青藤碱抑制人外周血CD4+T淋巴细胞内NF-AT和IFN-γ表达的体外研究

目的 通过研究青藤碱(SIN)对人外周血CD4+T淋巴细胞内核因子-AT(NF-AT)和γ干扰素(IFN-γ)表达的影响来探讨其免疫作用机制.方法 免疫磁珠法分选人外周血CD4+T淋巴细胞,并分为5组进行培养.(1)阴性对照组:细胞培养液中不加入任何药物;(2)阳性对照组:细胞培养液中加入终浓度为50ng/ml的环孢素A(CsA)溶液;(3)低浓度青藤碱(L-SIN)组:细胞培养液中加入终浓度为10μmol/L的青藤碱溶液;(4)中浓度SIN(M-SIN)组:细胞培养液中加入终浓度为200 μmol/L的青藤碱溶液;(5)高浓度SIN(H-SIN)组:细胞培养液中加入终浓度为1000 μmol/L的青藤碱溶液.观察各组CD4+T淋巴细胞的增殖情况,蛋白印迹法检测各组细胞内NF-AT的蛋白表达,流式细胞术检测各组细胞内IFN-γ的表达水平.结果 与阴性对照组比较,M-SIN和H-SIN组明显抑制了CD4+T淋巴细胞的增殖(P＜0.01).青藤碱呈浓度依赖性抑制CD4+T细胞内NF-AT和IFN-γ的表达(P＜0.01),而加入CsA培养的阳性对照组NF-AT和IFN-γ表达最低.CD4+T淋巴细胞内NF-AT的表达与细胞增殖抑制率之间存在负相关关系(rs=-0.969,P=0.000).结论 青藤碱抑制人外周血CD4+T淋巴细胞内NF-AT和IFN-γ表达;其免疫作用机制可能是通过降低NF-AT的表达以抑制T淋巴细胞的活化和增殖.     

Estimation of individual sensitivity to cyclosporin in children awaiting renal transplantation.

BACKGROUND: Optimal immunosuppressive drug therapy requires that efficacy be balanced against toxicity. We have performed in vitro assays of cyclosporin (CsA) efficacy in children awaiting renal transplantation. METHODS: Peripheral blood mononuclear cells (PBMC) from 13 children awaiting renal transplantation and 10 healthy paediatric controls ("responders") were incubated in the presence of CsA (0-250 ng/ml). Irradiated PBMC from a parent (prospective live donor) were cultured with those of the child in the presence of interleukin 2. Europium-labelled, non-irradiated phytohaemagluttinin-stimulated target cells from the parent were added to the culture after 7 days incubation. Target cell lysis was quantified by time resolved fluorometry. CsA-mediated inhibition of target cell lysis was calculated and used to compare individual responses to the drug. Two-colour flow cytometry was performed to identify activated subsets of lymphocytes at varying concentrations of CsA. RESULTS: Wide inter-individual variations in per cent lysis and per cent inhibition were observed in patients and controls. Immunophenotyping indicated expansion of CD8(+) and CD25(+) lymphocyte subsets following allo-stimulation that was inhibited by increasing concentrations of CsA. Eight out of 13 patients and four out of 10 controls were "sensitive" to CsA in vitro in that they achieved 50% inhibition of cell lysis (IC(50)) at low concentrations of the drug (<50 ng/ml). Eleven patients have subsequently received a renal transplant. Five out of seven of these patients with IC(50) <50 ng/ml have suffered problems with infection, nephrotoxicity and graft vasculopathy raising the possibility of "over-immunosuppression". CONCLUSION: The data imply a useful role for this model in the prediction of individual response to immunosuppression following allo-stimulation in the pre-transplant setting.     

In vitro study of expression of interleukin-2 receptors in T-lymphocytes from patients with IgA nephropathy

The present study was undertaken to examine the expression of interleukin-2 receptor (IL-2R)/CD25 antigen in cultured T-lymphocyte subsets in IgA nephropathy. Twenty-four IgA nephritic patients, 12 patients with chronic glomerulonephritis (non-IgA nephropathy), and 17 healthy controls were studied in an infection-free interval. T-cell subsets in peripheral blood mononuclear cells (PBMC) and activated T-lymphocyte subsets expressing IL-2R were determined by double immunofluorescence staining with fluorochromes conjugated to monoclonal antibodies against T-helper/inducer (CD4+) cell, T-suppressor/cytotoxic (CD8+) cell, B (CD20+) lymphocytes, and IL-2R. The percentages of CD4+ and CD8+ lymphocytes, CD4/CD8 ratio, and total activated lymphocytes (with IL-2R/CD25 antigen) did not differ between the IgA nephritic patients, patients with chronic glomerulonephritis, and healthy controls in freshly isolated, unstimulated lymphocytes or PBMC cultured with pokeweed mitogen. Following pokeweed mitogen stimulation for 5 days, 17.3 +/- 10.3%, 16.6 +/- 8.4%, and 16.7 +/- 9.4% of PBMC from IgA nephritic patients, patients with chronic glomerulonephritis, and controls respectively expressed IL-2R (p greater than 0.05). However, the individual T-cell subsets bearing IL-2R were distinctly different between the IgA nephritic patients and patients with chronic glomerulonephritis or healthy controls. IgA nephritic patients had increased activated CD4+ lymphocytes (with IL-2R) (p less than 0.025) and reduced activated CD8+ lymphocytes (p less than 0.025). Our study suggests a defective immunoregulation in IgA nephropathy with enhanced T-helper/inducer and reduced T-suppressor/cytotoxic activity when stimulated with mitogen and probably, during clinical exacerbation.     

中央记忆T细胞和效应记忆T细胞亚群在肝癌患者外周血中分布特点分析

【摘要】 目的 研究肝癌患者外周血中CD45RO+记忆T细胞(memory T cells, Tm)表面标志CD45RO、CD62L和CCR7的表达,以了解CD45RO+CD62L+CCR7+T细胞(中央型记忆T细胞, central memory T cells,TCM)和CD45RO+CD62L-CCR7-T细胞(效应型记忆T细胞,effector memory T cells, TEM)亚群在肝癌患者外周血中的分布特点。方法〓获取正常人健康志愿者(正常对照组)28例和2011年8月至2013年8月的初诊慢性乙型病毒性肝炎患者(乙肝对照组)28例以及同期患有乙肝的肝癌患者(乙肝肝癌组)30例的外周静脉血,分离外周血单个核细胞(PBMC)。用多色流式细胞仪检测技术(polychromatic flow cytometry, PFC)检测外周血PBMC中记忆T淋巴细胞表面标志CD3、CD4、CD8、CD45RO、CD62L、CCR7的表达情况。结果〓乙肝肝癌组外周血PBMC中,CD4+T淋巴细胞和CD8+T淋巴细胞分别占外周血PBMC的比例显著高于乙肝对照组(28.15±12.32 VS 11.56±5.78,29.33±15.54 VS 13.11±6.64)(P<0.001,P=0.019);而较正常对照组无明显差异。乙肝肝癌组外周血PBMC的T淋巴细胞中CD4+TEM占CD4+T记忆淋巴细胞比例显著高于乙肝对照组、正常对照组(93.78±5.63 VS 75.82±19.74,93.78±5.63 VS 68.25±15.65)(P=0.001, P=0.018),而CD4+TCM则显著低于乙肝对照组、正常对照组(0.55±1.02 VS 4.87±6.28,0.55±1.02 VS 4.16±3.49)(P均小于<0.001)。三组的CD4+TEM记忆细胞占外周血PBMC中CD4+T记忆淋巴细胞的比例显著高于CD4+TCM记忆细胞占外周血PBMC中CD4+T记忆淋巴细胞的比例(91.78±5.63 VS 0.55±1.02,75.82±19.74 VS 4.87±6.28,68.25±15.65 VS 4.16±3.49)(P值均小于0.001)。乙肝肝癌组外周血PBMC的T淋巴细胞中CD8+TEM和CD8+TCM占CD8+T记忆淋巴细胞比例与乙肝对照组、正常对照组相比,差异均无显著意义。三组的CD8+TEM记忆细胞占外周血PBMC中CD8+T记忆淋巴细胞的比例显著高于CD8+TCM记忆细胞占外周血PBMC中CD8+T记忆淋巴细胞的比例(80.12±13.56 VS 3.25±2.47,79.21±11.34 VS 4.44±7.19,73.33±16.88 VS 2.66±3.10)(P值均小于0.001)。结论〓在乙肝肝癌组、乙肝对照组和正常对照组中,无论是CD4+T记忆淋巴细胞,还是CD8+T记忆淋巴细胞,均以TEM占绝大多数,而TCM比例很小。乙肝肝癌组中,CD4+TEM显著高于乙肝对照组和正常对照组,CD4+TCM显著低于乙肝对照组和正常对照组,而CD8+TEM和CD8+TCM则与乙肝对照组、正常对照组均无明显差异。     

Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclosporine A vs. tacrolimus) in stable renal allograft recipients

Recent multicenter, randomized clinical trials have shown that in renal transplant patients tacrolimus (FK506) was more efficient than cyclosporine A (CsA) at preventing acute rejection. In order to try and evaluate whether this difference was related to a different in vivo T-cell suppression we assessed, in a prospective study, the frequencies of interleukin (IL)-2-, IL-4-, IL-5-, IL-6-, IL-10-, interferon-gamma (IFN-gamma)- and double-positive IL-2/IFN-gamma-producing whole T cells, CD4 + and CD8 + T-cell subsets by means of cytokine flow cytometry. This was performed after in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with phorbol myristate acetate (PMA) and ionomycin, in the presence of monensin, in 14 healthy volunteers (controls) and in 14 renal transplant patients. The immunosuppression of the latter was based either on CsA (n = 7) or on FK506 (n = 7). Cytokine-expressing T-cell frequencies were assessed immediately pretransplantation (DO), and subsequently 3 months (M3) and 6 months (M6) afterwards in fasting patients prior to the morning intake of the immunosuppressive drug. We found that at DO the frequencies of IL-2-(22 +/- 2% vs. 22.2 +/- 2%), IFN-gamma-(26 +/- 3% vs. 29 + 3.4%) and IL-4-(0.8 +/- 0.2% vs. 1.4 +/- 0.2%)-expressing T lymphocytes were not significantly different between the controls and the patients, respectively. Conversely, the frequency of IL-2/IFN-gamma double positive cells was higher in the latter (9.3 +/- 1.6%) than in the controls (5.6 +/- 0.8); p = 0.06. Finally, on D0 the frequencies of IL-5-, IL-6-, and IL-10-producing T lymphocytes were lower than 1%, in both groups, as well as after grafting, i.e. on M3 and M6. As compared to baseline (DO): (a) chronic immunosuppression significantly decreased the frequencies of IL-2-, IL-4- and IL-2/IFN-gamma-expressing T cells, whereas those of IFN-gamma, IL-5, IL-6, and IL-10 were not significantly affected; (b) the frequencies of cytokine-expressing T cells were not statistically different between M3 and M6; (c) the decrease in the frequencies of IL-2- and IL-2/IFN-gamma-expressing T cells affected CD4 + and CD8 + cells equally; (d) there was a marginal decrease in the frequency of IFN-gamma-expressing cells only in the CD4 + subset but not in the CD8 population; and (e) for CsA, but not for FK506, the frequency of the IL-2-expressing T cells was negatively correlated with the whole blood trough levels. When we compared the frequencies of cytokine-expressing cells in FK506- and CsA-treated patients, we found that the frequency of IL-2-expressing T cells was significantly lower with FK506 (10.9+/-1.61%) than with CsA (16.3 +/- 1.8%; p = 0.03), whereas the frequencies of the other cytokine-expressing cells were not statistically different between the two groups. In conclusion, our study clearly demonstrated that studied ex vivo, FK506 and CsA decrease the frequencies of cells expressing IL-2, IL-4 and IL-2/IFN-gamma in vivo but do not affect those expressing IFN-gamma. Meanwhile, the frequency of IL-2-producing T cells was more affected with FK506 than with CsA and was negatively correlated with the CsA trough level. Finally, our results regarding IL-2 might explain to some extent the higher efficiency of FK506 in vivo than CsA.     

Absence of CD4CD25 regulatory T cell expansion in renal transplanted patients treated in vivo with Belatacept mediated CD28-CD80/86 blockade

AIMS: Belatacept is a new recombinant molecule (CTLA4-Ig) that interferes with the second activation signal of T lymphocytes. CTLA4-Ig induced T cell allograft tolerance in rodents but not in primates. We examined the changes in peripheral lymphocyte subsets, including regulatory T cells, in renal transplant patients treated with Belatacept. METHODS: A cross-sectional immunological study was carried out 6 months after transplantation in 28 patients enrolled in the Belatacept phase II study. Eighteen patients received Belatacept, mycophenolate mofetil and steroids (Belatacept group), while the control group of 10 patients received cyclosporine, mycophenolate mofetil and steroids (CsA group). Lymphocyte subsets were examined by flow cytometry. Foxp3 mRNA expression was measured by quantitative PCR. RESULTS: The number of T lymphocytes and the percentage of CD3+ T cells were similar in both groups. However, the percentage of CD3+ CD4+ T cells was lower in the Belatacept group than in the control CsA group (B=42.5%+/-13.7 vs CsA=52.9%+/-9, p<0.005), and the percentage of CD3+ CD8+ cells was higher in the Belatacept group than in the control (B=32.9%+/-6.7 vs CsA=19.5%+/-8.2, p<0.0002). The percentage of CD19+ cells was similar in both groups. Among CD56+cells, only the percentage of CD16+ cells was significantly higher in the Belatacept group than in the control (B=82%+/-12 vs CsA=59.7%+/-25, p=0.01). Among CD4 and CD8 T cells the percentage of activated lymphocytes expressing CTLA4, HLA-DR or CD40L was similar in both groups. The percentage of CD4+CD25+ T cells was higher in the CsA group. The percentage of regulatory CD4+CD25+ cells with bright CD25 staining was similar in both groups (B=3.6+/-2.3% vs CsA=4.7+/-1.9%, ns) as was the expression of FoxP3. CONCLUSION: Our results indicated that Belatacept did not induce regulatory T cell expansion in vivo. We suggest that Belatacept treatment should be maintained after transplantation to allow graft acceptance.     

The clinical use of various blood compartments for cytomegalovirus (CMV) DNA quantitation in transplant recipients with CMV disease

BACKGROUND: The quantitation of cytomegalovirus (CMV) DNA is a cornerstone in the management of CMV disease in transplant recipients. However, a consensus as to what is the optimal blood compartment for the detection and quantitation of CMV DNA in peripheral blood is nonexistent. METHODS: With an automated quantitative assay, we have simultaneously quantified the CMV DNA load in whole blood (WB), plasma (PL), peripheral blood leukocytes (PBL), and peripheral blood mononuclear cells (PBMC) in 319 samples from 17 transplant recipients with 19 episodes of CMV disease that were treated with 2 weeks of intravenous ganciclovir. RESULTS: Higher levels of CMV DNA were observed in WB than PL (PL minus WB mean difference, 0.67 log; 95% confidence interval, -1.02 to -0.32; P=0.0009). This observation was most evident before treatment with intravenous ganciclovir (pretreatment geometric mean CMV DNA was 45,412 copies per ml of WB vs. 14,995 copies per ml of PL). In contrast, the CMV DNA levels between PBL and PBMC were highly comparable throughout the course of CMV disease and its treatment. Intravenous ganciclovir exerted a uniform effect on the four blood compartments with no statistically significant difference in the degree and rate of CMV DNA decline between WB and PL and between PBL and PBMC. CONCLUSIONS: Although our study demonstrates the adequacy of all blood compartments for CMV DNA quantification, the higher sensitivity of WB and its yield of higher CMV DNA render it an optimal sample for monitoring CMV DNA load during CMV disease in immunocompromised patients.     

大鼠心脏移植术后弓形虫感染时淋巴细胞亚群变化的观察

目的 观察大鼠器官移植术后机体免疫状态与环孢素A(CsA)使用和弓形虫发病的关系。方法 流式细胞术测定移植术后第5、10、15及20受体的T淋巴细胞亚群的变化。结果 CD4^ 和CD8^ T淋巴细胞酚率均有变化；使用环孢素组术后5d CID8^ T淋巴细胞较术前明显升高；术后10d，无论是否使用环孢素，CD8^ T淋巴细胞亦较术前明显升高；感染发作时CD8^ T淋巴细胞明显升高，CD4^ ／CD8^ 比值降低或倒置；移植术后供体携带病原体可致受体CD4^ ／CD8^ 比值低于受体隐性感染组的比值。结论 器官移植术后CsA使用导致免疫抑制，CD4^ 和CD8^ T淋巴细胞均参与对弓形虫的免疫作用；感染发作时CD8^ 明显升高，是主要的细胞毒细胞；CD4^ ／CD8^ 比值可用于评估机体免疫状态，预测弓形虫感染的发生。     

The effect of the CD28 activation pathway on the immunosuppressive action of cyclosporine.

The effect of the CD28 activation pathway on the immunosuppressive action of CsA was assessed. Human peripheral blood lymphocytes were stimulated with anti-CD3, bryostatin (Bryo) a novel activator of protein kinase C (PKC) and anti-CD28 singly or in combination, to which graded doses of CsA were added to determine relative sensitivity. Proliferation, IL-2 production, and IL-2 receptor expression were assessed and the IC50 determined. Lymphocytes stimulated with Bryo exhibited a marginal proliferative response but expressed the IL-2 receptor despite the presence of CsA. Addition of anti-CD3 or anti-CD28 to Bryo-stimulated lymphocytes promoted a vigorous proliferative response. CsA effectively inhibited the proliferative response and IL-2 production induced with anti-CD3 and Bryo but did not inhibit the response of cells stimulated with anti-CD28 and Bryo. However, II-2 receptor expression in both sets of cultures were comparable due to the induction of IL-2 receptor by Bryo and was not inhibited by CsA. Costimulation of lymphocytes with anti-CD3 plus anti-CD28 resulted in a 2-3-fold enhancement of proliferation compared with lymphocytes stimulated with anti-CD3 alone. Addition of CsA to lymphocytes stimulated with anti-CD3 resulted in the dose-dependent suppression of the proliferative response and IL-2 production (IC50 = 10-25 nM) but less so for IL-2 receptor expression (IC50 = 100-150 nM). In comparison, the proliferative response and IL-2 production elicited by anti-CD3 + anti-CD28 was more resistant to the effects of CsA (IC50 = 100-200 nM). However, IL-2 receptor expression exhibited comparable sensitivity to CsA (IC50 = 100-200 nM) in the presence of anti-CD28. Combination drug:drug studies revealed that CsA and the protein kinase C inhibitor H-7 were additive for both anti-CD3 and anti-CD3 plus anti-CD28 response. On the other hand, the cGMP-dependent protein kinase inhibitor H-8 was synergistic with CsA in inhibiting the response of lymphocytes to anti-CD3 plus anti-CD28 but only additive for responses to anti-CD3. Taken together, these data suggest that CsA inhibits T cell activation at two distinct levels, leading to inhibition of IL-2 production and inhibition of IL-2 receptor expression. Activation of the CD28 pathway partially overcomes the inhibitory activity of CsA on IL-2 production and may be mediated by indirect activation of a cGMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of rapamycin on cytokine profile in kidney transplant recipients treated with triple drug therapy

The aim of this study was to explore differences in the cytokine profile among de novo kidney transplant recipients treated with either Rapamycin (Rapa) + cyclosporine (CsA) + prednisone (P) or CsA + azathioprine (Aza) + P.

Patients and methods

Among the 13 adult kidney transplant recipients studied, seven received Rapa + CsA + P while the remaining six received CsA + Aza + P with their living donors serving as controls (n = 13). Spontaneous production of IL-2, IFNγ, IL-10, and TGF-β were measured by ELISA in supernatants from 24-hour cultured unstimulated peripheral blood mononuclear cell (PBMC) at time zero (the day before the transplant), and at 3 and 6 months posttransplant. Cytokines were also measured 1 month after CsA withdrawal in the Rapa + CsA + P group.

Results

From time zero to the end of the study, IL-2, IFNγ, and IL-10 were present at low or undetectable levels in all three groups. TGF-β tended to increase in supernatants from patients under Rapa + CsA + P at 6 months posttransplant and at 1 month after CsA withdrawal without correlation to Rapa blood levels. TGF-β remained stable throughout the study period for patients included in the CsA + Aza + P group. There was no difference in this cytokine level between these study groups at any given time.

Conclusions

This study showed no differences in the spontaneous cytokine profiles evaluated in patients treated with both therapeutic schemes.     

A limited sampling strategy for the estimation of 12-hour cyclosporine neoral area under the curve in Chinese cardiac transplant recipients

INTRODUCTION: This study determined the accuracy of a limited sampling strategy to predict the 12-hour cyclosporine neoral (CsA) area-under-the-curve (AUC) to provide a practical method for more accurate therapeutic drug monitoring (TDM) of CsA in Chinese heart transplant recipients. METHODS: Blood samples were collected at 0 (before the dose), and 0.5, 1, 2, 3, 4, 6, 8, 10, as well as 12 hours after CsA oral administration in 13 de novo heart recipients receiving oral CsA bid after rabbit antithymoglobulin sequential immuno-induction. Pharmacokinetics were analyzed for the first dose (PK-1) and the steady state dose (PK-2, 1 month after transplantation). The limited sampling strategies included single-point, 2-point, and 3-point prediction of AUC using multiple linear regression analyses. RESULTS: Comparing the AUC/mg dose, PK-1 was much lower than PK-2 (25.2 +/- 11.4 ng x h/mg x mL vs 45.4 +/- 12.9 ng x h/mg x mL; P = .0005 using paired t test). The correlations of each single-point blood level of PK-1 with the AUC were lower than those of the corresponding sampling time in PK-2. In the PK-2 study, C4 had the best correlation (r2 = 0.732; P = .00) as a single-point to predict AUC, but the 2-point C2 + C12 had a higher correlation (r2 = 0.937; P = .00). Among the 3-point combinations, C2 + C4 + C12 showen the best prediction (r2 = 0.982; P = .00) of the AUC in PK-2. CONCLUSION: The bioavailability of CsA was lower in PK-1 than in PK-2. At steady state, we recommend C2 + C12 to predict AUC because it is accurate and not labor-intensive.     

C0h/C2h monitoring of the pharmacodynamics of cyclosporin plus mycophenolate mofetil in human heart transplant recipients

Pharmacokinetic (PK) parameters like C2h have improved efficacy of immunosuppressive therapy. However, drug interactions, toxicities, and individual differences to drug effects still remain challenging. Therefore, this study was designed to assess pharmacodynamic (PD) effects of the combination cyclosporin (CsA) plus mycophenolate mofetil (MMF) on lymphocyte functions in peripheral blood of stable heart transplant recipients (HTx) using our established FACS assays. METHODS: Blood from 25 HTx patients was drawn before (C0h) and 2 hours after dosing (C2h). CsA and mycophenolic acid (MPA) concentrations were measured by EMIT. FACS assessed expression of cytokine production (IL-2, TNF-alpha), lymphocyte proliferation (PCNA), and T-cell activation (CD25, CD95). RESULTS: Evening doses of CsA (25/50/75 or 100 mg) and MMF (250/500 or 1000 mg) produced C0h levels as follows: CsA, 162 +/- 12 ng/mL; MPA, 1.7 +/- 0.2 mg/L. Morning doses of CsA (50/75 or 100 mg) and MMF (250/500/1000 or 1500 mg) produced C2h-levels as follows: CsA, 589 +/- 56 ng/mL and MPA, 7.4 +/- 1.3 mg/L. PD effects at C0h/C2h (% expression +/- SEM, all P < .05) were IL-2, 18 +/- 3/10 +/- 2; TNF-alpha, 12 +/- 2/7 +/- 1; PCNA, 8 +/- 1/5 +/- 1; CD25, 26 +/- 4/13 +/- 2; CD95, 23 +/- 4/11 +/- 2). Correlations (r2) at time point C2h between inhibition of lymphocyte functions (PD) with drug concentrations (PK) and with drug doses were CsA-PK, 0.71 to 0.91; MMF-PK, 0.55 to 0.76; CsA-dose, 0.73 to 0.87; MMF-dose, 0.61 to 0.80. CONCLUSION: For the first time, the immunosuppressive effects of the combination CsA plus MMF were quantified in whole blood of human HTx at different time points. PD assays may offer the opportunity to optimize clinical immunosuppressive drug therapy.     

Immunologic evaluation during the first year of life of infants born to cyclosporine-treated female kidney transplant recipients: analysis of lymphocyte subpopulations and immunoglobulin serum levels

BACKGROUND: In rodents, CsA has been shown to affect T-cell development, giving rise to an abnormal production of mature T cells and the absence of many T-cell subsets as well as to autoimmunity. Surprisingly, only a few studies investigated the effect of the immunosuppressive drug on the immune system of the human fetus. METHODS: We examined six infants born to female kidney transplant recipients who had received cyclosporine and methylprednisolone throughout their pregnancies. Peripheral blood was obtained 1 day and 2, 4, 6, and 12 months after birth, and two-color flow cytometric immunophenotyping of lymphocytes was performed. RESULTS: Total T cells, as well as CD4+ and CD8+ T cells, were low at birth, but normalized thereafter. Among T-cell activation markers, the expression of CD25, the alpha chain of the interleukin-2 receptor, was below the normal range or low range throughout the study period, and HLA-DR expression was extremely low at birth and failed to increase up to 12 months. The number of total B cells was lower than normal at birth, but steeply increased over time. In contrast, B-cell subset bearing CD5 antigen was severely depleted throughout the first year of life. Total IgG concentration was significantly lower than in controls at 2 months, mainly because of subnormal levels of IgG1 and IgG3 subclasses, which remained in the low range up to 6 months. Finally, infants showed normal numbers of true natural killer (NK) cells (CD3-CD16+CD56+), whereas the expression of CD57 antigen, defining non-MHC-restricted cytotoxic lymphocytes, was barely detectable at birth and failed to increase over time, in both CD8+ and CD8- subsets. Of note, none of the infants had clinical evidence of an immunodeficient state. CONCLUSIONS: continuous exposure to CsA in utero seemingly impairs T-, B-, and NK-cell development and/or maturation, and most of its effects are still apparent at 1 year, which might suggest that conventional vaccinations should be delayed in these infants.     

Declining intracellular T-lymphocyte concentration of cyclosporine a precedes acute rejection in kidney transplant recipients

BACKGROUND: We investigated cyclosporine A (CsA) concentrations at the site of action, inside T-lymphocytes, to evaluate its applicability as a new supplementary therapeutic drug monitoring method after renal transplantation. METHOD: In this prospective single-center study, 20 kidney transplant recipients, mean age 54 (range 21-74) years, on CsA-based immunosuppression were included within 2 weeks posttransplant and followed for 3 months. Nine patients also had one full 12-hour pharmacokinetic profile performed. T-lymphocytes were isolated from 7 ml whole blood using Prepacyte and intracellular CsA concentrations were determined using a validated liquid chromatography double mass spectrometry method. RESULTS: Seven patients (35%) experienced acute rejections (all biopsy verified) during the first three months posttransplantation. Intracellular CsA concentrations tended to decline 1 week prior to acute rejection and the decrease was significant (-27.1+/-14.6%, P=0.014) three days before the rejection episodes were recognized clinically. In addition, the intracellular CsA area under the curve 0-12 measured during stable phase was 182% higher in the rejection-free patients (P=0.004). There was no difference between patients experiencing rejection and the rejection-free patients with respect to CsA C2-levels, dose (mg/kg), human leukocyte antigen mismatch, donor age, recipient age, or ABCB1 genotyping. CONCLUSION: Intracellular CsA T-lymphocyte concentrations declined significantly 3 days prior to a rejection episode and there was a general lower intracellular exposure of CsA in recipients experiencing rejection. Intracellular measurement of CsA therefore seems to have a potential to further improve individualization of therapeutic drug monitoring. Larger studies are needed to elucidate the role for intracellular T-lymphocyte measurements in ordinary clinical care, for both CsA and other immunosuppressive drugs.     

In vitro analysis of verapamil-induced immunosuppression: potent inhibition of T cell motility and lymphocytic transmigration through allogeneic endothelial cells

BACKGROUND: Cyclosporine A (CsA) and tacrolimus prevent proliferation but not transendothelial migration of alloreactive lymphocytes into donor organs. As a result, serious adverse effects, such as nephrotoxicity and neurotoxicity, have been observed under CsA/tacrolimus therapy. The incorporation of new drugs with infiltration blocking properties might enhance the efficacy of the current immunosuppressive protocol, allowing lower CsA/tacrolimus dosage. Because Ca2+ plays a critical role in cell-cell interaction, the Ca2+-channel blocker verapamil might be a good cany. didate for supporting CsA/tacrolimus-based therapy. METHODS: A T-cell endothelial cell coculture model or immobilized immunoglobulin G globulin chimeras were employed to investigate how S- and R- verapamil interfere with the lymphocytic infiltration process. The expression and arrangement of membranous adhesion receptors and cytoskeletal F-actin filaments were analyzed by fluorometric method in the presence of. verapamil. RESULTS: Both verapamil enantiomers strongly inhibited lymphocyte infiltration. CD4+ and CD8+ T-cells were influenced to a similar extent with regard to horizontal locomotion (CD4+=CD8+), but to a different extent with regard to adhesion and penetration (CD4+ > CD8+). Moreover, penetration was blocked to a higher extent than was adhesion. ID50-values were 31 microM (CD4+-adhesion) and 11 microM (CD4+-penetration). Verapamil reduced P-selectin expression on endothelial cells and effectively down-regulated binding of T-cells to immobilized P-selectin immunoglobulin G globulins (ID50=4.4 microM; CD4+). A verapamil-induced reduction of intracellular F-actin in T-lymphocytes was proven to be mainly responsible for diminished cell locomotion. CONCLUSIONS: The prevention of CD4+ T-cell penetration by verapamil might argue for its use as an adjunct to CsA/tacrolimus-based immunosuppressive therapy.     

Management of Living Donor Liver Transplant Patients Using Twice-Daily 4-Hour Intravenous Cyclosporine Therapy

Oral administration of cyclosporine (CsA) is the currently favored route in most liver transplant centers. From October 1998 to January 2008, 86 living donor liver transplantations (LDLTs) were performed in 85 patients (46 adults and 39 children) at our institution. Seventy-three patients received tacrolimus (Tac), and 12 intravenous CsA twice daily at a dose of 3 mg/kg/d as a 4-hour continuous infusion. Thirteen of 73 Tac-based patients were switched to CsA because of side effects. Five were switched to intravenous CsA because they were unable to take the drug orally because of severe Tac-related complications. The remaining eight patients switched to oral CsA. We evaluated patients (11 adults and three children), including 12 with induction therapy and two with conversion therapy within 2 weeks of LDLT. The patients were given a 4-hour intravenous infusion of CsA at an initial dose of 3 mg/kg/d. Stable and adequate blood CsA concentrations were achieved by 4-hour intravenous CsA administration. Among several factors, only graft-to-recipient weight ratio (r = .743, P < .0001) showed significant correlations with initial blood CsA concentration. No adverse effects were observed after intravenous CsA. No patients developed biopsy-proven acute cellular rejection (ACR) during intravenous CsA administration, whereas two patients had histopathologically diagnosed episodes of ACR after conversion from intravenous to oral CsA. Our findings suggest that immediate administration of a 4-hour intravenous infusion of CsA at an initial dose of 3 mg/kg/d is practical and effective for routine clinical use.     

An increase in the survival of murine H-2-mismatched cultured fetal pancreas allografts using depleting or nondepleting anti-CD4 monoclonal antibodies, and a further increase with the addition of cyclosporine

Depletion of CD4+ T lymphocytes with monoclonal antibodies (mAbs) has been shown to prolong allograft survival in mice. In this study, two rat anti-CD4 mAbs, H129.19 and GK1.5, were administered either alone or in combination with cyclosporine (CsA) to recipients of MHC-mismatched (H-2k to H-2d) cultured fetal pancreas allografts to determine their effect on graft survival. When compared with control mice, splenic CD4+ cells of GK1.5-treated mice were depleted by greater than 95%, but in H129.19-treated mice no depletion of CD4+ cells occurred. Instead, rat Ig was present on the surface of CD4+ cells in H129.19-treated mice. Anti-CD4 therapy with either H129.19 or GK1.5 prolonged fetal pancreas allograft survival to a similar extent, but did not lead to indefinite survival. Blockade of the CD4 antigen by the mAb H129.19 was as effective as the depletion of CD4+ cells by GK1.5 in prolonging allograft survival. Rejection of grafts by day 28 posttransplantation occurred in the absence of CD4+ cells, as determined by both flow cytometric examination of spleen cells and immunoperoxidase staining of the graft site. CsA alone did not prolong graft survival, but its addition to either H129.19 or GK1.5 mAb treatment significantly increased the survival rate of grafts at 28 days compared with mAb treatment alone. These results suggest that CD4+ cell depletion is not essential for effective anti-CD4 mAb therapy--and, further, that CsA may have a direct inhibitory effect on CD8+ cells during allograft rejection.     

Effect of cyclosporine on T lymphocyte development. Relationship to syngeneic graft-versus-host disease

This report investigates the effects of cyclosporine on the reconstitution of T lymphocytes after syngeneic bone marrow transplantation and its role in the development of a novel T cell-mediated autoimmune disease, syngeneic graft versus host disease. We analyzed the effect of CsA treatment on T lymphocyte differentiation during reconstitution after bone marrow transplantation and correlated the maturation of CD4+ and CD8+ T cell subsets with the onset of syngeneic GVHD. Administration of CsA following syngeneic bone marrow transplantation leads to a developmental arrest of mature CD4+ and CD8+ T lymphocytes in the thymus and a marked reduction in cells expressing the alpha beta T cell receptor. The reduction of CD4+ and CD8+ T cell subsets is also reflected in the peripheral lymphoid compartment with an altered CD4/CD8 ratio. Functional assessment of the cells revealed that CD8+ cells respond normally to mitogenic signalling whereas CD4+ cells exhibit marginal proliferative responses. Both subsets of T lymphocytes respond to syngeneic B lymphoblasts, comparable to the response of T lymphocytes from non-CsA-treated syngeneic BMT recipients, suggesting that autoreactive cells are produced despite CsA treatment. Following discontinuation of CsA, T cell differentiation in the thymus is rapidly restored to normal. However, concurrent with the onset of syngeneic GVHD, a compensatory insurgence of CD4+ T helper cells is observed.     

Dyslipidemia can reduce the immunosuppressive effects of cyclosporine

AIMS: Dyslipidemia is a significant risk factor for the development of atherosclerotic disease and of chronic allograft rejection. Few data are available on the effects of dyslipidemia on the immunosuppressive action of immunosuppressive agents. We investigate the in vitro effects of lipids solution on the immunosuppressive action of cyclosporine (CsA). METHODS: Peripheral blood mononuclear cells (PBMC) were PHA or OKT3 activated in vitro with/without different concentrations of Intralipid solution (INT, range 0.5% to 15%). CsA inhibition of activation was measured after a 3 day incubation, by adding H3-thimidine. The intracellular concentration of CsA was measured by radioimmunoassay and related to the CsA inhibitory effects. RESULTS: Increasing INT concentration in the medium, CsA inhibition of PBMC activation by PHA or OKT3 was reduced from 72+/-13% to 8+/-2% and from 80+/-10% to 18+/-3%, respectively. A significant reduction of the intracellular CsA concentration was also evident with increasing INT concentrations and was related to the inhibitory activity of CsA. CONCLUSIONS: These results suggest that dyslipidemia may reduce the availability of intracellular CsA concentration to inhibit the immune activation process and may explain the relationship between dyslipidemia and chronic allograft loss.