Transplanted Rats Research Articles

Dilated ureter for esophageal substitution: A preliminary experimental study in the rat

Esophageal replacement using digestive organs such as the colon, stomach, or jejunum has been used to treat long-gap esophageal atresia and caustic esophageal strictures. Nevertheless, it presents a major challenge. Here, we report a preliminary experimental study that examined the use of a free dilated ureter as an option for esophageal substitution in a transplantation rat model. Ten 28-week-old male donor rats underwent distal ureteral ligation for 4 weeks, and the total dilated ureters were recovered. In each of the ten recipient 20-week-old male rats, a ureter was transplanted through the mediastinum into the esophageal bed, without vascular anastomosis. All rats received cyclosporine and cotrimoxazole for 10 days. On postoperative day 10, the rats were sacrificed, and the transplanted ureters were evaluated macroscopically and histopathologically. All procedures were achieved. In the early postoperative period, three transplanted rats died. Upon macroscopic evaluation, no evidence of complications was observed, and all transplanted ureters exhibited apparently good firm tissue. Histopathological examination showed a viable ureteral structure with good vascularity, low inflammation, and regenerated epithelium in all rats. As an option for esophageal substitution, heterotopic ureteral transplantation can be performed directly into the mediastinal location of the esophagus, without vascular anastomosis in a rat model. In the future, free dilated ureters might be useful for esophageal grafting or patching in humans; however, this procedure must be validated in additional large animal models before being attempted in humans.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Student t-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

Comparison of Infiltrating T Cells, B Cells and Macrophages During Experimental Renal Allograft Rejection

Despite substantial improvement of modern immunosuppression and reduced rejection rates, acute rejection still remains an obvious clinical problem after kidney transplantation. Acute rejection represents an ongoing immunological risk factor for subsequent interstitial fibrosis and tubular atrophy, whereby acute rejection scored on basis of the Banff classification focuses on the degree of lymphocyte infiltration to the interstitium, tubuli and arterial vessels. However, for the histological grading of acute rejection the specific subtypes of infiltrating cells are not considered. We performed an experimental renal transplantation model (Brown-Norway to Lewis) as standardized data on the infiltration pattern of CD3 positive T-cells, CD68 positive macrophages and CD20 positive B-cells after kidney transplantation are missing. We examined the dynamics of above named infiltrating cell populations 6 and 28 days after allogeneic transplantation. These data were compared with syngeneic transplanted rats and control rats. Cyclosporine A was administered as immunosuppression (5 mg/kg bw/day) and trough levels were comparable to clinical practice in humans. The expression of infiltrating cells was evaluated in interstitial, perivenous, periarterial, periglomerular and intraglomerular regions of kidney allografts under light microscope and was on the other hand analyzed by two different morphometric software programs. Resulting data were correlated with the corresponding kidney function as well as with histopathological classification according to Banff. In CsA treated control rats there was nearly no positive staining for CD3, CD20 and CD68 in the different compartments. Whereas the morphometric software based analysis of syngeneic transplanted rats demonstrated only a moderate 4-fold increase in CD4, a 1.6-fold increase in CD20 and a 5.4-fold increase in CD68 infiltration in comparison with control rats, a tremendous increase was seen in allogeneic transplanted rats in all infiltrating cell populations. The relative increase in CD20 infiltration (57-fold induction) was even more pronounced than the increase in CD3 (20.5-fold induction) or CD68 (5.4-fold induction) infiltration. 28 days after allogeneic transplantation, the number of infiltrating cells was still significantly increased, but to a lower extend than on day 6. The expression profile of infiltrating cells in the different cellular compartments was in accordance with the morphometric analysis. In conclusion, we established a highly standardized experimental model of renal transplantation to study the infiltrating patterns of CD3, CD20 and CD68 positive cells. After allogeneic transplantation we demonstrated a significant induction of CD3, CD20 and CD68 positive cells. The amount of infiltrating cells and the detailed localization of the infiltrating cells in different kidney compartments were evaluated for the first time with different immunohistochemical analyzing methods. Besides infiltration of CD3 and CD68 infiltrating cells, the robust infiltration of CD20 B-cells, even at early time points after transplantation might act as a silent subclinical trigger for subsequent chronic humoral rejection and premature graft loss.

Quantification of Islet Loss During Immune Rejection Using Iron-Labeled Islet Cells by 3T MRI in the Rat Model

Background: Monitoring the fate of transplanted islets remains a major challenge in clinical islet transplantation (IT). We developed a MRI imaging protocol (3D difference ultra-short echo-time = dUTE), resulting in positive contrast images of superparamagnetic iron-oxide (SPIO) nanoparticles-labeled islets transplanted in rat. Our aim was to compare the evolution of the MRI signal in 3 types of IT in the rat model. Methods: Syngeneic and allogeneic SPIO-labeled islets (ferucarbotran, 280μg/ml iron) were injected intraportally into streptozotocin-induced diabetic Lewis rats. Xenogeneic human islets were transplanted into normoglycaemic rats and graft functionality was evaluated by serum human C-peptide levels. Images were performed on a 3T clinical scanner, from day 0 up to day 106. An intensity threshold was applied within the liver region, giving automatically the number of dUTE-enhanced pixels, allowing quantification. Histological studies included insulin, CD4 and CD8 staining and iron detection. Results: Decay rates for the 3 types of transplantation were different (Figure 1). The syngeneic graft signal showed a 20% decrease during the first 2 weeks and remained stable up thereafter. For allogeneic transplantation, islet rejection (G>20 mmol/l) occurred at day 7.7±0.5 and 41%±12 of the initial signal was lost. In the xenogeneic model, 43%±8 of the initial signal was lost by day 3, when significant basal and stimulated human C-peptide levels were not detected any longer. Islet rejection was confirmed by islet CD4+ and CD8+ cell infiltration and loss of insulin staining. When allogeneic transplanted rats are treated with anti-lymphocytic serum, glycemia remains normal but MRI signal shows a slight decrease corresponding to sub-clinical immune rejection. Conclusion: After intra-portal IT and during immune rejection, the loss of SPIO-labeled islets can be monitored with clinical-grade 3T MRI imaging using a semi-automatic quantification method. The decay of the signal is correlated with the graft function and histological findings.

Transplantation of β-Endorphin Neurons into the Hypothalamus Promotes Immune Function and Restricts the Growth and Metastasis of Mammary Carcinoma

Neurobehavioral stress has been shown to promote tumor growth and progression and dampen the immune system. In this study, we investigated whether inhibiting stress hormone production could inhibit the development of mammary carcinoma and metastasis in a rat model of breast carcinogenesis. To enhance β-endorphin (BEP), the endogenous opioid polypeptide that boosts immune activity and decreases stress, we generated BEP neurons by in vitro differentiation from fetal neuronal stem cells and transplanted them into the hypothalami of rats subjected to breast carcinogenesis. BEP-transplanted rats displayed a reduction in mammary tumor incidence, growth, malignancy rate, and metastasis compared with cortical cells-transplanted rats. BEP neuron transplants also reduced inflammation and epithelial to mesenchymal transition in the tumor tissues. In addition, BEP neuron transplants increased peripheral natural killer (NK) cell and macrophage activities, elevated plasma levels of antiinflammatory cytokines, and reduced plasma levels of inflammatory cytokines. Antimetastatic effects along with stimulation of NK cells and macrophages could be reversed by treatment with the opiate antagonist naloxone, the β-receptor agonist metaproterenol, or the nicotine acetylcholine receptor antagonist methyllycaconitine. Together, our findings establish a protective role for BEP against the growth and metastasis of mammary tumor cells by altering autonomic nervous system activities that enhance innate immune function.

Transplantation of neurospheres derived from bone marrow stromal cells promotes neurological recovery in rats with spinal cord injury

Previous studies have revealed that cell therapy using bone marrow stromal cells (BMSCs) could promote motor functional recovery in animals with spinal cord injury (SCI). We describe here the development of cell biology technique and the experimental study of regeneration in SCI. The aim of this study was to investigate the potential for neurological recovery by transplantation neurospheres (NS) derived from BMSCs into thoracic SCI. Adult Fisher rats were used: 45 were subjected to complete thoracic SCI performed by the balloon compression method. BMSCs were cultured in vitro to obtain NS. Seven days after thoracic SCI, groups of 15 rats each received transplants of BMSCs-NS (group A), BMSCs (group B), or injection of medium only (group C) into the SCI lesion. Rats from each group were evaluated and compared longitudinally for motor function recovery. The spinal cords (SC) of injured rats were harvested at day 21 or day 42 and prepared for histological analysis. Five weeks after transplantation, many neuronal or axonal sproutings were observed and replaced by host cells in the SCI lesion of group A. Also, transplanted BMSCs-NS expressed neuronal lineage markers. Transplanted rats could walk with weight bearing and showed recovered motor evoked potentials (MEPs).

Optimal location and time for neural stem cell transplantation into transected rat spinal cord.

Implanted neural stem cells (NSC) could improve neurological functions following spinal cord injury (SCI), but the optimal conditions for NSC transplantation are largely unknown, especially in transected spinal cord. This study investigated the effect and fate of NSC engrafted into spinal cords at different locations and time points following T(9) spinal cord transection. Engrafted NSC could survive and migrate in host spinal cords. Significant improvement in hindlimb locomotor functions associated with NSC survival was found in rats receiving NSC transplantation in the spinal cords rostral to the transection site at the subacute stage (7 days post operation), compared with those caudal to the transection site at the acute stage (at the time of injury). At 4 weeks post operation, CD68 immunohistochemical staining confirmed that macrophages were less in rostrally transplanted sites and in subacute groups than seen in caudal and acute transplanted rats. The present findings indicated that NSC transplantation into spinal cords rostral to transection site at the subacute stage is an optimal strategy for engrafted NSC survival and host behavioral improvement. It therefore would be available to the usage of NSC for the treatment of SCI in the future clinic trial.

Anti‐fibrotic effect of chorionic plate‐derived mesenchymal stem cells isolated from human placenta in a rat model of CCl4‐injured liver: Potential application to the treatment of hepatic diseases

Translational studies have explored the therapeutic effects of stem cells, raising hopes for the treatment of numerous diseases. Here, we evaluated the therapeutic effect of chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from human placenta and transplanted into rats with carbon tetrachloride (CCl(4))-injured livers. CP-MSCs were analyzed for hepatocyte-specific gene expression, indocyanine green (ICG) uptake, glycogen storage, and urea production following hepatogenic differentiation. PKH26-labeled CP-MSCs were directly transplanted into the livers of rats that had been exposed to CCl(4) (1.6 g/kg, twice per week for 9 weeks). Blood and liver tissue were analyzed at 1, 2, and 3 weeks post-transplantation. The expression of type I collagen (Col I) and matrix metalloproteinases (MMPs) was analyzed in rat T-HSC/Cl-6 hepatic stellate cells co-cultured with CP-MSCs following exposure to TGF-β. The expression levels of α-smooth muscle actin (α-SMA) and Col I were lower in transplanted (TP) rats than in non-transplanted (Non-TP) animals (P < 0.05), whereas the expression levels of albumin and MMP-9 were increased. TP rats exhibited significantly higher uptake/excretion of ICG than non-TP rats (P < 0.005). In addition, collagen synthesis in T-HSC/Cl-6 cells exposed to TGF-β was decreased by co-culture with CP-MSCs, which triggered the activation of MMP-2 and MMP-9. These results contribute to our understanding of the potential pathophysiological roles of CP-MSCs, including anti-fibrotic effects in liver disease, and provide a foundation for the development of new cell therapy-based strategies for the treatment of difficult-to-treat liver diseases.

P48 Haematopoietic but not mesenchymal stem cells contribute to the stromal microenvironment in cholangiocarcinoma and do not transdifferentiate into malignant bile ducts

AimCholangiocarcinoma (CC) is characterised by a pronounced inflammatory stroma consisting of tumour associated myofibroblasts, macrophages, immune cells and a modified extracellular matrix. Furthermore, in animal models of gastric cancer, reports...

Mesenchymal Stem Cells Infusion Prevents Acute Cellular Rejection in Rat Kidney Transplantation

Mesenchymal stem cells (MSC) are multipotent cells that differentiate into various mature cell lineages. MSC show immunomodulatory effects by inhibiting T-cell proliferation. We evaluated the effect of the infusion of MSC in rats experimental kidney transplantation. Sprague-Dawley transgenic rats (SD) able to express the green fluorescent protein (EGFP) were used as MSC donors. Syngeneic (Lewis to Lewis, n = 10) and allogeneic (Fischer to Lewis, n = 10) kidney transplantations were performed after bilateral nephrectomy. Five transplanted rats who received syngeneic grafts, were treated with 3 × 10 6 MSC (Gr B), while the other 5 did not received MSC (Gr A). Five rats with allogenic grafts received 3 × 10 6 MSC (Gr C) and another 5 did not receive MSC (Gr D). The MSC were infused directly into the renal artery of the graft. No immunosuppressive therapy was provided. The animals were killed after 7 days. Biochemical analysis for renal function, histological (Banff criteria) and immunohistological analysis (ED1+ and CD8+) were performed on treated animals. MSC improved kidney function in Gr B and D vs Gr A and C. The tubular damage appeared to be less severe among Gr B and Gr D with respect to Gr A and C ( P < .01). Vasculitis was more accentuated in Gr A and C ( P < .01). MSCs reduced the inflammatory infiltrate; in Gr B and D, the number of ED1+ cells was lower than in Gr A and C ( P < .005), which was also observed for CD8+ cells ( P < .05). Our study demonstrated that the infusion of MSC attenuated histological damage from acute rejection by reducing the cellular infiltration.

Vancomycin lung concentration in acute and hyperacute rejection models of lung transplantation in rats☆☆☆

This study assesses vancomycin concentrations in allogenic transplanted rat lung tissue in acute and hyperacute rejection models of the lung. Left lung allotransplantation was performed from a male Brown Norway donor to a male Fisher F344 recipient in the case of an acute rejection model (the animals were sacrificed 5 days after transplantation) as well as a male Brown Norway donor to a male Wistar recipient in the case of hyperacute rejection (the animals were sacrificed 2 days after transplantation). Control rats were sham-operated and sacrificed on day 2 or 5, respectively, of the experimental model. Rejection was confirmed by blood gas assessment and lung histological examination. A single intraperitoneal dose of vancomycin 30 mg kg(-1) body weight was administered on the day of autopsy (day 2 or 5 from transplantation, respectively, of the experimental model), and then the blood and lung specimens were sampled at 0, 5, 1, 2, 4 and 6h from the time of drug injection. Arterial blood gas assessment (Wistar rats - pO(2): 44.33 ± 21.73 mmHg (mean ± standard deviation (SD)); Fisher rats - pO(2:) 50.67 ± 14.30 mmHg (mean ± SD)) as well as histopathologic examinations of lung grafts confirmed rejection. Vancomycin lung/plasma concentration ratio was significantly higher in transplanted rats than in sham-operated animals. Both acute and hyperacute lung rejection affect the lung/serum of vancomycin in the transplanted lung resulting in higher drug accumulation, especially in late post-dosage time.

Limited functional recovery in rats with complete spinal cord injury after transplantation of whole-layer olfactory mucosa

The olfactory mucosa (OM) consists of 2 layers, the epithelium and the lamina propria. Attempts have been made to restore motor function in rat models of spinal cord injury (SCI) by transplanting olfactory ensheathing cells from the lamina propria, but there has been no attempt to transplant the OM in animal models. To investigate the potential of the OM to restore motor function, the authors developed a rat model of SCI and delayed transplantation of syngenic OM. Two weeks after complete transection of the spinal cord at the T-10 level in Wistar rats, pieces of syngenic whole-layer OM were transplanted into the lesion. Rats that underwent respiratory mucosa transplantation were used as controls. The authors evaluated the locomotor activity according to the Basso-Beattie-Bresnahan scale for 8 weeks after transplantation. Obtained spinal cords were analyzed histologically. Results The OM transplantation rats showed significantly greater hindlimb locomotor recovery than the respiratory mucosa-transplanted rats. However, the recovery was limited according to the Basso-Beattie-Bresnahan scale. In the histological examination, the serotonergic raphespinal tract was regenerated. The pseudocyst cavity volume in the vicinity of the SCI lesion correlated negatively with the functional recovery. Transplantation of whole-layer OM in rats contributes to functional recovery from SCI, but the effect is limited. In addition to OM transplantation, other means would be necessary for better outcomes in clinical situations.

Visual restoration and transplant connectivity in degenerate rats implanted with retinal progenitor sheets.

The aim of this study was to determine whether retinal progenitor layer transplants form synaptic connections with the host and restore vision. Donor retinal sheets, isolated from embryonic day 19 rat fetuses expressing human placental alkaline phosphatase (hPAP), were transplanted to the subretinal space of 18 S334ter-3 rats with fast retinal degeneration at the age of 0.8-1.3 months. Recipients were killed at the age of 1.6-11.8 months. Frozen sections were analysed by confocal immunohistochemistry for the donor cell label hPAP and synaptic markers. Vibratome slices were stained for hPAP, and processed for electron microscopy. Visual responses were recorded by electrophysiology from the superior colliculus (SC) in 12 rats at the age of 5.3-11.8 months. All recorded transplanted rats had restored or preserved visual responses in the SC corresponding to the transplant location in the retina, with thresholds between -2.8 and -3.4 log cd/m(2). No such responses were found in age-matched S334ter-3 rats without transplants, or in those with sham surgery. Donor cells and processes were identified in the host by light and electron microscopy. Transplant processes penetrated the inner host retina in spite of occasional glial barriers between transplant and host. Labeled neuronal processes were found in the host inner plexiform layer, and formed apparent synapses with unlabeled cells, presumably of host origin. In conclusion, synaptic connections between graft and host cells, together with visual responses from corresponding locations in the brain, support the hypothesis that functional connections develop following transplantation of retinal layers into rodent models of retinal degeneration.

F-18 Fluorothymidine (FLT) Imaging of the Bone Marrow Compartment in Rats Following Whole Body Irradiation, Stem Cell Transplantation and Spontaneous Recovery.

Abstract 3529Poster Board III-466Pet imaging using F-18 glucose (FDG) is increasingly being used for evaluation and staging of malignancy. However, staging in hematopoietic tissue using this agent has been hampered by poor specificity. F-18 flourothymidine (FLT) is currently being evaluated clinically as an imaging technique for tumor detection and staging. Secondary to its inclusion in DNA during the S phase, FLT is much more specific to proliferative tissue and less hampered by inflammatory background. As FLT uptake occurs in proliferating cell populations, we attempted to determine if imaging could provide useful information for evaluating global hematopoietic injury and recovery following radiation and transplantation. Three major groups of Wistar-Furth rats were studied. Group 1 consisted of rats receiving 950cGy of Whole Body Irradiation (TBI). Group 2 consisted of rats transplanted with syngeneic bone marrow 24-48 hrs following irradiation. Group 3 consisted of 6 rats exposed to a potentially sub-lethal dose of 500cGy and not transplanted. FLT imaging was performed before irradiation (n=4), 24-48 hrs. following irradiation, and on day 4-5 post transplantation. Subsequent imaging was carried out in 4 transplanted rats on days 8 and 14. Comparative FDG studies were also performed in selected animals. Table 1 summarizes the imaging studies performed in various subsets of rats.Table 1Imaging Subsets of Experimental Animals and Histologic CorrelationsExperimental rat subsetsFLT # studies performedFDG # studies performedHistologic correlationNormal or baseline rat studiesn=10n=6Normal cellular marrow24-48hrs post 950 cGy TBIn=6n=4Marrow damage hypocellularityDay 7 post 950 cGy TBIn=4not doneAplastic marrowDay 6-7 post 950cGy TBI (4-5 days post transplantation)n=4n=4Focal areas of cellular regenerationDay 10 post 950 cGy TBI (and transplantation)n=4n=2Cellular marrowDay 6-7 post 500cGy TBI (No transplantation)n=6not doneModerate hypocellularityFLT imaging results were correlated with marrow histology and clinical survival in treated and control groups. Six of 6 irradiated control rats died with marrow aplasia during the second week following 950 cGy. Sub-lethally irradiated and transplanted rats animals showed clear evidence of definitive recovery as early as 6 days post irradiation or 4 days post transplantation respectively. FLT activity of all major marrow sites was easily identified and was superior to FDG images. Findings correlated with histologic evidence of early marrow repopulation and survival. Figure 1 illustrates FLT and FDG imaging performed in normal, post radiation and post transplanted rats. [Display omitted] We conclude that FLT imaging represents a practical noninvasive technique to evaluate marrow injury and early recovery following radiation and hematopoietic transplantation. Disclosures:No relevant conflicts of interest to declare.

An Easy and Reproducible Model of Kidney Transplantation in Rats

Objective Kidney transplantation in rats is an important research model. Various methods have been reported, but there is no “standard operation.” We investigated a 1-stage versus a 2-stage native nephrectomy and the type of ureteral anastomosis seeking to establish a standard, reproducible and successful method. Materials and Methods We used PVG (RT1c-RT1Ac: B/Dc) male rats, weighing approximately 200 to 250 g, that underwent transplantation after right recipient nephrectomy. Left recipient nephrectomy was performed either 10 days later or simultaneously. The ureteric anastomosis was fashioned 2 ways: using a ureteral stent or by bladder insertion. Results Urinary complications (obstruction or reflux) were observed in 77.8% when a ureteral stent was used for the ureteric anastomosis versus 28.6% when using the bladder insertion technique ( P = .0211). Transplanted rats with nephrectomy of both native kidneys at the time of grafting showed a perioperative mortality of 70%, whereas those hosts with a 2-stage nephrectomy displayed a mortality rate of 22% ( P = .0025). Conclusions The bladder insertion technique reduced the incidence of urological complications in rats. In addition, unilateral native nephrectomy at the time of operation with delayed contralateral nephrectomy was better tolerated than simultaneous bilateral nephrectomy. These 2 surgical variants allowed us to perform kidney transplantation with a high degree of success.

18F-FDG PET for Semiquantitative Evaluation of Acute Allograft Rejection and Immunosuppressive Therapy Efficacy in Rat Models of Liver Transplantation

Acute allograft rejection remains a major complication after liver transplantation. We report a semiquantitative imaging method of detecting acute allograft rejection with (18)F-FDG PET. Syngeneic and allogeneic transplanted rats, with or without immunosuppressive treatment, were subjected to serial PET. Autoradiography of the liver was conducted in both the syngeneic and the allogeneic rats. A significant increment of (18)F-FDG accumulation in liver allografts was observed by PET on day 2. The (18)F-FDG signal was concentrated in the area where inflammatory cells around the vessels were detected by autoradiography. Allotransplanted rats treated with an immunosuppressive agent displayed a marked decrease in hepatic (18)F-FDG uptake, compared with allotransplanted rats that were not treated. (18)F-FDG PET may be a valid method for facilitating the development of protocols to diagnose graft rejection and to monitor the efficacy of immunosuppressive therapy.

Upregulation of TNF Receptor Type 2 in Human and Experimental Renal Allograft Rejection

An important role of TNF interacting with TNFR2 has been shown in different models of ischemic, nephrotoxic and immune-mediated renal injury. To systematically evaluate the expression of TNFR2 in renal allograft rejection, we investigated human renal allograft biopsies and, in addition, established an experimental transplantation model in rats to verify the human data under standardized conditions. The expression of TNFR2 was analyzed in 96 human renal allograft biopsies with different disease entities. In a 6-day and a 28-day experimental protocol, TNFR2 was examined in kidney specimens and in the urine of control, uni-nephrectomized and transplanted rats +/- cyclosporine treatment (n = 114). In human biopsies and in rat allografts on day 6 with acute allograft rejection, significantly elevated expression of TNFR2 was observed in tubular epithelial cells, podocytes, B cells and monocytes/macrophages. The expression level was associated with renal function. The TNFR2 expression level at day 28 was significantly lower compared to day 6. TNFR2 is markedly upregulated both in human and experimental acute renal allograft rejection. Our data are robust and consistent between different species, suggesting a role for TNFR2 in the early course of rejection.

Multidrug resistance-related protein 2 genotype of the donor affects kidney graft function

We tested the effect of kidney-specific multidrug resistance-related protein (MRP2, ABCC2) deficiency on renal organic solute disposition as well as on renal protein and gene expression. Furthermore, we investigated whether a particular kidney donor ABCC2 genotype is associated with delayed graft function in patients. A new MRP2-deficient rat strain was established. Renal cross-transplantations were performed between congenic MRP2-deficient and wild-type rats. Renal disposition of MRP2 substrates was investigated in native and transplanted rats. Proteomic analyses and transcriptional profiling were performed in rat kidney graft cortices. Ninety-eight human kidney donor-recipient pairs were genotyped for five ABCC2 polymorphisms. The relationship between delayed graft function and ABCC2 genetic variants in donors and recipients was analyzed by backward stepwise logistic regression. In rats, the absence of renal MRP2 reduced renal bilirubin glucuronide excretion at pathologic plasma concentrations, modified renal p-aminohippurate excretion and did not affect renal morphine-6-glucuronide excretion. Renal MRP2 deficiency led to renal cortical protein or mRNA upregulation of glutathione transferase isoenzymes, glutaredoxin 2, and heme oxygenase-1. In patients, a particular donor ABCC2 genotype was associated with an increased incidence of delayed graft function. Kidney graft-specific MRP2 deficiency has mild effects on the renal excretion of some organic solutes under experimental conditions and induces a protein and gene expression pattern indicative of activated antioxidant defense mechanisms. This suggests that MRP2 is a determinant of the redox status in tubular epithelial cells and thus of the susceptibility to renal damage under conditions of treatment with multiple drugs and increased oxygen radical formation.

Rat Umbilical Cord Stem Cells Completely Abolish Rat Mammary Carcinomas with No Evidence of Metastasis or Recurrence 100 Days Post–Tumor Cell Inoculation

Genetically engineered stem cells efficiently deliver therapeutic proteins to cancer and other sites of inflammation. However, a major advantage would be realized if tumor-trafficking stem cells that have not been genetically modified exhibit an inherent antitumor effect, thus circumventing the necessity of the expression of exogenous genes by the cells. We transplanted Fisher 344 rat-derived mammary adenocarcinoma cells (Mat B III) orthotopically into syngeneic F344 rats with an intact immune system. Rat umbilical cord matrix stem (rUCMS) cells derived from Wharton's jelly were then administered intratumoral (i.t) or i.v. 4 days later. The tumor attenuation effect was significantly evident starting from day 14 in i.v. and i.t. rUCMS cell-transplanted rats compared with sham-transplanted rats. In addition, unmodified rUCMS cell-transplanted rats showed complete regression of tumors to undetectable levels by 34 to 38 days with no evidence of metastasis or recurrence 100 days post-tumor cell inoculation. Dye-loaded rUCMS cells were identified within tumors only 4 days after their i.v. transplantation. In vitro colony assays with rUCMS cells as feeder layers markedly reduced Mat B III colony size and number. Growth attenuation of Mat B III cells exposed to either rUCMS cells directly or to the conditioned medium derived from rUCMS cells was associated with apoptosis indicators, including increased activated caspase-3. In addition, rUCMS cells cocultured with Mat B III cells had a dose-dependent antiproliferative effect on Mat B III cells. These findings suggest that unmodified human UCMS cells could be used for targeted cytotherapy for breast cancer.

Ebselen Improves Ischemia-Reperfusion Injury After Rat Lung Transplantation

The heterocyclic organic compound ebselen (2-phenyl-1,2-benizsoselenazol-3(2H)-one) is a glutathione peroxidase mimick with protective properties against oxidative injury. Ebselen also has anti-inflammatory activity, including attenuation of tumor necrosis factor release and increase of interleukin-10, as shown in vivo, in inflammatory and ischemia-reperfusion injuries, including those of the lung. This study was designed to assess its effect on severe ischemia-reperfusion injury in a model of left-sided rat lung isotransplantation. Orthotopic single left-sided lung allotransplantation (Wistar to Wistar) was performed in female rats after a total ischemic time of 18 h. In nine recipients given 500 mg/kg oral ebselen 1 h before transplantation, graft PaO(2)/FiO(2) was improved 24 h after transplantation, as evidenced with a mean (standard deviation) PaO(2) of 139 (61) mmHg vs. eight controls with 65 (33) mmHg (p = 0.009). Bronchoalveolar PMN count was reduced to approximately 50% in the ebselen group compared with controls, whereas no difference in the tumor necrosis factor content was found. We conclude that the improvement of lung function in ebselen-treated transplanted rats is mainly the result of the anti-inflammatory activity of the drug during reperfusion.