Modulation of murine in vitro immune response by verapamil (V)

Functionally distinct lymphocyte subsets differ with regard to necessary activation signals. In selected circumstances lymphocyte activation has been shown to be critically dependent upon transcellular calcium influx. Whether calcium plays a central role in the activation of all lymphocytes remains to be determined. The effect of the calcium channel blocker verapamil on the induction of murine cytotoxic T lymphocytes (CTL), suppressor cells, T helper cells, and B cells was investigated. Verapamil (V) was found to inhibit the induction of cytotoxic effector cells. V acted primarily on the afferent limb of this immune response, was synergistic with cyclosporin A (CsA), and its effects could be largely reversed by the addition of exogenous helper factors. V also inhibited B cell proliferation in response to anti-mouse IgM in the presence of 2-mercaptoethanol, but in the absence of cognate or non-cognate T cell help. In contrast to this, V did not inhibit the activation of cells capable of inducing B cell proliferation nor did it inhibit the induction of suppressor cells. The selective suppression of V is discussed in terms of activation requirements of CTL, suppressor cells and helper cell subsets.     

注射用核糖核酸对小鼠免疫功能的影响

目的:研究脱氢卡维汀(YHL-DC)对四氯化碳(CCI4)致小鼠急性肝损伤的影响.方法:YHL-DC预防和治疗给药后观察其对CCl4致急性肝损伤小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)和总胆红素(TBIL)的影响,并对肝脏进行了病理组织学及电镜观察.结果:YHL-DC预防和治疗给药均能显著降低急性肝损伤小鼠ALT、AST和TBIL的升高,有效减轻CCl4引起的小鼠肝细胞变性、坏死、爽性细胞浸润和肝细胞超微结构的破坏.结论:YHL-DC对四氯化碳致小鼠急性肝损伤具有保护作用.     

Effects of mycotoxins on human immune functions in vitro.

Immunosuppressive and carcinogenic Fusarium mycotoxins may appear in domestic food products. Therefore, the immunological effects of Fusarium mycotoxins were tested on human peripheral blood mononuclear cells from different blood donors. In the present study we investigated deoxynivalenol (DON), 3-acetyldeoxynivalenol, fusarenon-X, T-2 toxin, zearalenone, alpha-zearalenol, beta-zearalenol and nivalenol for their effects on T and B cells in a proliferation assay, antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) cell activity on human peripheral blood mononuclear cells. The concentrations applied in our experiments were similar to those which can be found in normal human peripheral blood system (0.2--1800 ng/ml). Among the eight mycotoxins tested, T-2 toxin, fusarenon X, nivalenol and deoxynivalenol exerted the highest immunosuppressing effect on human peripheral blood mononuclear cells in vitro. Mycotoxin-induced immunosupression was manifested as depressed T or B lymphocyte activity. Furthermore, by virtue of inhibition of NK cell activity, the protection against tumor development may also be attenuated.     

Effect of short-term inhalation exposure to 1,3-butadiene on murine immune functions

Interest in 1,3-butadiene (BD) as a potential immunomodulator was prompted by reports of an increased incidence of neoplasia in humans exposed to BD during the manufacture of styrene-butadiene synthetic rubber, and by a recent study which demonstrated a high incidence of thymic lymphomas in B6C3F1 mice. B6C3F1 mice were exposed to 1250 ppm BD by inhalation 6 hr per day, 5 days per week, for 6 or 12 weeks. Immune function assays were selected to evaluate specific humoral and cell-mediated immunity and spontaneous cytotoxicity; lymphoid organ histopathology was also evaluated. A slight decrease in antibody plaque-forming cells (PFC) per spleen was observed in exposed mice, although PFC per 10(6) splenic lymphocytes was normal. Significant extramedullary hematopoiesis and erythroid hyperplasia was observed in spleens from exposed mice, and correlated with a twofold increase in thymidine incorporation in spontaneously proliferating splenocytes. No differences in proliferation to alloantigens were demonstrable between control and BD-exposed splenocytes. Mitogenesis by phytohemagglutinin, Concanavalin A, and lipo polysaccharide was suppressed in splenocytes from exposed mice, but may have been due to the cellular dilution effect of hematopoietic activity. Cytotoxic T-lymphocyte generation was suppressed after a 6-week exposure to BD, but was comparable to controls after 12 weeks of exposure. No differences in spontaneous cytotoxicity were observed between control and exposed mice. Overall, no persistent immunological defects were detectable after inhalation exposure to this tumorigenic agent.     

The immunomodulatory effects of the herbicide simazine on murine macrophage functions in vitro.

We examined the immunomodulating effects of simazine, a triazine herbicide, on murine peritoneal macrophages after in vitro pre-exposure. When thioglycollate-elicited macrophages pre-exposed to simazine were stimulated with lipopolysaccharide (LPS), the antitumor activity induced by LPS was suppressed by simazine. Simazine also inhibited poly I:C-induced antiviral activity and interferon (IFN) production in macrophages. In addition, the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) which have been known to be major effector molecules in macrophage-mediated cytotoxicity was decreased by simazine pretreatment in a dose-dependent manner. However, simazine had little effect on phagocytosis and the level of hydrogen peroxide (H(2)O(2)), interleukin-1 (IL-1) and IL-6 by LPS-stimulated macrophages. Taken together, these data indicate that simazine has a differential immunomodulating effect on macrophage secretory and cellular activities.     

In vivo and in vitro effects of 1,1-dimethylhydrazine on selected immune functions

The in vivo phase of the experiments reported here include the evaluation of immune function after short-or long-term treatment of mice with 1,1-dimethylhydrazine (UDMH). Long-term exposure (3 injections/week for 14 weeks) resulted in increased numbers of Jerne plaque-forming cells, a trend toward decreased induction of suppressor cell activity by concanavalin A (Con A), and no effects on mitogen-induced lymphocyte blast transformation (LBT), compared to saline-treated control mice. These effects were greatest at doses of 10 or 50 mg/kg, while higher doses had less of an effect. In vitro experiments were performed by adding UDMH to normal murine splenocytes in the LBT assay and con A-induced suppressor cell assay. The UDMH induced a significant enhanced response to lipopolysaccharide (LPS) at 10 and 50 micrograms/ml, and a suppressed response to both Con A and LPS at higher concentrations. The UDMH also caused a decrease in suppressor cell activity at 25 micrograms/ml. Selective abrogation of suppressor activity or alteration of the suppressor cell-helper ratio were suggested as possible mechanisms for the enhancement effect associated with UDMH.     

Assay of mitochondrial functions by resazurin in vitro

AIM: To study the mechanism of resazurin as indicator of mitochondrial function and to develop a rapid and sensitive assay for measuring metabolic activity of isolated mitochondria from rat liver in vitro. METHODS: The screening was carried out on 96-well microtitre plates by monitoring fluorescence intensity of resazurin reduced by mitochondria. Experimental conditions were optimized and influences of several inhibitors on mitochondrial function were observed. RESULTS: Fluorescence intensity increased in a linear manner when the mitochondrial protein concentration from 5 to 50 μg protein per well was incubated with resazurin (5μmol/L) during 230 min period at 37℃. Edetic acid could promote the reduction of resazurin in mitochondria. The fluorescence intensity decreased greatly after pretreatment with NAN3, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),and oligomycin compared with the control. However, the typical complex I inhibitor, rotenone enhanced the fluorescence intensity without mitochondria. CONCLUSION: Using resazurin to determine mitochondrial function is sensitive, inexpensive and could be easily automated for high throughput screening.     

In vitro suppressive effect of aflatoxin B1 on murine peritoneal macrophage functions.

We examined the immunosuppressive effects of aflatoxin B1 (AFB1), a toxic compound produced by the Aspergillus flavus, on murine peritoneal macrophages after in vitro pre-exposure. When thioglycollate-elicited macrophages pre-exposed to AFB1 were stimulated with lipopolysaccharide (LPS), antitumor activity induced by LPS was suppressed by 10 and 50 microM AFB1. In addition, the production of reactive intermediates including nitric oxide (NO), superoxide anion and hydrogen peroxide which have been known to be implicated in macrophage-mediated cytotoxicity, was decreased by AFB1 pretreatment in a dose-dependent manner. We also determined whether the macrophage-mediated cytokine production was altered by AFB1 in vitro pretreatment. AFB1 markedly inhibited TNF-alpha interleukin-1 (IL-1) and IL-6 production by LPS-stimulated macrophages. Taken together, these data indicate that AFB1 inhibits the killing ability of murine macrophages, decreases various secretory molecules in those cells and the macrophages would be one of many systems affected by AFB1.     

Hot water extract of the sclerotium of Polyporus rhinocerus Cooke enhances the immune functions of murine macrophages

Treatment of hot water extract of the sclerotium of Polyporus rhinocerus (PRW) with murine macrophages including RAW 264.7 cell line and primary macrophages (PMs) could enhance their functional activities. These include a significant up-regulation of pinocytosis; an increase in the production of reactive oxygen species (ROS) and nitric oxide (NO); an increase in tumor necrosis factor alpha (TNF-alpha) production and inducible nitric oxide synthase (iNOS) expression in both RAW 264.7 cells and PMs. Cell surface receptors for yeast-derived beta-glucan, including Dectin-1, CR3, and TLR2, were determined by flow cytometry, and the expression of Dectin-1+ cells on the cell surface decreased in the responses of PMs to PRW. PRW increased phosphorylation of IkappaBalpha, which could trigger the nuclear factor kappa B (NF-kappaB) signal pathway for macrophage activation in RAW 264.7 cells. Therefore, the immunomodulatory effect of PRW could be mediated by macrophage activation via the NF-kappaB signal pathway.     

The inhibitory effect of thioproline on carcinogenesis induced by N-benzylmethylamine and nitrite

Thioproline, which is readily nitrosated to form nitrosothioproline, is expected to act as a nitrite scavenger. The effect of thioproline as an inhibitor of the carcinogenesis induced by N-nitroso-N-benzylmethylamine precursors was examined. Two groups of male F-344 rats were given diet containing 0.25% N-benzylmethylamine (group I) or 0.25% N-benzylmethylamine plus thioproline (0.25% until wk 17 and then 0.5%; group II). Both groups were given drinking-water containing sodium nitrite (0.1% until wk 17 and then 0.2%). The experiment was continued for 717 days. Squamous cell carcinoma of the forestomach developed in six out of seven rats in group I and in significantly fewer, two out of nine rats, in group II. The degree of invasion by the tumours was also less in group II rats, given thioproline, than in group I. Thus thioproline suppressed carcinogenesis induced by N-benzylmethylamine and nitrite, possibly by inhibiting the in vivo nitrosation of N-benzylmethylamine.     

丹皮总苷体外对三类免疫细胞功能的影响

目的研究丹皮总苷（ＴＧＭ）体外对３类免疫细胞功能的影响。方法采用［３Ｈ］－ＴｄＲ参入法,检测Ｔ淋巴细胞增殖反应和分泌白介素２（ＩＬ－２）活性,Ｂ淋巴细胞增殖反应以及巨噬细胞产生ＩＬ－１。结果ＴＧＭ２～５０ｍｇ·Ｌ－１可明显促进刀豆蛋白Ａ诱导小鼠Ｔ淋巴细胞增殖反应和大鼠Ｔ淋巴细胞产生ＩＬ－２,ＴＧＭ还可促进脂多糖诱导Ｂ淋巴细胞增殖反应以及大鼠腹腔巨噬细胞产生ＩＬ－１,它们的浓度－效应曲线呈钟罩形。结论ＴＧＭ具有浓度依赖性双向免疫调节作用。     

Quercetin uptake and metabolism by murine peritoneal macrophages in vitro

Quercetin (Q), a bioflavonoid ubiquitously distributed in vegetables, fruits, leaves, and grains, can be absorbed, transported, and excreted after oral intake. However, little is known about Q uptake and metabolism by macrophages. To clarify the puzzle, Q at its noncytotoxic concentration (44μM) was incubated without or with mouse peritoneal macrophages for different time periods. Medium alone, extracellular, and intracellular fluids of macrophages were collected to detect changes in Q and its possible metabolites using high-performance liquid chromatography. The results showed that Q was unstable and easily oxidized in either the absence or the presence of macrophages. The remaining Q and its metabolites, including isorhamnetin and an unknown Q metabolite [possibly Q⁻ (O-semiquinone)], might be absorbed by macrophages. The percentage of maximal Q uptake by macrophages was found to be 2.28% immediately after incubation; however, Q uptake might persist for about 24 hours. Q uptake by macrophages was greater than the uptake of its methylated derivative isorhamnetin. As Q or its metabolites entered macrophages, those compounds were metabolized primarily into isorhamnetin, kaempferol, or unknown endogenous Q metabolites. The present study, which aimed to clarify cellular uptake and metabolism of Q by macrophages, may have great potential for future practical applications for human health and immunopharmacology.     

Improvement of cognitive functions by the acetylcholine releaser SM 21

The effect of administration of SM 21 on memory processes was evaluated in the mouse passive avoidance and in the rat social learning tests. SM 21 (10–20 mg kg⁻¹ i.p.) prevented amnesia induced by scopolamine and dicyclomine as tested by the mouse passive avoidance test and prevented memory disruption by AF‐64A and benehexol ascertained by the rat passive avoidance test. Both SM 21 enantiomers were able to abolish dicyclomine‐induced amnesia in mice. SM 21, starting from the dose of 10 mg kg⁻¹ i.p., antagonized the memory impairment produced by mecamylamine, baclofen, and diphenhydramine in mice, as well as amnesia induced by diazepam in rats. SM 21, at doses ranging between 10 and 30 mg kg⁻¹ i.p., prevented memory reduction in mice by hypoxia in the passive avoidance test. In the social learning test, SM 21 (10 mg kg⁻¹ i.p.) injected in adult rats reduced the duration of active exploration of a familiar partner in the second session of the test. SM 21 prevented amnesia in both mice and rats comparable to that of the cholinesterase inhibitor physostigmine (0.2 mg kg⁻¹ i.p.), the M1 selective agonist AF‐102B (10 mg kg⁻¹ i.p.), and the nootropic drug piracetam (30 mg kg⁻¹ i.p.). These results demonstrated the ability of SM 21 to modulate memory functions and suggests that SM 21 could be useful in the treatment of cognitive deficits. Drug Dev. Res. 47:118–126, 1999. © 1999 Wiley‐Liss, Inc.     

Bioactivation of clozapine by murine cardiac tissue in vivo and in vitro

Clozapine, an atypical neuroleptic, undergoes bioactivation to a chemically reactive nitrenium ion. This has been implicated in the pathogenesis of clozapine-induced agranulocytosis. Clozapine also causes myocarditis and cardiomyopathy, the mechanisms of which are unknown. To investigate this, we have evaluated whether clozapine undergoes bioactivation by murine cardiac tissue, in comparison to hepatic tissue. Mice were administered clozapine (5 and 50 mg/kg i.p.), and the extent of covalent binding was assessed by Western blotting. There was an increase in irreversible binding of clozapine to several proteins, ranging in mass from 30 to 250 kDa in both hepatic and cardiac tissue. Bioactivation by hepatic and cardiac microsomes was assessed by LC/MS using glutathione to trap the intermediate. Metabolism of radiolabeled clozapine to a glutathionyl conjugate by liver and cardiac microsomes was 30.5 +/- 3.3 and 3.6 +/- 0.3% of the initial incubation concentration, respectively. Ketoconazole (20 muM), a P450 inhibitor, significantly reduced binding in both hepatic and cardiac microsomes to 6.2 +/- 0.2 and 0.5 +/- 0.06%, respectively. These data indicate that clozapine undergoes bioactivation in the heart to a chemically reactive nitrenium metabolite that may be important in the pathogenesis of myocarditis and cardiomyopathy observed in man.     

妇炎清糖浆对小鼠免疫功能的影响

目的：研究妇炎清糖浆的免疫作用。方法：以健康早性KM小鼠和环磷酰胺致免疫低下模型小鼠为对象，观察妇炎清糖浆对小鼠单核细胞吞噬功能（采用碳粒廓清法）、2，4二硝基氯苯（2，4-dinitrochlorobenzene，DNCB）致小鼠迟发性过敏反应以及血清溶血素生成的影响。结果：妇炎清糖浆能显著增强小鼠巨噬细胞吞噬功能（P〈0.05）、胸腺指数（P〈0．05）、血清溶血素的含量和迟发性变态反应（P〈0．05）的作用。结论：妇炎清糖浆对非特异免疫及特异性免疫功能都有一定的促进作用，有助于盆腔炎疾病的恢复。     

The immune system and murine atherosclerosis

In this review the modulation of lipoprotein metabolism and atherogenesis by the innate and adaptive immune systems of the mouse is discussed. While recognizing the participation of all components of the immune system in atherogenesis, it is clear that robust atherogenesis may proceed without an adaptive immune response. But even when all components are active, the outcome reflects a balance between pro- and anti-inflammatory reactants and cells. This network of interactions is summarized in this review and is complemented by other reviews in this series. Also noted is the differential response of different vascular beds following manipulation of the components of the adaptive immune system. Further work is required to achieve a fuller understanding of the role of these systems in atherogenesis, especially with the prospect of favorably modulating atherogenesis by the manipulation of the participating immune components as for example in a vaccination approach.     

In vitro stimulation of murine peritoneal monocytes induced by alginates

In this trial we assessed the effect of soluble alginates on murine cells. Mouse peritoneal monocytes were stimulated in vitro with a solution of alginate. The production of TNF-alpha and nitric oxide (NO), the expression of surface molecules CD80 and CD86, and the ability of monocytes to phagocyte bacteria were assessed, in order to evaluate the effect of alginate on cell functionality. We showed that mouse peritoneal monocytes stimulated with alginate produce NO and TNF-alpha. In addition, alginate is able also to increase their phagocytic activity and to a lesser extent also to increase the expression of CD80. Even with different degrees, it implies that alginates per se act directly on immune response, being able to effectively stimulate proinflammatory activity. These findings corroborate the idea that alginates can represent interesting adjuvants to use to increase the efficacy of antigenic stimulation.