Examination of gene expression profile of functional human pancreatic islets after 2-week culture

Islet transplant faces significant challenges, mainly because of the high incidence of primary nonfunction of transplanted islets. Protocol modifications to improve the rate of islet function have included changes in pancreatic preservation and the introduction of short-term culture. Islet culture for 48 to 72 hours has become a standard part of most successful protocols for clinical islet transplantation. We have previously reported gene expression profiles associated with human pancreatic islet function. The aim of this study was to determine the change in gene expression profiles of functional islets after 2 weeks of culture in Memphis-serum free media. Human islets from four isolations were maintained in culture for 14 days in Memphis-serum free media. RNA was extracted from 10000 IEQ for analysis of the gene expression profiles using high-density Affymetrix U133A GeneChips and Genespring software. Islet function was assessed by measurements of human C-peptide at days 7 and 14 posttransplant into NOD-SCID mice. Human C-peptide levels were determined by radioimmunoassay. Our preliminary data showed that genes related to functionality, such as those directed toward insulin processing and secretion, did not vary over 14 days of culture, while genes related to exocrine pancreas and organ architecture and immune-associated genes decreased over time. The ability to maintain islets in culture is an important step toward the development of islet tissue repositories, as well as toward screening human islet preparations for additional pathogens.     

Thiazide-induced hypocalciuria is accompanied by a decreased expression of Ca2+ transport proteins in kidney

INTRODUCTION: Thiazide diuretics have the unique characteristic of increasing renal Na+ excretion, while decreasing Ca2+ excretion. However, the molecular mechanism responsible for this thiazide-induced hypocalciuria remains unclear. The present study investigates the effect of thiazides on the expression of the proteins involved in active Ca2+ transport as well as the role of extracellular volume (ECV) status. METHODS: Hydrochlorothiazide (HCTZ), 12 mg/24 hours, was administered during 7 days to Wistar rats by osmotic minipumps. In addition, ECV contraction was either prevented by Na+ repletion or induced by a low-salt diet. Expression levels of the proteins involved in active Ca2+ transport [i.e., epithelial Ca2+ channel (TRPV5/ECaC1), calbindin-D28K, Na+/Ca2+ exchanger (NCX1)], as well as the thiazide-sensitive Na+ Cl- cotransporter (NCC) were determined by real-time quantitative polymerase chain reaction (PCR) and semiquantitative immunohistochemistry. RESULTS: HCTZ significantly reduced urinary Ca2+ excretion (22%+/- 5% relative to controls). Hematocrit was significantly increased, confirming ECV contraction. In addition, Na+ depletion virtually abolished Ca2+ excretion (8%+/- 1%), while Na+ repletion during HCTZ treatment prevented both ECV contraction and hypocalciuria. HCTZ significantly decreased mRNA expression of TRPV5 (71%+/- 6%), calbindin-D28K (53%+/- 6%), NCX1 (51%+/- 8%) and NCC (50%+/- 11%), regardless of ECV status or calciuresis. Immunohistochemistry revealed reduced TRPV5 (43%+/- 2%), calbindin-D28K (59%+/- 1%) and NCC (56%+/- 4%) abundance. Furthermore, during HCTZ treatment, the subset of tubules coexpressing NCC and calbindin-D28K was significantly reduced (43%+/- 5%) and a disturbed cellular localization of NCC was observed. CONCLUSION: These data suggest that ECV contraction is a critical determinant of the thiazide-induced hypocalciuria, which is accompanied by a decreased expression of Ca2+ transport proteins.     

Human leukocyte antigen-G expression after kidney transplantation is associated with a reduced incidence of rejection

HLA-G is a non-classic Human Leukocyte Antigen (HLA-G) Class I of low polymorphism and restricted tissue distribution that displays tolerogenic functions. In heart transplantation and in combined liver/renal allograft transplantation, the expression of HLA-G has been associated with a lower incidence of acute graft rejection episodes and absence of chronic dysfunction. Since the expression of HLA-G in renal biopsies has been investigated only in few patients who received a combined kidney and liver transplant, in this study we performed a cross-sectional study, systematically comparing the expression of HLA-G in post-transplanted renal grafts, stratifying patients according to the presence or absence of rejection. PATIENTS AND METHODS: Seventy-three renal specimens (10 with acute rejection and 13 with chronic allograft nephropathy, and 50 with no signs of rejection) were immunohistochemically evaluated for HLA-G expression. RESULTS: In the group as a whole, HLA-G molecules were detected in 40 cases (54.8%). Among specimens that presented HLA-G expression, 2 out of 40 (5%) exhibited acute rejection, 2 (5%) exhibited chronic allograft nephropathy, and the remaining 36 (90%) exhibited no signs of rejection. The comparison between patients with rejection and those without rejection showed that the expression of HLA-G was significantly increased in specimens exhibiting no signs of rejection (p<0.0001). Considering only patients with acute rejection, 8 out of 10 patients showed no HLA-G expression in their kidney biopsies when compared to patients exhibiting no signs of rejection and absence of HLA-G was observed in 14 out of 50 (p=0.0032). Similarly, considering only patients with chronic allograft nephropathy, absence of HLA-G expression was observed in 11 out of 13 specimens, whereas in patients without rejection absence of HLA-G was observed in 14 out of 50 (p=0.003). Therapy with tacrolimus was significantly associated with the expression of HLA-G and a better graft prognosis. CONCLUSIONS: Our results suggest that HLA-G expression in the kidney allograft and the use of tacrolimus are associated with a lower frequency of acute renal rejection and chronic allograft nephropathy.     

β-榄香烯对肾癌放疗增敏相关基因表达谱的影响

目的探讨β-榄香烯对肾癌GRC-1细胞体外放射增敏相关基因表达谱的影响。方法体外培养GRC-1细胞,MTT法检测不同放射剂量(0、50、100、150及200 cGy)下,不同浓度β-榄香烯(空白组、空白乳组及10、20、30、40、50、60、70、80以及90μg/mlβ-榄香烯组)对GRC-1细胞体外生长的影响。流式细胞仪检测单纯放疗组(空白组,150 cGy)和放疗增敏组(20μg/mlβ-榄香烯,150 cGy)细胞周期变化与凋亡。选取包含4096个cDNA基因表达谱芯片分析2组间基因谱表达差异。结果β-榄香烯在20μg/ml时对GRC-1细胞有体外放射增敏作用。对肾癌细胞G2M阻滞作用随时间增加而增强,24 h时凋亡率为17.26%,48 h时作用达最高峰,凋亡率为24.34%。随时间和照射剂量增加细胞凋亡水平增高。基因芯片筛选出2组间差异表达基因360条,上调基因265条、下调基因95条。结论β-榄香烯乳对肾癌细胞的放疗增敏作用可能涉及原癌、抑癌基因等多种相关基因的变化,是一个多基因参与的复杂事件。     

Aldosterone resistance in kidney transplantation is in part induced by a down-regulation of mineralocorticoid receptor expression

BACKGROUND: After renal transplantation immunosuppressive drugs-like cyclosporin A (CsA) and FK506 induce either hypoaldosteronism or pseudo-hypoaldosteronism presenting with hyperkalemia and metabolic acidosis. We investigated the relationship between renal allograft function under CsA therapy and plasma aldosterone concentration, potassium- and water homeostasis and mineralocorticoid receptor (MR) expression level in peripheral leukocytes. METHODS: We studied 21 renal transplant patients under CsA therapy and 12 healthy controls. Transplant recipients were studied before and under fludrocortisone treatment. Using quantitative reverse-phase polymerase chain reaction (RT-PCR) specific for the MR, we analyzed the level of expression of MR in peripheral leukocytes. RESULTS: In acidotic transplant recipients (HCO(3) 18.5 +/- 1.2 mM) renal function was only slightly impaired with 2.0 +/- 0.2 mg creatinine/dL when compared with 1.8 +/- 0.3 mg/dL (ns) in non-acidotic patients (HCO(3) 23.0 +/- 2.8 mM). Mean plasma aldosterone levels in renal transplant recipients did not differ from control levels (150 +/- 33 pg/mL vs. 148 +/- 33 pg/mL, ns). In contrast, the expression level of MR in peripheral leukocytes of renal transplant recipients treated with CsA was significantly decreased when compared with healthy controls without renal disease (120 +/- 78 vs. 423 +/- 73 RNA molecules/0.5 microg total RNA, p < 0.01). The level of expression of MR in renal transplant recipients did not differ between acidotic patients and non-acidotic patients (ns). The application of fludrocortisone reversed hyperkalemia and metabolic acidosis without significant effect on MR expression. CONCLUSIONS: The present data demonstrate that hyperkalemia and metabolic acidosis following CsA treatment in kidney transplantation might be associated with a down-regulation of MR expression on peripheral leukocytes. Electrolyte imbalance is reversible on application of fludrocortisone. This observation supports fludrocortisone treatment in transplant patients with severe electrolyte disturbances.     

Bone morphogenetic protein-7 expression and activity in the human adult normal kidney is predominantly localized to the distal nephron

Bone morphogenetic protein-7 (BMP)-7 plays an important role during fetal kidney development. In the adult, BMP-7 is most strongly expressed in the kidney compared to other organs, but the exact expression pattern as well as the function of BMP-7 is unclear. The major aim of the present study was to define which parts of the human kidney do physiologically express BMP-7 and which cells appear to be targets of BMP activity by showing phosphorylated BMP-receptor-associated Smads 1, 5, or 8 and inhibitor of differentiation factor 1 (ID1) expression. BMP-7 expression was localized by immunohistology to the epithelia of the distal tubule as well as the collecting ducts (CDs). Phospho-Smads 1/5/8 and ID1 expression largely colocalized with BMP-7 and was also localized in the epithelia of the distal tubule and the CDs. This was confirmed by polymerase chain reaction-based mRNA expression analysis. In vitro, proximal tubular cells (PTCs) expressed BMP receptors and BMP-receptor-associated Smads and were reactive to BMP-7. Our data indicate that BMP-7 expression in the adult human kidney appears to be more restricted than in the fetal situation and predominantly found in the distal nephron. Also, evidence of in vivo BMP signalling (i.e. phospho-Smads and ID1 expression) was found there. These findings suggest that BMP-7 plays a physiological role mostly in this part of the kidney. Still, as reported previously, PTCs are responsive to BMP-7, but presumably not in an autocrine or paracrine mode in normal adult kidneys.     

Gonocyte transformation in a congenitally cryptorchid rat is normal and may be similar to the situation reported in human acquired cryptorchidism

Background

In congenital undescended testis (UDT) in humans, thermal insult damages early germ cell development during mini-puberty (3–6 months) causing increased risk of both cancer and infertility. In rodents however, UDT causes infertility but not cancer. In the TS rat with congenital UDT we hypothesized that early germ cell development would be normal as UDT only becomes manifest at 3–4 weeks (and the germ cells only become sensitive to thermal injury) after minipuberty is complete at 1 week.

肾移植术后早期受者外周血淋巴细胞基因表达谱研究

目的 从基因水平了解淋巴细胞在肾移植免疫反应过程中作用的分子机理。方法 分离16例未发生临床急性排斥反应的肾移植受者术前和术后第7天的外周血淋巴细胞，应用基因芯片技术，与包含4096个人eDNA克隆的微矩阵芯片杂交，分析2组细胞基因表达谱的差异。结果 肾移植术后第7天，外周血淋巴细胞差异表达基因有135个，其中下调基因53个，上调基因82个。这些基因主要涉及细胞信号转导、原癌基因类、DNA结合转录和蛋白翻译合成、细胞凋亡，另外也有离子通道和运输蛋白、细胞周期蛋白、细胞骨架和运动蛋白、代谢、发育相关基因等。结论 肾移植术后7d，受者外周血淋巴细胞一系列基因转录活化，说明肾移植术后早期免疫反应过程涉及多种类型的多个基因；基因芯片技术可有效筛选出肾移植术后受者外周血淋巴细胞的差异表达基因。     

Proliferation, migration, and differentiation of human neural stem/progenitor cells after transplantation into a rat model of traumatic brain injury

OBJECT: Cultures containing human neural stem and progenitor cells (neurospheres) have the capacity to proliferate and differentiate into the major phenotypes of the adult brain. These properties make them candidates for therapeutic transplantation in cases of neurological diseases that involve cell loss. In this study, long-term cultured and cryopreserved cells were transplanted into the traumatically injured rat brain to evaluate the potential for human neural stem/progenitor cells to survive and differentiate following traumatic injury. METHODS: Neural stem/progenitor cell cultures were established from 10-week-old human forebrain. Immunosuppressed adult rats received a unilateral parietal cortical contusion injury, which was delivered using the weight-drop method. Immediately following the injury, these animals received transplants of neural stem/progenitor cells, which were placed close to the site of injury. Two or 6 weeks after the procedure, these animals were killed and their brains were examined by immunohistochemical analysis. At both 2 and 6 weeks postoperatively, the transplanted human cells were found in the perilesional zone, hippocampus, corpus callosum, and ipsilateral subependymal zone of the rats. Compared with the 2-week time point, an increased number of HuN-positive cells was observed at 6 weeks. In addition, at 6 weeks post-injury/transplantation, the cells were noted to cross the midline to the contralateral corpus callosum and into the contralateral cortex. Double labeling demonstrated neuronal and astrocytic, but not oligodendrocytic differentiation. Moreover, the cortex appeared to provide an environment that was less hospitable to neuronal differentiation than the hippocampus. CONCLUSIONS: This study shows that expandable human neural stem/progenitor cells survive transplantation, and migrate, differentiate, and proliferate in the injured brain. These cells could potentially be developed for transplantation therapy in cases of traumatic brain injury.     

采用基因芯片筛选与汗腺发育相关基因的初步研究

目的：探讨人胚胎皮肤汗腺在不同发育胎龄时的基因表达变化特征及其可能的生物学意义。方法：收集12周（H1）、16周（H2）、24周（H3）等3组不同胎龄胎儿皮肤,组织常规切片,显微镜下观察胚胎皮肤不同发育阶段的汗腺结构特征,提取不同胎龄的胚胎表皮组织总RNA,用逆转录-聚合酶链反应（RT-PCR）方法和基因芯片技术检测胚胎皮肤汗腺在不同发育阶段的基因表达变化规律。结果：光镜下可见不同发育阶段的胎儿皮肤具有典型的组织学结构,胎龄24周时汗腺结构初步形成,数量稳定。在95%的可信区间内,H2与H1样本相比较（H2/H1）,差异表达基因有113个,其中下调的基因有45个,上调的基因有68个;H3与H2样本相比较（H3/H2）,差异表达基因有91个,其中下调的基因有66个,上调的基因有25个;差异表达的基因包括生长因子、细胞外基质分子和外胚层发育因子等,与汗腺发育进程密切相关。结论：将基因芯片运用于汗腺发生机制的研究,可以较全面地反映汗腺发育及成熟过程中基因表达变化规律,寻找特异性调控基因。通过对汗腺不同发育阶段的基因表达及功能研究,可以筛选关键步骤调控基因,指导实现分子基因水平调控皮肤创面修复和汗腺组织再生。     

Tubular gigantism of the kidney in a 41-mm, Streeter's 23rd horizon, human fetus with a comparative study of renal structures in normal human fetuses at a similar stage of development

This is a comparative study of the metanephric development of 1 abnormal and 7 normal human fetuses, all of them measuring approximately 41 mm in vertex-coccyx length (48-50 days of gestation). The abnormal fetus presented with dilatation of the collecting tubules and is, in accordance with our review of current literature, the first case in which this malformation has been described at such an early (embryonal) stage of development. Many theories have tried to explain the pathogenesis of this malformation. Our studies agree with the theory that proposes a delay in tubular canalization, to which changes in the structure of the tubular wall might be added.     

小鼠移植心心肌细胞转染白细胞介素10基因减轻术后免疫排斥反应

目的观察应用质粒载体转染白细胞介素10(IL-10)基因至小鼠移植心肌细胞内的转染效率和基因的表达效率,探讨IL-10分子在移植物急性排斥反应中的意义。方法以C57BL／6小鼠为供者,Balb／c小鼠为受者,建立异位心脏移植模型。根据移植前经供心升主动脉冠脉灌注试剂的不同,将受者分为4组,每组20只。A组:灌注30μl的生理盐水;B组:灌注30μl的空白质粒;C组:灌注30μl的IL-10基因;D组:灌注30μl的腺相关病毒(AAV)增强型IL-10。移植后1、3、5和7 d切取移植心脏,进行病理学检查、移植心心肌组织中基因产物的逆转录聚合酶链反应(RT-PCR)和IL-10的免疫组织化学检测。并观察各组存活受者的一般情况和移植心存活时间。结果病理学观察显示:A、B组移植心在术后第3 d起出现了明显的排斥反应改变,而C、D组第3、5和7 d移植心排斥反应改变程度均较A、B组明显减轻。移植后第1和3 d,A、B组移植心心肌细胞中可见低水平的IL-10 mRNA基因和IL-10蛋白的表达,而第5和7 d时几乎检测不到;C、D组在各个时间段都可见明显的IL-10 mRNA基因和IL-10蛋白表达。A、B组移植心存活时间分别为(8．125±0．991)d和(7．714±0．756)d;C、D组移植心存活时间分别为(15．714±2．498)d和(17．857±1．864)d。C、D组与A、B组比较,差异有统计学意义(P<0．01)。结论质粒载体能有效地将IL-10基因转染入小鼠心肌细胞并进行表达,而采用AAV的末端反向重复序列(ITRs)可以提高质粒转染效率和IL-10表达水平。局部高表达的IL-10能减轻移植心的排斥反应,延长其存活时间。