In Vivo Imaging of Neo-angiogenesis of Transplanted Metastases in Subrenal Capsule Assay Induced Rat Model.

BACKGROUND/AIM: Changes in the expression of neo-angiogenic molecules in the primary tumor and its metastases may significantly affect the efficacy of therapies. The aim of this study was to evaluate the alterations in aminopeptidase N (APN/CD13) and αvß3 integrin receptor expression in serially transplanted mesoblastic nephroma tumor (Ne/De) metastases using 68Gallium (68Ga)-labeled NOTA-cNGR and NODAGA-RGD radiotracers and preclinical positron emission tomography (PET) imaging. MATERIALS AND METHODS: Primary and metastatic mesoblastic nephroma (Ne/De) tumors were induced by subrenal capsule assay (SRCA) in Fischer-344 rats. In vivo PET imaging experiments were performed 8±1 days after the SRCA surgery using intravenously injected 68Ga-NOTA-c(NGR), 68Ga-NODAGA-RGD, and [18F]FDG radiotracers. RESULTS: Among the examined neo-angiogenic molecules, the expression of αvß3 integrin in the tumors was significantly lower than that of APN/CD13. This observation was confirmed by the PET data analysis, where a 2-6-fold higher APN/CD13-specific 68Ga-NOTA-cNGR accumulation was observed in both primary malignancies and metastases. However, a steadily increased accumulation of [18F]FDG, 68Ga-NODAGA-RGD, and 68Ga-NOTA-cNGR was observed in the tumors growing under the renal capsule and in the metastatic parathymic lymph nodes during serial transplantations. The observed increase in 68Ga- NOTA-cNGR accumulation during serial transplantations correlated well with the western blot analysis, where APN/CD13 protein levels were also elevated in the metastatic parathymic lymph nodes. CONCLUSION: The observed increase in glucose metabolism and the up-regulated expression of αvß3 integrin and APN/CD13 during serial transplantations of metastases may indicate enhanced malignancy.

Establishment and preclinical application of a patient-derived xenograft model for uterine cancer.

BACKGROUND: The patient-derived xenograft (PDX) model is a promising translational platform for duplicating the characteristics of primary tumors. Here, we established and characterized PDX models of uterine cancer to demonstrate their utility for preclinical drug testing. MATERIALS AND METHODS: We generated PDX tumors surgically derived from 58 cases of uterine cancer. Subrenal capsule xenografts and primary tumors were compared using microscopic examination, short tandem repeat analyses, and targeted sequencing analyses. A phosphatidylinositol 3-kinase (PI3K) inhibitor was administered to mice whose PDX tumors harbored a PTEN deletion or PIK3CA mutation. We also generated an orthotopic PDX model using uterine horn implantation. RESULTS: Thirty-three (56.9%) PDXs were successfully generated and passaged to maintain tumors. The histological features of the PDX tumors were stable over subsequent passages. By contrast, the proportions of epithelial and mesenchymal components of carcinosarcoma PDX models varied by generation. Targeted sequencing analyses revealed that all mutated cancer-related genes were stable during establishment and subgrafting. Treatment with a PI3K inhibitor cased a significant decrease in tumor weight in the clear cell carcinoma PDX harboring a frameshift PTEN deletion (p = 0.049) and in the serous carcinoma PDX harboring a missense PI3KCA mutation (p = 0.003) compared with matched controls. We also successfully established orthotopic PDX models (3/3; 100.0%). CONCLUSIONS: The histological and genetic features of PDXs were similar to those of primary tumors. This model is a promising translational platform for preclinical testing of new anticancer drugs and will enable the personalized development of therapeutic options for uterine cancer.

Tissue Recombination and Kidney Capsule Transplantation Assays for the Study of Epithelial-Mesenchymal Interactions.

Tissue interactions are crucial during the development of organs. Among the most studied tissue interactions are those that take place between the epithelial cells and the underlying mesenchymal cells, known as epithelial-mesenchymal interactions. Tissue recombination assay is a valuable model to study the mechanisms involved in the regulation of such interactions. Here, we describe how to dissociate and recombine the epithelial and mesenchymal components of the embryonic tooth. In addition, we explain how to transplant the recombined tissues under the kidney capsule of immunocompromised mice in order to allow their further development into a mature tooth.

Human iPSC-MSC-Derived Xenografts Modulate Immune Responses by Inhibiting the Cleavage of Caspases.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-ß1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732.

Patient-derived xenografts of gastrointestinal cancers are susceptible to rapid and delayed B-lymphoproliferation.

Patient-derived cancer xenografts (PDX) are widely used to identify and evaluate novel therapeutic targets, and to test therapeutic approaches in preclinical mouse avatar trials. Despite their widespread use, potential caveats of PDX models remain considerably underappreciated. Here, we demonstrate that EBV-associated B-lymphoproliferations frequently develop following xenotransplantation of human colorectal and pancreatic carcinomas in highly immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice (18/47 and 4/37 mice, respectively), and in derived cell cultures in vitro. Strikingly, even PDX with carcinoma histology can host scarce EBV-infected B-lymphocytes that can fully overgrow carcinoma cells during serial passaging in vitro and in vivo. As serial xenografting is crucial to expand primary tumor tissue for biobanks and cohorts for preclinical mouse avatar trials, the emerging dominance of B-lymphoproliferations in serial PDX represents a serious confounding factor in these models. Consequently, repeated phenotypic assessments of serial PDX are mandatory at each expansion step to verify "bona fide" carcinoma xenografts.

Diffuse large B-cell lymphoma patient-derived xenograft models capture the molecular and biological heterogeneity of the disease.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.

A patient-derived subrenal capsule xenograft model can predict response to adjuvant therapy for cancers in the head of the pancreas.

BACKGROUND: Although gemcitabine is commonly used as adjuvant therapy for pancreatic adenocarcinoma and pancreaticobiliary-type periampullary cancers, not all patients appear to benefit. This translational study evaluates the potential of a patient-derived subrenal capsule pancreatic cancer xenograft (SRCPCX) model to identify within eight weeks after surgery those tumours which will respond to gemcitabine. METHODS: SRCPCXs from 32 pancreatectomy patients were established in six to ten NOD/SCID mice per patient. After four weeks the mice were randomly assigned to receive gemcitabine or saline for four more weeks. After eight weeks, gemcitabine response in the grafts was evaluated by the percentage of tumour growth inhibition (%TGI), histological morphology and immunohistochemical markers (Ki-67, CK7 and cleaved caspase-3). These were collated into an Overall Response. Survival was assessed by Kaplan-Meier and Cox multivariate analyses. RESULTS: 375 of 450 pieces of tissue from 27 of 31 patients were evaluable. In 90% of patients, histopathological and immunostaining features of saline-treated control grafts were concordant with their original tumours. At follow up, six of 15 patients whose tumours had an Overall Response to gemcitabine died, compared with ten of 12 whose tumours did not respond (P = 0.025, Fisher's exact test). This was associated with improved survival on Kaplan-Meier analysis (P = 0.013). Cox multivariate analysis indicated that Overall Response, stage and grade were independent predictors of survival. CONCLUSION: This SRCPCX model retains major histopathological and immunohistochemical characteristics of the original tumour and when a combination of measures is used, enables early assessment of tumour sensitivity to gemcitabine in pancreatic cancers.

Decellularized liver scaffolds effectively support the proliferation and differentiation of mouse fetal hepatic progenitors.

Decellularized whole organs represent ideal scaffolds for engineering new organs and/or cell transplantation. Here, we investigate whether decellularized liver scaffolds provide cell-friendly biocompatible three-dimensional (3-D) environment to support the proliferation and differentiation of hepatic progenitor cells. Mouse liver tissues are efficiently decellularized through portal vein perfusion. Using the reversibly immortalized mouse fetal hepatic progenitor cells (iHPCs), we are able to effectively recellularize the decellularized liver scaffolds. The perfused iHPCs survive and proliferate in the 3-D scaffolds in vitro for 2 weeks. When the recellularized scaffolds are implanted into the kidney capsule of athymic nude mice, cell survival and proliferation of the implanted scaffolds are readily detected by whole body imaging for 10 days. Furthermore, epidermal growth factor (EGF) is shown to significantly promote the proliferation and differentiation of the implanted iHPCs. Histologic and immunochemical analyzes indicate that iHPCs are able to proliferate and differentiate to mature hepatocytes upon EGF stimulation in the scaffolds. The recellularization of the biomaterial scaffolds is accompanied with vascularization. Taken together, these results indicate that decullarized liver scaffolds effectively support the proliferation and differentiation of iHPCs, suggesting that decellularized liver matrix may be used as ideal biocompatible scaffolds for hepatocyte transplantation.

Detection of viability of transplanted beta cells labeled with a novel contrast agent - polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles by magnetic resonance imaging.

Islets can be visualized on MRI by labeling with superparamagnetic contrast agent during the transplantation procedure. However, whether the signal intensity reflects the cell number and cellular viability has not been determined. We used a self-synthesized novel superparamagnetic contrast agent -polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles (PVP-SPIO) - to label ß-TC-6 cells (a mouse insulinoma cell line) or primary islets with commercial Feridex as a control. The labeling efficiency of two agents was compared by Prussian blue staining, intracellular iron content determination and MR scanning. Cells were exposed to hypoxia, high-glucose or exogenous H2O2 stimulation before/after PVP-SPIO labeling. Normal and injured cells were also transplanted into renal subcapsule. A clinically used 3.0 T MR scan was performed in vitro and 24 h post-transplantation to investigate the correlation between cellular viability and signal. Our PVP-SPIO displayed superior biocompatibility and magnetic properties. All of the cells could be labeled at 100 µg/ml iron concentration after 24 h incubation. At 100 µg/ml iron concentration, 1 × 105 ß cells labeled with PVP-SPIO could already be visualized in vitro by MRI, less than the detection threshold of Feridex. There existed a linear correlation between the number of labeled cells and R2 value on the T2 -weighted images. The signal intensity and the intracellular iron content declined along with the decreased viability of labeled cells. There was also a significant difference in signal intensity between injured and normal labeled cells after transplantation. From these results, we concluded that PVP-SPIO possessed superior cell labeling efficiency, and ß cells could be labeled without compromising viability and function. The signal intensity on MRI might be a useful predictor to evaluate the number and the viability of PVP-SPIO-labeled cells.

Use of irinotecan for treatment of small cell carcinoma of the prostate.

BACKGROUND: Prostatic small cell carcinoma (SCC) is a rare variant of prostate cancer. It is extremely aggressive and resistant to available therapies with a median survival range of 5-17 months. No standard chemotherapeutic regimen has been established for its treatment. In search of a new therapeutic approach, we examined the response of patient-derived prostatic SCC tissue xenografts to irinotecan, a topoisomerase I inhibitor. METHODS: A tumor tissue line was established from a patient's prostatic SCC by subrenal capsule grafting using NOD-SCID mice. Mice carrying subcutaneous transplants of the tumor line were then treated for 2 weeks with irinotecan alone and in combination with cisplatin. The effect on tumor volume, histopathology, and apoptosis were determined. RESULTS: The prostatic SCC tissue line resembled the donor tissue in morphologic and immunohistochemical features. Irinotecan (20 mg/kg/day; days 1-3, 8-10) completely arrested xenograft growth with a small reduction in tumor volume and only minor weight loss of the hosts (7%); irinotecan (12 mg/kg; same schedule) + cisplatin (2.5 mg/kg/day; days 1 and 8) had a similar effect, but with lower weight loss. While the growth inhibition involved apoptosis, it was also associated with a marked increase in autophagy. CONCLUSIONS: Tumor tissue lines established via subrenal capsule xenografting provide models with clinical relevance and the present study suggests that irinotecan could be useful for therapy of refractory prostatic SCC, in particular in combination with cisplatin.

[Establishment of a xenograft model of human prostate cancer in mouse].

OBJECTIVE: To establish the murine xenograft model of human prostate cancer by grafting tumor tissues beneath the renal capsule of intact male athymic mouse. METHODS: Fifteen SCID mice were randomly divided into 3 groups (n = 5 each). Tissue recombinants were prepared in vitro with newborn BALB/c murine seminal vesicle mesenchyme (SVM) and surgical isolated human prostate cancer tissues by using recombination technique and then grafted beneath the renal capsule of intact male athymic mouse. At Week 4 after initial implantation, grafts were harvested and tumor sizes calculated. The expressions of human specific markers CK8/18 and vimentin were evaluated by immunohistochemistry to identify the human prostatic origin in grafts. P63 protein, a basal cell marker, was detected in prostate basal membrane to identify whether it was benign or malignant tissue. And the study control was prepared by implanting prostate cancer tissues alone under the renal capsule in SCID mouse. RESULTS: Of all 78 implantation cases in 15 mice, the tumor-forming rates were 100% (39/39) and 94.1% (37/39) respectively in the recombination and prostate cancer alone grafting groups. The recombination group was shown to be more efficient in terms of tumor size and weight in comparison with the prostate cancer alone group [(9.7 ± 3.1) vs (6.8 ± 2.0) mm(3), (12.1 ± 3.6) vs (8.2 ± 2.2) µg, P < 0.01]. There was no difference in serum PSA level between two groups. Grafts were confirmed as human prostate cancer tissues with the expressions of CK8/18 and vimentin. No expression of P63 was detected. CONCLUSION: The xenograft murine model of human prostate cancer is successfully established. It contains stroma components and is particularly suitable for studying the interaction of stroma and epithelia in the in vivo progression of prostate cancer.

Transfection with pax6 gene of mouse embryonic stem cells and subsequent cell cloning induced retinal neuron progenitors, including retinal ganglion cell-like cells, in vitro.

OBJECTIVE: It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). pax6 regulates early events in eye development, including the generation of retinal ganglion cells (RGCs). We previously reported the successful induction of corneal epithelial cells from ESCs transfected with the pax6 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the pax6 gene. METHODS: Undifferentiated mouse ESCs were transfected with pax6 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of pax6-transfected cells. We expanded the cloned pax6-transfected cells, which expressed nestin and musashi-1, for further characterization in culture media containing fibronectin. The cells were characterized using RT-PCR, immunostaining, electron microscopy, renal subcapsular transplantation assay and Ca imaging. RESULTS: We obtained clonally expanding pax6-transfected cells, all of which were positive for six3, sonic hedgehog (shh), math5, brn3, thy1 and melanopsin, by using several ESCs. When transplanted into a mouse renal capsule, they differentiated into neurons with elongated axons, expressing betaIII tubulin and neurofilament middle chain, and were free from teratoma development. Electron-microscopic examination showed neurotubules and neurofilaments in the axon-like processes of the cloned pax6-transfected cells. High KCl stimulation increased free Ca influx on Ca2+ imaging. CONCLUSIONS: ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the pax6 gene and subsequent limiting-dilution culture. Cloned cell lines may be useful to analyze the requirements for retinal progenitor cell differentiation, and our study suggests the clinical application of this cell type.

[Protective effect of heme oxygenase-1 induction in vivo to pancreas islet xenograft].

OBJECTIVE: To study the protective effect of islet xenograft and its possible mechanism of high expression of heme oxygenase-1 (HO-1) in donor pancreas islet induced by cobalt protoporphyrin (CoPP). METHODS: Male SD rats and C57BL/6 mouse were used as donors and recipients respectively. Donors were divided into 3 groups according to different pretreatment 24 hours before donation: control group (injected intraperitoneally with NaCl), induce group [injected intraperitoneally with cobalt-protoporphyrin (CoPP)], block group (injected intraperitoneally with CoPP and zinc protoporphyrin simultaneously). A modified approach was used for islet isolation.Recipients were rendered diabetic by intraperitoneal injection of streptozotocin. Islets were transplanted into mouse subrenal capsule. Postoperative mouse glycemia were monitored daily and normoglycemia time was compared among each group. The receptor mouse serum IL-10 was detected by ELISA approach, and real-time PCR was used to check the expression of IL-10 mRNA in islet graft tissues. The graft tissues were observed for the lymphocyte infiltration after HE staining. RESULTS: Diabetes mice accepted islets untreated, induced or blocked maintained the euglycemia for (9.3 +/- 1.4), (16.3 +/- 1.5) and (9.7 +/- 1.0) d respectively. The xeno-islets presented HO-1 over-expression survived much longer than that absent (P < 0.05), it was no significance between control group and block group (P > 0.05). The mouse islet serum IL-10 content after induction was (73.0 +/- 9.7) pg/ml, significantly higher than (30.6 +/- 3.9) pg/ml of the untreated group and (32.1 +/- 5.9) pg/ml of the blocked group (P < 0.05), there was no difference between control group and block group (P > 0.05). Moreover, the IL-10 mRNA expression up-regulated statistic significantly in HO-1 induced islet xeno-graft. Pathological examination showed that the graft lymphocyte infiltration of the induced group was obviously less serious than the other two groups. CONCLUSIONS: The higher expression of HO-1 induced by CoPP in vivo would significantly prolong graft survival time and its mechanism could be related to immune modulation of IL-10.

Bone marrow-derived mesenchymal stem cells improve islet graft function in diabetic rats.

Type 1 diabetes is associated with a progressive loss of beta cells and pancreatic islet transplantation could represent a cure for this disease. Herein we explored whether transplantation of bone marrow-derived mesenchymal stem cells (MSCs) allowed a reduced number of pancreatic islets to improve glycemic control in diabetic rats, by promoting islet vascularization. We transplanted 2000 syngenic islets alone or in combination with MSCs (10(6) cells) under the kidney capsules of diabetic Lewis rats. Animals transplanted with 2000 islets never reached normoglycemia. In contrast, rats transplanted with 2000 islets plus MSCs, showed a gradual fall in glycemia after transplantation, with normoglycemia maintained until killing. Comparable glycemic control was obtained with transplantation of 3000 islets alone. The MSC preparation used for in vivo experiments expressed high levels of vascular endothelial growth factor (VEGF(165)) and, at less extent, VEGF(189), as evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). In transplanted animals, vascularization was quantified by morphometric analysis of islet grafts with anti-RECA and anti-insulin antibodies. MSCs were stained with PKH-26. Mean capillary density was 1002 +/- 55 capillaries/mm(2) in islets transplanted alone. Co-infusion of MSCs with islets significantly increased the number of capillaries to 1459 +/- 66 capillaries/mm(2). In conclusion, our study indicated that co-transplantation of MSCs with pancreatic islets improved islet graft function by promoting graft vascularization.

Development of functional human immune system with the transplantations of human fetal liver/thymus tissues and expanded hematopoietic stem cells in RAG2-/-gamma(c)-/- MICE.

BACKGROUND: There is an increasing need for suitable animal models for the study of the human immune system and disease. The purpose of this study was to develop a practical in vivo model of human immune cell repopulation using ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells and subrenally coimplanted fetal liver/thymus tissues. METHODS: Freshly isolated fetal liver-derived CD34(+) hematopoietic stem cells were frozen until injected and ex vivo expanded with various cytokines for 7 days. After fetal liver/thymus tissues were subrenally coimplanted into preirradiated Rag2(-/-)gamma(c)(-/-) mice, frozen and ex vivo expanded CD34(+) cells were injected intravenously. The peripheral blood of the mice was monitored for the detection of human cell engraftment using flow cytometry. Then we confirmed human T-cell function by in vitro function assays. RESULTS: After fetal liver/thymus tissues were coimplanted into the irradiated Rag2(-/-)gamma(c)(-/-) mice, with frozen and ex vivo expanded CD34(+) hematopoietic stem cells, human cell engraftments were determined using hCD45 and multilineage markers. The cultured cells with the cytokine combination of stem cell factor, thrombopoietin, Flk2/Flk3 ligand (FL), and interleukin-3 showed stable and long-term engraftment compared to other combinations. The ex vivo expanded human fetal liver-derived CD34(+) hematopoietic stem cells, under our culture conditions, accomplished a large volume of expanded cells that were sustained, demonstrating self-renewal of the evaluated markers, which may have indicated long- term repopulation activity. CONCLUSION: The results of this study demonstrated a practical mouse model of expanded human immune cells especially T cells in Rag2(-/-)gamma(c)(-/-) mice.

Renal capsule-parathymic lymph node complex: a new in vivo metastatic model in rats.

BACKGROUND: The ultimate cause of cancer death is, in most cases, the appearance of metastases. The aim of the present study was to contribute to animal experimental investigations of metastatic tumor development. MATERIALS AND METHODS: Rat hepatocarcinoma (He/De), mesoblastic nephroma (Ne/De) cells, and in other cases tumor-bearing lymph nodes were transplanted under the renal capsule of F344 rats. Metastatic potential of tumor cells was examined by whole body autoradiography and phosphor image analysis. The organ distribution of cells was also investigated. RESULTS: Transplanted tumor cells resulted in metastases in the parathymic lymph nodes. Implanted India ink also demonstrated connection between the lymphatic vessels of the renal capsule and the parathymic lymph nodes. The metastatic potential was independent of the primary tumor growth rate. CONCLUSION: The renal capsule-parathymic lymph node complex seems to be suitable for the isolated in vivo examination of metastatic development and for the detailed analysis of secondary tumors.

Exendin-4 does not promote Beta-cell proliferation or survival during the early post-islet transplant period in mice.

Current pancreatic islet transplantation protocols achieve remarkable short-term success, but long-term insulin independence remains elusive. Hypoxic and inflammatory insults cause substantial early posttransplant graft loss while allo/autoimmunity and immunosuppressive drug toxicity threaten long-term graft mass and function. Exendin-4 (Ex4) is a GLP-1 receptor agonist that promotes beta-cell proliferation, survival, and differentiation. To determine whether Ex-4 displays potential as a graft-supportive agent, we transplanted 500 murine islets under the kidney capsule of syngeneic or allogeneic streptozocin-treated recipient mice and immediately initiated daily treatment with vehicle or Ex4. Graft beta-cell proliferation, death, and vascularity were assessed at 1, 3, and 10 days after syngeneic islet transplantation. For allogeneic recipients, blood glucose and body weight were assessed until glycemic deterioration. Ex-4 did not promote graft beta-cell proliferation, reduce beta-cell death, or enhance graft vascularity over the first 10 days after syngeneic islet transplantation. A trend toward prolongation of posttransplant euglycemia was observed with Ex4 treatment in nonimmune-suppressed allograft recipients, but its use in this setting was associated with frequent, severe hypoglycemia over the first 2 posttransplant days. Our findings do not support a beneficial effect of Ex-4 on islet grafts during the critical early posttransplant period, further, they demonstrate a significant hypoglycemic potential of Ex-4 in the first days after islet transplantation in mice. Optimal application of GLP-1 receptor agonists for long-term proliferative and survival benefits in transplantation may require earlier intervention prior to and/or during islet isolation for peri-transplant cytoprotection and administration beyond the period of engraftment.

The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development.

T cell development in the thymus depends on continuous colonization by hematopoietic precursors. Several distinct T cell precursors have been identified, but whether one or several independent precursor cell types maintain thymopoiesis is unclear. We have used thymus transplantation and an inducible lineage-tracing system to identify the intrathymic precursor cells among previously described thymus-homing progenitors that give rise to the T cell lineage in the thymus. Extrathymic precursors were not investigated in these studies. Both approaches show that the stream of T cell lineage precursor cells, when entering the thymus, selectively passes through the early T lineage precursor (ETP) stage. Immigrating precursor cells do not exhibit characteristics of double-negative (DN) 1c, DN1d, or DN1e stages, or of populations containing the common lymphoid precursor 2 (CLP-2) or the thymic equivalent of circulating T cell progenitors (CTPs). It remains possible that an unknown hematopoietic precursor cell or previously described extrathymic precursors with a CLP, CLP-2, or CTP phenotype feed into T cell development by circumventing known intrathymic T cell lineage progenitor cells. However, it is clear that of the known intrathymic precursors, only the ETP population contributes significant numbers of T lineage precursors to T cell development.

Immunoglobulin-like transcript 3-Fc suppresses T-cell responses to allogeneic human islet transplants in hu-NOD/SCID mice.

OBJECTIVE: The aim of our study was to explore the immunomodulatory activity of soluble immunoglobulin (Ig)-like transcript (ILT) 3-Fc in pancreatic islet transplantation and to determine its mechanism of action. RESEARCH DESIGN AND METHODS: NOD/SCID mice in which diabetes was induced by streptozotocin injection were transplanted with human pancreatic islet cells. Mice in which the transplant restored euglycemia were humanized with allogeneic peripheral blood mononuclear cells and treated with ILT3-Fc or control human IgG or left untreated. The blood glucose level was monitored twice a week, and rejection was diagnosed after two consecutive readings >350 mg/dl. Tolerated and rejected grafts were studied histologically and by immunostaining for human T-cells and insulin production. CD4 and CD8 T-cells from the spleen were studied for suppressor activity, expression of cytokines, and CD40L. RESULTS: Although human T-cell engraftment was similar in all groups, ILT3-Fc-treated mice tolerated the islets for the entire period of observation (91 days), whereas control mice rejected the graft within 7 weeks (P < 0.0001). ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human CD8(+) T suppressor cells that inhibited Th alloreactivity against graft HLA antigens. T-cells allostimulated in vitro in the presence of ILT3-Fc inhibited CD40L-induced upregulation of CD40 in human pancreatic islet cells. Histochemical studies showed dramatic differences between human pancreatic islets from tolerant, ILT3-Fc-treated mice and control recipients rejecting the grafts. CONCLUSIONS: The data indicated that ILT3-Fc is a potent immunoregulatory agent that suppressed islet allograft rejection in humanized NOD/SCID mice.

Sox9 is required for prostate development.

The mammalian prostate arises from the urogenital sinus and few factors have been identified to be important in the early stages of prostate development. In this study we show that the transcription factor Sox9 is expressed in the epithelia of all mouse prostatic lobes from the initial stages of their development. We used a conditional approach with mice expressing Cre recombinase under the control of Nkx3.1 regulatory sequences to delete Sox9 from the developing prostate. Mice with a prostate specific deletion of Sox9 showed a lack of ventral prostate development and abnormal anterior prostate differentiation. Analysis of these mutant animals revealed an early loss of expression of genes specific to the prostate epithelia such as Nkx3.1 and Shh and a marked reduction in proliferation in the ventral prostate but not in other lobes. Fgf signalling, through the MAPK pathway, has been shown to be important in prostate development and a lobe specific phenotype was reported for a prostate specific Fgfr2 mutant mouse model. Here we show that the levels of Fgfr2 and Sprouty2, a downstream target of Fgf signalling, were severely reduced in the ventral prostate of Sox9 mutant animals but not in other lobes. Prostate organ culture studies with a Mek inhibitor, U0126, and a Fgf receptor inhibitor, SU5402, indicate that the timing of expression of Cre in the mutant animals could account for the lobe specific phenotype in the Sox9 and Fgfr2 mutants. These studies imply that Sox9 is required for the early differentiation of the prostate bud epithelia.