Athymic Nude Rats Research Articles

CTNI-02. PRECLINICAL DATA AND INITIAL CLINICAL EXPERIENCE IN THE PHASE 1/2A DOSE ESCALATION TRIAL OF RHENIUM-186 NANOLIPOSOME (186RNL) IN LEPTOMENINGEAL METASTASES [LM]: THE RESPECT-LM TRIAL

Abstract INTRODUCTION Leptomeningeal metastases (LM) is a clinical complication that occurs when cancer cells invade the leptomeninges and cerebrospinal fluid of patients with malignant tumors. Once diagnosed, limited treatment options exist, and survival is poor. Rhenium-186 Nanoliposome (186RNL) is a liposomal encapsulated beta emitter with a short path length of 1.8 mm, thereby allowing high specific activity brachytherapy with limited exposure to surrounding tissues. METHODS Preclinical efficacy was assessed using C6-Luc glioma cells in Wistar rats or MDA-MB-231 in Athymic Nude Rats via intraventricular injection. At 15 days post inoculation the animals were treated with 0.69 mCi of 186RNL. Absorbed doses were assessed with gamma camera imaging at 0h, 24h, and 48h post-treatment. Tumor growth was assessed by luciferase bioluminescence. A multicenter phase 1/2a dose (3 + 3 design) escalation trial was then initiated with 186RNL in LM patients to determine the maximum feasible dose, overall response rate (ORR) and overall survival distribution. RESULTS A significant difference in survival between the control and treatment groups (n = 8 each) was observed in the C6/Wistar model at 2 weeks post treatment, with 50% survival in the control group and 100% survival in the treatment group (p = 0.0087). Similar efficacy was observed in the MDA-MB-236/Athymic model. Thus far two patients have received 186RNL with 6.6 mCi in 5ml via Ommaya reservoir. Post treatment imaging shows full CSF distribution by 4 hours and persistence of activity through 7 days following administration. The dose was well-tolerated with no related AEs through day 56 following treatment. CSF cell count by BioCept analysis was reduced by > 90% with durability through day 28. CONCLUSION Intraventricular delivered 186RNL is promising for treatment of LM, with very early evidence of efficacy in patients. Enrollment is ongoing.

Abstract 014: Gestational Diabetic Cd4+ T Cells Cause Increased Glucose, Blood Pressure And Placental Mitochondrial Dysfunction In A Novel Rat Model Of Diabetes During Pregnancy

Hypertensive (HTN) disorders complicate ~10% of all pregnancies worldwide and contribute to maternal and fetal morbidity and mortality. HTN increases risks for preeclampsia and preterm labor in women with gestational diabetes mellitus (GDM) and is characterized by hyperglycemia resulting from defects in insulin activity/secretion or both. Also, increased levels of maternal glucose, inflammatory mediators, abnormal placental vascular function, and cytokines are associated with the pathogenesis of GDM, however, we don’t know the importance of placental factors, inflammatory cells, and cytokines in causing HTN or other pathophysiological factors associated with GDM. We hypothesize placental CD4+T cells from pregnant DM patients (GDM) causes a DM phenotype in pregnant athymic nude rats. CD4+T cells were magnetically separated from placentas from GDM patients and injected into athymic nude rats on Gestation Day (GD) 12. On GD19, blood pressure (MAP), blood and tissues were collected and glucose was measured from GDM CD4+T cell recipients and pregnant nude control rats.Mitochondrial respiration and mtROS were measured as indicators of tissue function from isolated placental mitochondria using the Oxygraph 2K and fluorescent microplate reader, respectively. A student’s t-test was used for statistical analysis. On GD19, MAP increased to 117±5.3 mmHg (n=3) in GDM T cell recipients compared to control pregnant athymic nude rats 102±0.0 mmHg (n=5). Blood glucose levels were elevated with GDM CD4+ T cells (275 ± 80 mg/dl, n=3, p<0.05) compared to pregnant athymic nude rats (91 ± 16 mg/dl, n=5).Placental state 3 (10±1 vs 433±113 pmol/sec/mg, p<0.05), indicative of ATP production, was reduced with GDM CD4+ T cells (n=3) compared to control pregnant athymic nude rats(n=5). Placental mtROS (H2O2) was increased with GDM CD4+ T cells (264 ± 14 % gated, n=3, p<0.05) compared to pregnant athymic nude rats (100 ± 26 % gated, n=5).Collectively, these data indicate that GDM CD4+T cells cause the GDM phenotype during pregnancy by increasing glucose and blood pressure in association with placental mitochondrial dysfunction/ROS during pregnancy, thereby introducing a new model to investigate mechanisms and new therapies for diabetes during pregnancy.

IL17 administration in the Absence of T cells Results in Hypertension, NK cell Activation, and Reduced Pup Weight at Birth, but No Changes in Blood Pressure or Weight at Maturation of Offspring

Preeclampsia (PE) is characterized by new onset hypertension (HTN), fetal growth restriction (FGR), multi‐organ dysfunction, and increased inflammatory cytokines. Moreover, we have shown offspring of RUPP rats develop hypertension at different stages of life based on sex. More recent studies demonstrate a role for mitochondrial (mt) dysfunction/mtROS in the pathogenesis of the disease, however, causative factors for mt dysfunction are still being identified. Th17 cells stimulated by a rat model of PE have been shown to cause HTN, FGR, and mt dysfunction. One of the main cytokines produced by TH17 cells is IL‐17, which we have shown to cause HTN, mtROS, stimulate TH17 cell proliferation, and activate natural killer (NK) cells in pregnant Sprague Dawley rats. The athymic nude rat model lacks T cells, and therefore are useful in examining the differences in the roles of IL‐17 and TH17 cells in the pathophysiology of PE. We hypothesize that IL‐17 induces HTN and is associated with renal and placental mt dysfunction during pregnancy in the absence of T cells in pregnant athymic nude rats. We further hypothesize that offspring of these animals generate hypertension at maturation (16 weeks of age). IL‐17 (150 pg/day) was infused via osmotic minipumps on gestation day (GD) 14. Blood pressure (MAP) and mt function were measured on GD19 and were compared to normal pregnant (NP) rats. Pups were weaned 3 weeks after birth and weighed once a week for 16 weeks, and MAP was measured at 16 weeks. MAP increased in response to IL‐17 (NP+IL‐17, 115.3±1.5 mmHg in, n=6) compared to NP rats (95.5±3.85 mmHg, n=6, p<0.001). Interestingly, mtROS in the absence of T cells is reduced in both the placenta and kidney (46±6.6%fold change in NP+IL‐17 placental mitochondria; 48.8±2.8%fold change in the kidney mitochondria, n=5) compared to NP mtROS (100±6.6%fold change in NP placental mitochondria; 100±3.7% fold change in the kidney mitochondria, (n=4). ADP production in response to IL‐17 in the absence of T cells in the placenta or the kidney increases (105.5±91 pmol of O2/sec/mg, n=3NP+IL‐17 placental mitochondria, 2016±951.2 pmol of O2/sec/mg, n=4, in NP+IL‐17 kidney mitochondria) compared to NP (15.53±1.6 pmol of O2/sec/mg, n=3 NP placenta, 1196±459.5 pmol of O2/sec/mg, n=4 NP kidney (n=4)). Pup weight at GD19 decreased in response to IL‐17 from 1.46±0.2 g in NP (n=6) to 0.98±0.07g in NP+IL‐17 (n=6) in the absence of T cells (p<0.05). However, there was no change in weight after weaning and into adulthood in either male or female offspring. There was also no change in MAP in NP+IL17 (123± 2.7 mmHg, n=3) male offspring compared to NP offspring (122± 2.6 mmHg, n=4) at 16 weeks. There was also no change in female offspring MAP in response to IL‐17 (121± 12 mmHg, n=2) compared to NP (105 ± 21 mmHg, n=3) in offspring at 16 weeks. These results suggest that IL‐17 induces HTN, and FGR without the presence of T cells, and suggests that T cells induce mt dysfunction in PE. It also shows that IL‐17 may not cause hypertension of the offspring after birth.

The Role of B Cells in Mediating Hypertension in Preeclampsia or COVID‐19 Infection During Pregnancy

Preeclampsia (PE), new onset hypertension associated with placental ischemia during pregnancy, is associated with a pro‐inflammatory state characterized by increased T Helper cells and B cells secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1‐AA). We have previously shown that adoptive transfer of CD4+T cells from PE women causes a PE‐like phenotype in immunodeficient (nude athymic) pregnant rats. Moreover, our lab has shown that depletion of B Cells or inhibition of AT1‐AA, a product of B cell activation, attenuates hypertension in various rat models of PE. Interestingly, AT1‐AA has been found in patients with COVID‐19 (CV) infection. An increase incidence of PE has been associated with a history (hx) of COVID‐19 infection during pregnancy. Collectively, this information suggests not only that B cells play an important role in the production of AT1‐AA in PE, but also a hx of COVID‐19 infection during pregnancy may result in AT1‐AA, thereby, contributing to increased risk for PE. Although it is known that B cells are the only producers of antibodies and therefore produce AT1‐AA, recent studies have touted that B cells don’t play a role in the hypertension in response to placental ischemia. Current studies have only investigated B cells in animal models of PE; yet, no studies have examined the effects of B cells from PE patients to cause hypertension during pregnancy. We hypothesize that B cells play an important role to cause hypertension and other features of PE during pregnancy and that B cells in COVID hx pregnant patients contribute to AT1‐AA in pregnancy. To test this hypothesis, B cells were isolated from the placentas of normal pregnant control (NP), PE, normotensive CV hx, or PE CV hx patients upon delivery. Isolated B cells were transferred i.p. into pregnant Nude Athymic rats at gestation (GD) 12. On GD18 carotid catheters were inserted. Blood pressure (MAP) was measured, pups were weighed, and tissues were collected on GD19. Recipients of placental PE B cells had increased MAP (115±3 mmHg, n=6) compared to recipients of placental NP B cells (100±6 mmHg, n=4, p<0.05). There were no changes in placental:fetal weight ratio associated with PE B cells (0.42±0.02; n=9) compared to recipients of NP B cells (0.40±0.01, n=6). Also, recipients of placental PE B cells had elevated activated Natural Killer cells in their placentas (33±14, n=3) compared to recipients of NP B cells (24±12, n=2). Recipients of placental CV Hx PE B cells had elevated MAP (107±7 mmHg, n=2) compared to recipients of placental CV Hx NP B cells (99±8 mmHg, n=3). Animals that received CV hx PE B cells had reduced placental efficiency (0.430, n=1) compared to recipients of CV hx Normotensive B cells (0.38±0.01, n=3). Collectively, this study demonstrates an important role for PE B cells to cause hypertension during pregnancy; and indicates that B cells contribute to a higher incidence of PE in women with a history of COVID‐19 infection during pregnancy.

Transmural myocardial repair with engineered heart muscle in a rat model of heterotopic heart transplantation – A proof-of-concept study

Engineered heart muscle (EHM) can be implanted epicardially to remuscularize the failing heart. In case of a severely scarred ventricle, excision of scar followed by transmural heart wall replacement may be a more desirable application. Accordingly, we tested the hypothesis that allograft (rat) and xenograft (human) EHM can also be administered as transmural heart wall replacement in a heterotopic, volume-loaded heart transplantation model. We first established a novel rat model model to test surgical transmural left heart wall repair. Subsequently and in continuation of our previous allograft studies, we tested outcome after implantation of contractile engineered heart muscle (EHM) and non-contractile engineered connective tissue (ECT) as well as engineered mesenchymal tissue (EMT) allografts as transmural heart wall replacement. Finally, proof-of-concept for the application of human EHM was obtained in an athymic nude rat model. Only in case of EHM implantation, remuscularization of the surgically created transmural defect was observed with palpable graft vascularization. Taken together, feasibility of transmural heart repair using bioengineered myocardial grafts could be demonstrated in a novel rat model of heterotopic heart transplantation.

Placental CD4+ T cells from preeclamptic patients cause autoantibodies to the angiotensin II type I receptor and hypertension in a pregnant rat model of preeclampsia.

Preeclampsia (PE) is a hypertensive disorder of pregnancy associated with activated CD4+ T cells and autoantibodies to angiotensin II type 1 receptor (AT1-AA). We have previously shown that CD4+ T cells isolated from women with PE cause hypertension, increased tumor necrosis factor alpha (TNF-α), endothelin-1, and soluble fms-like tyrosine kinase-1 (sFlt-1) when injected into pregnant nude-athymic rats compared to CD4+ T cells from normal pregnant (NP) women. However, the role of PE CD4+ T cells to cause AT1-AA as a mechanism of hypertension is not known. Aim: Our goal was to determine if PE CD4+ T cells stimulate AT1-AA in pregnant nude-athymic rats. CD4+ T cells were isolated from human NP and PE placentasand injected into nude-athymic rats on gestational day (GD) 12. In order to examine the role of the PE CD4+ T cells to stimulate B cell secretion of AT1-AA, a subset of the rats receiving PE CD4+ T cells were treated with rituximab on GD 14 or anti-CD40 ligand (anti-CD40L) on GD 12. On GD 19, mean arterial pressure (MAP) and tissues were obtained MAP [114 ± 1 mmHg (n = 9)] and AT1-AA [19.8 ± 0.9 beats per minute (bpm, n = 4)] were increased in NP nude + PE CD4+ T cells compared to NP nude + NP CD4+ T cells [98 ± 2 mmHg (n = 7, P < 0.05) and 1.3 ± 0.9 bpm (n = 5, P < 0.05)]. Rituximab (103 ± 2 mmHg, n = 3, P < 0.05) and anti-CD40L (102 ± 1 mmHg, n = 3, P < 0.05) lowered MAP compared to NP nude + PE CD4+ T cells. Circulating a proliferation-inducing ligand (APRIL) and placental angiotensin-converting enzyme 2 (ACE-2) activity was increased in response to PE CD4+ T cells. These results show that placental CD4+ T cells play an important role in the pathophysiology of PE, by activating B cells secreting AT1-AA to cause hypertension during pregnancy.

A combined cell and growth factor delivery for the repair of a critical size tibia defect using biodegradable hydrogel implants.

The ability to repair critical‐sized long‐bone injuries using growth factor and cell delivery was investigated using hydrogel biomaterials. Physiological doses of the recombinant human bone morphogenic protein‐2 (rhBMP2) were delivered in a sustained manner from a biodegradable hydrogel containing peripheral human blood‐derived endothelial progenitor cells (hEPCs). The biodegradable implants made from polyethylene glycol (PEG) and denatured fibrinogen (PEG‐fibrinogen, PF) were loaded with 7.7 μg/ml of rhBMP2 and 2.5 × 106 cells/ml hEPCs. The safety and efficacy of the implant were tested in a rodent model of a critical‐size long‐bone defect. The hydrogel implants were formed ex‐situ and placed into defects in the tibia of athymic nude rats and analyzed for bone repair after 13 weeks following surgery. The hydrogels containing a combination of 7.7 μg/ml of rhBMP2 and 2.5 × 106 cells/ml hEPCs were compared to control hydrogels containing 7.7 μg/ml of rhBMP2 only, 2.5 × 106 cells/ml hEPCs only, or bare hydrogels. Assessments of bone repair include histological analysis, bone formation at the site of implantation using quantitative microCT, and assessment of implant degradation. New bone formation was detected in all treated animals, with the highest amounts found in the treatments that included animals that combined the PF implant with rhBMP2. Moreover, statistically significant increases in the tissue mineral density (TMD), trabecular number and trabecular thickness were observed in defects treated with rhBMP2 compared to non‐rhBMP2 defects. New bone formation was significantly higher in the hEPC‐treated defects compared to bare hydrogel defects, but there were no significant differences in new bone formation, trabecular number, trabecular thickness or TMD at 13 weeks when comparing the rhBMP2 + hEPCs‐treated defects to rhBMP2‐treated defects. The study concludes that the bone regeneration using hydrogel implants containing hEPCs are overshadowed by enhanced osteogenesis associated with sustained delivery of rhBMP2.

Fibroblast Growth Factor-Impregnated Collagen-Gelatin Sponge Improves Keratinocyte Sheet Survival.

Commercially available cultured epithelial keratinocyte sheets (KSs) have played an essential role in wound healing over the past four decades. Despite the initial uptake by the dermal elements, the survival rate of KS on the dermis-like tissue generated by conventional artificial dermis (AD) is low, making this method unsuitable for standard treatments. Therefore, an innovative AD such as collagen-gelatin sponge (CGS) that maintains the release of human recombinant basic fibroblast growth factor (bFGF) may promote wound healing. In this study, we examined whether combination therapy with KSs and CGS with bFGF (bFGF-CGS) could enhance KS survival by heterologous grafting by transplantation of human-derived KSs in an athymic nude rat wound model of staged skin reconstruction. The CGSs were implanted into skin defect wounds on athymic nude rats, which were then divided into two experimental groups: the bFGF group (CGSs containing bFGF, n = 8) and the control group (CGSs with saline, n = 8). Two weeks after implantation, human epithelial cell-derived KSs were grafted onto the dermis-like tissue, followed by assessment of the survival and morphology at 1 week later using digital imaging, histology (hematoxylin and eosin and Masson's trichrome staining), immunohistology (von Willebrand factor), immunohistochemistry (cytokeratin 1-5-6, Ki-67), and immunofluorescence (collagen IV, pan-cytokeratins) analyses. The bFGF group showed a significantly higher KS survival area (86 ± 58 mm2 vs. 32 ± 22 mm2; p < 0.05) and increased epidermal thickness (158 ± 66 μm vs. 86 ± 40 μm; p < 0.05) compared with the control group, along with higher dermis-like tissue regeneration, neovascularization, epidermal maturation, and basement membrane development. These results indicate that the survival rate of KSs in the dermis-like tissue formed by bFGF-CGS was significantly increased. Therefore, combination treatment of bFGF-CGS and KSs shows potential for full-thickness skin defect reconstruction in clinical situations. Impact statement This study highlights how using a combination of cultures, keratinocyte sheets, and collagen-gelatin sponge containing basic fibroblast growth factors can significantly improve cell survival in athymic nude rats with staged skin reconstruction. Our study makes a significant contribution to the literature because it highlights a novel and improved strategy for treating a very common condition such as skin wounds arising from many conditions. Clinical translation of this study may be useful for treating skin wounds.

IL-17-induced hypertension results in placental mitochondrial dysfunction and fetal growth restriction independent of T cells

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with fetal growth restriction (FGR), multi-organ dysfunction, increased Natural Killer cells, T helper cells, and interleukin 17 (IL-17). We have shown that IL-17 stimulates TH17 cells and NK cells in association with placental mitochondrial (mt) dysfunction, FGR and HTN in pregnant Sprague Dawley rats. However, our previous studies have not differentiated the effects of activated TH cells verses IL-17. The athymic nude rat lacks T cells but does have other components of the immune system, allowing us to examine the role of IL-17 in the absence of T cells in the pathophysiology of PE.

Application of Human Epineural Conduit Supported with Human Mesenchymal Stem Cells as a Novel Therapy for Enhancement of Nerve Gap Regeneration

Various therapeutic methods have been suggested to enhance nerve regeneration. In this study, we propose a novel approach for enhancement of nerve gap regeneration by applying human epineural conduit (hEC) supported with human mesenchymal stem cells (hMSC), as an alternative to autograft repair. Restoration of 20 mm sciatic nerve defect with hEC created from human sciatic nerve supported with hMSC was tested in 4 experimental groups (n = 6 each) in the athymic nude rat model (Crl:NIH-Foxn1rnu): 1 - No repair control, 2 - Autograft control, 3 - Matched diameter hEC filled with 1 mL saline, 4 - Matched diameter hEC supported with 3 × 106 hMSC. Assessments included: functional tests: toe-spread and pinprick, regeneration assessment by immunofluorescence staining: HLA-1, HLA-DR, NGF, GFAP, Laminin B, S-100, VEGF, vWF and PKH26 labeling; histomorphometric analysis of myelin thickness, axonal density, fiber diameter and myelinated nerve fibers percentage; Gastrocnemius Muscle Index (GMI) and muscle fiber area ratio. Best sensory and motor function recovery, as well as GMI and muscle fiber area ratio, were observed in the autograft group, and were comparable to the hEC with hMSC group (p = 0.038). Significant improvements of myelin thickness (p = 0.003), fiber diameter (p = 0.0296), and percentage of myelinated fibers (p < 0.0001) were detected in hEC group supported with hMSC compared to hEC with saline controls. At 12-weeks after nerve gap repair, hEC combined with hMSC revealed increased expression of neurotrophic and proangiogenic factors, which corresponded with improvement of function comparable with the autograft control. Application of our novel hEC supported with hMSC provides a potential alternative to the autograft nerve repair.Graphical

Abstract 11090: Therapeutic Effect of VEGF- and SDF-Nanoparticles in the Rat Monocrotaline Model of Pulmonary Hypertension

Introduction: Pulmonary vascular endothelial cell injury, dysfunction, and apoptosis are associated with the development of pulmonary arterial hypertension (PAH). Improving endothelial function may be therapeutically beneficial in this disorder. In the present study, we examined the effect of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α), proteins that support endothelial repair and tissue regeneration, in rats with monocrotaline-induced PAH. A polysaccharide nanoparticle was used as a carrier for VEGF and SDF-1α delivery, the formulation of which extends the retention time of the proteins in the lung by ~17-fold compared with free protein. Method: A crosslinked dextran sulfate-chitosan nanoparticle (XDSCS NP) was prepared as the carrier. Recombinant human VEGF and SDF-1α were incorporated in the XDSCS NP to generate VEGF-NP and SDF-NP, respectively. Athymic nude rats were injected with monocrotaline (50 mg/kg, IP) to induce PAH. Two weeks after the injection, VEGF-NP (8 μg/rat) and SDF-NP (4 μg/rat) were delivered to the lungs of rats via intratracheal aerosolization. At 4 weeks, catheterization of right and left heart was performed, and lung tissue collected for histological analysis. Results: Monocrotaline induced PAH in the nude rats, leading to right ventricular systolic pressure (RVSP) of 71±8 mmHg (mean±SD, n=8) vs. 29±2 mmHg in the untreated control rats. VEGF-NP and SDF-NP treatment reduced the RVSP to 48±13 mmHg. The Fulton indices of the three groups were 0.45±0.07, 0.22±0.01, and 0.25±0.06, respectively. The pulmonary vascular resistance indices of the groups were 3.2±0.7, 0.6±0.3, and 2.1±1.0 mmHg.min/ml/kg, respectively. Histological analysis showed monocrotaline-treated rats had prominent muscularization of precapillary vessels, which was markedly reduced by the VEGF-NP and SDF-NP treatment. Aerosolization of free VEGF and SDF-1α or of empty XDSCS NP carrier had no effect on monocrotaline-induce PAH. Conclusion: These data indicate that the delivery of VEGF and SDF to the lungs of rats in a NP-bound form had beneficial therapeutic effects in monocrotaline-induced PAH, and offer the potential for a novel therapeutic strategy for human pulmonary hypertensive disorders.

Proof of concept of a multimodal intravital molecular imaging system for tumour transpathology investigation

BackgroundTranspathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation.MethodsThe proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin–eosin (HE) stained for comparison with multimodal in vivo imaging.ResultsThe final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology.ConclusionsThe established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

A Prevascularized Polyurethane-Reinforced Fibrin Patch Improves Regenerative Remodeling in a Rat Right Ventricle Replacement Model.

Congenital heart defects (CHDs) affect 1 in 120 newborns in the United States. Surgical repair of structural heart defects often leads to arrhythmia and increased risk of heart failure. The laboratory has previously developed an acellular fibrin patch reinforced with a biodegradable poly(ether ester urethane) urea mesh that result in improved heart function when tested in a rat right ventricle wall replacement model compared to fixed pericardium. However, this patch does not drive significant neotissue formation. The patch materials are modified here and this patch is prevascularized with human umbilical vein endothelial cells and c-Kit+ human amniotic fluid stem cells. Rudimentary capillary-like networks form in the fibrin after culture of cell-encapsulated patches for 3 d in vitro. Prevascularized patches and noncell loaded patch controls are implanted onto full-thickness heart wall defects created in the right ventricle of athymic nude rats. Two months after surgery, defect repair with prevascularized patches results in improved heart function and the patched heart area exhibited greater vascularization and muscularization, less fibrosis, and increased M2 macrophage infiltration compared to acellular patches.

Abstract 26: IL-17 Causes Hypertension, Reduced Fetal Weight And Natural Killer Cell Activation In The Absence Of T Cells

Preeclampsia (PE) is characterized by new onset hypertension (HTN), intrauterine growth restriction (IUGR), multi-organ dysfunction, and is associated with increased inflammatory cytokines, such as interleukin 17 (IL-17). More recent studies demonstrate a role for mitochondrial (mt) dysfunction/mtROS in the pathogenesis of PE. Although we have shown T helper cells from a rat model of PE cause HTN and mt dysfunction the causative factors for mt dysfunction are still being identified. In addition, we have shown that IL-17 cause HTN, IUGR and activate natural killer (NK) cells, and cause mt dysfunction in pregnant Sprague Dawley rats. However, in our previous studies we couldn’t differentiate the effect of activated TH cells versus IL-17 to cause these characteristics of PE. The athymic nude rat model lacks mature T cells but does have other components of the immune system, and will thus allow us to examine the role of IL-17 in the absence of TH cells in the pathophysiology of PE. We hypothesize that in the absence of T cells IL-17 induces HTN, NK cell activation and IUGR which is associated with renal and placental mt dysfunction during pregnancy. To test our hypothesis, IL-17 (150 pg/day) was infused via osmotic minipumps inserted on gestation day (GD) 14. Blood pressure (MAP) and mt function were measured on GD19 and were compared to untreated pregnant (NP) athymic nude rats. In response to IL-17; MAP increased from 95±4mmHg in NP(n=6) to 115±2 in NP+IL-17(n=6) (p&lt;0.001); pup weight decreased from 1.46±0.2 g in NP (n=6) to 0.98±0.07g in NP+IL-17 (n=6) (p&lt;0.05); NK cell activation increased from 0±0 %lymphocytes in NP (n=3) to 0.4±0.1% lymphocytes in NP+IL-17 rats (n=6). Interestingly, placental mtROS reduced 54% fold compared to NP and renal mtROS reduced 51.2% compared to NP. ATP production increased from 15.53±1.6 pmol of O2/sec/mg in NP (n=3) to 105.5±91 pmol of O2/sec/mg in NP+IL-17 (n=3) in the placenta, and from 1196±460 pmol of O2/sec/mg in NP (n=4) to 2016±951 pmol of O2/sec/mg NP+IL17 (n=4) in the kidney. These results show that although IL-17 induces HTN, IUGR, and NK cell activation independent of T cells, T cells are necessary for reduced mitochondrial function observed in PE and in rat models of placental ischemia.

Freshly Thawed Cryobanked Human Neural Stem Cells Engraft within Endogenous Neurogenic Niches and Restore Cognitive Function after Chronic Traumatic Brain Injury.

Human neural stem cells (hNSCs) have potential as a cell therapy after traumatic brain injury (TBI). While various studies have demonstrated the efficacy of NSCs from ongoing culture, there is a significant gap in our understanding of freshly thawed cells from cryobanked stocks-a more clinically relevant source. To address these shortfalls, the therapeutic potential of our previously validated Shef-6.0 human embryonic stem cell (hESC)-derived hNSC line was tested after long-term cryostorage and thawing before transplant. Immunodeficient athymic nude rats received a moderate unilateral controlled cortical impact (CCI) injury. At four weeks post-injury, 6 × 105 freshly thawed hNSCs were transplanted into six injection sites (two ipsi- and four contra-lateral) with 53.4% of cells surviving three months post-transplant. Interestingly, most hNSCs were engrafted in the meninges and the lining of lateral ventricles, associated with high CXCR4 expression and a chemotactic response to SDF1alpha (CXCL12). While some expressed markers of neuron, astrocyte, and oligodendrocyte lineages, the majority remained progenitors, identified through doublecortin expression (78.1%). Importantly, transplantation resulted in improved spatial learning and memory in Morris water maze navigation and reduced risk taking in an elevated plus maze. Investigating potential mechanisms of action, we identified an increase in ipsilateral host hippocampus cornu ammonis (CA) neuron survival, contralateral dentate gyrus (DG) volume, and DG neural progenitor morphology as well as a reduction in neuroinflammation. Together, these findings validate the potential of hNSCs to improve function after TBI and demonstrate that long-term biobanking of cells and thawing aliquots before use may be suitable for clinical deployment.

Engineered neural tissue made using clinical-grade human neural stem cells supports regeneration in a long gap peripheral nerve injury model

A surgical autograft remains the clinical gold-standard therapy for gap repair following peripheral nerve injury, however, challenges remain with achieving full recovery and reducing donor-site morbidity. Engineered Neural Tissue (EngNT) manufactured using differentiated CTX0E03 human stem cells (EngNT-CTX) has been developed as a potential ‘off the shelf’ allogeneic autograft replacement. Ensheathed within a collagen membrane developed to facilitate biomechanical integration, EngNT-CTX was used to bridge a critical-length (15 mm) sciatic nerve gap injury in athymic nude rats. The effectiveness of EngNT-CTX was compared to an autograft using outcome measures that assessed neuronal regeneration and functional recovery at 8 and 16 weeks. At both time points EngNT-CTX restored electrophysiological nerve conduction and functional reinnervation of downstream muscles to the same extent as the autograft. Histological analysis confirmed that more motor neurons had successfully regenerated through the repair in EngNT-CTX in comparison to the autograft at 8 weeks, which was consistent with the electrophysiology, with the number of motor neurons similar in both groups by 16 weeks. The total number of neurons (motor + sensory) was greater in autografts than EngNT-CTX at 8 weeks, indicating that more sensory fibres may have sprouted in those animals at this time point. In conclusion, this study provides evidence to support the effectiveness of EngNT-CTX as a replacement for the nerve autograft, as the functional regeneration assessed through histological and electrophysiological outcome measures demonstrated equivalent performance. Statement of significanceFollowing injury a peripheral nerve has the capacity to regenerate naturally, however, in the case of severe damage where there is a gap the current gold-standard microsurgical intervention is an autograft. This is associated with serious limitations including tissue availability and donor-site morbidity.Tissue engineering aims to overcome these limitations by building a construct from therapeutic cells and biomaterials as a means to mimic and replace the autograft. In this study engineered neural tissue (EngNT) was manufactured using human stem cells (CTX) to bridge a critical-length gap injury. When compared to the autograft in an animal model the EngNT-CTX construct restored function to an equivalent or greater extent.

3D Multicellular Stem-Like Human Breast Tumor Spheroids Enhance Tumorigenicity of Orthotopic Xenografts in Athymic Nude Rat Model.

Simple SummaryBreast cancer presents a unique clinical problem because of the variety of cellular subtypes present, including cancer stem cells (CSCs). Breast CSCs can induce the formation of new blood vessels at the site of tumor growth and a develop metastatic phenotype by enhancing a stromal cell response, similar to that of the primary breast cancer. The aim of this study was to investigate breast cancer cells cultured in stromal stem cell factor-supplemented media to generate 3D spheroids that exhibit increased stem-like properties. These 3D stem-like spheroids reproducibly and efficiently established orthotopic breast cancer xenografts in the athymic nude rat. This approach enables a means to develop orthotopic tumors with a stem-like phenotype in a larger athymic rat rodent model of human breast cancer.Therapeutic targeting of stem cells needs to be strategically developed to control tumor growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes present, including cancer stem cells (CSCs). The development of 3D stem-like properties of human breast tumor spheroids in stem cell factor conditioned media was investigated in orthotopic xenografts for enhanced tumorgenicity in the athymic nude rat model. MCF-7, ZR-75-1, and MDA-MB-231 breast cancer cell lines were cultured in serum-free, stem cell factor-supplemented medium under non-adherent conditions and passaged to generate 3rd generation spheroids. The spheroids were co-cultured with fetal lung fibroblast (FLF) cells before orthotopic heterotransplantation into the mammary fat pads of athymic nude rats. Excised xenografts were assessed histologically by H&E staining and immunohistochemistry for breast cancer marker (ERB1), proliferation marker (Ki67), mitotic marker (pHH3), hypoxia marker (HIF-2α), CSC markers (CD47, CD44, CD24, and CD133), and vascularization markers (CD31, CD34). Breast cancer cells cultured in stem cell factor supplemented medium generated 3D spheroids exhibited increased stem-like characteristics. The 3D stem-like spheroids co-cultured with FLF as supporting stroma reproducibly and efficiently established orthotopic breast cancer xenografts in the athymic nude rat.

The Importance of B Cells in Causing Hypertension During Pregnancy; to B or Not to B

Preeclampsia (PE), new onset hypertension associated with placental ischemia during pregnancy, is associated with a pro-inflammatory state characterized by increased T Helper cells and B lymphocytes secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1-AA). We have shown that adoptive transfer of CD4+T cells from PE women causes a PE-like phenotype in immunodeficient pregnant rats. Moreover, our lab has shown that depletion of B Cells or inhibition of AT1-AA, a product of B cell activation, attenuates hypertension in various rat models of PE. Recent studies have touted that B cells don't play a role in the hypertension in response to placental ischemia. However, to date studies have only investigated B cells from animal models of PE, no studies have examined the effects of B cells from PE patients to cause hypertension during pregnancy. Therefore, we hypothesize that B cells and B cell communication with T cells, play an important role to cause hypertension and other features of PE during pregnancy. We tested our hypothesis in two separate studies involving adoptive transfer of PE cells into immunodeficient pregnant rats. First, placental CD4+T cells, isolated from either normal pregnant control (NP) or PE patients upon delivery, were transferred i.p. into Nude athymic rats at gestation day (GD) 12. Blood pressure (MAP) and circulating A Proliferation Inducing Ligand (APRIL), a cytokine necessary for B cell/plasma cell maturation and survival, were examined on GD19. In a separate group of Nude athymic rats, PE placental B cells were transferred on GD12 and MAP was determined at GD19. Recipients of placental PE CD4+ T cells had elevated MAP, 114±1 mmHg, compared to recipients of NP CD4+T cells, 97±3 mmHg (p<0.05). Recipients of PE CD4+T cells had smaller pups (1.23±0.075g) compared to recipients of NP CD4+T Cells (1.507±0.139g)(p=0.07)(ns). Importantly, recipient rats of PE CD4+T cells also had elevated APRIL (1.276±0.0.13 ng/mL) compared to recipients of NP CD4+ T cells (0.434±0.11 ng/mL)(p<0.01). MAP increased in Nude athymic pregnant rats that received placental PE B cells (114 ±5 mmHg)(n=3) compared to control Nude athymic pregnant rats (102±1 mmHg)(n=3). These studies show that 1)PE CD4+T cells stimulate factors necessary for B cell maturation which is associated with hypertension during pregnancy and that 2)PE B cells indeed cause hypertension in during pregnancy. Therefore, these studies demonstrate a role for B cells and T cell communication with B cells stimulated in response to PE to directly and indirectly play a role in hypertension during pregnancy.

Immunomodulation Therapy Using Tolerogenic Macrophages in a Rodent Model of Pulmonary Hypertension.

Inflammation plays a major role in the pathogenesis of pulmonary hypertension (PH). We sought to investigate the effects of a cell-based immunomodulation in a dysimmune model of PH. PH was induced in athymic nude rats using semaxinib (Su group, n = 6). Tolerogenic macrophages (toM) were generated from monocyte isolation and then injected either the day before semaxinib injection (Prevention group, n = 6) or 3 weeks after (Reversion group, n = 6). Six athymic nude rats were used as controls. In vivo trafficking of toM was investigated with bioluminescence imaging showing that toM were mainly located into the lungs until 48 h after injection. Right ventricular (RV) end-systolic pressure and RV systolic function were assessed at 4 weeks using echocardiography. Morphometric analysis and RNA sequencing of the lungs were realized at 4 weeks. Rats treated with toM (Prevention and Reversion groups) had a significantly lower RV end-systolic pressure at 4 weeks (respectively, 25 ± 8 and 30 ± 6 mmHg vs. 67 ± 9 mmHg, P < 0.001), while RV systolic dysfunction was observed in Su and Reversion groups. Mean medial wall thickness of small arterioles was lower in Prevention and Reversion groups compared with the Su group (respectively, 10.9% ± 0.8% and 16.4% ± 1.3% vs. 28.2% ± 2.1%, P < 0.001). Similarly, cardiomyocyte area was decreased in rats treated with toM (150 ± 18 and 160 ± 86 μm2 vs. 279 ± 50 μm2, P < 0.001). A trend toward upregulation of genes involved in pulmonary arterial hypertension pathobiology was found in Su rats, while KCNK3 was significantly downregulated (fold-change = 9.8, P < 0.001). Injection of toM was associated with a less severe phenotype of PH in rats exposed to angioproliferative stress. Preserved expression of KCNK3 may explain the protective effect of toM.

Characterization of ultrasound-mediated delivery of trastuzumab to normal and pathologic spinal cord tissue

Extensive studies on focused ultrasound (FUS)-mediated drug delivery through the blood–brain barrier have been published, yet little work has been published on FUS-mediated drug delivery through the blood-spinal cord barrier (BSCB). This work aims to quantify the delivery of the monoclonal antibody trastuzumab to rat spinal cord tissue and characterize its distribution within a model of leptomeningeal metastases. 10 healthy Sprague–Dawley rats were treated with FUS + trastuzumab and sacrificed at 2-h or 24-h post-FUS. A human IgG ELISA (Abcam) was used to measure trastuzumab concentration and a 12 ± fivefold increase was seen in treated tissue over control tissue at 2 h versus no increase at 24 h. Three athymic nude rats were inoculated with MDA-MB-231-H2N HER2 + breast cancer cells between the meninges in the thoracic region of the spinal cord and treated with FUS + trastuzumab. Immunohistochemistry was performed to visualize trastuzumab delivery, and semi-quantitative analysis revealed similar or more intense staining in tumor tissue compared to healthy tissue suggesting a comparable or greater concentration of trastuzumab was achieved. FUS can increase the permeability of the BSCB, improving drug delivery to specifically targeted regions of healthy and pathologic tissue in the spinal cord. The achieved concentrations within the healthy tissue are comparable to those reported in the brain.