Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.     

双歧杆菌对免疫抑制小鼠白色念珠菌感染的保护作用

为探讨双歧杆菌对白色念珠菌感染的保护作用,作者采用环磷酰胺腹腔注射方法复制了免疫抑制小鼠模型,研究白色念珠菌感染前后两歧双歧杆菌灌饲的保护作用。结果表明：白色念珠菌感染前灌饲两歧双歧杆菌能有效地抑制白色念珠菌在小鼠肠道中的定植,并且保护肠粘膜的完整性。对其机理的研究认为,两歧双歧杆菌可能是通过调整肠道菌群平衡、增加肠道中生理菌群的数量、降低肠道ｐＨ值、以及产生抗菌物质等途径发挥保护作用     

Characterization of effector cells with anti-Candida activity obtained from murine bone marrow cells cultured in the presence of rhG-CSF: comparison between normal and CY-treated mice

Bone marrow cells (BMC) obtained from normal and cyclophosphamide (CY)-treated mice were cultured in the presence of recombinant human granulocyte-colony stimulating factor (rhG-CSF) and their effector cell activities inhibiting growth of C. albicans were examined. When BMC from CY-treated mice were preincubated with 0.05 ng/ml of rhG-CSF, effector cells with enhanced anti-C. albicans activity were recovered in the adherent cell population, whereas anti-C. albicans activity of BMC from normal mice was found in the non-adherent cell population. During culture without the presence of rhG-CSF, nonadherent BMC, seemingly granulocytes, from normal mice showed apoptotic change, but addition of rhGCSF clearly inhibited this change. On the other hand, when BMC from CY-treated mice were cultured with rhG-CSF, adherent cells as the main effector had the appearance of monocytes. These differences between the effectors with anti-C. albicans activity obtained from normal and CY-treated mice are discussed.     

FK506 reduces abnormal prion protein through the activation of autolysosomal degradation and prolongs survival in prion-infected mice

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNP^Sc) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, ATG12–ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNP^Sc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNP^Sc in addition to attenuation of microgliosis and neuroprotection.     

Cloning and sequence analysis of a rapamycin-binding protein-encoding gene (RBP1) from Candida albicans.

Rapamycin (Rm) and FK506 are macrolide antifungal agents that exhibit potent immunosuppressive properties in higher eukaryotes which are mediated through interaction with specific receptor proteins (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. We previously isolated a gene encoding an RBP from Saccharomyces cerevisiae, and demonstrated that null mutations in this gene (called RBP1) result in a recessive Rm-resistant (RmR) phenotype. We now have cloned the Candida albicans RBP1 gene via complementation of the RmR phenotype in S. cerevisiae. The predicted C. albicans RBP exhibits 61%, 52% and 49% amino acid (aa) sequence identity with RBPs (FKBPs) from S. cerevisiae, Neurospora crassa and human cells (FKBP-12), respectively. Furthermore, several of the aa residues identified as being important for drug binding in human FKBP-12 are conserved within the C. albicans RBP.     

Equilibrium studies of a fluorescent tacrolimus binding to surfactant protein A

Tacrolimus (FK506) is a hydrophobic immunosuppressive agent used in kidney, liver, and lung transplantation. The objective of this study was to characterize the binding of FK506 to surfactant protein A (SP-A), an abundant lipoprotein found in the alveolar fluid that functions as part of the innate immune system in the lung. We have synthesized a novel derivative of FK506 in which a dansyl moiety was covalently bound via cadaverine to the C22 position of the FK506 molecule (DNS-FK). Using the fluorescence and anisotropy properties of DNS-FK, we demonstrated that tacrolimus avidly binds to SP-A with an apparent equilibrium association constant (K(app)) of 10(7)M(-1) and a Gibbs binding free energy of -40 kJ mol(-1)K(-1). Derivatization of FK506 at the C22 position did not block FK506 binding to the cytosolic immunophilin FK506-binding protein (FK-BP) or human serum albumin (HSA), both used as controls of tacrolimus-binding proteins. K(app) for FK-BP/DNS-FK and HSA/DNS-FK complexes were 1.5 x 10(7) and 10(7)M(-1), respectively. The high sensitivity of this analytical technique makes it suitable for binding analysis of FK506 to proteins.     

Cyclosporine downregulates Fas ligand expression on vascular endothelial cells: implication for accelerated vasculopathy by immunosuppressive therapy.

Although the introduction of cyclosporine A (CyA) and FK506 for immunosuppressive therapy has dramatically enhanced the early survival of organ transplant recipients, administration of these immunosuppresants is correlated with high incidence of transplant arteriosclerosis. Transplant-associated arteriosclerosis is believed to result from recipient inflammatory responses to the allograft, as it is characterized by early mononuclear cell infiltration of the transplanted vessel. We reported that vascular endothelial cells naturally express a death factor, Fas ligand, that may function to inhibit detrimental leukocyte infiltration. Here, we show that CyA or FK506 downregulates FasL expression on endothelial cells with accompanying decrease in the cytotoxicity toward Fas-bearing cells. Our findings not only demonstrate a novel biological action of these drugs, but also suggest a mechanism by which immunosupressive treatment contributes to atherogenesis.     

Chorionic gonadotropin-like proteins in the obplacental giant cells of the rabbit

Diabetes mellitus was induced in 40 male C57BL6 mice by injection of a low dose of streptozocin (45 mg/kg body weight) on 5 consecutive days. Twenty four of the mice were immunosuppressed by administration of 1.5 mg FK506/kg body weight daily for 10, 15, 18 and 24 days. Administration of FK506 almost completely inhibited the streptozocin-induced islet damage, and consequently glycaemia remained normal. In FK506-treated animals any inflammatory infiltrate was very sparse and was limited to the vascular pole of the islets. Immunocytochemical results demonstrated that infiltrating cells were Ia-immunoreactive, but were not activated. Ultrastructural observations confirmed the absence of B cell necrosis and degranulation in FK506-treated mice; the few infiltrating elements encountered did not contain phagocytic vesicles or show other signs of activation.     

Clonal expansion of superantigen-reactive T cells is resistant to FK506 in mice with AIDS.

Subcutaneous injection of FK506 (10 mg/kg of body weight) completely blocked the clonal expansion of staphylococcal enterotoxin A (SEA)-reactive T cells in healthy (control) mice after SEA injection but did not disturb it in mice with murine AIDS (MAIDS) caused by infection with LP-BM5 murine leukemia virus. MAIDS mice are characterized by utilization of a FK506-insensitive pathway for clonal expansion of superantigen-reactive T cells.     

Effects of gamma-interferon and FK506 on resting B cell proliferation of New Zealand black/white F1 mice.

We examined the effects of gamma-interferon (gamma-IFN) and the new immunosuppressant FK506 on resting B cell proliferation of New Zealand black/white F1 hybrid (B/W F1) mice, an animal model of human systemic lupus erythematosus (SLE). gamma-IFN and FK506 inhibited in a dose-dependent manner both B cell proliferation and autoantibody production of resting B cells respectively. There was a synergistic interaction between gamma-IFN and FK506 in their inhibition and they did not exhibit cell cytotoxicity. This in vitro synergism of gamma-IFN and FK506 may have clinical application in that low doses of gamma-IFN and FK506 combinations may be effective to correct polyclonal B cell activation of patients with SLE.     

Enhanced resistance againstEscherichia coli infection by subcutaneous administration of the hot-water extract ofChlorella vulgaris in cyclophosphamide-treated mice

Summary The effects ofChlorella vulgaris extract (CVE-A) on the recovery of leukocyte number and the augmentation of resistance to bacterial infection were examined in CDF1 mice made neutropenic by cyclophosphamide (CY). They were treated intraperitoneally with CY (150 mg/kg) on day 0, and were given CVE-A (50 mg/kg) subcutaneously (s. c.) every other day from day 1 to day 13 after CY treatment. CVE-A accelerated the recovery of polymorphonuclear leukocytes (PMN) in the peripheral blood in CY-treated mice. The number of granulocyte/monocyte-progenitor cells (CFU-GM) in the spleen increased rapidly and highly after the administration of CVE-A in CY-treated mice, in contrast to the absence of change due to CVE-A in the number of bone marrow cells in CY-treated mice. Administration of CVE-A in CY-treated mice enhanced the accumulation of PMN in the inflammatory site and the activity of the accumulated leukocyte cells in luminol-dependent chemiluminescence. The mice became highly susceptible to an intraperitoneal infection withE.coli on day 4 after CY treatment, whereas the mice given CVE-A showed an enhanced resistance againstE.coli infection, irrespective of the timing of challenge. The bacterial number in CY-treated mice increased explosively after inoculation, resulting in death within 24 h. A progressive elimination of bacteria was observed from 6 h in the peritoneal cavity, spleen and liver of CY-treated mice given CVE-A s.c. These results indicate that CVE-A can be used as a potent stimulant of nonspecific resistance to infection in neutropenic mice.     

益气补肾方药对化疗荷瘤小鼠免疫功能的影响

目的　探讨益气补肾方药 (由金匮肾气方加味人参、黄芪组成 ,以下均称方药 ,TS)对环磷酰胺 (CY)处理的荷H2 2 瘤小鼠非特异免疫功能的影响。方法　用CY处理荷H2 2 瘤小鼠 ,建立免疫力低下动物模型。用乳酸脱氢酶 (LDH)释放法分别检测NK细胞、巨噬细胞 (M)的活性 ,用RT PCR法检测白细胞介素 12 (IL 12 )mRNA在脾脏细胞中的表达。结果　TS CY组小鼠吸光度A值为 1 332± 0 5 10 ,明显高于CY组小鼠 (P <0 0 1)。TS CY组小鼠A值为 1 12 9± 0 2 80 ,明显高于CY组小鼠 (P <0 0 1)。此方药能明显提高CY所致免疫力低下小鼠NK细胞、M的活性 ,增加IL 12在脾细胞中的表达。结论　该方药可以明显提高环磷酰胺所致免疫力低下荷瘤小鼠的非特异免疫功能。     

Resistance to Friend murine leukemia virus infection conferred by the Fv-4 gene is recessive but appears dominant from the effect of the immune system

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). However, the resistance caused by Fv-4 is recessive in nude mice, which suggests that immunological effects play important roles in this resistance in vivo (K. Higo, Y. Kubo, Y. Iwatani, T. Ono, M. Maeda, H. Hiai, T. Masuda, K. Kuribayashi, F. Zhang, T. Lamin, A. Adachi, and A. Ishimoto, J. Virol. 71:750-754, 1997). To determine the immunological effect on the resistance in vivo, we infected immunologically immature newborn mice homozygous (Fv-4(r/r)) and heterozygous (Fv-4(r/-)) for Fv-4. Although the Fv-4(r/r) mice showed complete resistance to F-MuLV whether infected neonatally or as adolescents, the Fv-4(r/-) mice showed high sensitivity to viral proliferation and disease induction when infected as newborns but complete resistance when infected as adolescent mice. To confirm the immunological effect on the resistance in adolescent mice with the Fv-4(r/r) and Fv-4(r/-) genotypes, we examined the effect of an immunosuppressant drug, FK506, on the resistance. The mice with the Fv-4(r/r) genotype treated with FK506 still showed resistance, but the mice with the Fv-4(r/-) genotype became highly sensitive to F-MuLV infection. Flow cytometric analysis to detect the Fv-4 gene product showed that the Fv-4 gene product was expressed on the cells from newborn and adolescent mice. The Fv-4 gene product was also detected on the cells from the FK506-treated mice as well as on those from untreated mice. However, a quantitative difference in the gene product between the cells with the Fv-4(r/r) and Fv-4(r/-) genotypes was detected by indirect staining for flow cytometry. These results show that the resistance to F-MuLV infection conferred by the Fv-4 gene is originally recessive, but it looks dominant in adolescent mice mainly because of the effect of the immune system.     

Candida albicans infection enhances immunosuppression induced by cyclophosphamide by selective priming of suppressive myeloid progenitors for NO production

Systemic infections caused by fungi after cytoreductive therapies are especially difficult to deal with in spite of currently available antimicrobials. However, little is known about the effects of fungi on the immune system of immunosuppressed hosts. We have addressed this by studying the in vitro T cell responses after systemic infection with Candida albicans in cyclophosphamide-treated mice. After cyclophosphamide treatment, a massive splenic colonization of the spleens, but not lymph nodes, by immature myeloid progenitor (Ly-6G(+)CD11b(+))cells is observed. These cells are able to suppress proliferation of T lymphocytes via a nitric oxide (NO)-dependent mechanism. Systemic infection with a sublethal dose of C. albicans did not cause immunosuppression per se but strongly increased NO-dependent suppression in cyclophosphamide-treated mice, by selective priming of suppressive myeloid progenitors (Ly-6G(+)CD11b(+)CD31(+)CD40(+)WGA(+)CD117(low/-)CD34(low/-)) for iNOS protein expression. The results indicate that systemic C. albicans infection can augment the effects of immunosuppressive therapies by promoting functional changes in immunosuppressive cells.     

Long-Term Acceptance of Major Histocompatibility Complex-Mismatched Cardiac Allograft Induced by a Low Dose of CTLA4IgM Plus FK506

The immunosuppressant FK506 prolongs allograft survival. However, at therapeutic doses it has significant side effects. A fusion protein consisting of the extracellular portion of CTLA4 and the Fc portion of human IgG (CTLA4IgG) also prolongs allograft survival, but large doses of CTLA4IgG are required for the induction of cardiac allograft acceptance. Therefore, we constructed a pentameric form of a new CTLA4 fusion protein, CTLA4IgM. We tested whether low doses of CTLA4IgG or CTLA4IgM in combination with subtherapeutic doses of FK506 can prolong allograft survival in a synergistic fashion. C57BL/6 (H-2^b) neonatal hearts were transplanted to CBA/J (H-2^k) mice in a heterotopic, nonvascularized cardiac allograft model. The findings demonstrate that a combination of low doses of FK506 plus a pentameric form of CTLA4Ig, CTLA4IgM, leads to significant graft survival, while a combination of FK506 plus CTLA4IgG does not.     

Improved therapeutic effects of interleukin 2 after the accumulation of lymphokine-activated killer cells in tumor tissue of mice previously treated with cyclophosphamide

Summary We investigated the combined effects of human recombinant interleukin 2 (IL-2) and cyclophosphamide (CY) on s.c. transplanted 3LL lung carcinoma in C57BL/6 mice. A total of 95% of the tumors were competely cured when CY (150 mg/kg, i.v.) was given on day 5 (5 days after tumor implantation) and IL-2 (5×10⁴ Jurkat Units/day, i.p.) was then combined with it between day 6 and day 15. CY alone brought about the complete regression of tumors, although 60% of the mice died of local recurrence and pulmonary metastasis; IL-2 alone had no therapeutic effect. Satisfactory effects from the combination of CY and IL-2 were also obtained by 5 days administration of IL-2 between days 11 and 15, initiated 6 days after CY treatment, but not by that given before CY (days 1–5) or 1 day after CY (days 6–10). No therapeutic effects from IL-2 were observed when it was combined with other types of chemotherapy that showed not therapeutic effects by themselves. Nor were we able to observe any transplantation resistance to the rechallenge of 3LL tumor in cured mice. We particularly examined the lymphokine-activated killer (LAK) cells as we suspected that these were responsible for the development of active effector cells in the treated mice. LAK cell activity in fresh spleen cells was detected in mice treated with IL-2 alone but not in untreated mice nor in those treated with CY alone or CY plus IL-2. The number of LAK precursor cells in the spleen had increased on day 8 and on day 13 in untreated mice with 3LL, as compared with the incidence in normal mice, while the number of cells had decreased by day 18. On the other hand LAK precursor cells were suppressed on day 8 and tended to recover thereafter in CY-treated mice. Adoptively transferred LAK cells were found to accumulate in CY-treated tumors 2.5 times more densely than in untreated tumors. The preferential accumulation of LAK cells that had been activated systemically by the appropriately timed administration of IL-2 in tumor tissue was followed by the improved effects obtained by combined treatment with CY and IL-2.Supported in part by Grants-in-Aid for Cancer Research from the Japanese Ministry of Education, Science and Culture and from the Japanese Ministry of Health and Welfare     

Cellular elements in the resistance to candida infection in mice. I. Contribution of T lymphocytes and phagocytes at various stages of infection.

Live organisms (cfu) of Candida albicans per organ were counted 1 hr and 1 to 20 days after an intravenous inoculation into various groups of mice which had distinct levels of immunologic or non-immunologic defense mechanisms. a) The number of cfu in the liver decreased progressively in normal mice, but those in the kidney maintained a constant level during the observation period. b) The number of cfu in the liver decreased progressively also in nude mice. In their kidneys, however, cfu increased progressively at a late stage of infection. c) In lethally irradiated AKR of nude mice in which phagocyte functions were severely depressed, the number of cfu increased progressively in both liver and kidney from the initial stage of infection. d) In immunized AKR mice, growth of C. albicans was suppressed at late stages of infection. Such protective immunity could be transferred partly with immune lymphoid cells but not with hyperimmune serum in the experimental system employed. In protection against candida infection, non-immune phagocytosis and T cell-mediated immunity appear to be required at the early and late stages of infection, respectively.     

Dose-dependent effects of FK506 on neuroregeneration in a rat model

This study explored the effects of different doses of FK506 on peripheral nerve regeneration, to determine whether neuroregeneration could be enhanced without the toxicity of systemic immunosuppression. In the first part of the study, subimmunosuppressive doses of FK506 were determined by examining skin allograft survival in a rat model. Full-thickness skin grafts (2 cm2) from Wistar rats were grafted to recipient Lewis rats. The procedure was performed for six groups (n = 6). The control group received no FK506, and the other five groups received daily doses of FK506 of 0.125, 0.25, 0.5, 1.0, or 2.0 mg/kg. Animals that received 2.0 mg/kg FK506 per day exhibited complete skin graft take, whereas all other groups demonstrated complete rejection. After determination of the immunosuppressive dose of FK506, the neuroregenerative effects of different doses of FK506 were explored by assessing nerve regeneration in 80 rats after tibial nerve transection and repair. The control group received no FK506, whereas the other four groups were given daily doses of FK506 of 0.25, 0.5, 1.0, or 2.0 mg/kg. Rats were euthanized at three time points (25, 30, and 35 days), to fully investigate the effects of different FK506 dosing regimens on neuroregeneration. Histomorphometric analyses performed on postoperative days 30 and 35 demonstrated statistically significant improvements in neuroregeneration with subimmunosuppressive FK506 doses of 0.5 and 1.0 mg/kg per day. Therefore, the study demonstrated that neuroregeneration was enhanced at low doses of FK506 that were not sufficient to prevent skin allograft rejection.