In pig-to-primate xenotransplantation, multiple transgenic pigs are required to overcome a series of transplant rejections. The generation of multiple transgenic pigs either by breeding or the introduction of several mono-cistronic vectors has been hampered by the differential expression patterns of the target genes. To achieve simultaneous expression of multiple genes, a poly-cistronic expression system using the 2A peptide derived from the Thosea asigna virus (T2A) can be considered an alternative choice. Before applying T2A expression system to pig generation, the expression patterns of multiple genes in this system should be precisely evaluated. In this study, we constructed several bi-cistronic T2A expression vectors, which combine target genes that are frequently used in the xenotransplantation field, and introduced them into porcine fibroblasts. The proteins targeted to the same or different subcellular regions were efficiently expressed without affecting the localization or expression levels of the other protein. However, when a gene with low expression efficiency was inserted into the upstream region of the T2A sequences, the expression level of the downstream gene was significantly decreased compared with the expression efficiency without the insertion. A small interfering RNA targeting one gene in this system resulted in the significant downregulation of both the target gene and the other gene, indicating that multiple genes combined into a T2A expression vector can be considered as a single gene in terms of transcription and translation. In summary, the efficient expression of a downstream gene can be achieved if the expression of the upstream gene is efficient.
Neural stem cells (NSCs) are among the most promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. One of the remaining obstacles for NSC therapy is to overcome the alloimmune response on NSCs by the host.
To investigate the mechanisms of immune modulatory function derived from the interaction of human NSCs with allogeneic T cells, we examined the immune regulatory effects of human NSCs on allogeneic T cells in vitro.
Significantly, NSCs induced apoptosis of allogeneic T cells, in particular CD4+ T cells. Interaction of CD70 on NSCs and CD27 on CD4+ T cells mediated apoptosis of T cells. Thus, blocking CD70–CD27 interaction prevented NSC-mediated death of CD4+ T cells.
We present a rational explanation of NSC-induced immune escape in two consecutive stages. First, CD70 constitutively expressed on NSCs engaged CD27 on CD4+ T cells, which induced Fas ligand expression on CD4+ T cells. Second, CD4+ T-cell apoptosis was followed by Fas–Fas ligand interaction in the CD4+ T cells.
Neural stem cells; Co-stimulatory molecules; Immune escape mechanism
Urinary tract infection (UTI) occurs in 30%-50% of individuals with autosomal dominant polycystic kidney disease (ADPKD). However, the clinical relevance of asymptomatic pyuria in ADPKD patients remains unknown.
We retrospectively reviewed medical records of 256 ADPKD patients who registered to the ADPKD clinic at Seoul National University Hospital from Aug 1999 to Aug 2010. We defined the asymptomatic pyuria as more than 5-9 white blood cells in high-power field with no related symptoms or signs of overt UTI. Patients were categorized into 2 groups depending on its duration and frequency: Group A included non-pyuria and transient pyuria patients; Group B included recurrent and persistent pyuria patients. The association between asymptomatic pyuria and both the development of overt UTI and the deterioration of renal function were examined.
With a mean follow-up duration of 65.3 months, 176 (68.8%) out of 256 patients experienced 681 episodes of asymptomatic pyuria and 50 episodes of UTI. The annual incidence of asymptomatic pyuria was 0.492 episodes/patient/year. The patients in group B showed female predominance (58.5% vs. 42.0%, P=0.01) and experienced an upper UTI more frequently (hazard ratio: 4.612, 95% confidence interval: 1.735-12.258; P=0.002, adjusted for gender and hypertension). The annual change in estimated glomerular filtration rate (ΔeGFR) was significantly larger in magnitude in group B than in group A (-2.7±4.56 vs. -1.17±5.8, respectively; P=0.01). Age and Group B found to be the independent variables for ΔeGFR and developing end-stage renal disease (16.0% vs. 4.3%, respectively; P=0.001).
Chronic asymptomatic pyuria may increase the risk of developing overt UTI and may contribute to declining renal function in ADPKD.
Polycystic kidney disease; Chronic renal failure; Glomerular filtration rate; Pyuria; Urinary tract infection
Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.
Renal failure is one of the most serious complications associated with autosomal dominant polycystic kidney disease (ADPKD). To date, early markers have failed to predict renal function deterioration at the early stages. This 1-year prospective study evaluated N-acetyl-β-D-glucosaminidase (NAG) as a new surrogate marker for renal function in ADPKD.
A total of 270 patients were enrolled in the study, and we measured urinary NAG, β2-microglobulin, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) prospectively for 1 year to compare their predictive values for renal function.
Baseline urinary NAG/Cr was negatively correlated with estimated glomerular filtration rate (GFR) (r2 = 0.153, P < 0.001) and positively correlated with total kidney volume (TKV) (r2 = 0.113, P < 0.001). Among other biomarkers, urinary NAG/Cr better discriminated patients with decreased renal function from those with conserved renal function, showing the largest area under the curve (AUC 0.794). Immunohistochemical study revealed strong staining along the cyst-lining epithelial cells as well as the nearby compressed tubular epithelial cells. However, both single and repeated measurements of urinary NAG/Cr failed to predict renal function decline in 1 year.
Urinary NAG/Cr may be a useful surrogate marker for renal function in ADPKD patients.
Autosomal dominant polycystic kidney disease; Biomarkers; Renal function
Administration of an ICAM-1–specific antibody arrests dendritic cells in a semi-immature state and facilitates antigen-specific T cell tolerance to islet allografts in humanized mice and Rhesus monkeys.
Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti–ICAM-1–induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.
Cardiovascular disease (CVD) is the leading cause of death in renal allograft recipients with functioning graft. Our study aimed to determine the incidence and the risk factors of cardiovascular disease after renal transplantation in Korea. We retrospectively analyzed 430 adult recipients who underwent kidney transplantation between January 1997 and February 2007. CVD was defined as a composite outcome of ischemic heart disease, cerebrovascular accident and peripheral vascular disease. Mean age of recipients was 40.0±11.8 yr. Mean duration of follow-up was 72±39 months. The cumulative incidence of CVD after renal transplantation was 2.4% at 5 yr, 5.4% at 10 yr and 11.4% at 12 yr. Multivariate analysis revealed that recipient's age, diabetes mellitus and duration of dialysis before transplantation were associated with post-transplant CVD (hazard ratio 1.843 [95% CI, 1.005-3.381], 3.846 [95% CI, 1.025-14.432] and 3.394 [95% CI, 1.728-6.665] respectively). In conclusion, old age, duration of dialysis and diabetes mellitus are important risk factors for post-transplant CVD, although the incidence of post-renal transplant CVD is lower in Korea than that in western countries.
Cardiovascular Diseases; Incidence; Kidney Transplantation; Risk Factors; Diabetes Mellitus; Koreans
Anti-erythropoietin antibodies usually cross-react with all kinds of recombinant erythropoietins; therefore, erythropoiesis-stimulating agent (ESA)-induced pure red-cell aplasia (PRCA) is not rescued by different ESAs. Here, we present a case of ESA-induced PRCA in a 36-yr-old woman with chronic kidney disease, whose anemic condition improved following reintroduction of darbepoetin-α. The patient developed progressive, severe anemia after the use of erythropoietin-α. As the anemia did not improve after the administration of either other erythropoietin-α products or erythropoietin-β, all ESAs were discontinued. Oxymetholone therapy failed to improve the transfusion-dependent anemia and a rechallenge with ESAs continuously failed to obtain a sustained response. However, her anemia improved following reintroduction of darbepoetin-α at 3 yr after the initial diagnosis. Interestingly, anti-erythropoietin antibodies were still detectable, although their concentration was too low for titration. In conclusion, darbepoetin-α can improve ESA-induced PRCA when the anti-erythropoietin antibody titer declines and its neutralizing capacity is lost.
Red-Cell Aplasia, Pure; Kidney Failure, Chronic; Erythropoietin, Recombinant; Darbepoetin-alfa
Marginal grafts should be used more actively in Asian countries where deceased donor transplantation is unpopular. We modified a quantitative donor scoring system proposed by Nyberg and his colleagues and developed a donor scoring system in order to assess the quality of deceased donor grafts and their prognostic value as an initial effort to promote usage of marginal donors.
Materials and Methods
We retrospectively evaluated 337 patients.
A scoring system was derived from six donor variables [age, 0-25; renal function, 0-4; history of hypertension, 0-4; Human Leukocyte Antigen (HLA) mismatch, 0-3; body weight, 0-1; cause of death, 0-3 points]. Donor grafts were stratified by scores: grade A, 0-10; grade B, 11-20; grade C, 21-30; and grade D, 31-40 points. Donor grades significantly correlated with estimated glomerular filtration rate (eGFR) at 6 months (A, 64.0 mL/min/1.73 m2; B, 57.0 mL/min/1.73 m2; C, 46.8 mL/min/1.73 m2; p < 0.001). The five-year graft survival rate was also lower in grade C than grade A (74% vs. 93%, p = 0.002). Donors in grade C and D were regarded as marginal donors. The proportion of marginal donors was much lower in Korea, compared with data from the United Network for Organ Sharing (15.2% vs. 29%).
Considering the scarcity of deceased donor kidneys and the relatively better graft outcome with lower grade-donors in Korea, it is worth increasing the usage of marginal grafts.
Kidney transplantation; cadaver; donor selection
The long-term clinical benefits of vascular access blood flow (VABF) measurements in hemodialysis (HD) patients have been controversial. We evaluated whether early VABF may predict long-term vascular access (VA) patency in incident HD patients. We enrolled 57 patients, of whom 27 were starting HD with arteriovenous fistulas (AVFs) and 30 with arteriovenous grafts (AVGs). The patients' VABF was measured monthly with the ultrasound dilution technique over the course of the first six months after the VA operation. During the 20.4-month observational period, a total of 40 VA events in 23 patients were documented. The new VA events included 13 cases of stenosis and 10 thrombotic events. The lowest quartile of average early VABF was related to the new VA events. After adjusting for covariates such as gender, age, hypertension, diabetes, VA type, hemoglobin levels, body mass index, parathyroid hormone, and calcium-phosphorus product levels, the hazard ratio of VABF (defined as <853 mL/min in AVF or <830 mL/min in AVG) to incident VA was 3.077 (95% confidence interval, 1.127-8.395; P=0.028). There were no significant relationships between early VABF parameters and VA thrombosis. It is concluded that early VABF may predict long-term VA patency, particularly VA stenosis.
Renal Dialysis; Blood Flow Velocity; Vascular Patency; Indicator Dilution Techniques
Immune activation via TLRs is known to prevent transplantation tolerance in multiple animal models. To investigate the mechanisms underlying this barrier to tolerance induction, we used complementary murine models of skin and cardiac transplantation in which prolonged allograft acceptance is either spontaneous or pharmacologically induced with anti-CD154 mAb and rapamycin. In each model, we found that prolonged allograft survival requires the presence of natural CD4+Foxp3+ T regulatory cells (Tregs), and that the TLR9 ligand CpG prevents graft acceptance both by interfering with natural Treg function and by promoting the differentiation of Th1 effector T cells in vivo. We further demonstrate that although Th17 cells differentiate from naive alloreactive T cells, these cells do not arise from natural Tregs in either CpG-treated or untreated graft recipients. Finally, we show that CpG impairs natural Treg suppressor capability and prevents Treg-dependent allograft acceptance in an IL-6-independent fashion. Our data therefore suggest that TLR signals do not prevent prolonged graft acceptance by directing natural Tregs into the Th17 lineage or by using other IL-6-dependent mechanisms. Instead, graft destruction results from the ability of CpG to drive Th1 differentiation and interfere with immunoregulation established by alloreactive natural CD4+Foxp3+ Tregs.
We determined the relationship between the progression of immunoglobulin A nephropathy (IgAN) and the A1818T polymorphism in intron 2 of Angiotensin II type 2 receptor (AT2R) gene, which might play protective roles in the pathogenesis of IgAN. Patients with biopsy-proven IgAN were recruited from the registry of the Progressive REnal disease and Medical Informatics and gEnomics Research (PREMIER) which was sponsored by the Korean Society of Nephrology. A1818T polymorphism of AT2R gene was analyzed with PCR-RFLP method and the association with the progression of IgAN, which was defined as over 50% increase in baseline serum creatinine level, was analyzed with survival analysis. Among the 480 patients followed for more than 10 months, the group without T allele had significantly higher rates of progression of IgAN than the group with T allele (11.4% vs. 3.9%, p=0.024), although there were no significant differences in the baseline variables such as initial serum creatinine level, the degree of proteinuria, and blood pressure. In the Cox's proportional hazard model, the hazard ratio of disease progression in the patients with T allele was 0.221 (95% confidence interval for Exp(B): 0.052-0.940, p=0.041) compared to that of without T allele. In conclusion, A1818T polymorphism of AT2R gene was associated with the progression of IgAN.
Polymorphism, Single Nucleotide; Glomerulonephritis, IGA; Receptor, Angiotensin, Type 2
T cell Ig mucin 1 (TIM-1) plays an important role in regulating immune responses in autoimmune and asthma models, and it is expressed on both Th1 and Th2 cells. Using an antagonistic TIM-1–specific antibody, we studied the role of TIM-1 in alloimmunity. A short course of TIM-1–specific antibody monotherapy prolonged survival of fully MHC-mismatched vascularized mouse cardiac allografts. This prolongation was associated with inhibition of alloreactive Th1 responses and preservation of Th2 responses. TIM-1–specific antibody treatment was more effective in Th1-type cytokine–deficient Stat4–/– recipients as compared with Th2-type cytokine–deficient Stat6–/– recipients. Subtherapeutic doses of rapamycin plus TIM-1–specific antibody resulted in allograft acceptance and prevented the development of chronic allograft vasculopathy. Allograft survival via this treatment was accompanied by a Th1- to Th2-type cytokine switch. Depletion of natural Tregs abrogated the graft-protecting effect of the TIM-1–specific antibody. Importantly, CD4+CD25+ Tregs obtained from long-term survivors had enhanced regulatory activity as compared with naive CD4+CD25+ Tregs. Consistent with this, TIM-1–specific antibody treatment both preserved Tregs and prevented the expansion of alloreactive effector Th1 cells in an alloreactive TCR transgenic adoptive transfer model. These studies define previously unknown functions of TIM-1 in regulating alloimmune responses in vivo and may provide a novel approach to promoting transplantation tolerance.
Macrophage infiltration has been observed in the renal biopsy specimens of diabetic nephropathy (DN), and hyperglycemic state stimulates the renal expression of RANTES (regulated upon activation, normal T-cell expressed and secreted) and MCP-1 (monocyte chemoattractant protein-1). Upregulation of RANTES and MCP-1 with infiltrating macrophages may play a crucial role in the development and progression of DN. Genetic polymorphisms of RANTES and its receptors were reported to be independent risk factors for DN. We genotyped single nucleotide polymorphism (SNPs) in the MCP-1 G-2518A, CCR2 G46295A, RANTES C-28G and G-403A in 177 diabetic end-stage renal disease (ESRD) patients and 184 patients without renal involvement (controls) in order to investigate the effects of these SNPs on DN in Korean patients with type 2 DM. There were no differences in the frequencies of SNPs and the distribution of haplotypes of RANTES promoter SNPs between two groups. In conclusion, there were no associations of MCP-1, CCR2 and RANTES promoter SNPs with diabetic ESRD in Korean population. Prospective studies with clearly-defined, homogenous cohorts are needed to confirm the effect of these genetic polymorphisms on DN.
Diabetic Nephropathies; Kidney Failure, Chronic; Monocyte Chemoattractant Proteins; RANTES; Polymorphism, Single Nucleotide Polymorphisms; Diabetes Mellitus, Type 2