Kidney Capsule Research Articles

Abstract 3099: Biological evaluation of a novel AKR1C3 inhibitor in patient-derived prostate cancer cell line and xenograft models

Abstract INTRODUCTION AND OBJECTIVES: Prostate cancer is a highly heterogeneous cancer type with distinct genomic and phenotypic characteristics that drive tumorigenesis and the differential response to drug therapies. A limit number of prostate cancer cell lines and patient-derived xenograft (PDX) models hinders research to improve disease outcome. Some currently available PDX models were derived from the primary tumor samples are insufficient to recapitulate the clinical response at more advanced stages. In this study, we developed patient-derived models from patients with advanced disease and evaluated a novel AKR1C3 inhibitor in these models. METHODS: Samples received from our Pathology Biorepository Shared Resource were divided into four groups and subjected to pathological staining, RNA extraction, xenografting in NSG mice via renal capsule and subcutaneous implantation in SCID mice and conditional reprogramed cultures (CRCs) or organoid culturing. The AKR1C3 inhibitor PB was modified from celecoxib. Androgen receptor (AR), AR-V7 and AKR1C3 expression were determined by western blot. The effects of the AKR1C3 inhibitor on enzalutamide sensitivity were characterized by growth assay and colony formation assay. RESULTS: Eight PDX models have been developed from prostate cancer patients with high Gleason score and/or at the castration-resistant stages. Among the PDX models, one spontaneous indefinite cell line PS1172 was established. Early passage CRCs showed the epithelial morphology with AR positive expression. Through serially passaging PS1172 PDX with castration in SCID mice, the castration resistant cell line 1172CR was re-cultured from castration-resistant PS1172 PDX tumors. 1172CR cells were resistant to enzalutamide treatment and expressed high level of AKR1C3 and AR-V7. A novel AKR1C3 inhibitor (PB) which displayed superior potential to inhibit AKR1C3 activity and suppress enzalutamide resistant prostate cancer cell growth was tested in these models. At the same dose, PB significantly suppressed 1172CR cell growth and colony formation compared to indomethacin and enzalutamide. PB also significantly suppressed AR/AR-V7 protein expression compared to indomethacin in 1172CR cells. CONCLUSION: PS1172 and castration-resistant 1172CR cells are novel models with significant characteristics such as AR-V7 and AKR1C3. These novel prostate cancer models are ideal for small molecule testing and resistant mechanism investigating. Citation Format: Joy C. Yang, Shu Ning, Hans Adomat, Martin Gleave, Allen Gao, Christopher P. Evans, Chengfei Liu. Biological evaluation of a novel AKR1C3 inhibitor in patient-derived prostate cancer cell line and xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3099.

Abstract 6206: Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging

Abstract Tumor growth requires a functioning vascular network and offers an attractive therapeutic target. The invasive proliferating neovasculature of tumors lacks pericyte support and exhibits increased permeability thereby offering a unique, potentially selective target for anticancer therapy. Most vascular disrupting agents (VDAs) inhibit microtubule formation, causing rapid morphological changes in endothelia cells resulting in dramatically increased vessel permeability, cellular detachment, vessel occlusion, and vessel wall damage [doi: 10.3390/molecules26092551]. Highly vascular kidney tumors are expected to be particularly sensitive to VDAs. We are evaluating OXi8007, a water soluble indole phosphate pro-drug derivative of the first generation VDA combretastatin. The prodrug OXi8007 is stable in saline but releases active OXi8006 is the presence of alkaline phosphate. Preliminary pharmacokinetic data benchmarked using 13C-labeled OXi8006 and OXi8007 show rapid vascular clearance and conversion to the glucuronide conjugate in vivo. Doses up to 700 mg/kg were tolerated by mice. Luciferase-expressing RENCA tumor cells were implanted orthotopically in the right kidney capsule of syngeneic BALB/c mice. Bioluminescence Imaging (BLI) was applied weekly to assess tumor growth and acute response to OXi8007. Following addition of luciferin substrate subcutaneously BLI signal from the tumor reached a maximum within 10 minutes. When BLI was repeated 4 hrs after 250 or 350 mg/kg OXi8007 IP, the generated signal was <1% of baseline signal indicating acute vascular disruption. Multispectral Optoacoustic Tomography (MSOT) revealed matching acute changes in tumor vascular oxygenation and additionally showed distinct heterogeneity. An oxygen gas breathing challenge indicated lower oxygen saturation in the tumor following treatment. Histology showed massive hemorrhage following both doses. BLI signal recovered after 1 to 2 days and repeated administration of OXi8007 again caused acute vascular shutdown. These results match previous observations in breast tumor models [doi: 10.1016/j.canlet.2015.08.021] indicating opportunities for therapeutic application. Citation Format: Hashini I. Wanniarachchi, Regan Schuetze, Lorena Arango, Khagendra Hamal, Graham J. Carlson, Cyprian Pavlich, Yuling Deng, Mary L. Trawick, Kevin G. Pinney, Li Liu, Ralph P. Mason. Discerning vasculature destruction caused by novel vascular disrupting agents examined by optical and multispectral optoacoustic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6206.

Titanium Nanosurface with a Biomimetic Physical Microenvironment to Induce Endogenous Regeneration of the Periodontium.

The periodontium supports the teeth by dentoalveolar fibrous joints that serve unique oral functions. Endogenous regeneration of the periodontium around artificial teeth (dental implants) provides a cost-effective solution for the extension of healthy life expectancy but remains a challenge in regenerative medicine. Biomimetics can create smart biomaterials that tune endogenous cells at a tissue-material interface. Here, we created a smart titanium nanosurface mimicking the surface nanotopography and micromechanical properties of the tooth root cementum (TRC), which is essential for the induction of dentoalveolar fibrous joints to regenerate the periodontium. After transplantation into the rat renal capsule, only the titanium artificial tooth with the TRC-mimetic nanosurface formed a complex dentoalveolar fibrous joint structure, with bone tissue, periodontal ligament (PDL), and TRC, in the decellularized jawbone matrix. TRC-mimetic titanium implants induce the formation of functional periodontium, even in a jawbone implantation model, which generally causes osseointegration (ankyloses). In human PDL cells, TRC analogousness in the surface mechanical microenvironment regulates matrix mineralization through bone sialoprotein expression and phosphorus metabolism, which are critical for cementogenesis. Therefore, the titanium nanosurfaces with nanotopographical and mechanical microenvironments mimicking the TRC surface induce dentoalveolar fibrous joints for periodontal regeneration by interfacial tuning of endogenous cells.

Persistent Aseptic Pyelonephritis After Acute Bacterial Pyelonephritis: Possible Role of Corticosteroids

Persistent Aseptic Pyelonephritis After Acute Bacterial Pyelonephritis: Possible Role of Corticosteroids

822-P: The Harmine and Exendin4 Combination Markedly Expands Human Beta-Cell Mass In Vivo in Diabetic Mice

Diabetes results from diminished functional β-cell mass. Small molecule inhibitors of Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A) markedly increase human β-cell replication in vitro and in vivo. Furthermore, combination of DYRK1A inhibitors such as Harmine (H) with widely clinically used glucagon-like peptide 1 receptor agonists such as Exendin-4 (E) further enhance human β-cell proliferation. However, whether H+E combination increases human β-cell mass and improves glucose homeostasis in diabetic immunosuppressed mice transplanted with a marginal number of human islets is unknown. Human islets from 5-6 different adult healthy donors were transplanted under the kidney capsule (200 islets per kidney) of streptozotocin-induced diabetic immunodeficient Rag1-/- mice. At the time of transplantation, osmotic Alzet minipumps were implanted for continuous delivery of vehicle V, H, E and H+E for a period of 12 weeks. At the end of the study, human islet-bearing kidneys were harvested, labeled with insulin antisera and cleared using a modification of the iDISCO+ method. Imaging and analysis of the β-cell volume was done using the Light-sheet Ultramicroscope II and Imaris software. Transplanted diabetic mice who received the combination of H+E showed a significant increase of human β-cell volume up to 600% compared with mice treated with V or the drugs alone. This increase correlated with a significant increase in plasma human insulin levels, sustained normalization of blood glucose levels and significantly improved glucose tolerance. Our studies provide unequivocal documentation that treatment with the harmine and exendin-4 combination can lead to actual increases in human β-cell mass in diabetic conditions suggesting that β-cell replenishment might be possible in humans with diabetes. Disclosure K.A.Beliard: None. S.Stanley: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. C.Rosselot: None. Y.Li: None. A.Alvarsson: None. P.Wang: None. K.Thakkar: None. D.Guevara: None. R.J.Devita: None. A.F.Stewart: None. Funding NIH/NIDDK 1R01DK105015-05.

1458-P: Lipophagy Is Constitutively Active in Pancreatic Beta Cells and Maintains Insulin Secretion

Lipid droplets (LDs) , organelles important for intracellular lipid metabolism, are actively formed in rat and human adult beta cells and affect islet function and health. Acute mobilization of LDs by adipose triglyceride lipase in beta cells is known to support insulin secretion. As LD degradation by lysosomal acid lipase (LIPA) through lipophagy is an alternative pathway for LD catabolism, we addressed whether LIPA regulates beta cell function. LDs in human beta cells are associated with autophagy (LC3) and lysosomal (LAMP1) markers. LIPA suppression via siRNA and LIPA inhibitor (lalistat2) in INS1 cells increased LD size (DMSO 0.5 vs. Lali2 1.5 µm2/cell, p<0.05, p value by Student’s t test unless specified otherwise) and number (Scr 2/cell vs. siLIPA 14/cell, p<0.05; DMSO 4.5/cell vs. Lali2 16/cell, p<0.05) . Rat and human beta cells treated with Lali2 also showed increased in LD area and/or number of LDs per cell (p<0.05) . In all three models, LIPA suppression increased TG contents confirming constitutively active lipophagy. While acute inhibition of LIPA by Lali2 had little effect on glucose-stimulated insulin secretion (GSIS) , chronic (over 48 h) LIPA suppression by Lali2 or siRNA reduced GSIS from INS1 cells, rat islets, and human islets. Lentiviral shRNA transduction in human pseudoislets decreased insulin secretion in response to glucose (41 ± 10.5% of Scr, p<0.05) and to KCl (61.3 ±16.1% of Scr, p<0.05) in vitro. When Scr and shLIPA human pseudoislets were transplanted under kidney capsules of NSG mice on Western diet, human insulin secretion in response to intraperitoneal glucose load was blunted in mice carrying shLIPA islets compared with shScr control (2-Way ANOVA, p<0.05) . In summary, lipophagy is constitutively active in human and rat beta cells and serves as a pathway for LD catabolism that is acutely insensitive to glucose. However, LIPA mediated LD catabolism is needed for beta cell function in a long-term. Disclosure S.Liu: None. B.R.Brennecke: None. U.Yang: None. J.A.Ankrum: None. Y.Imai: None. Funding National Institute of Diabetes andDigestive and Kidney Diseases (R01=DK090490) U.S. Department of Veterans Affairs (IBX005107)

211-LB: The Islet Tester Mouse, a Stable and Reproducible In Vivo Model to Study Human Islet Function

The current gold standard for assessing the function of human islet in vivo, is to engraft islets under the kidney capsule of hyperglycemic, immunodeficient mice. Current models, such as Streptozotocin (STZ) treatment or NSG-Akita mice, are limited due to instable hyperglycemia and/or morbidity of the mouse strain. The glycemia in STZ treated mice is variable and can even result in death or spontaneous recovery of normoglycemia, making it difficult to discern small differences in human islet function. The NSG-Akita mouse is difficult to breed and maintain, and thus has reduced practicality. To address these limitations, we developed a stable, highly reproducible immunodeficient Islet Tester mouse via CRISPR-Cas9 mediated gene editing of glucokinase (GCK) directly in NSG zygotes. Islet Tester mice are heterozygous for the Arg345->stop variant and present with stable hyperglycemia (∼250mg/dl) , without the severe age-dependent morbidity of the NSG-Akita mice. This mouse will allow us to study human islet biology over time and under different physiological conditions and could provide a useful preclinical platform to investigate drugs targeting islet cells. Disclosure E. Waite: None. C. L. May: None. Human pancreas analysis program (hpap) : n/a. K. H. Kaestner: None.

Impact of atrazine and nitrate on liver and kidney of egyptian toad Sclerophrys regularis: bioindicator alarming on ecosystem

Atrazine and nitrate have been shown to act as potent oxidative stressors in amphibians either alone or in combination under stable laboratory conditions, causing histopathological alternations in liver and kidney structures at the sub-lethal concentrations. A control group and three treatments groups were tested; atrazine, nitrate, atrazine-nitrate treatments with doses of 300 μg L-1, 200 mg L-1 and their combination respectively. Sever distortion in liver and kidney tissues were shown related to the different treatments. The most hepatic lesions were observed depletion in glycogen content, degeneration of hepatocytes, hemorrhage, necrosis, vasodilatation, congestion in blood vessels, cloudy swelling in the hepatocytes and aggregation of melanomacrophage cells in between the hepatocytes that increased in combination treatment group. In kidney, the most lesions were represented in degeneration of renal tubules, fibrosis, hemorrhage, leucocytes infiltration, thickness in the wall of the renal capsule, atrophy of glomerulus, deformation of Bowman’s epithelium. These negative impacts may be a bioindicator alarming the ecosystem disrupting caused by the uncontrolled apply of these chemicals in agriculture.

Renal adaptation in relation to insectivorous feeding habit in the greater mouse-eared bat, Myotis myotis (Chiroptera: Vespertilionidae).

We examined the histological structure of the kidneys of Myotis myotis to better understand their structural adaptations to dietary habits. M. myotis is an insectivorous bat species that belongs to the family Vespertilionidae. The kidneys of M. myotis are unilobular, bean-shaped, and surrounded by a renal capsule. The two parts are distinguished by a thin cortex and a thicker medulla. Renal corpuscles consist of the glomerulus and Bowman's capsule. The proximal tubule consists of cubic cells with a well-developed brush border, whereas the distal tubule is lined with a simple cubic epithelium without a brush border. The Henle's loop, located in the medullary region, was composed of flat cells. The microvilli of proximal tubule epithelial cells and basal lamina of proximal and distal tubule epithelial cells were periodic acid Schiff (PAS)-positive. The PAS-positive reaction of the microvilli of proximal tubular epithelial cells and basal lamina of proximal and distal tubule epithelial cells is due to the presence of glycogen, which may be used as an energy substrate during absorption. The presence of acidic glycoconjugates in the papilla was demonstrated by Alcian blue (pH2.5)-PAS staining. According to the result of silver impregnation staining, it was determined that reticular fibers form a dense fibrillary network in the kidney parenchymal tissue. Reticular fibers are responsible for demarcating and supporting the borders of cells by forming a thin network of fibrils beneath the basal lamina of the cells. Structural features in the kidney, such as a thin cortex and thicker medulla, long conical papilla, and division of the thick medulla into inner and outer regions, of M. myotis may be an adaptation to produce concentrated urine, thereby reducing water loss associated with insectivorous feeding habits.

Rare malignant kidney (renal) tumor – leiomyosarcoma of the renal vein (clinical case)

Soft tissue sarcomas are a rare group of malignant tumors that develop from different types of connective tissue. One of the histological variants of primary renal sarcoma is renal leiomyosarcoma. Leiomyosarcoma of kidneys can develop from the renal vasculature, renal veins, pelvic muscles, smooth muscle components of the renal capsule. Clinical and radiologic signs of leiomyosarcoma are nonspecific. Diagnosis is based on histological and immunohistochemical studies after surgical intervention. This article presents a clinical case of diagnosis and treatment of leiomyosarcoma of the renal vein.

V10-09 SUTURELESS PURELY OFF-CLAMP ROBOTIC PARTIAL NEPHRECTOMY: TECHNIQUE AND PERIOPERATIVE OUTCOMES

You have accessJournal of UrologyCME1 May 2022V10-09 SUTURELESS PURELY OFF-CLAMP ROBOTIC PARTIAL NEPHRECTOMY: TECHNIQUE AND PERIOPERATIVE OUTCOMES Leonardo Misuraca, Riccardo Mastroianni, Gabriele Tuderti, Umberto Anceschi, Alfredo Maria Bove, Aldo Brassetti, Mariaconsiglia Ferriero, Salvatore Guaglianone, Michele Gallucci, and Giuseppe Simone Leonardo MisuracaLeonardo Misuraca More articles by this author , Riccardo MastroianniRiccardo Mastroianni More articles by this author , Gabriele TudertiGabriele Tuderti More articles by this author , Umberto AnceschiUmberto Anceschi More articles by this author , Alfredo Maria BoveAlfredo Maria Bove More articles by this author , Aldo BrassettiAldo Brassetti More articles by this author , Mariaconsiglia FerrieroMariaconsiglia Ferriero More articles by this author , Salvatore GuaglianoneSalvatore Guaglianone More articles by this author , Michele GallucciMichele Gallucci More articles by this author , and Giuseppe SimoneGiuseppe Simone More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002623.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: In the last years, sutureless approach has gaining popularity for its feasibility and safety, together with promising functional outcomes and shorter operative time and length of stay compared to renorraphy technique. In this video we describe surgical steps, perioperative and short term functional outcomes of sutureless off clamp robotic partial nephrectomy. METHODS: This was the case of an endophytic 3 cm left renal mass (RENAL score: 9). Preoperatively, a mixture of lipiodol and indocyanine green was delivered transarterially to mark the lesion. Gerota's capsule was opened and NIRF was used to easily locate the tumor. Tumor margins were scored with monopolar coagulation, following ICG guidance to define tumor edges. The enucleation plane was progressively developed, cauterizing all arterial feeders. Two suction devices with irrigation were used simultaneously to maximize the visualization of the enucleation plane developed following the ICG guidance. Monopolar coagulation of the resection bed started from the outer part of the cortical layer, moving inwards to the renal medulla. Once haemostasis was achieved, near infrared fluorescence was used to inspect renal parenckhyma perfusion. Renal capsule was closed with hem-o-lok and a drain was left in place. Perioperative outcomes were compared to a propensity score matched cohort of renorraphy cases. RESULTS: 180 procedures over 548 were sutureless. Trifecta achievement rate was significantly higher in the sutureless group (93% vs 83%). A propensity score matched analysis, generated 2 cohorts of 80 patients homogeneous for age, gender, ASA score, baseline eGFR, tumor size and surgical complexity.Patients receiving sutureless partial nephrectomy had shorter hospital stay, negligible complications and increased likelihood of achieving Trifecta. Kaplan Maier analysis showed significant CKD stage migration free survival probabilities comparable between groups. CONCLUSIONS: Patients receiving sutureless off-clamp robotic partial nephrectomy had negligible complications and minimal incidence of perioperative renal function impairment. This approach increased the probability to achieve the Trifecta compared to renorraphy cohort. further studies are needed to assess long-term outcomes. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e851 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Leonardo Misuraca More articles by this author Riccardo Mastroianni More articles by this author Gabriele Tuderti More articles by this author Umberto Anceschi More articles by this author Alfredo Maria Bove More articles by this author Aldo Brassetti More articles by this author Mariaconsiglia Ferriero More articles by this author Salvatore Guaglianone More articles by this author Michele Gallucci More articles by this author Giuseppe Simone More articles by this author Expand All Advertisement PDF DownloadLoading ...

MP27-08 EVALUATING THE EFFICACY OF A NOVEL AKR1C3 INHIBITOR IN PATIENT-DERIVED PROSTATE CANCER CELL LINE AND XENOGRAFT MODELS

You have accessJournal of UrologyCME1 May 2022MP27-08 EVALUATING THE EFFICACY OF A NOVEL AKR1C3 INHIBITOR IN PATIENT-DERIVED PROSTATE CANCER CELL LINE AND XENOGRAFT MODELS Joy C. Yang, Shu Ning, Hans Adomat, Martin Gleave, Allen C. Gao, Christopher P. Evans, and Chengfei Liu Joy C. YangJoy C. Yang More articles by this author , Shu NingShu Ning More articles by this author , Hans AdomatHans Adomat More articles by this author , Martin GleaveMartin Gleave More articles by this author , Allen C. GaoAllen C. Gao More articles by this author , Christopher P. EvansChristopher P. Evans More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002570.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is a highly heterogeneous cancer type with distinct genomic and phenotypic characteristics that drive tumorigenesis and the differential response to drug therapies. A limit number of PCa cell lines and patient-derived xenograft (PDX) models hinders research to improve disease outcome. Some currently available PDX models were derived from the primary tumor samples are insufficient to recapitulate the clinical response at more advanced stages. In this study, we developed patient-derived models from patients with advanced disease and evaluated a novel AKR1C3 inhibitor in these novel models. METHODS: Samples received from our Pathology Biorepository Shared Resource were divided into four groups and subjected to pathological staining, RNA extraction, xenografting in NSG mice via renal capsule and subcutaneous implantation in SCID mice and conditional reprogramed cultures (CRCs) or organoid culturing. The AKR1C3 inhibitor PB was modified from celecoxib. AR and AR-V7 expression were determined by western blot. The effects of the AKR1C3 inhibitor on enzalutamide sensitivity were characterized by growth assay and colony formation assay. RESULTS: Eight PDX models have been developed from patients with high Gleason score and/or at the castration-resistant stages. Among the PDX models, one spontaneous indefinite cell line PS1172 was established. Early passage CRCs showed the epithelial morphology with AR positive expression. Through serially passaging PS1172 PDX with castration in SCID mice, the castration resistant cell line 1172CR was re-cultured from castration resistant PS1172 PDX tumors. 1172CR cells were resistant to enzalutamide treatment and expressed high level of AKR1C3 and AR-V7. A novel AKR1C3 inhibitor (PB) which displayed superior potential to inhibit AKR1C3 activity and suppress enzalutamide resistant prostate cancer cell growth was tested in these models. At the same dose, PB significantly suppressed 1172CR cell growth and colony formation compared to indomethacin and enzalutamide. PB also significantly suppressed AR/AR-V7 protein expression compared to indomethacin in 1172CR cells. CONCLUSIONS: PS1172 and castration-resistant 1172CR cells are novel models with significant characteristics such as AR-V7 and AKR1C3. These models are ideal for small molecule testing and resistant mechanism investigating. Source of Funding: This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu), R01CA251253 (C, Liu), and Paul Calabresi Clinical Oncology K12 Program (C, Liu) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e452 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joy C. Yang More articles by this author Shu Ning More articles by this author Hans Adomat More articles by this author Martin Gleave More articles by this author Allen C. Gao More articles by this author Christopher P. Evans More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF DownloadLoading ...

Bone Marrow-Derived Cells Contribute to the Maintenance of Thymic Stroma including TECs.

In paradox to critical functions for T-cell selection and self-tolerance, the thymus undergoes profound age-associated atrophy and loss of T-cell function, further enhanced by cancer therapies. Identifying thymic epithelial progenitor populations capable of forming functional thymic tissue will be critical in understanding thymic epithelial cell (TEC) ontogeny and designing strategies to reverse involution. We identified a new population of progenitor cells, present in both the thymus and bone marrow (BM) of mice, that coexpress the hematopoietic marker CD45 and the definitive thymic epithelial marker EpCAM and maintain the capacity to form functional thymic tissue. Confocal analysis and qRT-PCR of sorted cells from both BM and thymus confirmed coexpression of CD45 and EpCAM. Grafting of C57BL/6 fetal thymi under the kidney capsule of H2BGFP transgenic mice revealed that peripheral CD45+ EpCAM+ GFP-expressing cells migrate into the developing thymus and contribute to both TECs and FSP1-expressing thymic stroma. Sorted BM-derived CD45+ EpCAM+ cells contribute to reaggregate thymic organ cultures (RTOCs) and differentiate into keratin and FoxN1-expressing TECs, demonstrating that BM cells can contribute to the maintenance of TEC microenvironments previously thought to be derived solely from endoderm. BM-derived CD45+ EpCAM+ cells represent a new source of progenitor cells that contribute to thymic homeostasis. Future studies will characterize the contribution of BM-derived CD45+ EpCAM+ TEC progenitors to distinct functional TEC microenvironments in both the steady-state thymus and under conditions of demand. Cell therapies utilizing this population may help counteract thymic involution in cancer patients.

A mediator complex subunit 12 gain-of-function mutation induces partial leiomyoma cell properties in human uterine smooth muscle cells

To clarify whether a mediator complex subunit 12 (MED12) gain-of-function mutation induces leiomyoma cell properties in human uterine smooth muscle cells (USMCs). Experimental study. Academic research laboratory. Women undergoing hysterectomy for leiomyoma. CRISPR/Cas9-mediated genome editing to introduce an MED12 gain-of-function mutation (G44D) into human USMCs. Cell proliferation, collagen production, and invivo tumorigenicity of USMCs with vs. without the MED12 mutation. Uterine smooth muscle cells isolated from the uterine myometrium of a 44-year-old patient were subjected to lentiviral vector-mediated gene transduction of the fluorescent protein Venus, followed by long-term passage. Uterine smooth muscle cells with a normal female karyotype, high cell proliferative activity, and Venus expression, but without stem/progenitor cell populations, were obtained and designated as USMC44. Using CRISPR/Cas9-mediated genome editing, mtUSMC44 (MED12, 131G>A, p.G44D) and mock USMC44 without MED12 mutation (wtUSMC44) were established from USMC44. wtUSMC44 and mtUSMC44 showed similar cell proliferation activity, even in the presence of estradiol and progesterone (EP) together with transforming growth factor-beta 3 (TGFB3). In addition, wtUSMC44 and mtUSMC44 generated similar tiny smooth muscle-like tissue constructs when xenotransplanted beneath the kidney capsule in immunodeficient mice treated with EP alone or TGFB3. In contrast, mtUSMC44 produced more collagen type I than wtUSMC invitro, and this production was likely enhanced by EP and TGFB3. The results suggest that the MED12 gain-of-function mutation is involved in collagen production. Although approximately 70% of leiomyomas have MED12 mutations, additional factors and/or events other than MED12 and/or myometrialstem/progenitor cells may be required for fully inducing leiomyoma cell properties, including transformation, in USMCs.

Poly(4-Styrenesulfonic Acid-co-maleic Anhydride)-Coated NaGdF4:Yb,Tb,Nd Nanoparticles with Luminescence and Magnetic Properties for Imaging of Pancreatic Islets and β-Cells.

Novel Yb,Tb,Nd-doped GdF3 and NaGdF4 nanoparticles were synthesized by a coprecipitation method in ethylene glycol (EG) in the presence of the poly(4-styrenesulfonic acid-co-maleic anhydride) stabilizer. The particle size and morphology, crystal structure, and phase change were controlled by adjusting the PSSMA concentration and source of fluoride anions in the reaction. Doping of Yb3+, Tb3+, and Nd3+ ions in the NaGdF4 host nanoparticles induced luminescence under ultraviolet and near-infrared excitation and high relaxivity in magnetic resonance (MR) imaging (MRI). In vitro toxicity of the nanoparticles and their cellular uptake efficiency were determined in model rat pancreatic β-cells (INS-1E). As the NaGdF4:Yb,Tb,Nd@PSSMA-EG nanoparticles were non-toxic and possessed good luminescence and magnetic properties, they were applicable for in vitro optical and MRI of isolated pancreatic islets in phantoms. The superior contrast was achieved for in vivo T2*-weighted MR images of the islets transplanted under the kidney capsule to mice in preclinical trials.

AMG487 inhibits PRRSV replication and ameliorates lung injury in pig lung xenografts by down-regulating the expression of ANXA2

Porcine reproductive and respiratory syndrome (PRRS) is a pig disease caused by the PRRS virus (PRRSV) that is characterized with diffuse interstitial pneumonia and lung edema. High expressions of chemokine CXCL10 and its receptor CXCR3 are reported in infected porcine lungs. Since CXCR3 is a key player in host inflammatory response, it might be a therapeutic target to treat lung damage caused by PRRSV infection. The size of pigs has long hampered research into molecular mechanisms of PRRS and validating the potential pharmaceutical targets. In this study, a porcine lung xenograft model with PRRSV infection was generated in immunodeficient mice to evaluate the therapeutic effects of the CXCR3 antagonist AMG487 on PRRSV infection-induced lung injury. The porcine lung tissues developed normally two weeks after xeno-transplantation in the mouse kidney capsule. Infection of PRRSV resulted in its efficient replication in the xenografts and histological damage to the porcine lung tissue structure, with no or little effects on mouse lungs. AMG487 administration dramatically reduced the number of PRRSV genome copies and significantly alleviated the porcine lung injury. Furthermore, treatment of AMG487 in cultured porcine macrophages consistently suppressed PRRSV replication with significant downregulation of Annexin A2 (ANXA2), a cellular protein facilitating viral replication. These findings provide a suitable model for evaluating new antiviral therapies as well as a possible therapeutic option for virus infection-induced lung injury.

Syngeneically transplanted insulin producing cells differentiated from adipose derived stem cells undergo delayed damage by autoimmune responses in NOD mice

Insulin-producing cells (IPCs) generated by our established protocol have reached the non-clinical ‘proof of concept’ stage. Our strategy for their clinical application is the autotransplantation of IPCs into patients with type 1 diabetes mellitus (T1DM). In this context, the autoimmunity that characterized T1DM is important, rather than allorejection. We aimed to determine how these IPCs respond to T1DM autoimmunity. IPCs were generated from the subcutaneous fat tissue of non-obese diabetic (NOD) mice using our protocol. IPCs derived from NOD mice were transplanted under the kidney capsules of NOD mice at the onset of diabetes and the subsequent changes in blood glucose concentration were characterized. Blood glucose decreased within 30 days of transplantation, but increased again after 40–60 days in three of four recipient NOD mice. In tissue samples, the numbers of CD4+ and CD8+ T cells were significantly higher 60 days after transplantation than 30 days after transplantation. In conclusion, IPCs significantly ameliorate the diabetes of mice in the short term, but are damaged by autoimmunity in the longer term, as evidenced by local T cells accumulation. This study provides new insights into potential stem cell therapies for T1DM.

Magnetic Resonance Imaging of Transplanted Porcine Neonatal Pancreatic Cell Clusters Labeled with Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles.

Recently, we have shown that manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) conjugated with exendin-4 (Ex4) act as a contrast agent that directly trace implanted mouse islet β-cells by magnetic resonance imaging (MRI). Here we further advanced this technology to track implanted porcine neonatal pancreatic cell clusters (NPCCs) containing ducts, endocrine, and exocrine cells. NPCCs from one-day-old neonatal pigs were isolated, cultured for three days, and then incubated overnight with MnMEIO-Ex4 NPs. Binding of NPCCs and MnMEIO-Ex4 NPs was confirmed with Prussian blue staining in vitro prior to the transplantation of 2000 MnMEIO-Ex4 NP-labeled NPCCs beneath the left renal capsule of six nondiabetic nude mice. The 7.0 T MRI on recipients revealed persistent hypointense areas at implantation sites for up to 54 days. The MR signal intensity of the graft on left kidney reduced 62–88% compared to the mirror areas on the contralateral kidney. Histological studies showed colocalization of insulin/iron and SOX9/iron staining in NPCC grafts, indicating that MnMEIO-Ex4 NPs were taken up by mature β-cells and pancreatic progenitors. We conclude that MnMEIO-Ex4 NPs are excellent contrast agents for detecting and long-term monitoring implanted NPCCs by MRI.

Effect of Aerobic Treadmill Exercise Regulating Glucose Transporter 9 on Renal Function in Hyperuricemia Rats

<bold>Objective</bold> To explore the possible mechanism of aerobic treadmill exercise in improving renal function in hyperuricemia (HUA) rats. <bold>Methods</bold> A total of 24 male SD rats were randomly divided into the Control group, the Model group and the Exercise group according to the random number table method, with 8 rats in each group. The Control group was given 2 g/(kg·d) distilled water intragastric administration, the Model group and the Exercise group were given 2 g/(kg·d) oxonic aid potassium salt (OAPS) intragastric administration, continuous intragastric administration for 10 days. From the 11th day, the Control group was given distilled water intragastric administration every other day, and the Model group was given OAPS intragastric administration every other day to maintain high uric acid status, with a dose of 2 g/(kg·d). In both groups, diet and water were not restricted, exercise freely, without other intervention, lasted for three weeks. The Exercise group was given intragastric administration as same as the Model group, and was given aerobic treadmill training simultaneously, the speed was 12 m/min, 20 minutes a time, once a day, continuous training for six days and rest for one day, continuous training for three weeks. Hematoxylin-eosin (HE) was used to stain the renal histopathological section, and the microscope (×400) was used to observe the structural changes of renal tubules and glomerulus; the peroxidase method was used to detect the renal function indexes such as uric acid (UA), creatinine (Cre) and urea nitrogen (BUN); enzyme-linked immunosorbent assay (ELISA) method was used to determine the contents of interleukin-1β (IL-1β) in serum and kidney tissues; Western blot was used to detect the expressions of Caspase-1, IL-1β and glucose transporter 9 (GLUT-9) in the kidney tissues. <bold>Results</bold> ① Pathological examination results: there was not obvious pathological changes were observed in the kidney tissues of the Control group; in the Model group, the renal tissue capsule was incomplete, the glomerulus was deformed and atrophic, and the renal tubules were disordered; in the Exercise group, the number of inflammatory cell infiltration was significantly decreased, the renal tubular structure was relatively intact, and the pathological changes such as cytoplasmic vacuolation of renal tubular epithelial cells were significantly decreased. ② UA, Cre and BUN content of serum: compared with the Control group, UA, BUN and Cre content in serum of the Model group and Exercise group was significantly increased (<italic>P</italic>&lt;0.05); compared with the Model group, UA, BUN and Cre content in serum of the Exercise group were significantly decreased (<italic>P</italic>&lt;0.05). ③ Serum and kidney IL-1β content: compared with the Control group, the expression of IL-1β in serum and kidney of the Model group and the Exercise group were significantly increased (<italic>P</italic>&lt;0.05); compared with the Model group, the expression of IL-1β in serum and kidney of the Exercise group was significantly decreased (<italic>P</italic>&lt;0.05). ④ Expressions of Caspase-1, IL-1β and GLUT-9 protein: compared with the Control group, the expressions of Caspase-1, IL-1β and GLUT-9 protein in kidney tissues of the Model group and the Exercise group were significantly increased (<italic>P</italic>&lt;0.05); compared with the Model group, the expressions of Caspase-1, IL-1β, and GLUT-9 protein in kidney tissues of the Exercise group were significantly decreased (<italic>P</italic>&lt;0.05). <bold>Conclusion</bold> Aerobic treadmill exercise can effectively reduce the level of uric acid and inflammatory response of hyperuricemia rats, and improve renal function, which may be related to the regulation of GLUT-9 protein expression.

Identification and Validation of a New Peptide Targeting Pancreatic Beta Cells.

Noninvasive targeted visualization of pancreatic beta cells or islets is becoming the focus of molecular imaging application in diabetes and islet transplantation studies. In this study, we aimed to produce the beta-cell-targeted peptide for molecular imaging of islet. We used phage display libraries to screen a beta-cell-targeted peptide, LNTPLKS, which was tagged with fluorescein isothiocyanate (FITC). This peptide was validated for targeting beta-cell with in vitro and in vivo studies. Immunocytochemistry (ICC) and fluorescence-activated cell sorting (FACS) analysis were used to validate the target specificity of the peptide. FITC-LNTPLKS displayed much higher fluorescence in beta cells vs. control cells in ICC. This discrimination was consistently observed using primary rodent islet. FACS analysis showed right shift of peak point in beta cells compared to control cells. The specific bind to in situ islet was verified by in vitro experiments using rodent and human pancreatic slices. The peptide also showed high affinity of islet grafts under the renal capsule. In the insulinoma animal model, we could find FITC-LNTPLKS accumulated specifically to the tumor, thus indicating a potential clinical application of molecular imaging of insulinoma. In conclusion, LNTPLKS showed a specific probe for beta-cells, which might be further utilized in targeted imaging/monitoring beta cells and theragnosis for beta-cells-related disease (diabetes, insulinoma, etc.).