Glycyrrhizin protects mice from concanavalin A-induced hepatitis without affecting cytokine expression.

The administration of concanavalin A (Con A) to mice induces cytokine-dependent hepatitis. In the present study, the effect of glycyrrhizin on Con A-induced hepatitis was examined. Treatment of mice with Con A (0.2 mg/mouse, i.v.) induced elevation of the plasma transaminase activities at 24 h. Mice were treated with glycyrrhizin (100, 200 and 400 mg/kg, i.p.), and glycyrrhizin at the doses of 200 and 400 mg/kg inhibited the Con A-induced elevation of the plasma transaminase activities. At 1 h after Con A treatment, interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-6 proteins were released into the plasma. Although treatment with glycyrrhizin at 200 mg/kg inhibited Con A-induced hepatitis, it did not affect the release of any of these Con A-induced cytokines into the plasma. The present results clearly show that glycyrrhizin inhibited Con A-induced hepatitis without affecting cytokine expression.     

Concanavalin A-induced hepatitis in mice is prevented by interleukin (IL)-10 and exacerbated by endogenous IL-10 deficiency

One single intra-venous (i.v.) injection of Concanavalin A (Con A) into mice provokes a cell-mediated immunoinflammatory hepatitis. We have presently evaluated the immunopharmacological effects of exogenous interleukin (IL)-10 and the role of endogenous IL-10 in this model by using exogenous IL-10, anti-IL-10 monoclonal antibody (mAb) and mice with disrupted IL-10 gene (IL-10 KO mice). Whilst exogenous IL-10 administered in a prophylactic (1 h prior to Con A) and even "early" therapeutic fashion (30 min after Con A) reduced the elevation of transaminase activities in plasma in a dose-dependent manner, observed in control mice, these biochemical markers of liver injury were significantly increased both in IL-10 KO mice as well as in those receiving anti-IL-10 mAb. Interestingly, doses of Con A lower than 20 mg/kg that were only capable of inducing slight serological signs of hepatitis in mice, exerted marked hepatitic effects when administered to either anti-IL-10 mAb-treated mice or to IL-10 KO mice. The disease modulating effects of exogenous IL-10 and either genetical or pharmacologically-induced IL-10 deficiency were associated with profound and opposite modifications of the Con A-induced increase in the circulating levels of IFN-gamma and TNF-alpha. Relative to control animals, the blood levels of these cytokines were diminished in IL-10-treated mice and augmented in both IL-10 KO mice and anti-IL-10 mAb-treated mice. These results prove the physiological antiinflammatory role of endogenous IL-10 in Con A induced hepatitis and the beneficial effects of IL-10 treatment to prevent this condition.     

Zaltoprofen inhibits concanavalin A-induced decrease of body weight in mice

Treatment of mice with concanavalin A (Con A) (12.5 mg/kg, i.v.) decreased the body weight at 24 h. This Con A-induced body weight decrease was accompanied by reduction in food and water intake. Zaltoprofen is a non-steroidal anti-inflammatory drug. The administration of Zaltoprofen (10 mg/kg) at 8 h after Con A treatment was found to inhibit the Con A-induced reduction in body weight. The food intake in mice treated with Con A plus Zaltoprofen (10 mg/kg) was four times greater than that in mice treated with only Con A. The present results showed inhibition of the Con A-induced body weight loss by Zaltoprofen and suggested its possible effectiveness for the treatment of "sickness behavior".     

Prevention of concanavalin A-induced mice hepatitis by molsidomine

Molsidomine is effective in reducing portal hypertension in cirrhosis, but its effect on hepatitis is not known. In the present study, the effect of molsidomine on hepatitis was examined using mouse concanavalin A (Con A)-induced hepatitis and mouse anti-Fas antibody-induced hepatitis. Treatment of mice with Con A caused elevation of plasma alanine aminotransferase (ALT) at 8 and 24 h (n=4). Pretreatment of mice with molsidomine (3, 10, 30 and 100 mg/kg, i.p.) prevented Con A-induced hepatitis. Treatment of mice with anti-Fas antibody (150 microg/kg, i.v.) caused elevation of plasma ALT at 3.5 h. Pretreatment mice with molsidomine (10 mg/kg, i.p.) showed only 47% inhibition of anti-Fas antibody caused elevation of plasma ALT. The present results showed effectiveness of molsidomine in preventing Con A-induced mice hepatitis.     

NC-1500 prevents concanavalin A-induced mice hepatitis without affecting cytokine gene expression.

NC-1500 is a dihydropyridine type calcium channel blocker. The effect of NC-1500 on mice concanavalin A-induced hepatitis was examined. Treatment of mice with concanavalin A (Con A) caused elevation of plasma transaminase. Pretreatment of mice with NC-1500 (3, 10 and 30 mg/kg, p.o.) prevented this Con A-induced elevation of plasma transaminase. Treatment of mice with Con A induced tumor necrosis factor-alpha (TNF-alpha) mRNA expression in the liver. However, NC-1500 (30 mg/kg, p.o.) did not affect this Con A-induced TNF-alpha mRNA expression in the liver. The present results showed that NC-1500 inhibited Con A-induced hepatitis without affecting TNF-alpha mRNA expression in the liver.     

Immunomodulatory effects of neurotropin through the recovery of interleukin-2 production in autoimmune-prone (NZB/NZW) F1 mice

The immunomodulatory effects of Neurotropin, a substance extracted from inflammatory skin of rabbits inoculated with vaccinia virus, were assessed in autoimmune-prone (NZB/NZW) F1 (B/W F1) mice. The concanavalin A (Con A)-induced proliferative response of spleen cells was markedly decreased in aged B/W F1 mice as compared with young B/W F1 mice. Neurotropin, when administered i.p. to aged B/W F1 mice, significantly increased the Con A-induced proliferative response. In aged B/W F1 mice, interleukin-2 (IL-2) production by Con A-stimulated spleen cells was severely impaired and IL-2 responsiveness of Con A-activated spleen cells was partially decreased in comparison with young B/W F1 mice. Neurotropin, administered to the aged B/W F1 mice, restored IL-2 production by Con A-stimulated spleen cells to the level of young B/W F1 mice. Furthermore, Neurotropin completely restored the IL-2 responsiveness of Con A-activated spleen cells from aged B/W F1 mice. To test whether Neurotropin exerts its immunoregulatory activities in B/W F1 mice by restoring IL-2 production, we directly examined the effect of recombinant IL-2 on the immune functions of spleen cells in vitro. Recombinant IL-2 markedly enhanced Con A-induced proliferative response of aged B/W F1 mice. Furthermore, the suppressive activity of spleen cells which had been activated by Con A in the presence of rIL-2 was significantly increased. These results indicate that some immunoregulatory functions of aged B/W F1 mice can be corrected by IL-2 and suggest that Neurotropin restores immunoregulatory activity in B/W F1 mice by the recovery of IL-2 production.     

Protection from concanavalin A (Con A)-induced T cell-dependent hepatic lesions and modulation of cytokine release in mice by sodium fusidate

The immunomodulatory effects of the antibiotic sodium fusidate (SF) were tested in a model of T cell-dependent hepatic injury that can be induced in normal mice by a single i.v. injection of Con A. Signs of hepatitis with elevated transaminase activities in plasma, severe infiltration of the liver by neutrophil granulocytes, lymphocytes and monocytes, and necrotic areas were observed in control mice treated intraperitoneally with PBS 24 h and 1 h before Con A challenge. T cell- and macrophage-derived cytokines (IL-2, interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), IL-1β, IL-6) were released with different kinetics in the circulation of these mice. SF, 20, 40 or 80 mg/kg, administered 24 h and 1 h before Con A challenge, protected the mice against the hepatitic effects of Con A. The protective effects of SF were dose-dependent and accompanied by profound modifications of blood levels of cytokines induced by Con A, so that, relative to control mice, SF (80 mg/kg)-treated animals showed markedly diminished plasma levels of IL-2, IFN-γ and TNF-α, along with augmented levels of IL-6. These results suggest that SF might be useful in the treatment of immunoinflammatory liver diseases in humans.     

Synthetic derivatives of osthole for the prevention of hepatitis

Prevention of hepatitis is a worldwide issue. For most patients with liver disease, hepatoprotective drugs are required. But there are only a few hepatoprotective drugs available. Osthole is a coumarin compound and protects the liver from hepatitis by preventing the development of apoptosis. However, osthole exhibits low water solubility, and some structural modifications are required for sufficient hepatoprotection upon oral administration. We synthesized 28 osthole derivatives, and then studied their effects by using mice concanavalin A (Con A) -induced hepatitis. The osthole derivatives No.1, 9 and 19 showed stronger inhibition of Con A-induced elevation of plasma alanine aminotransferase (ALT). Oral administration of osthole at the dose of 100 mg/kg (n=10) inhibited 38.0% of the Con A-induced elevation of plasma ALT. In contrast, oral administration of Nos. 1, 9 and 19 at the dose of 100 mg/kg (n=5) caused 68.7%, 62.5% and 88.3% inhibition of the Con A-induced elevation of plasma ALT, respectively. These synthetic osthole derivatives could contribute to the development of hepatoprotective drugs effective for various types of liver diseases on oral administration.     

Augmentation of mitogen-induced interleukin 2 production by oral administration of polysaccharide SPR-901.

The effects of oral administration of antitumor polysaccharide SPR-901 (RBS), an alpha-1,3 branched alpha-1,6 glucan, on concanavalin A (Con A)-induced interleukin 2 (IL-2) production of splenocytes were studied. The augmentation effect on Con A-induced IL-2 production was evident when more than 30 mg/kg of SPR-901 was administered orally to mice. On the other hand, oral administration of B512 dextran, an analogous alpha-1,6 glucan, did not show any augmentation effects on IL-2 production. The augmentation effect of SPR-901 on IL-2 production seemed to be mediated mainly by macrophages stimulated with SPR-901.     

Tamoxifen alleviates disease severity and decreases double negative T cells in autoimmune MRL-lpr/lpr mice

Previous study suggested that MRL-lpr/lpr mice treated with tamoxifen (TAM) had less severe proteinuria, reduced serum titre of anti-dsDNA autoantibodies and an increased survival rate. To investigate further the regulatory mechanisms of TAM on MRL-lpr/lpr female mice, a total dose of 200 microg per mice (5.5 mg/kg) was given every 2 weeks subcutaneously, while the control mice were injected with oil only. After being treated with TAM four times, the mice were killed and cellular functions were evaluated. The TAM-treated groups had smaller sized spleen and lymph nodes. Flow cytometric analysis of splenocytes had a significantly lower percentage of cell number of T cells and double negative T cells (CD4- CD8- T cells). There was no difference in cytokine production (interleukin (IL)-2, IL-4, IL-5, IL-10 and interferon-gamma (IFN-gamma)) from splenocytes stimulated with concanavalin A (Con A) or cytokines (IL-6) secreted by peritoneal exudate cells when stimulated with lipopolysaccharide (LPS). However, IL-2 from lymph node cells was significantly higher on TAM-treated mice. Finally, splenocytes or purified T cells stimulated with anti-CD3 antibody plus cross-linking immunoglobulin G (IgG) of the TAM-treated group had higher 3H-incorporation of proliferation assay compared with that of control groups. In vitro study further demonstrated that IL-2-activated proliferation of lymph node double negative (DN) T cells can be inhibited by TAM treatment in a dose-dependent manner. Our finding demonstrated that TAM may potentially influence T cells and modulate the immune function, which offers a novel approach to explore the feasibility of hormone therapy for autoimmune diseases.     

Immunomodulatory Activity of Wy-18, 251 (3-(P-Chlorophenyl) Thiazolo [3,2-A]Benzimidazole-2-Acetic Acid)

The in vitro and in vivo effects of the experimental immunomodulatory agent Wy-18,251 (3-(p-chlorophenyl) thiazolo [3,2-a] benzimidazole-2-acetic acid) were studied in comparison with levamisole and indomethacin. Levamisole (4 mg/kg, i.v.) but not Wy-18, 251 (≤ 10 mg/kg, i.v.) enhanced carbon clearance rates in vivo in mice. Both Wy-18, 251 and levamisole (100 mg/kg, p.o.) significantly suppressed the symptoms of experimental allergic encephalomyelitis (EAE) in rats injected with spinal cord emulsion, but neither were as effective as tilorone in this model. Wy-18, 251 and levamisole (1-100 mg/kg, p.o.) suppressed the in vivo generation of plaque-forming cells (PFC) in mice immunized with sheep red blood cells while indomethacin (9 mg/kg, p.o.) enhanced PFC formation. All 3 agents (10^-5-10^-6 M) enhanced the in vitro ovalbumin (0A)-specific and Con A- or PHA-induced proliferative response and Con A-stimulated interleukin 2 (IL-2) synthesis of rat spleen cells. Furthermore, in vivo treatment of rats with 1-10 mg/kg (p.o.) of Wy-18, 251 and levamisole but not indomethacin increased the subsequent in vitro mitogen or antien (OA) responsiveness of spleen cells. None of the drugs (10^-5-10^-7 M) influenced the natural killer cell (NK) activity of rat spleen cells when incorporated directly into the ⁵¹Cr release NK assay.     

胸腺五肽对鼠免疫功能的影响

本文研究了合成胸腺五肽(TP-5)对正常小鼠和老龄大鼠免疫功能的影响。TP-50.5,1,2,5mg/kg ip×3d可明显增强脾淋巴细胞的增殖反应,选择性地抑制脾细胞溶血空斑数(PFC),TP-52,5mg/kg可显著增加ICR小鼠胸腺的重量,用5mg/kg TP-5可对抗50mg/kg环孢霉素A(CysA)对淋巴细胞增殖的抑制作用。体外脾细胞培养中,TP-51,5,10,20μg/ml与ConA协同作用能明显增强小鼠脾淋巴细胞的增殖,同样,对老龄大鼠TP-5具有相同的刺激作用。     

Immunological abnormalities in rats with adjuvant-induced arthritis--II. Effect of antiarthritic therapy on immune function in relation to disease development

The effects of the experimental immunomodulatory agent tilomisole (Wy-18,251; (3-(p-chlorophenyl) thiazolo [3,2-a]benzimidazole-2-acetic acid) on disease development and immune function in rats with adjuvant-induced arthritis was assessed in comparison with indomethacin and levamisole. Daily p.o. administration of tilomisole (100-200 mg/kg/day) to M. butyricum-injected rats significantly reduced both edema and bone erosion in the uninjected paw. Moreover, tilomisole treatment restored to normal the diminished Con A-induced proliferative response and IL 2 synthesis observed in spleen cells from arthritic rats, but had no effect on macrophage IL 1 production. In contrast, levamisole treatment (25 mg/kg/day) of arthritic rats improved splenic immune function but did not influence paw edema or bone erosion. Conversely, indomethacin (1 mg/kg/day) significantly reduced paw edema and bone erosion but did not improve the deficient proliferative response or IL 2 synthesis by "arthritic" spleen cells. These results indicate that tilomisole possesses combined antiinflammatory and immunomodulatory activity in adjuvant-arthritic rats which is distinctly different from the effects of either indomethacin or levamisole. Moreover, these data suggest that tilomisole has potential disease-modifying activity in arthritis, which is currently being more closely examined in clinical trials.     

Effect of Ladasten on the Content of Cytokine Markers of Inflammation and Behavior of Mice with Experimental Depression-Like Syndrome

The effects of ladasten and reference product imipramine (10 mg/kg) on the content of cytokines TNF-α, IL-6, IL-10, IL-1α, IL-2, IL-4, IL-5, IL-17, IFN-γ, and GM-CSF and behavior of male C57Bl/6 mice were studied on the model of a depression-like state induced by a single intraperitoneal injection of bacterial LPS in a dose of 100 μg/kg. Ladasten was injected 5 times in doses of 30 and 50 mg/kg. LPS was administered 1 h after the last injection of the test agents. Behavioral disturbances and significant increase in the concentration of TNF-α and IL-6 in blood plasma were observed 2 h after LPS treatment. Ladasten was more potent than imipramine in decreasing the content of proinflammatory cytokines TNF-α and IL-6 and preventing the development of behavioral disturbances in mice. Antiasthenic drug ladasten was shown to decrease the elevated level of proinflammatory cytokines TNF-α and IL-6 after LPS treatment. Further studies should be performed to develop new indications for the use of ladasten in adjuvant therapy of depression.     

The Effect of Tp-3 (Arg-Lys-Asp), Tp-4 (Arg-Lys-Asp-Val), and Tp-5 On the Metastatic Capacity of Intravenously Injected Lewis Lung Tumor Cells

The oligopeptides Arg-Lys-Asp (TP-3), Arg-Lys-Asp-Val (TP-4), and Arg-Lys-Asp-Val-Tyr (TP-5) considered as the active short fragments of thymopoietin were administered (lo mg/kg) to C₅₇BI mice 24 hours before the intravenous inoculation of Lewis lung tumor (LLT) cells. A substantial decrease in lung metastasis number was observed as a result of treatment with all of the three oligopeptides. TP-3, TP-4, and TP-5 treatment decreased the immunosuppressive activity of Cyclophosphamid (240 mg/kg) given 96 hours before the inoculation of LLT cells.

After thymectomy, performed eight days before the LLT inoculation, only TP-3 treatment resulted in the decrease of Cyclophosphamid immunotoxicity. A stimulating effect of TP-3 on T helper cell activity is assumed. The oligopeptides TP-3, TP-4, and TP-5 are recommended for clinical trial in case of malignant tumors and immunodeficiency.     

In vivo effect of isoprinosine on interleukin-2 production, lymphocyte mitogenesis and NK activity in normal and cyclophosphamide immunosuppressed mice

The in vivo effect of isoprinosine on IL-2 production, mitogen-induced proliferation and NK activity of lymphocytes from normal as well as cyclophosphamide (CY) treated mice has been investigated. Isoprinosine was given in a single dose (50 mg/kg or 5 mg/kg) to normal or CY treated mice (250 mg/kg i.p. simultaneously to isoprinosine). An enhancement of T lymphocyte proliferation and IL-2 production was observed in both cases. There was no correlation between the small effect observed in T lymphocyte proliferation and the enhancement of IL-2 levels found in the supernatants of Con A activated spleen cells, specially in normal mice. Administration of isoprinosine every day (50 mg/kg) augmented Con A induced mitogenesis, IL-2 production and NK activity in animals treated with cyclophosphamide, but not in normal mice. Isoprinosine could be of interest in a combined treatment with immunosuppressants for the restoration of certain immune functions. The effect of isoprinosine on immune responses may be mediated in part by changes in IL-2 activity.     

Effect of feeding a diet with half of the recommended levels of all vitamins on the natural and inducible levels of cytotoxic activity in mouse spleen cells.

Groups of 6-week-old female C57Bl/6 mice were fed a normal diet with recommended levels of all vitamins or a vitamin-deficient (VD) diet containing half of the recommended level of each vitamin. At different time periods (1-11 weeks) after the initiation of diets, basal natural killer (NK) activity, interleukin-2 (IL-2) and concanavalin A (Con A)-induced cytotoxic activity, Con A-induced IL-2 production and levels of allospecific cytotoxic T cell activity generated in a mixed lymphocyte culture (MLC), were studied in spleen cells derived from control and VD mice. Results indicated that: (i) spleen NK activity remained normal until 2 weeks after the initiation of VD diet, fell steeply to low levels at the 4 and 5 week time points and remained depressed thereafter; (ii) IL-2- and Con A-induced levels of cytotoxic activity in spleen cells derived from VD mice declined at 4 weeks after the institution of VD diet, and then remained low throughout the study; (iii) the capacity of spleen cells from VD mice to generate IL-2 in response to Con A and cytotoxic T cells in response to allogeneic spleen cells, was normal at 1 and 4 weeks after initiation of the VD diet and was markedly depressed at the 6 and 9 week time points. These results suggest that partial combined deficiencies of dietary vitamins strongly influence assays of immune function.     

Stimulation of splenic T-lymphocyte function by endogenous serotonin and by low-dose exogenous serotonin.

The modulatory effects of endogenous serotonin on splenic T-cell activity were investigated using two distinct approaches. The first approach showed that pretreatment of mice with p-cholorphenylalanine (PCPA) to deplete intracellular stores of serotonin reduced the capacity of their splenic T cells to proliferate and to express interleukin-2 receptor (IL-2R) in response to concanavalin A (Con A). These responses could be restored by the addition of serotonin to the spleen cell cultures. In contrast, PCPA treatment did not effect stimulation of spleen cells to produce IL-2. The second approach showed that T-cell proliferation to Con A as well as to IL-2 was diminished by the presence of antagonists to the serotonin-2 receptor (5-HT2R). The effects of low doses (100 ng/ml) of exogenously added serotonin on functions of normal spleen cells were also examined. At this low dose, serotonin stimulated splenic T-cell proliferation in response to IL-2, and enhanced both proliferation and IL-2 production in response to a suboptimal concentration of Con A. These results show autologous serotonin to be required for T-cell activation and that the activation of suboptimally stimulated T cells can be augmented with low doses of exogenously added serotonin. These data also suggest that the positive regulation of T-cell function by serotonin is mediated through 5-HT2R.     

Augmentation of Mouse Natural Killer (NK) Activity by GM-1/P, A Processed form of Monosialoganglioside GM-1

We describe the immunomodulatory activity of GM-1/P a processed form of GM-1 (monosialoganglioside) extracted from ox brain, purified and physically modified. We examined the effect of in vivo and in vitro treatment of GM-1/P on natural (NK) activity and its ability to induce the production of interleukin-2 (IL-2) in the mouse. In vivo treatment with GM-1/P (1 mg/Kg, i.v., day-1) resulted in a marked increase and in a change of distribution of NK activity, which was associated with lower density Percoll fractions. Marked increase was already observed at 18 hrs and then declined by day 4. In vitro treatment with GM-1/P (2ug/ml) enhanced NK activity of B6 spleen cells, already after 6 hours of incubation, remaining at plateau levels within 18 hours. A role of IL-2 in this enhancement was suggested by the ability of an anti-IL-2 rabbit antiserum to abolish in vitro increased cytotoxicity. the presence of IL-2 in the supernatants of splenocytes from GM-1/P (lmg/Kg, i.v., day-1) treated mice stimulated with Con A or Con A plus TPA for 48 hrs was evaluated by proliferation of an IL-2 dependent CTLL cell line. GM-1/P by itself was unable to stimulate IL-2 production; however it markedly increased IL-2 production induced by Con A or Con A plus TPA.