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ABSTRACT: To investigate the feasibility of bone marrow mesenchymal stem cells (BMSCs) in inducing immune tolerance and to establish the mouse model of reverse chimerism in xeno-skin transplantation.
The mouse model of bone marrow-chimerism was established with immuncompromised BALB/C-nu/nu female mice by receiving the transplantation of BMSCs from green fluorescent protein (GFP)-C57BL/10 male mice, the optimized chimeric time was identified by RT-PCR testing of SRY gene and immunohistochemistry measurement of GFP expression. In the experiment group, GFP-C57BL/10 male mice received the transplantation of the skin from immuncompromised BALB/C-nu/nu female mice with BMSCs bone marrow-chimerism. In the rejection group, GFP-C57BL/10 male mice received the transplantation of the skin from immuncompromised BALB/C-nu/nu female mice without BMSCs bone marrow-chimerism. In the control group, allo-transplantation of skin was performed in GFP-C57BL/10 male mice. Histological study was performed to investigate the survival rate and angiogenesis of the transplanted skin. RESULTS :The bone marrow chimeric model was established, the expressions of SRY gene and GFP protein reached the highest level at four weeks (1.22 +/- 0.10; 458.0 +/- 3.4) post-transplanted with BMSCs (10(6)), which was significantly different in comparison with those at one week, two weeks and six weeks posttransplantation(P < 0.05). Four-weeks after transplantation was further confirmed as the optimized chimeric time. The mean survival time of donor skin graft > 14 d in the experimental group, while it only was 5 d in the rejection group. CONCLUSION :The bone marrow-chimerism can be formed in the recipient by donor BMSCs transplantation, which can further induce tolerance of mice xeno-skin transplantation by reverse chimerism.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 03/2013; 44(2):165-9.
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Lei Luo, Jun Lu,
Wen C Li,
Juan Shan,
Fu S Li,
Dan Long,
Jia Y Guo,
Qiao W Wu,
Tao Lin,
Ping Y Li,
Li Feng
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ABSTRACT: BACKGROUND: RNA interference targeting RelB can significantly attenuate ischemia/reperfusion (I/R)-induced renal dysfunction, but its roles in liver I/R injury remain to be defined. We have investigated whether siRNA targeting of RelB in liver could also elicit protection against I/R injury. MATERIALS AND METHODS: A RelB miRNA RNAi expression plasmid, a scrambled plasmid, or phosphate-buffered saline (50 μg of the plasmid diluted in phosphate-buffered saline, 8% wt/vol) were rapidly injected, within 6-8 s, into mouse tail veins 24 h before liver I/R. Mice were subjected to 30 min of 70% hepatic ischemia or to a sham operation. Six h after reperfusion, blood and liver tissue samples were collected for subsequent assays. RESULTS: The expression level of RelB was reduced in the RelB RNAi group compared with the control group, while it was increased in the I/R group. In the sham group, malondialdehyde, myeloperoxidase (MPO), and superoxide dismutase serum levels were almost the same, but the alanine aminotransferase level in the untreated group was 20- to 25-fold lower than in the other groups. In I/R-treated mice, although alanine aminotransferase, malondialdehyde, and MPO serum levels in the RelB RNAi group were lower than in other groups, all were higher than in the sham group. Silencing RelB could inhibit the decrease of superoxide dismutase activity and the upregulation of MPO and tumor necrosis factor α induced by I/R injury. CONCLUSIONS: Silencing RelB can protect the liver against I/R-induced damage. Therefore, it is a promising therapeutic target for protection against I/R injury in the liver.
Journal of Surgical Research 09/2012; · 2.25 Impact Factor
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Lei Luo,
Chengwen Li,
Wenqiao Wu, Jun Lu,
Yanni Zhou,
Juan Shan,
Shengfu Li,
Dan Long,
Yingjia Guo,
Youping Li,
Li Feng
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ABSTRACT: Memory T cells (T(M)s) exhibit differential susceptibility to many immunomodulatory strategies that induce immunologic tolerance in naïve T cells, which are believed to be an important barrier to inhibiting rejection and inducing tolerance. As skin grafts are a common model for acquiring T(M)s, we evaluated function of T(M)s derived from skin grafts. We also assessed the modulatory effects on memory T cells function of the microRNAs miR-155 and miR-181a, which are involved in regulating cytokine secretion and TCR sensitivity to antigen, respectively.
Memory CD4(+) T cells derived from skin-graft recipient mice, and naïve CD4(+) T cells from untreated mice, were isolated by negative magnetic selection, and then stimulated with dendritic cells pulsed with donor-specific antigens. Effector function and regulating mechanisms were assessed.
In contrast to naïve CD4(+) T cells, CD4(+) T(M)s stimulated with donor-specific antigen could quickly generate effector function in terms of proliferation and cytokine secretion; miR-155 and miR-181a levels in CD4(+) T(M)s rapidly increased during immune response compared to naïve CD4(+) T cells.
Memory CD4(+) T cells derived from skin grafts could be used as an experimental tool for evaluating effects of different immune-modulating strategies on T(M)s. Levels of miR-155 and miR-181a up-regulated quickly in T(M)s, which could be an important mechanism by which T(M)s mediate immune responses rapidly.
Journal of Surgical Research 11/2011; 176(2):649-56. · 2.25 Impact Factor
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ABSTRACT: The ability of DCs to induce immune tolerance depends on its maturation status. RelB plays a pivotal role in DCs differentiation. A therapeutic protocol of DCs-based not only induces hyporesponsiveness in T(N)s, but also in alloreactive T(M)s is required. Thus, it is urgent to assess modulatory effects of RelB-silenced DCs on T(M)s and T(N)s. In this study, we constructed lentiviral vector which could efficiently silenced the RelB in DCs (DCs-miR RelB) to keep them immature. These DCs induced antigen-specific hyporesponsiveness in CD4(+) T(N)s. In contrast, upon re-stimulation with mature DCs, CD4(+) T(M)s primed by DCs-miR RelB maintained hyporesponsiveness in terms of proliferation and cytokine production. And these may be associated with micro155 and micro181a expression levels in T(M)s and T(N)s. These results may help developing the DCs-based therapeutical protocols by inducing hyporesponsiveness in CD4(+) T(N)s and T(M)s.
Cellular Immunology 11/2011; 273(1):85-93. · 1.97 Impact Factor
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ABSTRACT: Cytokine response modifier A protein is a caspase inhibitor that inhibits caspase activity and protects cells from apoptosis. Chronic cyclosporine nephropathy is a significant cause of chronic graft dysfunction. We explored cytokine response modifier A protein-alleviated chronic cyclosporine nephropathy for ways of improving chronic graft dysfunction.
Cytokine response modifier A protein-transferring HK-2 cells were cultured with different concentrations of cyclosporine. Cytokine response modifier A protein mRNA and proteins were detected by real-time polymerase chain reaction and Western blot, cell viability was detected by (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide), and apoptosis was detected by flow cytometry.
Cyclosporine caused a concentration-dependent and time-dependent loss of cell viability in HK-2 cells. Cytokine response modifier A protein mRNA was expressed at 48 and 72 hours (P < .05), while protein was detected at 72 hours. Cell viability in the cytokine response modifier A protein-transfected group was significantly greater than that of the control group when treated with 1 µg/mL, 10 µg/mL, or 20 µg/mL cyclosporine at 24 or 48 hours (P < .05). The apoptosis in cytokine response modifier A protein-transfected cells was significantly lower than that of controls (P < .05).
Cytokine response modifier A protein protects renal cells from cyclosporine injury by inhibiting activated caspases. Cytokine response modifier A protein transfection may improve chronic cyclosporine nephropathy and provide for improving chronic graft dysfunction.
Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation. 10/2011; 9(5):302-9.
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Dan Long, Jun Lu,
Lei Luo,
Yingjia Guo,
Chengwen Li,
Wenqiao Wu,
Juan Shan,
Lulu Li,
Shengfu Li,
Youping Li,
Tao Lin,
Li Feng
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ABSTRACT: Of solid organ transplantations, pancreas transplantation is associated with the highest incidence of pancreatic fibrosis in the early post-transplantation period. Activated pancreatic stellate cells (PSCs) are the main source of pancreatic fibrosis. Octreotide is widely used as a prophylactic for postoperative complications in pancreas transplant recipients. Recent studies have shown that it can inhibit liver fibrosis. This study investigated the effect of octreotide in pancreas graft fibrosis in rats.
Isolated PSCs from Sprague Dawley rats were co-cultured with different doses of octreotide (1.25, 2.5, 5, 10, 20, and 40 ng/mL). PSC proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 48, 72, and 96 h. The α-smooth muscle actin (α-SMA) and collagen I expressions of PSCs were detected by immunohistochemistry and reverse-transcriptase polymerase chain reaction. Rat heterotopic pancreaticoduodenal transplantation was performed with and without octreotide treatment (0.01 mg/kg). Pancreas grafts were harvested at postoperative d 1, 3, 5, and 7. Hematoxylin-eosin staining, Masson's trichrome staining, and immunohistochemical staining for α-SMA, collagen I, and tumor growth factor-β1 (TGF-β1) were performed.
Octreotide at a concentration of >20 ng/mL significantly inhibited PSC activation and proliferation in vitro. Inflammatory infiltration was reduced in the octreotide group in vivo, and the expression levels of α-SMA, collagen I, and TGF-β1 were also lower, with statistic significant difference or not. Masson's trichrome staining showed a decrease in collagen deposition with octreotide treatment.
Octreotide effectively inhibits PSC activation and proliferation in vitro, but has a limited inhibitory effect on the development of pancreas graft fibrosis.
Journal of Surgical Research 07/2011; 176(1):248-59. · 2.25 Impact Factor
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Jun Lu,
Lei Luo,
Yingjia Guo,
Dan Long,
Liang Wei,
Juan Shan,
Li Feng,
Shengfu Li,
Xiaoyan Yang,
Yiping Lu,
Sheri Krams,
Youping Li
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ABSTRACT: Background and Objective: Human major histocompatibility complex class I-related gene A (MICA) is reportedly associated with poor transplant outcomes and a high risk of acute and chronic rejection in solid organ transplantation. However, studies on these risks have found conflicting results. In order to identify areas in which additional research is needed, we have undertaken the first systematic review of evidence concerning the risk of anti-MICA antibodies in recipients’ sera.Methods: We searched MEDLINE, EMBASE, and the Cochrane Library for original reports of clinical studies involving detection of MICA abs in transplant recipients’ sera which used survival rate, acute rejection, and/or chronic rejection as outcome measures. RevMan 5.0.15 was used to calculate relative risk (RR), odds ratios (ORs), and 95% confidence intervals (95%CIs).Results: We found 18 relevant articles, with a total of 6,607 recipients. Follow-up duration ranged from 1 to 15 years. In studies with more than 2 years of follow-up, anti-MICA abs positive in kidney recipients’ post-transplant sera was associated with a lower graft survival rate (4 years: RR = 2.04, 95%CI 1.30 to 3.22; 3 years: OR = 3.56, 95%CI 1.47 to 8.62; 2 years: RR = 2.17, 95%CI 1.09 to 4.31) and a higher acute rejection rate (RR = 1.92, 95%CI 1.27 to 2.91), but there was no clear association with chronic rejection. Similar conclusions could not be drawn for heart or liver transplantation due to possible confounding by anti-HLA abs and the small sample sizes of the available studies.Conclusion: Anti-MICA antibodies in recipients’ sera may associated with poor graft survival rates and high risk of acute and chronic rejection in solid organ transplantation, but more rigorous studies are needed to confirm or refute this relationship. Current immunosuppressive therapy may fail to suppress the harmful effect of MICA antigens.
Journal of Evidence-Based Medicine 05/2011; 4(2):106 - 121.
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Juan Shan,
Yinjia Guo,
Lei Luo, Jun Lu,
Chengwen Li,
Chuntao Zhang,
Yuchuan Huang,
Li Feng,
Wenqiao Wu,
Dan Long,
Shengfu Li,
Youping Li
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ABSTRACT: Regulatory T cells (Tregs) are considered to be critical for the induction of transplant tolerance. Tregs counts were measured in blood, biopsy and urine sample after transplantation in many studies. Although not unanimous, some studies have suggested that Tregs is associated with better outcome and can also serve as an immune marker to predict the individual risk of rejection and identify tolerant patients. In this study, we systematically reviewed the correlation between Tregs and transplant outcomes, identifying if Tregs can predict transplant rejection and tolerance. A total of 22 articles were included and assessed, the results showed that Tregs in recipients are helpful to maintain a stable graft function, reduce acute/chronic rejection rate. And the Tregs in graft and urine, rather than in PBL, may have a better diagnostic value for transplant outcomes. However, since the low quality of included studies, results may be influenced by bias. More high quality studies with bigger sample size are still needed in future.
Cellular Immunology 05/2011; 270(1):5-12. · 1.97 Impact Factor
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ABSTRACT: The complexity of surgical procedure in mouse heterotopic heart transplantation (HHT) has prevented its widespread use. The present study reported a modified technique - splint tubing technique (STT) based on cuff technique (CT).
C57BL/10 and BALB/c mice were performed in syngeneic and allogeneic HHT using STT and CT. The main improvement is that the recipient external jugular vein and common carotid artery were independently opened a mouth and inserted a cannula to avoid the difficult operations of sleeved and everted tube. Graft function was assessed by pulse palpation, echocardiography and histopathologic examination.
Ten syngeneic and thirty allogeneic HHT using STT were performed with six graft losses. Ten allogeneic HHT using CT were carried out with two graft losses. Technically successful syngeneic grafts have survived to the pre-specified 30days endpoint with strong contraction. STT significantly shortened operation time compared with CT (32.33±4.21min vs 45.15±4.89min, P<0.05). No significant difference was observed in survival time between two methods.
STT is easily learned. It reduces the operation difficulty and makes the operation possible for the beginner to master this skill within 1-2weeks. Shorter operation time leads higher operative success rate.
Transplant Immunology 04/2011; 25(1):82-7. · 1.46 Impact Factor
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ABSTRACT: Ginsenoside Rb1 could prevent ischemic neuronal death and focal cerebral ischemia, but its roles to liver warm I/R injury remain to be defined. We determined if Rb1 would attenuate warm I/R injury in mice.
Mice were divided into sham, I/R, Rb1+I/R (Rb1 postconditioning, 20mg/kg, i.p. after ischemia), sham+L-NAME, I/R+L-NAME, and Rb1+I/R+L-NAME groups using 60min of the liver median and left lateral lobes ischemia. Serum levels of alanine aminotransferase (ALT) were measured and morphology changes of livers were evaluated. Contents of nitric oxide (NO) and nitric oxide synthase (NOS), malondialdehye (MDA) and activity of superoxide dismutase (SOD) were measured. Expressions of Akt, p-Akt, iNOS, HIF-1alpha, tumor necrosis factor-a (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were also determined by western blot or immunohistochemistry.
Rb1 postconditioning attenuated the dramatically functional and morphological injuries. The levels of ALT were significantly reduced in Rb1 group (p<0.05). Rb1 upregulated the concentrations of NO, iNOS in serum, iNOS, and activity of SOD in hepatic tissues (p<0.05), while it dramatically reduced the concentration of MDA (p<0.05). Protein expressions of p-Akt, iNOS and HIF-1alpha were markedly enhanced in Rb1 group. Protein and mRNA expressions of TNF-α and ICAM-1 were markedly suppressed by Rb1 (p<0.05).
We found that Rb1 postconditioning could protect liver from I/R injury by upregulating the content of NO and NOS, and also HIF-1alpha protein expression. These protective effects could be abolished by L-NAME. These findings suggested Rb1 may have the therapeutic potential through ROS-NO-HIF pathway for management of liver warm I/R injury.
Life sciences 02/2011; 88(13-14):598-605. · 2.56 Impact Factor
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ABSTRACT: Chronic cyclosporine A (CsA) nephrotoxicity (CCN) is a major cause of chronic renal dysfunction and has no effective clinical interventions yet.
To reveal the mechanisms of renal cell apoptosis in CCN, we analyzed all in vitro studies of such mechanisms.
We collected all in vitro studies about the mechanisms of renal cell apoptosis induced by CsA in Medline (1966 to July 2010), Embase (1980 to July 2010) and ISI (1986 to July 2010), evaluated their quality according to in vitro standards and extracted data following the PICOS principles and synthesized the data.
First,CsA could upregulate Fas and Fas-L expression, increase FADD and apoptosis enzymes (caspase-2, -3, -4, -7, -8, -9 and -10) and downregulate the Bcl-2 and Bcl-xL. Second, CsA could induce oxidative stress and damage the antioxidant defense system. Third, CsA could increase the expression of HERP, GRP78 and CHOP. Fourth, CsA could induce renal cell apoptosis and increase their iNOS and p53 expression in cultured cells.
At least four pathways are involved in renal cell apoptosis induced by CsA in different cell species. Caspases might be their final common pathway in vitro. They might all provide potential points for interventions, but these need to be confirmed in vivo.
American Journal of Nephrology 01/2011; 33(6):558-66. · 2.54 Impact Factor
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ABSTRACT: To investigate whether hypoxia-inducible factor (HIF) 1alpha and cyclosporin A (CsA) can regulate MICA/B expression and affect NK cytotoxicity during ischemia/reperfusion (I/R) injury.
We generated an HIF-1alphaDeltaODD-expressing adenovirus which can functionally and steadily express HIF-1alpha during normoxia and transfected human cardiomyocytes (HCMs) to investigate whether HIF-1alpha, as a single factor, can upregulate MICA/B expression. Alternatively, HCMs were treated with HIF-1alpha RNAi or CsA, and then cultured under hypoxia/reoxygenation (H/R) condition to simulate I/R injury in vitro. Cells were collected at different time points and used for studies of gene expression and NK cytotoxicity.
Expression of MICA/B in HCMs is upregulated through HIF-1alpha overexpression in normoxia, and inhibited by HIF-1alpha RNAi treatment during hypoxia-reoxygenation (H/R). NK cytotoxicity towards HCMs shows a positive correlation with HIF-1alpha expression. Moreover, CsA can inhibit HIF-1alpha and MICB expression but upregulates MICA expression during H/R.
These findings suggest that proper control of HIF-1alpha expression via CsA dose may be a potential therapeutic approach for avoiding MIC expression, and improving the function and long-term survival of heart allografts.
Life sciences 07/2010; 87(3-4):111-9. · 2.56 Impact Factor
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ABSTRACT: To review the effects of different immunosuppressive drugs on proliferation and function of regulatory T cells (Tregs).
We searched MEDLINE, Embase (from inception to September 2009), and the Cochrane Library (Issue 4, 2009) for clinical and basic research about the effects of various immunosuppressive drugs on Tregs. Data were extracted and methodological quality was assessed by two independent reviewers. Outcome measures for clinical research included blood Tregs levels, acute rejection episodes, and graft function. Outcomes for basic research included percentage of Tregs proliferation, function, Tregs phenotype, and evidence for possible mechanisms. We analyzed data qualitatively.
Forty-two studies, including 19 clinical trials and 23 basic studies, were included. The immunosuppressive drugs studied were calcineurin inhibitors (CNIs), Rapa, anti-metabolism drugs, IL-2 receptor-blocking antibodies, T-cell depleting antibodies, and co-stimulation blockade antibodies. Most of the studies were on Rapa and CNIs. Eight basic studies on Rapa and CNIs showed that Rapa could promote the proliferation and function of Tregs, while CNIs could not. Five clinical trials involving a total of 158 patients showed that patients taking Rapa had higher blood concentration of Tregs than patients taking CNIs, but no difference was found in graft function (6-42 months follow-up).
There is substantial evidence that Rapa favors Tregs survival and function. However, the higher numbers of blood Tregs in patients treated with Rapa do not show any association with better graft function. Larger clinical studies with longer follow-up are needed to more thoroughly assess the efficacy of immunosuppressive drugs on Tregs, and reveal whether a relationship exists between Tregs and graft function.
Journal of Evidence-Based Medicine 05/2010; 3(2):117-29.
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ABSTRACT: It is an urgent need to induce and keep the donor-specific immune tolerance without affecting the function of normal immune defense and immune surveillance in clinical organ transplantation. Large number of studies showed that both the establishment of donor-recipient chimerism and the application of antibodies or drugs could obtain the donor-specific immune tolerance in animal transplantation model. However, the former as treatment of clinical practice has a poor feasibility, the latter has a very low success rate in clinical organ transplantation. There is a group of naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) that mediate immune tolerance by suppressing alloreactive T cells in vivo. These cells are unable to curb the occurrence of allograft rejection owing their low content. And donor-specific Tregs amplified in vitro alone can not induce donor-specific immune tolerance for recipient. Rapamycin (RPM) as a proliferation signal inhibitor, studies have shown it can effectively inhibit allograft rejection and maybe contribute to induction of immune tolerance. But there exist still many dose-dependent adverse reactions which could prevent the establishment of immune tolerance and reduce the life quality of recipients in the clinical application of RPM. Therefore, we speculate a small amount of RPM combined with donor-specific Tregs amplified in vitro may be not only induce the achievement of donor-specific tolerance, but also reduce or eliminate the side effects of RPM in clinical organ transplantation.
Cellular Immunology 01/2010; 264(2):111-3. · 1.97 Impact Factor
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ABSTRACT: Rapamycin (RPM), a powerful agent used clinically in transplant recipients, induces CD4(+)CD25(+) regulatory T cells (Tregs) which play an important role in induction of immune tolerance. However, long-term use of RPM has negative side effects. In this report, we found that combination with the low dose RPM and high dose IL-2 did not affect antigen presentation of rat B cells to Tregs, and could efficiently promote Tregs proliferation and enhance their inhibitory activities in vitro. In addition, the combination of low dose RPM and high dose IL-2 enhanced mRNA expression of Foxp3, TGF-beta1 and Pim-2 in Tregs but not in CD4(+)CD25(-) T effector cells (Teffs). The Tregs inhibitory activity is positively associated with mRNA expressions of TGF-beta1 and Pim-2 while unrelated to the Foxp3 mRNA expression. Our present study offers one approach to expand functional Tregs in vitro, which maybe used for clinical immune tolerance induction.
Cellular Immunology 01/2010; 264(2):180-5. · 1.97 Impact Factor
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ABSTRACT: Cyclosporine A(CsA) causes significant nephrotoxicity that contribute to kidney graft loss in the long-term, it can induce cell apoptosis in renal cortex and medulla, reduce kidney function. The mechanisms are complex and involved in six apoptosis pathways including classical pathway, mitochondrial pathway, endoplasmic reticulum pathway, angiotensin II pathway, NO- and hypertonicity-related pathway. All these pathways may converge to a single way by activated Caspase-3, -6, -8, -9 and -12 that may become a potential new target for intervention. CrmA protein belonging to one of serine protease inhibitor family members, it widely inhibits the inflammatory Caspases and apoptotic Caspases and has a strong function on inhibiting apoptosis induced by many chemical inducers through effectively blocking Caspase-1, -3, -4, -5, -8, -9, and -10 enzymes. It is not reported if CrmA is resistant to apoptosis induced by immunosuppressant so far. Therefore we speculate CrmA can block down CsA-induced renal cell apoptosis through inhibiting the Caspase-3, -6, -8, -9 and -12 activated and further to eliminate CRD induced by CsA.
Cellular Immunology 01/2010; 265(1):6-8. · 1.97 Impact Factor
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ABSTRACT: Human major histocompatibility complex class I-related chain A (MICA) plays a dual role in adaptive and innate immune responses. Increasing evidence demonstrates that MICA is closely correlated with acute and chronic kidney allograft rejection. Therefore, understanding the activation mechanisms of MICA is important in kidney transplantation. We previously demonstrated that ischemia/reperfusion injury (IRI) could up-regulate MICA expression on mouse kidney allografts. Since hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular adaptive responses to hypoxia during IRI, here we investigate whether HIF-1 could up-regulate MICA expression and its influence on NK cell cytotoxicity.
We find that HIF-1alpha plays an important role in up-regulating MICA expression, inducing IFNgamma secretion and NK cell cytotoxicity during hypoxia/reoxygenation. First, we generated a HIF-1alphaDELTAODD-expressing adenovirus to stably and functionally express HIF-1alpha in human renal proximal tubular epithelial (HK-2) cells under normoxia conditions. HIF-1alpha over-expression in HK-2 cells induces MICA expression and enhances NK cell cytotoxic activity towards cells that express HIF-1alpha. Second, we used a hypoxia/reoxygenation cell model to simulate IRI in vitro and found that the suppression of HIF-1alpha by RNAi induces down-regulation of MICA expression and inhibits NK cytotoxicity. In antibody blocking experiments, an anti-MICA mAb was able to down-regulate NK cell cytotoxic activity towards HK-2 cells that over-expressed HIF-1alpha. Moreover, when NK cells were co-cultured with the HK-2 cells expressing MICA, which was up-regulated by over-expression of HIF-1alpha, there was a significant increase in the secretion of IFNgamma. In the presence of the blocking MICA mAb, IFNgamma secretion was significantly decreased.
These results demonstrate that hypoxia/reoxygenation-promoted MICA expression on HK-2 cells is through a HIF-1 pathway. The increased IFNgamma secretion and enhanced NK cell cytotoxicity was mainly due to the surface expression of MICA induced by over-expression of HIF-1alpha. This study enhances our understanding of MICA activation mechanisms during kidney transplantation and provides insights into how IRI can influence transplant outcome. Moreover, these findings might be also important for developing strategies to reduce the effect of MICA in kidney transplant outcomes in the future.
BMC Cell Biology 01/2010; 11:91. · 2.59 Impact Factor
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ABSTRACT: The expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in heart allografts is an important mechanism in response to ischemia/reperfusion (I/R) injury which represents the single major non-immunologic factor implicated in pathogenesis of chronic graft dysfunction (CGD). Adenoviral mediated overexpression of HIF-1alpha is a useful way to investigate the molecular mechanisms of I/R injury and the cardiac function during heart transplantation. The oxygen-dependent degradation (ODD) domain of HIF-1alpha can lead to degradation of the HIF-1alpha protein in normoxia. This will be an obstacle to steady expression of HIF-1alpha in heart allograft after transduction. In this study, we obtained the coding sequence of HIF-1alpha without ODD domain (HIF-1alphaDeltaODD) through a PCR-based method, and then generated the HIF-1alphaDeltaODD-expressing adenovirus. In normoxia, adenoviral mediated expression of HIF-1alphaDeltaODD shows constitutive activity in human cardiomyocytes, and can up-regulate heme oxygenase (HO)-1 mRNA levels significantly compared with the group transduced with HIF-1alpha-expressing adenovirus. The constructed HIF-1alphaDeltaODD-expressing adenovirus can be used to transduce allografts in animal studies to investigate the mechanism of CGD and provide a useful model to study the regulation mechanisms of genes regulated by HIF-1alpha alone.
Plasmid 09/2009; 63(1):20-6. · 1.52 Impact Factor
