铁离子沉积与心脏移植急性排斥反应相关性研究

目的：探讨铁离子在心脏移植急性排斥反应的作用。 方法： 建立小鼠颈部异位心脏移植模型，实验分为同系移植组（C57BL/6→C57BL/6）和同种异体移植组（BALB/c→C57BL/6），普鲁士蓝染色观察铁离子在心肌组织的沉积情况，免疫组化法观察HO-1在心肌组织的表达。 结果： 铁离子在浸润的巨噬细胞中沉积，HO-1主要在浸润的炎性细胞中表达，两者随急性排斥反应的加重表达上调。 结论： 铁离子沉积与心脏移植急性排斥反应的病理过程相关，且可作为心脏移植急性排斥反应的监测指标。     

CD40-deficient dendritic cells producing interleukin-10, but not interleukin-12, induce T-cell hyporesponsiveness in vitro and prevent acute allograft rejection.

The induction of an immune response or tolerance is mediated by corresponding subsets of dendritic cells (DC). However, the property of tolerogenic DC is not clear. Recently, we have characterized a population of CD11c+ splenic DC derived from long-term mixed leucocyte culture (LT-MLC), which are able to proliferate upon stimulation and have a strong primary mixed leucocyte reaction (MLR)-stimulating activity in conventional MLR. In this study, we show that, in contrast to the irradiated ones, non-irradiated LT-MLC-derived DC induce polyclonal antigen-specific T-cell hyporesponsiveness when cocultured with allogeneic splenocytes for 3-11 days. The degree of the hyporesponsiveness increased with the length of coculture. Although these DC expressed major histocompatibility complex class II and B7 costimulatory molecules, which are down-regulated during coculture, they expressed very low or undetectable CD40 before and after coculture, respectively. The CD40-deficient DC spontaneously produce interleukin-10 (IL-10), but not IL-12. The skewed balance between IL-10 and IL-12 is associated with their capability to induce T-cell hyporesponsiveness, because a neutralizing antibody to IL-10, exogenous recombinant IL-12 or lipopolysaccharide (LPS) significantly blocked the hyporesponsiveness. Accordingly, infusion of a small number of non-irradiated LT-MLC-derived DC (5x105) significantly prolonged the survival of a vascularized heterotopic murine heart transplant, whereas irradiated DC accelerated graft rejection. These data suggest that CD40-deficient DC producing IL-10, but not IL-12 can induce T-cell hyporesponsiveness in vitro and in vivo.     

Comparison of thymocyte development and cytokine production in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice

CD7 and CD28 are Ig superfamily molecules expressed on thymocytes and mature T cells that share common signaling 0mechanisms and are co-mitogens for T cell activation. CD7-deficient mice are resistant to lipopolysaccharide (LPS)-induced shock syndrome, and have diminished in vivo LPS-triggered IFN-gamma and tumor necrosis factor (TNF)-alpha production. CD28-deficient mice have decreased serum Ig levels, defective IgG isotype switching, decreased T cell IL-2 production and are resistant to Staphylococcus aureus enterotoxin-induced shock. To determine synergistic roles CD7 and CD28 might play in thymocyte development and function, we have generated and characterized CD7/CD28 double-deficient mice. CD7/CD28-deficient mice were healthy, reproduced normally, had normal numbers of thymocyte subsets and had normal thymus histology. Anti-CD3 mAb induced similar levels of apoptosis in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient thymocytes as in control C57BL/6 mice (P = NS). Similarly, thymocyte viability, apoptosis and necrosis following ionomycin or dexamethasone treatment were the same in control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice. CD28-deficient and CD7/CD28-deficient thymocytes had decreased [3H]thymidine incorporation responses to concanavalin A (Con A) stimulation compared to control mice (P < or = 0.01 and P < or = 0.05 respectively). CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 double-positive cells compared to freshly isolated wild-type, CD7-deficient and CD28-deficient thymocytes. Con A-stimulated CD4/CD8 double-negative (DN) thymocytes from CD7/CD28 double-deficient mice expressed significantly lower levels of CD25 when compared to CD4/CD8 DN thymocytes from wild-type, CD7-deficient and CD28-deficient mice (P < 0.05). Anti-CD3-triggered CD7/CD28-deficient thymocytes also had decreased IFN-gamma and TNF-alpha production compared to C57BL/6 control, CD7-deficient and CD28-deficient mice (P < or = 0.05). Thus, CD7 and CD28 deficiencies combined to produce abnormalities in the absolute number of B7-1/B7-2-expressing cells in the thymus, thymocyte IL-2 receptor expression and CD3-triggered cytokine production.     

Verapamil does not prevent isoproterenol-induced cardiac hypertrophy

To assess the role of verapamil (VER)-sensitive calcium (Ca2+) channels in the regional development and pathogenesis of isoproterenol (ISO)-induced cardiac hypertrophy, male Fischer rats (150-175 g, N = 51) were divided into control (C) and ISO-treated (I) and VER (V1, 100 mg/L drinking water; V2, 10 mg/kg ip twice daily) and no-VER subgroups. Rats began VER treatment 1 week before isoproterenol which was given subcutaneously by minipump (3 mg/kg/d) for 7 days. Mean total heart weight of all I rats was larger than C rats (p less than 0.05). Regionally, percent hypertrophy of the right ventricle (RV) was greater (p less than 0.05), while that of the atria tended to be greater (p = ns) than the left ventricle plus septum in ISO-treated rats (LVS: I, 20 +/- 5%; V1I, 19 +/- 3%; V2I, 22 +/- 6%; RV: I, 37 +/- 10%; V1I, 40 +/- 11%; V2I, 34 +/- 7%; atria: I, 64 +/- 21%; V1I, 54 +/- 56%; V2I, 51 +/- 28%). The total and regional hypertrophy of the heart produced by ISO was not affected by VER treatment. Isoproterenol produced diffuse and focal myocardial fibrosis in the subendomyocardium of the LVS with occasional foci of fibrosis in the RV and the subepimyocardium of the LVS. Quantitative analysis of picrosirius red-stained tissue sections demonstrated an increased volume percent of myocardial connective tissue limited to the subendomyocardium of the LVS in response to ISO (p less than 0.05). Verapamil partially reduced the ISO-induced fibrosis (p less than 0.05) (I, 7.04 +/- 1.01%; V1I, 4.06 +/- 0.28%; V2I, 3.87 +/- 0.27%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of B-cell death does not restore T-cell-dependent immune responses in CD40-deficient mice

Signalling through CD40 is essential for the development of immunoglobulin G (IgG) antibody responses, germinal centres and B-cell memory against T-dependent antigens. In addition, engagement of CD40 in B cells promotes cell survival by inducing the expression of anti-apoptotic members of the bcl-2 family of cell-death regulators. In the present study we analysed whether T-dependent immune responses can be developed in mice deficient in CD40 if the anti-apoptotic activity mediated by the engagement of CD40 in B cells is compensated by the constitutive over-expression of anti-apoptotic genes of the bcl-2 family. We showed that the over-expression of either hbcl-2 or hbcl-xL transgenes in B cells is not sufficient to restore IgG antibody responses and germinal centre formation in CD40-deficient mice. These results indicate that CD40 functions, other than those mediated through survival, are required for the establishment of T-dependent B-cell responses.     

肾移植急性排斥反应时B7-2/CD28信号通路的表达*☆

背景：以往动物研究表明,在器官移植急性排斥反应时共刺激分子的表达与急性排斥反应密切相关。 目的：观察急性排斥反应时患者移植肾脏组织和外周血中B7-2/CD28信号通路的表达。 方法：对53例同种异体肾移植患者于移植前1 d、移植后1,3,7,14,21,28 d分别取外周血以及在临床诊断急性排斥反应当天和抗排斥治疗1周后额外采血,用流式细胞仪检测共刺激分子B7-2/CD28在外周血淋巴细胞中的表达;同时,行经皮肾穿刺活检供肾修整结束时、移植后7 d、1个月、6个月、1年或以上获取活检肾脏组织,用免疫组织化学方法检测活检组织中B7-2/CD28的表达情况。 结果与结论：移植后1,3 d内所有患者外周血中CD28+,CD4+/CD28+,CD8+/CD28+细胞比率均有显著下降(P < 0.05),一二周后恢复到术前水平;移植后7 d未发生急性排斥反应的患者肾脏组织B7-2阳性表达率显著上升(P < 0.05),1个月后下降至移植前水平(P > 0.05)。移植后发生急性排斥反应的患者外周血CD28+,CD4+/CD28+,CD8+/CD28+细胞比率及肾脏组织B7-2阳性表达率明显上升(P < 0.05),经抗排斥治疗1周后均好转。结果证实,在肾移植后出现急性排斥反应时,肾脏组织以及外周血中共刺激分子B7-2/CD28的表达上调与急性排斥反应的发生密切相关。       

环孢菌素A抑制小鼠同种心脏移植急性排斥反应中Lymphotactin表达的研究

目的：研究小鼠同种心脏移植急性排斥反应中淋巴细胞趋化因子（lptn）的表达情况及环孢菌素A（cyclosporine A, CsA）的抑制作用。 方法： 改良Banff评分系统判断同种小鼠移植心急性排斥反应程度，RT-PCR检测移植心组织内淋巴细胞趋化因子表达水平，ELISA方法检测心脏移植小鼠脾细胞活化T细胞核因子(NFAT)活性。 结果： C57BL/6-Balb/c急性排斥组小鼠移植术3 d后脾脏显著增大。术后第5、7 d移植心肌间淋巴细胞浸润程度评分分别为2.667±0.577和2.333±0.577。C57BL/6-Balb/c+CsA组小鼠移植术后脾脏肿大明显减轻，术后第5、7 d心肌间淋巴细胞浸润程度评分分别为1.000±0.000和1.333±0.577。急性排斥组和CsA处理组小鼠移植心脏在术后第5 d和第7 d都可检测到Lptn mRNA阳性表达，但CsA处理组Lptn mRNA的表达明显弱于急性排斥组。治疗剂量的CsA可以完全抑制NFATc1活性。 结论： Lptn在早期移植免疫事件中具有重要的作用，CsA仅能部分抑制Lptn mRNA的表达。活化T细胞Lptn的表达调控存在NFAT以外的途径。     

Prior exercise training does not prevent acute cardiac alterations after myocardial infarction in female rats

OBJECTIVE:

This study aimed to investigate whether previous exercise training could prevent or attenuate acute cardiac alterations after myocardial infarction.

METHODS:

Female rats were submitted to swim training (1 h/day; 5 days/week) or allowed to remain sedentary for 8 weeks. Afterwards, they were randomly assigned to left coronary artery occlusion or sham surgery. After this procedure, the rats remained sedentary for one week until euthanasia. Cardiac structural and functional analyses were performed using Doppler echocardiography. The rats that had a moderate or large infarct size were included in the evaluations. The data (mean ± SEM) were analyzed using a two-way ANOVA model followed by Tukey''s post-hoc test.

RESULTS:

After the surgery, no significant difference between the exercise and sedentary groups was observed in the left ventricular infarct sizes (34.58±3.04 vs. 37.59±3.07). In another group of rats evaluated with Evans blue 1 h after myocardial infarction, no siginificant difference in the area at risk was observed between the exercised and sedentary rats (49.73±1.52 vs. 45.48±3.49). The changes in the left ventricular fractional areas for the exercised and sedentary myocardial infarction groups (36±2% and 39±3%, respectively) were smaller than those for the exercise sham surgery (ES, 67±1%) and sedentary sham surgery (SS, 69±2%) groups. The E/A was higher in the sedentary myocardial infarction (4.4±0.3) and exercised myocardial infarction (5.5±0.3) rats than in the SS (2.4±0.1) and ES (2.2±0.1) rats.

CONCLUSION:

Previous swim training of female rats does not attenuate systolic and diastolic function alterations after myocardial infarction induced by left coronary artery occlusion, suggesting that cardioprotection cannot be provided by exercise training in this experimental model.     

The host response of CD28-deficient mice to Pneumocystis infection

Pneumocystis infection leads to a life threatening pneumonia in susceptible individuals. While depletion or dysfunction of CD4+T cells is a key determinant of susceptibility to Pneumocystis, the host response that leads to resolution of infection or lung injury is less well understood. We had previously shown that mice deficient in the T cell costimulatory molecule CD28 are susceptible to infection with Pneumocystis. A detailed analysis revealed that they clear Pneumocystis with delayed kinetics. This is associated with an influx of naïve CD8+T cells. Depletion of CD8+T cells did not alter organism burden, suggesting these cells are not responsible for clearance. Analysis of the cytokine milieu demonstrated a consistent increase in mRNA for IL-10 and IFN-γ in the CD28-deficient mice. These data suggest that CD28 function in important in the efficiency of the host response to Pneumocystis pneumonia.     

Cimetidine does not accelerate skin graft rejection in mice.

The influence of cimetidine on skin graft rejection was studied in a well defined transplantation model of inbred mice. Four allogeneic transplantation combinations with increasing antigenic disparity and one xenograft combination were studied. Cimetidine (25 mg/kg body weight) was administered intraperitoneally at 8-hr intervals until rejection occurred. No differences in graft survival were observed between cimetidine-treated groups and saline-treated controls in any of the combinations studied.     

Linomide does not prevent spontaneous autoimmune thyroiditis in NOD mice

Linomide is a potent immunomodulator and has been reported to prevent type 1 diabetes mellitus in non-obese diabetic (NOD) mice and to reduce the incidence of other autoimmune diseases in animal models. The mechanisms of action seem to involve antigen expression by down regulation of macrophage activity and to antagonise the activation of Th1 cells during the cellular immune response. With the purpose to investigate the effect of Linomide on the incidence of spontaneous autoimmune thyroiditis (AIT) in female NOD mice we administered Linomide in drinking water (100 mg/kg/day) to NOD mice from 5th to 19th week of age. The mice were sacrificed at the end of week 19. None of the mice developed diabetes during the study period. The incidence of thyroiditis was evaluated on paraffin HE-stained sections and graduated on a scale from 0 to 4. Thirty-two percent of 37 mice treated with Linomide developed thyroiditis compared to 45% of 22 controls (p=0.31, chi2 =1.00). Among the mice who developed thyroiditis no difference in the degree of thyroiditis was found. Therefore no beneficial effect of Linomide on the incidence of spontaneous AIT in NOD mice could be demonstrated.     

Experimental autoimmune meningitis: a novel neurological disease in CD28-deficient mice

C57BL/6 mice develop T-cell-mediated experimental autoimmune encephalomyelitis (EAE) after immunization with the neuroantigen myelin oligodendrocyte glycoprotein. (MOG). We immunized CD28-deficient C57BL/6 mice to determine the role of T cell costimulation in the immune response to MOG. CD28-/- mice developed experimental autoimmune meningitis (EAM). EAM is a fatal, acute disease characterized by simultaneous weakness in all limbs, photophobia, irritability, and spatial disorientation. Histologically, EAM consisted of an infiltrate of myeloid, monocytic, and lymphocytic leukocytes within the leptomeninges. In contrast, the brain parenchyma was unaffected. EAM was mediated by CD4+ T cells since CD4 depletion prevented the disease. Upon rechallenge, mice in which EAM was prevented by CD4+ cell depletion developed EAE not EAM. Therefore, the presence or absence of CD28 determines the initial phenotype of the immune response to MOG. EAM, which develops in the absence of CD28, is a unique experimental model for immune-mediated aseptic meningitis.     

The significance of eosinophils in mild and moderate acute cardiac allograft rejection

The presence of eosinophils has previously been associated with severe acute cardiac allograft rejection. This appears to be a relatively uncommon finding, judging from our experience and the paucity of information appearing in literature. We report three cases where a prominent infiltrate of eosinophils was noted on endomyocardial biopsy following cardiac transplantation. There was no evidence of severe acute rejection in any of these three patients, and one patient had only mild acute rejection without even focal myocardial necrosis. An infiltrate, which includes eosinophils, does not appear to be restricted to severe acute cardiac allograft rejection. Therefore, when eosinophils are noted in endomyocardial biopsy specimens, decisions to revise the immunosuppressive regimen of cardiac transplant recipients should continue to be based upon established conventional histologic criteria.     

Helicobacter hepaticus does not induce or potentiate colitis in interleukin-10-deficient mice

Helicobacter hepaticus has been reported to induce colitis, hepatitis, and hepatocellular carcinoma in several different murine models. The aim of this study was to determine if H. hepaticus will cause colitis in monoassociated mice lacking the interleukin-10 gene (IL-10(-/-) mice) and potentiate colitis in specific-pathogen-free (SPF) IL-10(-/-) mice. Germfree IL-10(-/-) mice on either a mixed (C57BL/6 x 129/Ola) or inbred (129/SvEv) genetic background were monoassociated with H. hepaticus ATCC 51448 by oral feeding and rectal enemas. In a second experiment, germfree IL-10(-/-) mice were colonized with stool from SPF mice that harbored or did not harbor endogenous H. hepaticus. After 7 to 9 weeks of colonization, weight loss and mortality were assessed, the colon was isolated for histology and IL-12 secretion, and mesenteric lymph node cells were assessed for T-cell activation markers. It was found that IL-10(-/-) mice monoassociated with H. hepaticus for up to 16 weeks showed almost no histologic colitis or increased IL-12 production. SPF IL-10-knockout mice had no significant difference in weight loss, mortality rate, histologic scores, colonic IL-12 secretion, or T-cell activation with or without H. hepaticus. We conclude that H. hepaticus does not induce or potentiate disease in our IL-10(-/-) mice and therefore is not required to induce colitis in genetically susceptible hosts.     

CD28 gene polymorphisms and acute cellular rejection after liver transplantation

The co-stimulatory molecule CD28 plays an important role in T-cell-mediated immune response like acute cellular liver transplant rejection. The aim of the retrospective case- control study was to examine whether the single nucleotide polymorphisms (SNPs) rs3116487, rs3116494, and rs3116496 of the CD28 gene are associated with acute cellular liver transplant rejection. The mentioned SNPs were genotyped in 147 liver transplant recipients without acute cellular rejection and 144 liver transplant recipients with acute cellular rejection by real-time endpoint genotyping. The genotype and allele frequencies of the SNPs did not show any significant differences between both groups. Haplotype analyzes of the SNPs also showed no association. Our data suggest that the analyzed SNPs are not major contributors to the susceptibility of acute cellular liver transplant rejection.     

CD28 activation does not down-regulate Cbl-b expression in aged rat T-lymphocytes

It is well known that T-lymphocyte proliferation declines ex vivo with age, and is associated with decreased expression and/or activity of stimulatory intracellular signaling proteins. However, the role of inhibitory intracellular signaling molecules like the ubiquitin ligase Cbl-b in regulating T-lymphocyte function in aging is largely unknown. Therefore, we tested the hypothesis that T-lymphocyte proliferation declines with age, in part, due to increased expression of Cbl-b. We show that young splenic T-lymphocytes reduced Cbl-b expression when stimulated with anti-CD3 and anti-CD28 antibodies, while in aged T-lymphocytes the CD28-dependent Cbl-b down-regulation did not occur. This effect did not appear to be due to reduced CD28 receptor expression on aged T-lymphocytes. The mechanism for lack of Cbl-b down-regulation may involve the proteasome since blocking proteasomal activity in young T-lymphocytes prevented Cbl-b down regulation while there was no effect in aged T-lymphocytes on Cbl-b expression. These data provide evidence for a novel mechanism by which aging reduces T-lymphocyte function.     

Treatment of acute kidney allograft rejection with a non-mitogenic CD3 antibody

T3/4.A is a non-mitogenic murine IgA mAb to human CD3 that was selected for clinical studies to provide an alternative for the mitogenic, T cell-activating, therapeutic mAb OKT3. Previously, we reported that T3/4.A is better tolerated in humans than the IgG2a-CD3 mAb T3/4.2a. Here we report the results of a phase II clinical trial to assess the immunosuppressive potential of T3/4.A. Eighteen first kidney transplant recipients with a first rejection episode were included. Baseline immunosuppression consisted of cyclosporin and prednisolone. Rejection treatment consisted of 5 mg mAb per day during 10 days. Fourteen patients responded, of whom four experienced a second rejection within 2 weeks, one experienced chronic rejection after 2.5 years, whereas the others remained rejection-free after treatment (median duration of follow-up 42 months). Four patients did not respond and eventually lost their graft. These results are similar to treatment results with OKT3, as reported in the literature. Following the first dose of T3/4.A, side effects were limited, and reduced compared to OKT3-treated controls. On the second day, 15 patients developed transient vomiting and/or diarrhoea, which coincided with elevated serum levels of proinflammatory cytokines. Minimal or even no side effects occurred during the remaining days, which is in sharp contrast to that seen generally during OKT3 treatment. Both T cell numbers and TCR expression were reduced during the therapy. We conclude that T3/4.A is a good alternative for OKT3 to treat rejection episodes in renal transplant recipients.     

SAHA,an HDAC inhibitor,synergizes with tacrolimus to prevent murine cardiac allograft rejection

Suberoylanilide hydroxamic acid (SAHA), as a histone deacetylase (HDAC) inhibitor (HDACi), was recently found to exhibit an immunosuppressive effect. However, whether SAHA can synergize with calcineurin inhibitors (CNIs) to inhibit allograft rejection and its underlying mechanism remain elusive. In this study, we demonstrated the synergistic effects of SAHA and non-therapeutic dose of tacrolimus (FK506) in prolonging the allograft survival in a murine cardiac transplant model. Concomitant intragraft examination revealed that allografts from SAHA-treated recipients showed significantly lower levels of IL-17 expression, and no discernable difference for IL-17 expressions was detected between SAHA- and SAHA/FK506-treated allograft as compared with allografts from FK506-treated animals. In contrast, administration of FK506 significantly suppressed interferon (IFN)-γ but increased IL-10 expression as compared with that of SAHA-treated animals, and this effect was independent of SAHA. Interestingly, SAHA synergizes with FK506 to promote Foxp3 and CTLA4 expression. In vitro, SAHA reduced the proportion of Th17 cells in isolated CD4⁺ T-cell population and decreased expressions of IL-17A, IL-17F, STAT3 and RORγt in these cells. Moreover, SAHA enhances suppressive function of regulatory T (Treg) cells by upregulating the expression of CTLA-4 without affecting T effector cell proliferation, and increased the proportion of Treg by selectively promoting apoptosis of T effector cells. Therefore, SAHA, a HDACi, may be a promising immunosuppressive agent with potential benefit in conjunction with CNI drugs.     

Overexpression of Polycomb-group gene rae28 in cardiomyocytes does not complement abnormal cardiac morphogenesis in mice lacking rae28 but causes dilated cardiomyopathy

The Polycomb-group genes (PcG) are widely conserved from Drosophila to mammals and are required for maintaining positional information during development. The rae28 gene (rae28) is a member of the mouse PcG. Mice deficient in rae28 (rae28(-/-)) demonstrated that rae28 has a role not only in anteroposterior patterning but also in cardiac morphogenesis. In this study we generated transgenic mice with ubiquitous or cardiomyocyte-specific exogenous rae28 expression. Genetic complementation experiments with these transgenic mice showed that ubiquitous expression of rae28 could reverse the cardiac anomalies in rae28(-/-), whereas cardiomyocyte-specific expression of rae28 could not, suggesting that rae28 is involved in cardiac morphogenesis through a noncardiomyocyte pathway. Interestingly, however, cardiomyocyte-specific overexpression of rae28 caused dilated cardiomyopathy, which was associated with cardiomyocyte apoptosis, abnormal myofibrils, and severe heart failure. Cardiac expression of rae28 was predominant in the early embryonic stage, whereas that of the other PcG members was relatively constitutive. Because rae28 forms multimeric complexes with other PcG proteins in the nucleus, it is presumed that constitutive cardiomyocyte-specific rae28 overexpression impaired authentic PcG functions in the heart. rae28-induced dilated cardiomyopathy may thus provide a clue for clarifying the direct role of PcG in the maintenance of cardiomyocytes.