BCG)-induced macrophage cytotoxicity in a bladder cancer cell, MBT-2, was studied in vitro. BCG stimulated thioglycollate-elicited murine peritoneal exudate cells (PEC) to induce cytotoxic activity and to produce cytokines such as interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and PGE2. NS398, a specific COX-2 inhibitor, and indomethacin (IM), a COX-1 and COX-2 inhibitor, enhanced viable BCG-induced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. However, NS398 and IM did not enhance these activities induced by killed BCG. Enhanced cytotoxicity was mediated by increased amounts of IFN-gamma and TNF-alpha. Exogenous PGE2 reduced cytotoxic activity and IFN-gamma and TNF-alpha production of PEC. These results suggest that PGE2 produced by BCG-activated macrophages has a negative regulatory effect on the cytotoxic activity of macrophages. Accordingly, a PG synthesis inhibitor may be a useful agent to enhance BCG-induced antitumour activity of macrophages.

Cytotoxic effect of an anti-liver monoclonal autoantibody obtained after neonatal thymectomy in mice.

A monoclonal autoantibody, LSA-1, against murine liver antigen was obtained by fusing spleen cells from a neonatally thymectomized BALB/c mouse with SP2/0 murine myeloma cells. The LSA-1 isotype was IgG2b and kappa. LSA-1 was specific to the liver, especially, to a liver-specific membrane lipoprotein (LSP) fraction. By Western blotting analysis, LSA-1 mainly detected a 100 kDa protein of LSP fraction. LSA-1 stained cytoplasm of the cryostat sections of liver in immunohistochemical analysis. Furthermore, the antigen recognized with LSA-1 was highly expressed on the surface of a murine hepatoma cell line, MH134, slightly on a murine normal liver cell line, C1469, and on freshly prepared hepatocytes, but not on spleen cells. LSA-1 had a cytotoxic activity on liver cell lines in the presence of a complement in vitro. Furthermore, injection of LSA-1 into mice-induced liver injury. These results suggest that anti-liver autoantibody plays an important role in the induction of autoimmune hepatitis. Accordingly, this antibody will be a useful tool for the analysis of the pathogenesis of autoimmune hepatitis.

Phenytoin promotes Th2 type immune response in mice.

The effects of chronic administration of phenytoin, a common anticonvulsive drug, on immune responses were studied in mice. Anti-keyhole limpet haemocyanin (KLH) IgE antibody response after KLH-immunization was enhanced in phenytoin-treated mice. Proliferative responses of spleen cells induced with KLH, concanavalin A (ConA), lipopolysaccharide and anti-CD3 antibody were reduced in phenytoin-treated mice. Accessory function of spleen adherent cells on ConA-induced T cell proliferative response was reduced in phenytoin-treated mice. KLH-induced IL-4 production of spleen cells was enhanced, while IFN-gamma production was reduced in phenytoin-treated mice. In addition, production of IL-1 alpha, but not IL-6 and IL-12 by spleen adherent cells from phenytoin-treated mice was reduced. Natural killer cell activity was reduced in phenytoin-treated mice. These results suggest that phenytoin treatment preferentially induces a Th2 type response. We also observed that plasma ACTH and corticosterone levels were increased in phenytoin-treated mice, and speculated that phenytoin might act directly and indirectly, through HPA axis activation, on the immune system to modulate Th1/Th2 balance.

Suppression of macrophage interleukin-12 and tumour necrosis factor-alpha production in mice infected with Toxocara canis.

Toxocara canis induces a predominantly Th2 type response with enhanced amounts of interleukin (IL)-4 and reduced amounts of interferon (IFN)-gamma in infected mice. In this study, we investigated the macrophage function of T. canis-infected mice from the standpoint of cytokine production. C3H/HeN mice were infected with T. canis by oral administration of embryonated eggs. Ten days after infection, macrophages were obtained from spleen and peritoneal cavity, were cultured with lipopolysaccharide, and cytokines in the culture supernatant were evaluated with enzyme-linked immunosorbent assay. Macrophages from T. canis-infected mice produced IL-1 and IL-6 equivalent to macrophages from normal mice. The production of IL-10 and tumour growth factor (TGF)-beta was enhanced, but IL-12 and tumour necrosis factor (TNF)-alpha production was diminished. The addition of IFN-gamma, anti-IL-10 antibody, anti-TGF-beta antibody or indomethacin did not restore the production of IL-12 and TNF-alpha by macrophages from T. canis-infected mice. Furthermore, the stimulation of normal macrophages with T. canis antigen in vitro induced IL-1alpha, IL-6, IL-10 and TGF-beta, but not IL-12 and TNF-alpha. These results indicate that cytokine producing pattern of macrophages is altered by T. canis-infection, and this altered macrophage function may play an important role in the modification of the immune response to T. canis.

Cyclooxygenase-2 expression in the hippocampus of genetically epilepsy susceptible El mice was increased after seizure.

It has been suggested that cyclooxygenase (COX)-2 and prostaglandin play a role in epilepsy. We studied the expression of COX-2 in the hippocampus and the effect of oral administration of indomethacin, a COX inhibitor, on seizure activity in genetically seizure-susceptible El mice. COX-2 protein significantly increased in the hippocampi of El mice after epileptic seizure. Indomethacin did shorten the duration from seizure onset to full recovery in El mice although the threshold and the duration of seizure were not changed.

Prostaglandin E2 up-regulates macrophage-derived chemokine production but suppresses IFN-inducible protein-10 production by APC.

PGE(2) has been known to suppress Th1 responses. We studied the role of PGE(2) in two representative chemokines, macrophage-derived chemokine (MDC) and IFN-inducible protein-10, production by LPS- or CD40-stimulated spleen cells. The production of MDC, one of the ligands for CCR4 preferentially expressed on Th2, was enhanced in nonstimulated, LPS-, CD40-, or CD3-stimulated spleen cells by the pretreatment with PGE(2), while the production of IFN-inducible protein-10, a representative ligand for CXC chemokine receptor 3 expressed on Th1, was suppressed. MDC production was also enhanced by IL-4, IL-5, and intracellular cAMP-elevating agents such as dibutyryl cAMP and 3-isobutyl-1-methylxanthine, and the effect of IL-4, IL-5, and PGE(2) was additive. However, the pretreatment with IL-6, IL-10, or TGF-beta, or the neutralization of IFN-gamma or IL-12 had no effect on MDC production. B cells, macrophages, and dendritic cells were main producers of MDC, while T cells produced only a small amount of MDC. MDC production by B cells was equally stimulated by LPS and anti-CD40 Ab, while that by macrophages and dendritic cells was more markedly stimulated by anti-CD40 Ab, and PGE(2) further enhanced MDC production by these stimulated cells. These results indicate that PGE(2) regulates Th1/Th2-related chemokine production by B cells, macrophages, and dendritic cells, and that this is a new function of PGE(2) for the regulation of Th2 immune responses at the induction and activation stages.

Role of TGF-beta in the retinoic acid-induced inhibition of proliferation and melanin synthesis in chick retinal pigment epithelial cells in vitro.

The purpose of this study was to investigate the effects of all-trans retinoic acid (RA) on the induction of transforming growth factor-beta (TGF-beta) that is concerned with the proliferation and melanin synthesis of chick retinal pigment epithelial (RPE) cells in vitro. Chick RPE cells were cultured in the presence or absence of RA and anti-TGF-beta antibody for 7 days. The effects of RA and pan-specific TGF-beta antibody on RPE cell proliferation were assessed by counting the number of cells, and their effects on melanin synthesis were evaluated by measuring the melanin content of the cells. TGF-beta activity in the culture supernatant of RPE cells was measured using CCL-64 cells. RA significantly inhibited RPE cell proliferation and increased melanin synthesis. The addition of pan-specific TGF-beta antibody to the culture blocked the inhibition of RPE cell proliferation and the increased melanin synthesis. RA induced TGF-beta production in the culture supernatant of RPE cells. These findings indicate that RA regulates the proliferation and melanin synthesis of RPE cells via induction of TGF-beta.

Effect of mite antigens on antigen presenting cells/macrophages in mice.

The effect of mite antigens on murine lymphocytes and macrophages was studied in vitro. Antigens prepared from Dermatophagoides farinae bodies (Dfb) or recombinant Mag3, glutathione-S transferase (GST)-fused mite antigen, stimulated murine spleen cells to proliferate. The responder cells were B cells, because the response was sensitive to anti-Ig antibody and C treatment, but not to anti-Thy 1 antibody and C treatment. The response was not due to lipopolysaccharide contamination, a representative B-cell mitogen, because polymyxin B column-passed Dfb significantly stimulated B cells, and GST protein alone did not stimulate them. Alloantigen presenting activity was increased in mite antigen-treated B cells and spleen adherent cells. Mite antigens stimulated CD80 and the major histocompatibility complex (MHC) class II molecule expression, but suppressed CD86 expression on B cells and spleen adherent cells that were detected by a flow cytofluorometer. Antibodies to the MHC class II molecules, CD80 and CD86 blocked the alloantigen-presenting activity. Furthermore, mite antigens stimulated B cells and spleen adherent cells to produce cytokines. These results suggest that mite antigens have a stimulating activity on antigen-presenting cells/macrophages and modulate immune responses.

Macrophage-colony stimulating factor inhibits the growth of human ovarian cancer cells in vitro.

The effect of macrophage-colony stimulating factor (M-CSF), which regulates the growth and differentiation of haematopoietic progenitor cells on the growth of ovarian cancer cells was investigated in three ovarian cancer cell lines in vitro. The spontaneous growth of these cells was significantly inhibited by the addition of M-CSF in a concentration-dependent manner over 96 h of culturing. The maximum response was obtained with 10 ng/ml (3857 U/ml) of M-CSF by counting the viable cell number using the trypan blue exclusion assay. [(3)H]-thymidine incorporation by these cells was also suppressed following a 96-h incubation with M-CSF. The inhibitory effect of M-CSF was reversed by the addition of anti-M-CSF monoclonal antibody. Flow cytometric analysis revealed that the treated ovarian cancer cells arrested at the G0/G1 phase of the cell cycle. These cells expressed M-CSF receptors on their surface as detected by Scatchard plot analysis using (125)I-labelled M-CSF. These results indicate that M-CSF has an antitumour activity for ovarian cancer cells and suggest that it can be applied for the treatment of this disease.

Murine IL-2 secreting recombinant Bacillus Calmette-Guerin augments macrophage-mediated cytotoxicity against murine bladder cancer MBT-2.

PURPOSE: This study was to establish a more effective anti-cancer immunomodulating agent by constructing recombinant (r) Bacillus Calmette-Guérin (BCG) secreting alpha-antigen (alpha-Ag) fused murine (m) interleukin (IL)-2, and to study its biological activity on cell-mediated cytotoxicity against murine bladder cancer cell, MBT-2, in vitro. MATERIALS AND METHODS: pSO246 plasmid vector ligated with mIL-2 gene was introduced into BCG by electroporation. Thioglycollate-elicited murine peritoneal exudate cells (PEC) were stimulated in vitro with parental BCG or rBCG and their cytotoxic activity and the cytokine production was studied. Cytokines were assayed by an enzyme-linked immunosorbent assay (ELISA) and L929 bioassay. Cytotoxicity was measured by 51Cr releasing assay. RESULTS: rBCG (alpha-Ag-IL-2) secreted functional IL-2 and augmented more efficient cytotoxicity to MBT-2 and cytokines such as IL-12, tumor necrosis factor and interferon (IFN)-gamma in PEC than parental BCG did. rBCG (alpha-Ag) had the same activity as BCG. Anti-IL-2 antibody reduced rBCG (alpha-Ag-IL-2)-mediated cytotoxicity and IFN-gamma production. Exogenous IL-2 also enhanced BCG-mediated cytotoxicity, but 100 times more IL-2 was required to express the same activity as rBCG (alpha-Ag-IL-2). Anti-IL-12 neutralizing antibody and the depletion of T cells and NK cells reduced IFN-gamma production by PEC stimulated with rBCG (alpha-Ag-IL-2), suggesting that T cells, NK cells and IL-12 participate in the enhancement of IFN-gamma production. CONCLUSIONS: rBCG secreting IL-2 showed significant antitumor activity and cytokine production and this will be a promising agent for bladder cancer patient to reduce both clinical dose and side effects of BCG for immunotherapy.

Enhancing effect of an inhibitor of nitric oxide synthesis on bacillus Calmette-Guerin-induced macrophage cytotoxicity against murine bladder cancer cell line MBT-2 in vitro.

We studied the effect of an inhibitor of nitric oxide (NO) synthesis, N(G)-monomethyl-L-arginine (L-NMMA), on the Bacillus Calmette-Guérin (BCG)-induced antitumor activity of murine peritoneal exudate cells (PEC) against murine bladder cancer cell line MBT-2 in vitro. L-NMMA enhanced BCG-induced cytotoxic activity of PEC, as well as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production. The L-NMMA-induced enhancement was due to the prolonged survival of BCG in macrophages, because no enhancement of cytotoxicity was observed and neither IFN-gamma nor TNF-alpha production was significantly enhanced by killed BCG. Anti-TNF-alpha antibody (Ab) and anti-IFN-gammaAb reduced the L-NMMA-induced enhancement of the cytotoxicity. The depletion of T cells from PEC reduced the production of both IFN-gamma and TNF-alpha, as well as the enhancement of cytotoxicity induced by viable BCG plus L-NMMA. These results suggest that L-NMMA has an enhancing effect on BCG-induced macrophage cytotoxicity and the enhancement is partially mediated by T cells and their soluble products. Accordingly, NO inhibitor should be a valuable adjunct to BCG immunotherapy for bladder cancer.

Reduced interleukin-1 responsiveness in immune system and central nervous system of inbred polydipsic (STR/N) mice.

Inbred polydipsic mice (STR/N strain) have primary polydipsia. The previous studies found abnormalities in the central nervous system (CNS), especially in the hypothalamus and circumventricular organ. As a part of pursuing to find the cause of the polydipsia, we investigated immunological characteristics of STR/N mice, using the ICR strain of mice as control. Their thymic subset cells showed that CD4+CD8+ double positive cells were increased, CD4+ single positive cells were decreased and CD5 expression was deficient, compared to ICR mice. T cell proliferative response and interleukin (IL)-2 production caused by IL-1beta stimulation were reduced in STR/N mice than those in the ICR mice. In in vivo studies the degree of thymic atrophy and the increases in serum level of ACTH and corticosterone induced by intraperitoneal IL-1beta injection were much less in STR/N mice than those in controls. Furthermore, adipsic response also induced by IL-1beta injection was greatly reduced compared to their control mice. All these results suggest that the responsiveness to IL-1 is impaired both in the immune system and the CNS of STR/N mice.

Chinese medicinal herb, Acanthopanax gracilistylus, extract induces cell cycle arrest of human tumor cells in vitro.

We investigated the effect of a Chinese medicinal herb, Acanthopanax gracilistylus (AG), extract (E) on the growth of human tumor cell lines in vitro. AGE markedly inhibited the proliferation of several tumor cell lines such as MT-2, Raji, HL-60, TMK-1 and HSC-2. The activity was associated with a protein of 60 kDa, which was purified by gel-filtration chromatography. Cell viability analyses indicated that the treatment with AGE inhibits cell proliferation, but does not induce cell death. The mechanism of AGE-induced inhibition of tumor cell growth involves arrest of the cell cycle at the G(0) / G(1) stage without a direct cytotoxic effect. The cell cycle arrest induced by AGE was accompanied by a decrease of phosphorylated retinoblastoma (Rb) protein. Furthermore, cyclin-dependent kinases 2 and 4 (Cdk2 and Cdk4), which are involved in the phosphorylation of Rb, were also decreased. These results suggest that AGE inhibits tumor cell growth by affecting phosphorylated Rb proteins and Cdks.

Involvement of the protein kinase pathway in melanin synthesis by chick retinal pigment epithelial cells.

Protein kinases are involved in a variety of cellular functions and cell proliferation in eyes. We have explored the involvement of protein kinase C (PKC) in cell proliferation and melanin synthesis by chick retinal pigment epithelial (RPE) cells in vitro. This was achieved by incubation of confluent RPE cells with known inhibitors of protein kinase, H-7, W-7, H-8, and staurosporine. Chick RPE cells were cultured in the presence or absence of the protein kinase inhibitors for a 10-day period. Effects of the inhibitors on cell proliferation and melanin synthesis, as an indication of cell differentiation, were assessed by counting the number of surviving cells and by measuring the melanin content in the cells, respectively. H-7, W-7, and staurosporine inhibited cell proliferation and increased melanin synthesis in a concentration-dependent manner during culture; however, H-8 did not produce these cellular effects. These findings indicate that PKC and calcium/calmodulin-dependent kinase pathways are involved in the proliferation and differentiation of chick RPE cells.

Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice.

PGE2 has been shown to play a prominent role in regulating Th1 and Th2 type responses. We studied the role of PGE2 in IFN-gamma production by Staphylococcus aureus Cowan I-stimulated spleen cells from several mouse strains such as BALB/c, C3H/HeN, and C57BL/6. When spleen cells were pretreated with indomethacin (cyclooxygenase (COX)-1 and COX-2 inhibitor) or NS-398 (COX-2-specific inhibitor), S. aureus Cowan I -induced IFN-gamma production was increased more markedly in spleen cells from BALB/c mice than from C3H/HeN and C57BL/6 mouse. However, PGE2 production was not significantly different among spleen cells from three mouse strains. When various concentrations of PGE2 were exogeneously added to spleen cells, PGE2 showed a stronger suppressive effect on IFN-gamma production in spleen cells from BALB/c mice than from other strains of mice. This suppressive effect of PGE2 in BALB/c mice mainly depended on IL-12p70 production by APCs. More PGE2 binding sites were found in BALB/c spleen cells than in C3H/HeN spleen cells, indicating that the sensitivity difference to the suppressive effect of PGE2 was due to the difference of the number of PGE2 receptors. The administration of NS-398 into BALB/c mice enhanced Ag-specific IFN-gamma production, but not IL-4 production. This effect is the same as IL-12 administration in vivo. From these results, we propose that the modulation of PGE2 is important for Th1 activation via IFN-gamma and IL-12p70 production in vitro and in vivo and that PGE2 is one of the pivotal factors in the Th2-dominant immune response in BALB/c mice.

B cell stimulating activity of metallothionein in vitro.

The effect of metallothionein (MT) on lymphocytes was studied in vitro. Rabbit MT induced the proliferative responses of mouse splenocytes at concentrations of 1-25 microg/ml. MT synergistically enhanced Con A- or LPS-induced proliferative response of splenocytes. A similar effect was also observed for rabbit splenocytes at a similar concentration. Free heavy metals such as Cd and Zn only weakly stimulated splenocytes. 2-mercaptoethanol (2-Me) abrogated the effect of MT, suggesting that thiols in MT play an important role for splenocyte response. MT stimulated splenocytes from MT-knockout mice as well as those from normal control mouse. The responder cells for MT stimulation were B cells and MT also induced B cell differentiation to produce Ig. MT induced the calcium influx of B cells, but not T cells. These results indicate that MT has a potent immunomodulating activity, especially on B cells.

Suppressive effect of Chinese medicinal herb, Acanthopanax gracilistylus, extract on human lymphocytes in vitro.

We studied the effect of a Chinese medicinal herb, Acanthopanax gracilistylus, extract (AGE), on human lymphocytes in vitro. AGE markedly suppressed the proliferative responses of human peripheral blood lymphocytes stimulated with mitogens concanavalin A (Con A) and Staphylococcus aureus Cowan I (SAC). Both T cell and B cell activities-production of interferon-gamma and immunoglobulin-were suppressed by AGE. The mechanism of AGE-induced suppression of lymphocytes is to arrest the cell cycle at the G0/G1 stage without a direct cytotoxic effect. AGE also suppressed the alloantigen-specific cytotoxic T lymphocyte response. However, natural killer cell activity was less sensitive to the suppressive activity of AGE. In contrast, AGE markedly enhanced monocyte function to produce cytokines. These activities of AGE were associated with a 60-kD protein which was sensitive to treatment with pronase E, but not with NaIO4. These results suggest that AGE has an immunomodulating activity on human lymphocytes and its properties could be clinically applied in the treatment of several diseases such as autoimmune and allergic diseases.

CD44 stimulation induces integrin-mediated adhesion of colon cancer cell lines to endothelial cells by up-regulation of integrins and c-Met and activation of integrins.

For cancer metastasis, tumor cells present in the circulation must first adhere to the endothelium. Integrins lymphocyte function-associated antigen (LFA) 1 and very late antigen 4 play a central role in leukocyte adhesion to the endothelium and subsequent migration into tissues. The majority of tumor cells derived from solid cancers including colorectal cancer do not express suitable adhesion receptors, LFA-1 and very late antigen 4. We investigated the mechanisms of adhesion and transendothelial migration of cancer cells using colorectal carcinoma cell lines. Our results showed the following novel features of CD44 on the cells: (a) colon cancer cells express high levels of CD44; (b) stimulation of cancer cells by CD44 cross-linking or fragmented hyaluronan markedly induces the expression of LFA-1s, some of which reveal an activation epitope on the cells; (c) CD44 cross-linking induces F-actin polymerization in the cell cortex; (d) fragmented hyaluronan induces up-regulation of the activation epitope of LFA-1, which is mediated through protein kinase C; (e) stimulation of CD44 augments the LFA-1-mediated adhesion of cancer cells to endothelial cells and intercellular adhesion molecule 1-transfected cells and facilitates transendothelial migration; (f) stimulation of CD44 also induces expression of the hepatocyte growth factor (HGF) receptor c-Met on cancer cells; and (g) HGF further amplifies the LFA-1-mediated adhesion of cells prestimulated by CD44-derived signaling. Our results indicated that stimulation by CD44 induces "outside-in signaling," which consists of a direct pathway via CD44 and an alternate pathway through the induction of c-Met expression via HGF. Such stimuli augment the expression and trigger the function of integrins via "inside-out signaling" in colon cancer cells, which leads to amplification of integrin-mediated adhesion to the vessel wall and subsequent transendothelial migration.

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.