Astilbin inhibits contact hypersensitivity through negative cytokine regulation distinct from cyclosporin A

BACKGROUND: IL-10 is known as a negative regulator for inflammatory diseases, including contact dermatitis. However, only a few drug candidates are reported to induce endogenous IL-10. OBJECTIVE: We sought to elucidate a new mechanism underlying the immunosuppressive properties of astilbin through negative cytokine regulation in comparison with the effective pattern with cyclosporin A. METHODS: Contact hypersensitivity was induced in mice with picryl chloride. Lymph node cells were isolated for adoptive transfer and cytokine assays. RESULTS: Astilbin significantly inhibited contact hypersensitivity when given in the elicitation phase but not in the sensitization phase, whereas cyclosporin A inhibited both phases. Lymph node cells from donor mice administered astilbin failed to adoptively transfer the hypersensitivity. Astilbin in vivo remarkably induced IL-10 expression in lymph node cells at an earlier time and decreased TNF-alpha and IFN-gamma expression at a later time. Furthermore, the in vivo neutralization of IL-10 significantly impaired the effect of astilbin on contact hypersensitivity. In the isolated lymphocytes sensitized with picryl chloride in vivo and challenged with trinitrobenzene-sulfonic acid in vitro, astilbin did not affect the cell proliferation but modulated the above cytokine profiles as its in vivo effect in a concentration-dependent manner and furthermore significantly enhanced the expressions of suppressor of cytokine signaling 1 and 3. On the other hand, cyclosporin A strongly inhibited proinflammatory cytokine production but influenced neither IL-10 nor downstream suppressor of cytokine signaling 1 and 3 expression. CONCLUSION: Astilbin alleviates contact hypersensitivity through a unique mechanism involving a negative cytokine regulation through stimulating IL-10, which is distinct from the immunosuppressant cyclosporin A.     

The effects of cyclosporin A on the induction, expression and regulation of the immune response to herpes simplex virus.

Investigations were conducted to determine the effect of cyclosporin A (CsA) on the delayed type hypersensitivity (DTH) and antibody response of mice to Herpes simplex virus (HSV). Given only at the time of priming, the drug had little effect on the subsequent DTH response in mice receiving a 'DTH immunogenic' inoculation regime. However, CsA restored normal responsiveness in groups receiving a 'DTH tolerogenic' regime implying the abrogation of T suppressor (Ts) cells. Ts cell induction was insensitive to cyclophosphamide. Antibody responses were not suppressed after giving CsA with either of these regimes and enhancement was shown in some groups. DTH was substantially reduced by CsA when the drug was given repeatedly between the time of priming and challenge, or when previously primed mice received the drug shortly before challenge.     

Identification and properties of Trichomonas vaginalis proteins involved in cytadherence.

Trichomonas vaginalis NYH286 surface proteins which are candidates for mediating parasite cytadherence (adhesins) were identified. At least four trichomonad protein ligands ranging in relative molecular mass from 65 to less than or equal to 21 kilodaltons were found to selectively bind to chemically stabilized HeLa cells. The proteins were present on the surfaces of 10 different isolates of T. vaginalis examined; however, the nonpathogenic trichomonad T. tenax did not possess similar HeLa cell-binding proteins under identical experimental conditions, suggesting that these proteins are unique to the pathogenic human trichomonads. The surface nature of the candidate adhesins was confirmed by the ability of the proteins on intact, live organisms to be radioiodinated and to be removed with trypsin treatment. Rabbit antiserum (immunoglobulin G fraction) generated against adhesin proteins electroeluted from acrylamide preparations inhibited cytadherence compared with control immunoglobulin G. An adherence-negative subpopulation of T. vaginalis NYH286 organisms was also isolated. These nonadherent trichomonads did not synthesize the adhesin proteins. Interestingly, absence of adhesins from these parasites paralleled expression of a major immunogen known to undergo phenotypic variation. Revertant organisms derived from the adherence-minus subpopulation synthesized the adhesins and attached to HeLa cells. The emergence of revertant adherent T. vaginalis organisms also corresponded with the appearance of parasites which were without the major immunogen on their surface. Finally, it was determined that only those parasites lacking the major surface immunogen were capable of adherence and toxicity to HeLa cells.     

Effects of cyclosporin A on the cells responsible for the anticryptococcal cell-mediated immune response and its regulation.

Cyclosporin A (CsA), a potent immunosuppressive drug, was used to explore further the induction, expression, and regulation of lymphoid cells involved in the delayed-type hypersensitivity (DTH) response to cryptococcal antigen(s). We found that the induction of the cells responsible for DTH (TDH cells) was not affected by CsA, but their expression was inhibited in CsA-treated mice. The inhibition of expression of the TDH cells could not be attributed to the Cryptococcus neoformans-specific suppressor T (Ts) cells, even though the Ts cells were induced in CsA-treated mice. Instead, the suppressed expression of the TDH cells in CsA-treated mice was a direct effect of CsA or its products. Our studies with CsA also resulted in the first identification of a population of cells that significantly amplify the anticryptococcal DTH response. The amplifier cells were induced in mice that were given a primary immunizing dose of cryptococcal antigen in complete Freund adjuvant, and they amplified the anticryptococcal DTH response in recipient mice when they were transferred at the time of immunization of the recipient. The amplifier cell population was distinct from the TDH cells in that CsA inhibited the production of the amplifying cells but did not affect the induction of TDH cells. Amplification of the DTH response was a cell-mediated event, since cells but not serum from immunized mice mediated the amplified response in recipient mice. Thus, CsA enabled us to characterize anticryptococcal TDH and Ts cells further and to add to the immune cell circuit of the cryptococcal system a distinct population of cells that amplifies the anticryptococcal DTH response.     

Antimalarial activity of cyclosporin A

Cyclosporin A has been shown to possess antimalarial activity in mice infected withPlasmodium berghei NK 65 orPlasmodium chabaudi. Significant antimalarial effects were obtained with five daily oral doses of 25 mg/kg. Cyclosporin A treatment started concurrently with the inoculation of parasites was less effective than treatment started when parasitaemia was already established. Evidence so far suggests that the antimalarial action results from a direct toxic effect on the parasite. Combined treatment with Cyclosporin A and Pyrimethamine indicated that the two compounds may act synergistically.     

Antischistosomal effects of cyclosporin A

Administration of cyclosporin A, a new selective immunosuppressive agent, to mice infected withSchistosoma mansoni resulted in a significant reduction in the number of mature and immature male and, to a greater extent, female worms. With lower, subeffective, doses a reduction in hemoglobinase activity and protein content of female schistosomes is produced. Evidence available so far suggests that the antischistosomal effects of cyclosporin A are mediated through a stimulation of host mechanisms directed against the parasite.Supported by grants from NIH (#GM 16492-12) and the Agency for International Development (#AID/ta-C-1312).     

Effects of cyclosporin A on sex hormone and estrogen receptor in male rat with special reference to cyclosporin A-induced osteoporosis

The mechanisms of high turnover bone loss induced by Cyclosporin A (CsA) are not clearly understood. Deficiencies in sex hormones result in high turnover osteoporosis, and not only androgen but also estrogen plays an important role in maintaining bone mass in men. To study whether or not there are any changes in the levels of sex hormones, aromatization, and the expression of estrogen receptors in CsA-induced osteoporosis, we treated 39 rats with vehicle, low-dose CsA (5 mg/kg) and high dose CsA (15 mg/kg) for 28 days, and measured sex hormone levels by radioimmunoassay. Aromatase activities in ROS cells and 3T3-L1 cells were determined by measuring the conversion rate of 3H-androstenedione into 3H-estrone. ER and ER mRNA were measured by competitive RT-PCR in collected marrow cells and ROS cells. The levels of free testosterone in the serum in low-dose CsA-treated rats were unchanged, but the levels were significantly decreased in those treated with high-dose CsA as previously reported. The levels of total estradiol in the serum were significantly increased in the low-dose CsA-treated group (5 mg/kg) and were comparable to levels of the control group in the high-dose CsA-treated group (15 mg/kg). CsA increased the conversion of 3H-androstenedione to 3H-estrone in ROS cells, but not in 3T3-L1 cells. Meanwhile, CsA treatment did not change the rates of ER or ER mRNA expression in ROS cells or in collected bone marrow cells. In conclusion, CsA treatment decreased the level of free testosterone in the serum, but did not decrease the level of serum estradiol by enhancing aromatization. High-turnover osteoporosis induced by clinical dosage CsA treatment may not be caused by lowering the levels of circulating estrogen or by decreasing the expression of estrogen receptors.     

Effect of cyclosporin A on immunity to Listeria monocytogenes.

The effect of the immunosuppressive drug cyclosporin A (CS-A) on immunity to the facultative intracellular bacterium Listeria monocytogenes was investigated in unprimed and primed mice. Different treatment protocols were followed to evaluate the time dependence of CS-A-mediated immune suppression and the effect of CS-A on immunological memory to L. monocytogenes. The effect of CS-A was observed only during and after activation of T cell-mediated immunity, whereas early resistance exerted by macrophages assessed 6 and 70 min after challenge remained unaffected. CS-A suppressed efficient elimination of L. monocytogenes even when given after day 3 of a primary infection. This contrasts with findings in other models, including viral infections, where CS-A must be administered very early in an immune response to suppress it. CS-A suppressed antibacterial resistance in mice primed at various times before challenge; suppression of protection was time dependent and was virtually complete in livers, whereas CS-A-resistant memory persisted in spleens for up to 10 months.     

Different susceptibilities to cyclosporin A of the mitogenic and potentiating activities of interleukins

A dissociation was observed between the mitogenic and potentiating activities of interleukins (ILs)-1 or -2 in murine thymocyte cultures treated with drugs. The direct mitogenic effects of the ILs were unaffected by cyclosporin A (CsA) at concentrations which abolished the potentiating activities of these mediators, i.e. their synergy with lectins. Conversely to CsA, dexamethasone was more inhibitory to the mitogenic activity of the ILs than to their synergistic reactions with the lectins. The resistance to CsA of the mitogenic activity of IL-1 was unexpected since this response is assumed to be mediated by newly formed IL-2 and CsA inhibits IL-2 production. This resistance was further tested by coculturing thymocytes with the IL-2-dependent CT6 cells; net gains of thymidine uptake by the cocultures were attributed to IL-2 release. Such net gains were observed in cocultures stimulated with IL-1 alone and were relatively resistant to CsA. On the other hand, net gains stimulated by mitogenic lectins, alone or with IL-1, were eliminated by CsA. These results support the notion that IL-1 direct activity on thymocytes is mediated by newly released IL-2 and show that this IL-1 activity is unusual in being resistant to CsA. Low levels of protection against CsA were also observed in cultures potentiated by IL-1: lymphocytes stimulated by lectins or antigens and IL-1 were inhibited by CsA less than lymphocytes stimulated without IL-1. Yet, this partial protection by IL-1 was achieved only at CsA concentrations about 100 fold lower than those resisted by thymocytes directly stimulated by IL-1.     

GapA and CrmA coexpression is essential for Mycoplasma gallisepticum cytadherence and virulence

It was previously demonstrated that avirulent Mycoplasma gallisepticum strain R(high) (passage 164) is lacking three proteins that are expressed in its virulent progenitor, strain R(low) (passage 15). These proteins were identified as the cytadhesin molecule GapA, the putative cytadhesin-related molecule CrmA, and a component of a high-affinity transporter system, HatA. Complementation of R(high) with wild-type gapA restored expression in the transformant (GT5) but did not restore the cytadherence phenotype and maintained avirulence in chickens. These results suggested that CrmA might play an essential role in the M. gallisepticum cytadherence process. CrmA is encoded by the second gene in the gapA operon and shares significant sequence homology to the ORF6 gene of Mycoplasma pneumoniae, which has been shown to play an accessory role in the cytadherence process. Complementation of R(high) with wild-type crmA resulted in the transformant (SDCA) that lacked the cytadherence and virulence phenotype comparable to that found in R(high) and GT5. In contrast, complementation of R(high) with the entire wild-type gapA operon resulted in the transformant (GCA1) that restored cytadherence to the level found in wild-type R(low). In vivo pathogenesis trials revealed that GCA1 had regained virulence, causing airsacculitis in chickens. These results demonstrate that both GapA and CrmA are required for M. gallisepticum cytadherence and pathogenesis.     

Anti-schistosomal activity of cyclosporin A: studies on murine spleen cells and the influence of a cyclosporin antagonist on resistance to infection.

Mice were treated with 5-day courses of cyclosporin A (CsA) around the time of infection with Schistosoma mansoni. Recovery of lung-stage worms 4-8 days post-infection (p.i.) was substantially reduced (80%) and no sexually mature adults were recovered from the hepatic portal system at 7 weeks p.i. Flow cytometric analysis of spleen cells from CsA-treated animals during the period of maximal parasite attrition revealed transient reductions in CD3+ and CD4+ cells and in the CD4+: CD8+ ratio compared with drug vehicle-treated, infected controls. No significant numerical changes in B cells, macrophages or eosinophils were detected relative to vehicle-treated infected mice. Transfer of spleen cells from CsA-treated donors 8 days after infection failed to confer increased resistance to S. mansoni infection on untreated recipients. Moreover, concomitant administration of CsA and an inducer of interleukin-2 production (ADA-202-718) did not interfere with the anti-schistosomal effect of CsA. Despite our incomplete understanding of the in vivo properties of CsA and reports of its paradoxical effects on immune responses, these new data indicate that the influence of CsA in schistosomiasis is unlikely to be mediated by modulation of host cell mediated immunity. This contrasts with certain other anti-parasitic effects of CsA which appear to be mediated by an action on T cells.     

Renal injury induced by long-term administration of cyclosporin A to rats.

Chronic administration of cyclosporin A (CyA) to animals and humans results in renal damage characterized by tubulointerstitial lesions and renal insufficiency. This nephrotoxicity limits the use of CyA in the management of graft rejection. Despite recent investigations in this area, relatively few studies correlate structural and functional abnormalities in animals undergoing long-term CyA treatment. The authors therefore treated 30 rats with CyA at the dose of 40 mg/kg/48 hr for up to 5 months. An additional group of 30 rats was given the vehicle alone and was considered as a control group. Renal morphology and function were studied. For evaluation of the effect of drug withdrawal, a third group of 25 animals received CyA for 3 months and was followed for another 2 additional months after drug withdrawal. The results show that the chronic administration of CyA to rats induced complex renal morphologic changes, associated with renal insufficiency, polyuria, and enhanced sodium excretion. Withdrawal of the drug resulted in almost complete normalization of morphologic and functional parameters. The early morphologic expression of CyA nephrotoxicity was isometric vacuolization and loss of brush border involving the proximal tubular cells, followed by a peculiar lesion in distal tubular cells due to glycogen accumulation. Glomerular and interstitial damage was mild and appeared only after 3 months of CyA administration. No vascular abnormalities were found in rats treated with CyA for 5 months. A CyA-induced decrease in the glomerular filtration rate (GFR) correlates with brush border loss but not with isometric vacuolization. The distal tubular glycogen accumulation was associated with the development of polyuria and enhanced sodium excretion. Given the high blood sugar level and severe glycosuria in animals treated chronically with CyA, it is also concluded that CyA possesses a diabetogenic effect which is likely to be responsible for glycogen accumulation at the tubular level.     

Treatment of Graves' ophthalmopathy with cyclosporin A

Six patients with Graves' ophthalmopathy (2 with acute and 4 with chronic alterations) were treated with cyclosporin A (10 mg/kg/day) for 5 weeks. This treatment had no effect on either the ocular manifestations (protrusion, eye muscle function) or subjective well-being of the patients. In contrast, creatinine clearance decreased from 83.5 to 55.5 ml/min during treatment, but normalized (94.9 ml/min) after cessation of the drug. A transient increase in serum 4-androstenedione was observed in 3 patients. We conclude that cyclosporin A has no convincing effect in the treatment of Graves' ophthalmopathy, but rather exerts serious renal effects.     

Interaction of cyclosporin A with antineoplastic agents

Summary A synergistic effect of etoposide and cyclosporin A was observed in a patient with acute T-lymphocytic leukemia in relapse. The concomitant administration of etoposide and cyclosporin A resulted in eradication of hitherto refractory leukemic infiltration of bone marrow. Severe side effects in terms of mental confusion and progressive hyperbilirubinemia, however, point to an enhancement not only of antineoplastic effects but also of toxicity in normal tissues. This report demonstrates for the first time that the pharmacodynamic properties of cyclosporin A may not be confined strictly to suppression of normal T-cell functions.Abbreviations ALL Acute lymphatic leukemia - AUL Acute undifferentiated leukemia - Cy A Cyclosporin A Supported by BDMFT grant PTB 03 8468     

Exacerbation of experimental pyelonephritis by cyclosporin A

An athymic rat strain lacking functional T cells was used to assess the role of cell-mediated immunity (CMI) in host defence against renal infection. CMI was ruled out as a relevant host defence component, but when cyclosporin A (CsA) was administered to athymic animals, renal infection was exacerbated. CsA is thought to affect T lymphocyte function and, in the absence of a target cell, cellular defences in the athymic animal were not expected to be compromised by CsA. An effect on non-cellular defence mechanisms was therefore considered but our studies did not support this explanation--rather they indicated a depression of either cellular defences or of a specific cellular component. The present experiments have provided additional information on the relationship between CsA administration and the depression of host defence mechanisms but further studies will be necessary to identify the components affected.     

儿童肺炎支原体抗体检测与肺炎支原体培养对照研究

目的通过肺炎支原体抗体检测与肺炎支原体培养的对照研究，探讨肺炎支原体检测方法。方法通对以呼吸道感染住院的252例儿童患者，同时应用肺炎支原体抗体检测和肺炎支原体培养，观察两种方法的优缺点。结果肺炎支原体抗体检出率是86、5％，肺炎支原体培养检出率是43．6％，而252例儿童患者临床确诊为肺炎支原体感染者为116例，临床符合率分别是70，6％和94．8％，说明肺炎支原体抗体检测具有一定的假阳性。结论肺炎支原体培养具有方便、简单、准确，且可以用于早期检测等优点，尤其适合于不易抽静脉血的新生儿和危重病人。     

Mutation of two Mycoplasma arthritidis surface lipoproteins with divergent functions in cytadherence

Mycoplasma arthritidis is a natural pathogen of rats, causing an acute polyarthritis. Previous studies identified two membrane-bound lipoproteins, Maa1 and Maa2, thought to be associated with cytadherence of M. arthritidis strain 158p10p9. We have since confirmed that Maa1 is a major adhesin, although the role of Maa2 has proven more elusive. Both proteins were capable of eliciting protective immunity in rats against challenge with the virulent strain 158p10p9, suggesting that they may be important in pathogenesis. The purpose of this study was to better understand the roles of Maa1 and Maa2 in cytadherence in vitro. Insertion mutants were created for both genes by transposon mutagenesis. In vitro adherence of the Maa1 mutant KOMaa1 to rat L2 lung cells was reduced to the level previously reported for a spontaneous low-adherence mutant of 158p10p9 in which Maa1 is truncated and nonfunctional. Surprisingly, adherence of the Maa2 mutant KOMaa2 was approximately fivefold greater than that of the wild type. Complementation of KOMaa1 and KOMaa2 with wild-type alleles of maa1 and maa2, respectively, returned adherence to wild-type levels. This work confirms our earlier observation that Maa1 is a major adhesin for M. arthritidis strain 158p10p9. Maa2, on the other hand, may play a suppressive or modulatory role, possibly serving to release organisms from microcolonies at certain stages of infection.     

Chemosensitization to adriamycin by cyclosporin A and verapamil in human retinoblastoma cell lines.

The chemosensitizing effects of cyclosporin A and verapamil on the cytotoxicity of adriamycin were investigated using MTT assay against two human retinoblastoma cell lines, Y79 and WERI-Rb-1. Y79 and WERI-Rb-1 were totally resistant to doses up to 5.0 micrograms/ml of verapamil. Cyclosporin A inhibited the survival of Y79 and WERI-Rb-1 dose-dependently, however, the maximum inhibition at the highest concentration tested (5.0 micrograms/ml) was less than 50% (% survival at 5.0 micrograms/ml of cyclosporin A: 65.6% and 66.9% in Y79 and WERI-Rb-1, respectively). Combination of cyclosporin A and verapamil did not further inhibit the survival of Y79 and WERI-Rb-1 compared with cyclosporin A alone. Adramycin inhibited the survival of Y79 and WERI-Rb-1 dose-dependently. The chemosensitizing effects of cyclosporin A and verapamil on the cytotoxicity of adriamycin were evaluated in terms of sensitizing index (SI: the ratio of IC50 to adriamycin alone to IC50 to adriamycin in the presence of cyclosporin A and/or verapamil). Cyclosporin A significantly enhanced SI and the addition of verapamil enhanced SI further: SI values at 5.0 micrograms/ml of cyclosporin A, 5.0 micrograms/ml of cyclosporin A plus 1.5 micrograms/ml of cyclosporin A plus 1.5 micrograms/ml of verapamil, 5.0 micrograms/ml of cyclosporin A plus 3.0 micrograms/ml of verapamil were 2.0, 2.6 and 2.8 in Y79 and 2.6, 5.8 and 9.7 in WERI-Rb-1, respectively. These results suggest that cyclosporin A and verapamil are promising chemosensitizers to adriamycin in the treatment of retinoblastoma.