Cyclosporine in transplantation - a history of converging timelines

Discovery and pharmacological development of cyclosporine was conducted by Jean Borel and colleagues in the 1970s. Cyclosporine is the first compound to inhibit the lymphocytes specifically and reversibly, and represents the prototype of a new generation of immunosuppressive drugs: the calcineurine inhibitors. Historical chronology of successes in clinical application of cyclosporine and development of solid-organ transplantation are retraced here, underscoring the converging timelines of this drug and these interventions. In 1978-79 the first successful results of the use of cyclosporine in kidney were reported. Cyclosporine was the first single drug able to control rejection. In 1982-83 first trials demonstrated the benefit from treatment with cyclosporine in kidney recipients compared to azathioprine and steroids. In the 1980s solid-organ transplantation entered the cyclosporine era with unhoped-for results in heart transplantation. The present review focuses also on cyclosporine-based regimen of immunosuppression, adverse side effects and safety in pregnancy in subjects under treatment with cyclosporine.     

Acute renal failure after cardiac transplantation: a case report and review of the literature.

Acute renal failure (ARF) is a relatively frequent complication associated with heart transplantation. It develops in the first few days postoperatively and is characterized by oliguria with laboratory and urinary indices typical of pre-renal azotemia. Cyclosporine, especially with higher doses, is one of the many factors which play an integral part in the nephrotoxicity following cardiac transplant. Poor preoperative renal function and perioperative hemodynamic compromise may also contribute to ARF. The actual incidence of ARF now encountered by transplant centers may be lower than previously reported, the result of lower cyclosporine doses. Currently, management is entirely supportive, but novel therapeutic approaches with atrial natriuretic peptide-like substances are being explored. A case illustrating the typical clinical presentation of ARF after heart transplant will be presented and the clinical features will be reviewed.     

Chronomodulatory effect of cyclosporine upon survival of DBA mice with L1210 leukemia

A circadian stage-dependent anti-tumor effect of cyclosporine was tested on 268 female DBA mice, 9-10 weeks of age. The mice were kept in 6 different environmental chambers on regimens of 12h of light alternating with 12h of darkness, staggered by 4h: they were inoculated intraperitoneally with 2 X 10(5) L1210 cells at one of 6 different circadian stages. At the same circadian stage, starting 48h after inoculation, for 4 days, each mouse received the vehicle, a fixed dose of cyclosporine (15 mg/kg b.w.), a varying dose of cyclosporine 5, 10, 20 and 25 mg/kg b.w.) or no treatment. Cyclosporine prolonged survival time in a circadian stage dependent fashion (p less than 0.01), as shown by an analysis of variance and by cosinor analysis (mesor = 8.45h; amplitude = 5.45h; acrophase = 12 HALO). Cyclosporine thus acts, in a feed-sideward, as a chronomodulator of the interaction between the tumor and its host.     

GM-CSF restores innate,but not adaptive,immune responses in glucocorticoid-immunosuppressed human blood in vitro

Infection remains the major complication of immunosuppressive therapy in organ transplantation. Therefore, reconstitution of the innate immunity against infections, without activation of the adaptive immune responses, to prevent graft rejection is a clinically desirable status in transplant recipients. We found that GM-CSF restored TNF mRNA and protein expression without inducing IL-2 production and T cell proliferation in glucocorticoid-immunosuppressed blood from either healthy donors or liver transplant patients. Gene array experiments indicated that GM-CSF selectively restored a variety of dexamethasone-suppressed, LPS-inducible genes relevant for innate immunity. A possible explanation for the lack of GM-CSF to restore T cell proliferation is its enhancement of the release of IL-1betaR antagonist, rather than of IL-1beta itself, since exogenously added IL-1beta induced an IL-2-independent Con A-stimulated proliferation of glucocorticoid-immunosuppressed lymphocytes. Finally, to test the in vivo relevance of our findings, we showed that GM-CSF restored the survival of dexamethasone- or cyclosporine A-immunosuppressed mice from an otherwise lethal infection with Salmonella typhimurium. In addition to this increased resistance to infection, GM-CSF did not induce graft rejection of a skin allotransplant in cyclosporine A-immunosuppressed mice. The selective restoration potential of GM-CSF suggests its therapeutic use in improving the resistance against infections upon organ transplantation.     

Cyclosporine has a direct effect on the differentiation of a mucin-secreting cell line

Cyclosporine is a potent immunosuppressant used in the treatment of ulcerative colitis and keratoconjunctivitis sicca. Neither the etiologies of these diseases nor the mechanism by which cyclosporine exerts its therapeutic effect is well understood. Since both diseases are linked by a common decrease in mucin-filled goblet cells, this study tests a hypothesis that cyclosporine acts directly on goblet cells to promote their differentiation and production of secretory mucins. The HT29-18N2 human colon adenocarcinoma cell line, which is capable of forming monolayers of well-differentiated goblet cells, was used as a model system. Cyclosporine induced a dose-dependent increase in intracellular mucin stores. A 2-week exposure to 1 microM cyclosporine resulted in an average increase in mucin volume of 94%. This increase resulted from both a higher percentage of cells with mucin stores and an increased volume of mucin per cell. PSC-833, a nonimmunosuppressive analog of cyclosporine, also increased mucin production. The intracellular accumulation of mucin was not a result of reduced secretion, since the time required for the release of pulse-radiolabeled glycoproteins was similar for both control and cyclosporine-treated monolayers. The effect of cyclosporine was not mediated by the drug's previously documented abilities to decrease cellular proliferation rates, inhibit calmodulin, antagonize prolactin receptor binding, or modulate prostaglandin production.     

Association of IL-15 and IP-10 Serum Levels with Cytomegalovirus Infection,CMV Viral Load and Cyclosporine Level after Kidney Transplantation

Background:Cytomegalovirus (CMV) infection is the most common complications following kidney transplantation. Natural killer (NK) cells demonstrated critical anti-viral role in controlling and elimination of CMV after transplantation. Interleukin-15 (IL-15) is a pleiotropic cytokine that promotes the activity of NK cells and strengthens the acquired immune system. Also, IP10 (CXCL10) is a chemotactic factor which regulates NK cell recruitment and antiviral immune response. We aimed to determine the correlation between the serum levels of IL-15 and IP-10 cytokines with CMV infection, CMV viral load, and cyclosporine as a major immunosuppressive treatment after transplantation.Methods:Fifty-eight kidney transplant recipient patients without evidence of CMV virus disease before transplantation surgery were included in the study. From the day of transplant surgery, the patients were evaluated based on the presence of CMV Ag pp65, CMV viral load, serum levels of IL-15 & IP-10, Cyclosporine levels (C0 & C2), Glomerular Filtration Rate (GFR), and hematological & biochemical Index, up to 75 days.Results:Comparison analysis of serum levels of IL-15 and IP-10 showed no significant association with CMV infection in kidney transplant recipients. In addition, CMV viral load and cyclosporine levels at C0 and C2 did not affect patients'' IL-15 and IP-10 levels.Conclusion:The levels of IP-10 and IL-15 cytokines are not affected with CMV infection, even if a viral infection occurs in the early days after transplantation or long afterwards. In addition, taking the different levels of cyclosporine did not affect the cytokines levels. Other mechanisms may play a role in maintaining the levels of these cytokines.Key Words: Cytokine, Cytomegalovirus, IP-10, Interleukin-15, Transplantation     

Immunity related to cytomegalovirus after allogenic bone marrow transplantation: role of donor immunity to cytomegalovirus in the incidence of cytomegalovirus infection in recipients

Cytomegalovirus infections are severe and frequent after BMT. This study included 34 bone marrow transplant recipients (23 aplastic anaemias and 11 leukaemias), their marrow donors and 125 related or non related normal controls. Assays were performed before transplantation and every 30 days between D 0 and D 90, and then every six months. They included detection of CMV induced lymphocyte proliferation in vitro, CMV antibody determinations by complement fixation and reverse haemagglutination, viraemia and/or viruria. Similarly, cellular immunity to mitogens and to other specific antigens was evaluated. During the period of study, 22 patients developed CMV infection. The diagnostic was confirmed by virus isolation from the 12th to the 96th day after the graft. Development of positive CMV proliferation test occurred from the 9th to the 84th day after virus isolation (30 to 120th day after the graft). In one case, the CMV infection was only proved by the lymphocyte proliferation to CMV in vitro and only 60 days later by viruria and 105 days later by detection of CMV antibodies. For the other 12 patients (7 aplasies and 5 leukaemias) and 10 of their bone marrow donors, no CMV infection was proved, before or after transplant, by any of the assays performed. By selecting a donor without previous CMV infection, we hope to reduce the incidence of CM infection in recipients.     

Cyclosporine A: Review of genotoxicity and potential for adverse human reproductive and developmental effects: Report of a working group on the genotoxicity of cyclosporine A,August 18, 1993

Cyclosporine is an important therapeutic agent for transplant recipients and for a growing number of autoimmune diseases. Experimental animal and human data has indicated that cyclosporine is unlikely to be genotoxic. In contrast, azathioprine, an agent often given with cyclosporine, is considered to be genotoxic making the assessment of the independent effects of cyclosporine difficult. Cyclosporine does appear to be related to the development of tumors, primarily lymphomas, in animals and humans, but the basis of its potential carcinogenicity is not completely understood. In terms of reproductive and developmental toxicity, cyclosporine produces some adverse effects in both experimental animals and humans. In animals, the effects are seen at high doses sufficient to cause maternal toxicity. In humans, outcomes such as growth retardation have been noted, but the confounding effects of renal toxicity and resultant pregnancy complications cloud the interpretation. An increase in congenital anomalies and genetic disease have not been found reported in human studies that are limited in sample size.     

Tolerance induction in composite facial allograft transplantation in the rat model

Clinical application of composite tissue allograft transplants opened discussion on the restoration of facial deformities by allotransplantation. The authors introduce a hemifacial allograft transplant model to investigate the rationale for the development of functional tolerance across the major histocompatibility complex barrier. Eighteen rats in three groups were studied. The composite hemifacial allotransplantations including the ear and scalp were performed between Lewis-Brown Norway (RT1l+n) and Lewis (RT1l) rats and isotransplantations were performed between Lewis rats. Isograft controls (n = 6) and allograft controls (n = 6) did not receive treatment. Allografts in treatment group (n = 6) were treated with cyclosporine A 16 mg/kg/day during the first week; this dose was tapered to 2 mg/kg/day over 4 weeks and maintained at this level thereafter. Functional tolerance to face allografts was evaluated clinically and histologically. Donor-specific chimerism was assessed at days 21 and 63 by flow cytometry. In vitro evaluation of donor-specific tolerance was performed by mixed lymphocyte reaction at day 160 after transplantation. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 7 days after transplantation, as confirmed by histopathologic analysis. Five of six face allografts under the cyclosporine A protocol showed no signs of rejection for up to 240 days and remained alive and under evaluation, whereas one animal showed signs of rejection at day 140. This was reversed by adjustment of the cyclosporine A dose. At day 21 after transplantation, flow cytometric analysis of the donor-specific chimerism showed 1.11 percent of double-positive CD4FITC/RT1Ac-Cy7 and 1.43 percent of double-positive CD8PE/RT1Ac-Cy7 T-cell populations in the peripheral blood of hemiface allotransplant recipients. The chimerism level of double-positive CD4FITC/RT1Ac-Cy7 T cells increased to 3.39 percent, whereas it remained stable for the double-positive CD8PE/RT1Ac-Cy7 T-cell population at day 63 after transplantation (1.00 percent). The mixed lymphocyte reaction assay at day 160 after transplantation revealed donor-specific tolerance to donor (Lewis-Brown Norway) antigens and strong reactivity to the third-party (ACI) alloantigens. In this study, donor-specific chimerism and functional tolerance were induced in hemifacial allograft transplants across the major histocompatibility complex barrier under cyclosporine A monotherapy protocol. This model will allow further studies on tolerance induction protocols.     

Pretreatment with phenobarbitone modifies suppressant effect of cyclosporine on wound healing

Cyclosporine has been reported to suppress the tensile strength of healing incision wound. Prednisolone, an inducer of hepatic microsomal enzymes, abolished the tensile strength suppressant effect of cyclosporine. Cyclosporine is metabolized by the hepatic cytochrome P-450 enzymes. Induction of microsomal enzymes with phenobarbitone was evaluated for its effect upon the wound healing suppressant effect of cyclosporine. Pretreatment of male rats with phenobarbitone (75 mg/kg/day, ip) for 3 days resulted in alleviating the tensile strength suppressant effect of cyclosporine (5 mg/kg/day, po for 10 days). Phenobarbitone, per se, did not affect the tensile strength. That phenobarbitone prevents cyclosporine induced nephrotoxicity without affecting the humoral immunosuppressant action of cyclosporine has recently been reported. The possibility of modulating microsomal enzymes with phenobarbitone offers another approach in preventing cyclosporine-associated toxicities during immunosuppression.     

Host resistance to cyclosporine induced syngeneic graft-versus-host disease. Requirement for two distinct lymphocyte subsets

Cyclosporine is crucial for the prevention of organ allograft rejection and allogeneic graft-vs-host disease (GVHD). Despite its potent immunosuppressive activity, cyclosporine elicits a T cell-mediated autoimmune syndrome after autologous or syngeneic bone marrow transplantation, which has been termed syngeneic GVHD (SGVHD). Recent studies have shown that for disease manifestation, a cytoxan and radiation-sensitive T cell dependent host resistance mechanism must be eliminated, allowing the clonal expansion of autoreactive cells. This report characterizes the autoregulatory lymphocyte population, present in normal animals, capable of inhibiting the adoptive transfer of SGVHD. First, twice the number of unfractionated splenocytes from normal animals to those from autoimmune donors ensured complete inhibition of the adoptive transfer of immune reactivity. Second, the phenotype of this host resistance mechanism in normal splenocytes involves dual regulatory T cell subsets. A helper/inducer subset (W3/25+) must be cotransferred with a cytotoxic/suppressor subset (OX8+) in a ratio that approximates the normal ratio in normal unfractionated splenocytes in order to affect inhibition of the transfer of SGVHD. Moreover the specific inducer regulatory activity resides in the OX22-, W3/25+ subset of Th cells.     

Th1 cytokines and NK cells participate in the development of murine syngeneic graft-versus-host disease.

Syngeneic graft-vs-host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic bone marrow cells followed by a short course of therapy with the immunosuppressive agent cyclosporine A. Following cessation of cyclosporine A therapy, animals develop clinical symptoms of SGVHD: weight loss, runting, and diarrhea. While it has been suggested that T cells are responsible for the induction and effector phases of SGVHD, the role of nonspecific effector cells and cytokine mediators has yet to be examined in the disease process. Mice with SGVHD had increased levels of message for IL-12p40, IFN-gamma, and TNF-alpha in the target organs of SGVHD as compared with transplant controls and asymptomatic cyclosporine A-treated mice. Concomitant with the increase in Th1 cytokines was an enhanced cellular infiltrate in the target organs of SGVHD mice as determined by histological analysis. To directly examine the role of IL-12 in the development of SGVHD, in vivo neutralization of IL-12 was performed. Treatment of mice with Abs to IL-12 inhibited SGVHD-mediated tissue pathology and mortality. Because IL-12 has been shown to activate both T cells and NK cells to secrete IFN-gamma and to become more cytolytic, studies were initiated to ascertain which lymphocyte populations play a role in the development of murine SGVHD. Depletion of NK cells inhibited clinical symptoms of SGVHD. In contrast, T cell depletion did not alter the disease process. Therefore, these findings collectively demonstrate a role for IL-12 and NK cells in the effector phase of murine SGVHD.     

Differential effect of cyclosporine A on respiratory burst by several types of human leukocytic cells.

The effect of cyclosporine on PMA-stimulated superoxide production has been studied on human alveolar macrophages, human neutrophils, cytoplasts and Epstein-Barr-virus-transformed B lymphocytes. Cyclosporine inhibits superoxide production in alveolar macrophages but not in neutrophils and cytoplasts. The respiratory burst of B-lymphocytes was scarcely inhibited by cyclosporine. The activity of NADPH oxidase from macrophages and neutrophils was not directly affected by cyclosporine. These data are considered in relation with the proposed mechanism for cyclosporine action and the stimulation of the respiratory burst.     

Effect of cyclosporine on the lymphoid organs of CBA mice]

The influence of cyclosporine which is an immunodepressant on morphology of the lymphoid organs was studied on CBA mice. The immunodepressant was administered intramuscularly in a single LD50 and in a dose of 7 mg/kg for the treatment course of 21 days. The examinations were performed in various periods within 28 days after discontinuation of the drug use. It was shown that cyclosporine induced cell depletion in the thymus cortical and medullar zones, inhibition of lymphocyte mitotic activity, alteration of the Hassall corpuscles and impairment of their formation. It also induced devastation of the thymus-dependent zones in the mesenteric lymph nodes and spleen. When cyclosporine was used in the course doses the morphological changes in all the lymphoid organs were more marked. The morphological changes were reversible.     

Immunostimulatory action of AC II--an ayurvedic formulation useful in HIV

Immunostimulatory activity of AC II, a registered ayurvedic preparation prepared at Amala Ayurvedic Research Centre for treating HIV and AIDS is reported. AC II administration could significantly enhance the mitogen-induced proliferation of lymphocytes of spleen cells. It was also found to increase cell-mediated immune responses in normal and tumor-bearing control animals. Oral administration of AC II significantly enhanced Natural Killer cell activity in normal and tumor-bearing animals on the 7th day, which was observed earlier than the tumor-bearing control animals and normal animals. Antibody dependent cellular cytotoxicity (ADCC) was also increased in AC II treated normal and tumor-bearing animals. An early enhancement of antibody-dependent complement-mediated cytotoxicity was also observed by the administration of AC II in normal as well as tumor-bearing animals. Treatment with AC II elevated the levels of IL-2, TNF-alpha and IFN-gamma in normal mice. Administration of AC II was also found to increase the cytotoxic T lymphocyte production in EL4 treated mice. These studies support the use of this immune stimulatory preparation in HIV patients.     

Study of the immunotropic activity of aminoglycoside antibiotics

The action of some aminoglycoside antibiotics on the immune system was studied on both intact mice and the animals with immune deficiency caused by administration of cyclophosphamide. The following tests were used: local hemolysis (the Herne test), lymphocyte transformation (LT), delayed hypersensitivity to sheep red blood cells and the local graft versus host reaction (GVHR). Amikacin was shown to have no significant action on the activity of lymphocytes in the intact mice and stimulated both cellular (LT and GVHR) and humoral (the Herne test) immunity in the animals with lowered immunological reactivity. Sisomicin had no significant action on the immune system of the animals. Gentamicin suppressed the immune response only in the intact mice. Kanamycin and streptomycin induced inhibition of humoral and cellular immunity in both the intact mice and animals with immune deficiency. On the basis of the results it was concluded that gentamicin, amikacin and sisomicin may be used in the treatment of diseases developing in the presence of immune deficiency whereas streptomycin and kanamycin should be recommended when inhibition of the immunity is needed.     

Effect of Immunosuppressive Agents on Hepatocyte Apoptosis Post-Liver Transplantation

Introduction

Immunosuppressants are used ubiquitously post-liver transplantation to prevent allograft rejection. However their effects on hepatocytes are unknown. Experimental data from non-liver cells indicate that immunosuppressants may promote cell death thereby driving an inflammatory response that promotes fibrosis and raises concerns that a similar effect may occur within the liver. We evaluated apoptosis within the liver tissue of post-liver transplant patients and correlated these findings with in vitro experiments investigating the effects of immunosuppressants on apoptosis in primary hepatocytes.

Methods

Hepatocyte apoptosis was assessed using immunohistochemistry for M30 CytoDEATH and cleaved PARP in human liver tissue. Primary mouse hepatocytes were treated with various combinations of cyclosporine, tacrolimus, sirolimus, or MMF. Cell viability and apoptosis were evaluated using crystal violet assays and Western immunoblots probed for cleaved PARP and cleaved caspase 3.

Results

Post-liver transplant patients had a 4.9-fold and 1.7-fold increase in M30 CytoDEATH and cleaved PARP compared to normal subjects. Cyclosporine and tacrolimus at therapeutic concentrations did not affect hepatocyte apoptosis, however when they were combined with MMF, cell death was significantly enhanced. Cell viability was reduced by 46% and 41%, cleaved PARP was increased 2.6-fold and 2.2-fold, and cleaved caspase 3 increased 2.2-fold and 1.8-fold following treatment with Cyclosporine/MMF and Tacrolimus/MMF respectively. By contrast, the sirolimus/MMF combination did not significantly reduce hepatocyte viability or promote apoptosis.

Conclusion

Commonly used immunosuppressive drug regimens employed after liver transplantation enhance hepatocyte cell death and may thus contribute to the increased liver fibrosis that occurs in a proportion of liver transplant recipients.     

Effect of gamma irradiation on the immunological properties of the brucellar protective antigen

It was shown that the splenocytic response to mitogens in guinea pigs was activated 7 days following immunization thereof with a gamma-irradiated brucellar protective agent (gamma-BPA), while nonirradiated BPA inhibited lymphocyte proliferation under the effect of mitogens. gamma-BPA, as compared with BPA, circulated in blood for a longer time, induced a more rapid and prolonged synthesis of antibodies, and provided the development of a more intensive immunity.     

Cyclosporine A, an in vitro calmodulin antagonist, induces nuclear lobulations in human T cell lymphocytes and monocytes

Cyclosporine A is a noncytotoxic, natural, 11 amino acid cyclic peptide used clinically as an immunosuppressant to prevent organ rejection after transplantation. Cyclosporine A is an in vitro calmodulin antagonist. At the low concentrations required to inhibit calmodulin-dependent phosphodiesterase in vitro, cyclosporine A causes a dramatic alteration in the nuclear morphology of 23% of human peripheral blood mononuclear leukocytes in vitro without loss of viability. The shape of the nucleus changes from ovoid to a distinctive, radially splayed lobulated structure. The changes occur in a dose-dependent manner in 60 min at 37 degrees C. Specific monoclonal antibodies to human leukocytes identify the cells susceptible to nuclear lobulation by cyclosporine A as OKT4 antigen-positive T cell lymphocytes and monocytes. The lobulated nuclei are 2N as determined by flow cytometric measurement of ethidium bromide fluorescence of DNA. The cyclosporine A-induced lobulation of T cell nuclei requires both physiologic temperature and metabolic energy. Although structurally different than cyclosporine A, the calmodulin antagonists R24571 and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide] also produce T cell nuclear lobulations that are indistinguishable from the nuclear lobulations caused by cyclosporine A. These data indicate that nonmitotic structural elements that govern normal nuclear morphology in a subset of mononuclear leukocytes appear to require a calmodulin-mediated process. Cyclosporine A may be a useful noncytotoxic inhibitor of calmodulin-dependent systems that influence nuclear structure and function.     

IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究

将白细胞介素-2基因和猪细小病毒VP2基因主要抗原区克隆至pCI-neo真核表达载体中,构建了pCIneo-IL2-VP2重组质粒,用脂质体将其转染到PK-15细胞中,利用免疫荧光方法检测在体外表达情况。并以小鼠为动物模型,将pCIneo-IL2-VP2重组质粒、对照组pCI-neo和猪细小病毒活疫苗通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-IL2-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-IL2-VP2质粒1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-IL2-VP2能够有效诱导机体产生体液免疫和细胞免疫。