Testing Of New Therapeutic Approaches Research Articles

Steatosis drives monocyte-derived macrophage accumulation in human metabolic dysfunction-associated fatty liver disease

Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common complication of obesity with a hallmark feature of hepatic steatosis. Recent data from animal models of MAFLD have demonstrated substantial changes in macrophage composition in the fatty liver. In humans, the relationship between liver macrophage heterogeneity and liver steatosis is less clear. Liver tissue from 21 participants was collected at time of bariatric surgery and analysed using flow cytometry, immunofluorescence, and H&E microscopy. Single-cell RNA sequencing was also conducted on a subset of samples (n= 3). Intrahepatic triglyceride content was assessed via MRI and tissue histology. Mouse models of hepatic steatosis were used to investigate observations made from human liver tissue. We observed variable degrees of liver steatosis with minimal fibrosis in our participants. Single-cell RNA sequencing revealed four macrophage clusters that exist in the human fatty liver encompassing Kupffer cells and monocyte-derived macrophages (MdMs). The genes expressed in these macrophage subsets were similar to those observed in mouse models of MAFLD. Hepatic CD14+ monocyte/macrophage number correlated with the degree of steatosis. Using mouse models of early liver steatosis, we demonstrate that recruitment of MdMs precedes Kupffer cell loss and liver damage. Electron microscopy of isolated macrophages revealed increased lipid accumulation in MdMs, and exvivo lipid transfer experiments suggested that MdMs may serve a distinct role in lipid uptake during MAFLD. The human liver in MAFLD contains macrophage subsets that align well with those that appear in mouse models of fatty liver disease. Recruited myeloid cells correlate well with the degree of liver steatosis in humans. MdMs appear to participate in lipid uptake during early stages of MALFD. Metabolic dysfunction associated fatty liver disease (MAFLD) is extremely common; however, the early inflammatory responses that occur in human disease are not well understood. In this study, we investigated macrophage heterogeneity in human livers during early MAFLD and demonstrated that similar shifts in macrophage subsets occur in human disease that are similar to those seen in preclinical models. These findings are important as they establish a translational link between mouse and human models of disease, which is important for the development and testing of new therapeutic approaches for MAFLD.

Modeling Traumatic Brain Injury in Human Cerebral Organoids.

Traumatic brain injury (TBI) is a head injury that disrupts the normal brain structure and function. TBI has been extensively studied using various in vitro and in vivo models. Most of the studies have been done with rodent models, which may respond differently to TBI than human nerve cells. Taking advantage of the recent development of cerebral organoids (COs) derived from human induced pluripotent stem cells (iPSCs), which resemble the architecture of specific human brain regions, here, we adapted the controlled cortical impact (CCI) model to induce TBI in human COs as a novel in vitro platform. To adapt the CCI procedure into COs, we have developed a phantom brain matrix, matching the mechanical characteristics of the brain, altogether with an empty mouse skull as a platform to allow the use of the stereotactic CCI equipment on COs. After the CCI procedure, COs were histologically prepared to evaluate neurons and astrocyte populations using the microtubule-associated protein 2 (MAP2) and the glial fibrillary acidic protein (GFAP). Moreover, a marker of metabolic response, the neuron-specific enolase (NSE), and cellular death using cleaved caspase 3 were also analyzed. Our results show that human COs recapitulate the primary pathological changes of TBI, including metabolic alterations related to neuronal damage, neuronal loss, and astrogliosis. This novel approach using human COs to model TBI in vitro holds great potential and opens new alternatives for understanding brain abnormalities produced by TBI, and for the development and testing of new therapeutic approaches.

Stimulation of Treg Cells to Inhibit Osteoclastogenesis in Gorham-Stout Disease.

Gorham-Stout disease (GSD) is a very rare syndrome displaying excessive bone erosion and vascular lesion. Due to the rarity of the disease and to the limited studies, its etiopathogenesis is not entirely known. The involvement of immune system in the progressive osteolysis was recently suggested. Indeed, extensive reciprocal interactions between the immune and skeletal systems have been demonstrated. This study aimed to evaluate alterations of immune cells in GSD. An increase of CD8+ cells and reduction of CD4+ and CD4+CD25+CD127low cells was revealed in patients. Interestingly, patients’ regulatory T cells maintain the ability to respond to extracellular stimuli and to regulate osteoclastogenesis; GSD cells proliferate under aCD3/CD28 signal reaching similar levels to those observed in control culture and exert their immunomodulatory activity on effector T cells. GSD Treg cells preserved their inhibitory effects on the osteoclastogenesis. These results suggest that stimulation of Treg cells could open the way for the identification and testing of new therapeutic approaches for patients affected by GSD.

In vivo Imaging With 18F-FDG- and 18F-Florbetaben-PET/MRI Detects Pathological Changes in the Brain of the Commonly Used 5XFAD Mouse Model of Alzheimer's Disease.

Imaging biomarkers of Alzheimer's disease (AD) that are able to detect molecular changes in vivo and transgenic animal models mimicking AD pathologies are essential for the evaluation of new therapeutic strategies. Positron-emission tomography (PET) using either 18F-Fluorodeoxyglucose (18F-FDG) or amyloid-tracers is a well-established, non-invasive tool in the clinical diagnostics of AD assessing two major pathological hallmarks. 18F-FDG-PET is able to detect early changes in cerebral glucose metabolism and amyloid-PET shows cerebral amyloid load. However, the suitability of 18F-FDG- and amyloid-PET in the widely used 5XFAD mouse model of AD is unclear as only a few studies on the use of PET biomarkers are available showing some conflicting results. The aim of this study was the evaluation of 18F-FDG-PET and amyloid-PET in 5XFAD mice in comparison to neurological deficits and neuropathological changes. Seven- and 12-month-old male 5XFAD mice showed a significant reduction in brain glucose metabolism in 18F-FDG-PET and amyloid-PET with 18F-Florbetaben demonstrated an increased cerebral amyloid deposition (n = 4–6 per group). Deficits in spatial reference memory were detected in 12-month-old 5XFAD mice in the Morris Water Maze (n = 10–12 per group). Furthermore, an increased plaque load and gliosis could be proven immunohistochemically in 5XFAD mice (n = 4–6 per group). PET biomarkers 18F-FDG and 18F-Florbetaben detected cerebral hypometabolism and increased plaque load even before the onset of severe memory deficits. Therefore, the 5XFAD mouse model of AD is well-suited for in vivo monitoring of AD pathologies and longitudinal testing of new therapeutic approaches.

Bidirectional gut-to-brain and brain-to-gut propagation of synucleinopathy in non-human primates.

In Parkinson's disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the CNS. Here, we compare, in baboon monkeys, the pathological consequences of either intrastriatal or enteric injection of α-synuclein-containing Lewy body extracts from patients with Parkinson's disease. This study shows that patient-derived α-synuclein aggregates are able to induce nigrostriatal lesions and enteric nervous system pathology after either enteric or striatal injection in a non-human primate model. This finding suggests that the progression of α-synuclein pathology might be either caudo-rostral or rostro-caudal, varying between patients and disease subtypes. In addition, we report that α-synuclein pathological lesions were not found in the vagal nerve in our experimental setting. This study does not support the hypothesis of a transmission of α-synuclein pathology through the vagus nerve and the dorsal motor nucleus of the vagus. Instead, our results suggest a possible systemic mechanism in which the general circulation would act as a route for long-distance bidirectional transmission of endogenous α-synuclein between the enteric and the central nervous systems. Taken together, our study provides invaluable primate data exploring the role of the gut-brain axis in the initiation and propagation of Parkinson's disease pathology and should open the door to the development and testing of new therapeutic approaches aimed at interfering with the development of sporadic Parkinson's disease.

A way to understand idiopathic senescence and apoptosis in primary glioblastoma cells\xa0\u2013 possible approaches to circumvent these phenomena

BackgroundGlioblastoma (GB) is considered one of the most lethal tumors. Extensive research at the molecular level may enable to gain more profound insight into its biology and thus, facilitate development and testing of new therapeutic approaches. Unfortunately, stable glioblastoma cell lines do not reflect highly heterogeneous nature of this tumor, while its primary cultures are difficult to maintain in vitro. We previously reported that senescence is one of the major mechanisms responsible for primary GB cells stabilization failure, to a lesser extent accompanied by apoptosis and mitotic catastrophe-related cell death.MethodsWe made an attempt to circumvent difficulties with glioblastoma primary cultures by testing 3 different approaches aimed to prolong their in vitro maintenance, on a model of 10 patient-derived tumor specimens.ResultsTwo out of ten analyzed GB specimens were successfully stabilized, regardless of culture approach applied. Importantly, cells transduced with immortalizing factors or cultured in neural stem cell-like conditions were still undergoing senescence/apoptosis. Sequential in vivo/in vitro cultivation turned out to be the most effective, however, it only enabled to propagate cells with preserved molecular profile up to 3rd mice transfer. Nevertheless, it was the only method that impeded these phenomena long enough to provide sufficient amount of material for in vitro/in vivo targeted analyses. Interestingly, our data additionally demonstrated that some subpopulations of several stabilized GB cell lines undergo idiopathic senescence, however, it is counterbalanced by simultaneous proliferation of other cell subpopulations.ConclusionsIn the majority of primary glioma cultures, there has to be an imbalance towards apoptosis and senescence, following few weeks of rapid proliferation. Our results indicate that it has to be associated with the mechanisms other than maintenance of glioblastoma stem cells or dependence on proteins controlling cell cycle.

Experimental models and tools to tackle glioblastoma.

ABSTRACTGlioblastoma multiforme (GBM) is one of the deadliest human cancers. Despite increasing knowledge of the genetic and epigenetic changes that underlie tumour initiation and growth, the prognosis for GBM patients remains dismal. Genome analysis has failed to lead to success in the clinic. Fresh approaches are needed that can stimulate new discoveries across all levels: cell-intrinsic mechanisms (transcriptional/epigenetic and metabolic), cell-cell signalling, niche and microenvironment, systemic signals, immune regulation, and tissue-level physical forces. GBMs are inherently extremely challenging: tumour detection occurs too late, and cells infiltrate widely, hiding in quiescent states behind the blood-brain barrier. The complexity of the brain tissue also provides varied and complex microenvironments that direct cancer cell fates. Phenotypic heterogeneity is therefore superimposed onto pervasive genetic heterogeneity. Despite this bleak outlook, there are reasons for optimism. A myriad of complementary, and increasingly sophisticated, experimental approaches can now be used across the research pipeline, from simple reductionist models devised to delineate molecular and cellular mechanisms, to complex animal models required for preclinical testing of new therapeutic approaches. No single model can cover the breadth of unresolved questions. This Review therefore aims to guide investigators in choosing the right model for their question. We also discuss the recent convergence of two key technologies: human stem cell and cancer stem cell culture, as well as CRISPR/Cas tools for precise genome manipulations. New functional genetic approaches in tailored models will likely fuel new discoveries, new target identification and new therapeutic strategies to tackle GBM.

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

Murine model and mechanisms of treatment-induced painful diabetic neuropathy

Diabetes mellitus represents a group of metabolic diseases that are characterized by hyperglycemia caused by either lack of insulin production or a reduced ability to respond to insulin. It is estimated that there were 347 million people worldwide who suffered from diabetes in 2008 and incidence is predicted to double by 2050. Neuropathy is the most common complication of long-term diabetes and approximately 30% of these subjects develop chronic neuropathic pain. A distinct acute, severe form of neuropathic pain, called insulin neuritis or treatment-induced painful neuropathy of diabetes (TIND), may also occur shortly after initiation of intensive glycemic control, with an incidence rate of up to 10.9%.The pathological mechanisms leading to TIND, which is mostly unresponsive to analgesics, are not yet understood, impeding the development of therapies. Studies to date have been clinical and with limited cohorts of patients. In the current study, we developed chronic and acute insulin-induced neuropathic pain in mice with type 2 insulin-resistant diabetes. Furthermore, we determined that insulin-induced acute allodynia is independent of glycemia levels, can also be induced with Insulin-like Growth Factor 1 (IGF1) and be prevented by inhibition of AKT, providing evidence of an insulin/IGF1 signaling pathway-based mechanism for TIND. This mouse model is useful for the elucidation of mechanisms contributing to TIND and for the testing of new therapeutic approaches to treat TIND.

Role and Mechanisms of Rhoa G17V in the Pathogenesis of AITL

Peripheral T cell lymphomas (PTCL) are a heterogeneous group of lymphoid tumors resulting from the malignant transformation of mature T-cells. Among these, angioimmunoblastic T-cell lymphoma (AITL), which accounts for almost 20% of PTCL cases, represents one of the most aggressive forms of non-Hodgkin lymphoma with a dismal survival rate of 30% at 5 years. AITL tumors are characterized by loss of function mutations in the Ten-Eleven Translocation 2 (TET2) epigenetic tumor suppressor and a highly recurrent mutation (p.Gly17Val, G17V) in the RHOA small GTPase gene, present in almost 70% of AITL cases. Immunohistochemical analyses and gene expression profiling studies have established follicular T helper cells (Tfh), a as the cell of origin of AITL. Tfh cells are a CD4+ T-cell population characterized by high levels of expression of the chemokine receptor CXCR5, the inhibitory receptor PD-1 and the transcription factor BCL6. To investigate the specific role and mechanisms of RHOA G17V in AITL we first generated a conditional inducible Rhoa G17V knock-in mouse by introducing a floxed Rhoa wild type minigene upstream of the Rhoa G17V mutant exon in the endogenous RhoA locus by homologous recombination. RhoA G17V conditional knockin mice express wild type RhoA but switch to express the mutant G17V allele upon expression of Cre recombinase. Crossing this line with a CD4CreERT2 strain, which expresses a tamoxifen-inducible form of Cre, results in tamoxifen inducible selective activation of Rhoa G17V specifically in CD4 T-cells. Given the close association of the RHOA G17V mutation with AITL we hypothesized that expression of mutant Rhoa G17V could promote Tfh cell polarization and transformation, explaining the high prevalence of this mutation in AITL cases. Notably, tamoxifen-induced expression of Rhoa G17V in CD4CreERT2 RhoA G17V knockin mice resulted in spontaneous accumulation of Tfh cells expressing Cxcr5, Pd-1 and Bcl6 in absence of immunization. Mechanistically, RhoA G17V Tfh cells expressed increased levels of the ICOS receptor, a major factor in Tfh development, and showed enhanced ICOS signaling. Moreover, suppression of ICOS signaling with an anti-ICOSL blocking antibody impaired Rhoa G17V-induced Tfh polarization. Next, we analyzed the specific interaction of RHOA G17V and loss of Tet2 in AITL. Expression of mutant RHOA G17V in hematopoietic progenitors from Tet2 knockout mice resulted in the specific development of AITL with a median latency of 14 months. Tet2-/- RHOA G17V tumors are clonal and transplantable and present typical AITL Tfh-like features including expression of CXCR5, PD-1, BCL6 and ICOS, and are critically dependent on ICOS signaling for growth and proliferation in vivo. In all, our results demonstrate an instructive role for the RHOA G17Vmutation in Tfh cell fate specification and in AITL transformation and support an oncogenic role for ICOS signaling in the pathogenesis of AITL. The AITL animal model reported here may facilitate the preclinical testing of new therapeutic approaches for the treatment of this disease. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1461: A panel of patient derived gastrointestinal stromal tumors (GIST) xenograft models for in vivo preclinical drug testing

Abstract Objective: Most GIST are dependent on the KIT/PDGFRA signaling pathway and therefore can be treated with specific tyrosine kinase inhibitors (TKI). With time GIST develop resistance to TKI. We need reliable models which can be used for preclinical drug testing. Methods: Patient-derived xenografts were established in nu/nu NMRI mice by subcutaneous implantation of fresh, surgically resected tumor specimens from consenting patients with GIST treated at the University Hospitals Leuven. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of mice. At each passage tumor fragments were collected for histological and molecular characterization. A model was considered to be established after observing stable histological and molecular features for at least two passages. Results: At present we have access to five successfully established GIST models, with different KIT mutations and different sensitivity to standard TKI (Table). Untreated tumors from subsequent passages show morphological and immunohistochemical features resembling the original patient biopsy, verifying the stability of GIST xenografts growth. KIT mutational status has also been confirmed in samples obtained from all models. These models are used for pre-clinical testing of new therapeutic approaches, and current results served as a rationale for at least four prospective clinical trials. If needed, we parallel our patient-derived models with in vitro or in vivo studies using one of the few established GIST cell lines. Conclusion: Our established GIST xenografts maintain the histological and molecular characteristics of the original patient sample and are a very useful and reliable preclinical platform for testing the new targeted approaches in GIST. The platform is available for collaboration with academic and commercial partners (contact patrick.schoffski@uzleuven.be). The overview of patients-derived GIST xenograftsModel*KIT mutationIn vivo efficacy [% of relative tumor volume after three weeks]PrimarySecondaryImatinib (50mg/kg, BID)Sunitinib (40mg/kg, QD)UZLX-GIST1p.V560Dn/a2020UZLX-GIST2p.A502_Y503dupn/a17080UZLX-GIST3p.W557_V559delinsFn/a2525UZLX-GIST4p.K558_G565delinsRn/a4520UZLX-GIST9p.P577delp.W557LfsX5, p.D820G250170*models which showed tumor growth for at least two passages; n/a-not applicable Citation Format: Agnieszka Wozniak, Thomas Van Looy, Giuseppe Floris, Yemarshet K. Gebreyohannes, Jasmien Wellens, Haifu Li, Jasmien Cornillie, Ulla Vanleeuw, Daphne Hompes, Marguerite Stas, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. A panel of patient derived gastrointestinal stromal tumors (GIST) xenograft models for in vivo preclinical drug testing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1461. doi:10.1158/1538-7445.AM2015-1461

A Panel of Gastrointestinal Stromal Tumours (Gist) Xenograft Models for in Vivo Preclinical Drug Testing

ABSTRACT Aim: Most GIST are dependent on the KIT/PDGFRA signalling pathway and therefore can be treated with specific tyrosine kinase inhibitors (TKI). With time GIST develop resistance to the TKI. We need reliable models which can be used for preclinical drug testing. Methods: Patient-derived xenografts were established in nu/nu NMRI mice by subcutaneous implantation of fresh, surgically resected tumour specimens from consenting patients with GIST treated at the University Hospitals Leuven. Once tumour growth was observed, pieces of tumour were re-transplanted to next generations of mice. At each passage tumour fragments were collected for histological and molecular characterization. A model was considered to be established after observing stable histological and molecular features for at least two passages. Results: At present we have access to five successfully established GIST models, with different KIT mutations and different sensitivity to standard TKIs (Table). Established GIST xenograft models and their sensitivity to standard treatment. In vivo efficacy was assessed by % of relative tumour volume after 3 weeks under either imatinib (50mg/kg BID) or sunitinib (40mg/kg QD) Model* KIT mutation In vivo efficacy Imatinib Sunitinib UZLX-GIST1 p.V560D 20 20 UZLX-GIST2 p.A502_Y503dup 170 80 UZLX-GIST3 p.W557_V559delinsF 25 25 UZLX-GIST4 p.K558_G565delinsR 45 20 UZLX-GIST9 p.P577del; p.W557LfsX5; p.D820G 250 170 *models which showed tumor growth for at least two passages Untreated tumours from subsequent passages show morphological and immunohistochemical features resembling the original patient biopsy, verifying the stability GIST xenografts growth. KIT mutational status has also been confirmed in samples obtained from all models. These models are used for subsequent testing of new therapeutic approaches and current results have had an impact on at least four prospective clinical trials. If needed, we parallel our patient-derived models with in vitro or in vivo studies using one of the few established GIST cell lines. Conclusions: Our established GIST xenografts maintain the histological and molecular characteristics of the original patient sample and are a very useful and reliable preclinical platform for testing the new targeted approaches in GIST. Disclosure: All authors have declared no conflicts of interest.

An Innovative Humanized Mouse Model of Multiple Myeloma (MM) for Studying MM in Its Natural Environment Using Multi-Color Light Sheet Fluorescence Microscopy (LSFM)

AbstractAbstract 1809Various imaging platforms are well established in hematology research. Nevertheless, the three-dimensional architecture of the bone marrow and tumor growth within this microenvironment remain largely uncharacterized. To date the major hindrance to microscopically image tumor engraftment and the immune response in the bone marrow on a single cell level is the compact structure of the bone that is almost impossible to image through.Therefore, we developed a novel bioluminescent mouse model that recapitulates the clinical characteristics of MM using the new human UMM3 cell line (CD38+, CD56+, CD138+, CD19−, CD20−), from the pleural effusion of a patient with an IgG lambda myeloma (ISS stage I) as well as the well-characterized RPMI8226 cell line transduced to express eGFP and firefly luciferase (UMM3eGFPluc and RPMIeGFPluc). 1×106 MM cells were injected intravenously into NOD.Cg-Prkdcscid IL2rg (NSG) mice and disease progression and bone marrow (BM) engraftment were monitored twice weekly by in vivo bioluminescence imaging. Both cell lines homed to the BM compartment, reflecting MM pathophysiology. Histological analysis confirmed BM engraftment and showed multiple osteolytic lesions for both UMM3 and RPMI cells.Since we were interested in imaging the interactions between human MM cells and the bone marrow microenvironment on a single cell level, we employed the multi-color LSFM after decalcification, specific deep-tissue antibody staining and clearing of the bone structures. With this innovative microscopy technique, we were able to establish a novel tool to display tumor cell engraftment in the bone marrow compartment in three dimensions through the intact bone.We recorded 1500 optical sections for three individual channels each (488, 532, and 647 nm) with an increment of 5μm which allows scanning the whole bone marrow compartment of the sternum within minutes in single-cell resolution. Using higher magnification enabled us to even visualize subcellular components within the bone marrow.Moreover, tissue autofluorescene, recorded mainly in the 488 nm channel, displayed detailed microanatomical structures which allowed for the localization of individual cells within their anatomical context. We could establish protocols for various fluorophore-coupled antibodies and successfully stained CD138+ cells in relation to CD3+ cells and to the microenvironment in the bone marrow. The CD138-positive cells infiltrated the bone marrow in a number of small clusters and comprising about 15% of cellular elements in total. Ex vivo bioluminescence imaging of the sternum from UMM3 tumor-bearing mice revealed massive infiltration of luciferase-expressing cells into the bone marrow compartment. This could also be confirmed by flow cytometrical analysis of bone marrow cells which showed eGFP+hCD138+ cells.We have successfully introduced a novel technique to study MM cell engraftment and progression in a humanized mouse model. We were able to track the tumor cells both in the living animal by in vivo bioluminescence imaging and on single-cell resolution by multi-color LSFM within the intact bone. Our model may lead to better insights into the pathogenesis of MM and could serve as a model for preclinical testing of new therapeutic approaches for the treatment of MM patients. Disclosures:No relevant conflicts of interest to declare.

Targeting the lateral but not the third ventricle induces bone loss in ewe

Osteoporosis is a chronic disease characterized by bone loss and increased skeletal fragility. Large animal models are required for preclinical testing of new therapeutic approaches. We have recently demonstrated that continuous intracerebroventricular (ICV) application of leptin into the lateral ventricle (LV) induces bone loss in ewe. On the basis of these findings, we reasoned that the third ventricle (TV) is an even better target because of its closer location to the hypothalamus that mediates leptin effects on bone. Corriedale sheep were randomly mixed to four groups of four ewe each: control entire (control), ovarectomy plus ICV application of cerebrospinal fluid (OVX), OVX plus ICV application of leptin into the LV (leptin-LV); and ICV application of leptin into the TV (leptin-TV). After 3 months, histomorphometric characterization and bone turnover parameters were analyzed. Highly significant loss of trabecular bone was observed only in leptin-LV group. Increased osteoclast indices and urinary cross-lap excretion were observed in OVX and leptin-TV group. In contrast, serum parameters of osteoblast activity were only significantly decreased in leptin-LV group. Autopsy of ewe brain showed fibrosis around the stainless steel cannula in leptin-TV group. ICV application of leptin into the LV strongly reduces bone formation and leads to a highly significant trabecular bone loss in ewe. In contrast, ICV application of leptin into the TV is technically more demanding and results are unpredictable, because the required use of stainless steel cannula induces peri-implant fibrosis that might prevent leptin to enter the cerebrospinal fluid.

Modeling HIV-associated neurocognitive disorders in mice: new approaches in the changing face of HIV neuropathogenesis

It is well established that infection with the human immunodeficiency virus (HIV) leads to immune suppression. Less well known is the fact that long-term, progressive HIV disease is associated with the development of cognitive deficits. Since the introduction of combined antiretroviral therapy (cART), the clinical presentation of HIV infection has evolved into a chronic illness with very low levels of viral replication and chronic immune activation, with compliant affected individuals surviving for decades with a high quality of life. Despite these advances, many HIV-infected individuals develop some degree of neurodegeneration and cognitive impairment. The underlying pathophysiological mechanisms are not well understood, and there are no effective treatments. Thus, there is an unmet need for animal models that enable the study of HIV-associated neurocognitive disorders (HAND) and the testing of new therapeutic approaches to combat them. Here, we review the pros and cons of existing mouse models of HIV infection for addressing these aims and propose a detailed strategy for developing a new mouse model of HIV infection.

Experimental Model of Large Pulmonary Embolism Employing Controlled Release of Subacute Caval Thrombus in Swine

Purpose To develop a catheter-based model of large pulmonary embolism (PE) in swine based on in situ venous thrombus formation. Materials and Methods Ten Yorkshire swine underwent transjugular implantation of a retrievable inferior vena cava (IVC) filter. A thrombin and collagen mixture was injected into a confined space created by two balloons inflated proximal and distal to the IVC filter. Animals were left to survive for 7 days ± 3 to allow thrombus to organize in situ. The caval thrombus was released on transcatheter retrieval of the IVC filter and embolized into the main and branch pulmonary arteries. The severity of PE was scored based on digital subtraction angiography with the Miller index. At necropsy, thrombi were recovered and analyzed histopathologically. Results Large PE was induced in all animals (Miller index score of 15 ± 5). Two animals developed saddle embolus with bilateral pulmonary artery occlusion, and five developed proximal occlusion of the left or right pulmonary artery. Nevertheless, no animal exhibited significant hemodynamic compromise. Large tubular thrombi were explanted in the size range of 5–10 cm long and 0.5–1 cm wide. Histologic analysis indicated an organized thrombus with infiltration of white blood cells and fibrin deposition. Conclusions Large caval thrombi can be formed in vivo and released at a predetermined time to induce large PE in a large animal model. This may help in the development and testing of new therapeutic approaches for PE.

A Unique New Humanized Mouse Model for Multiple Myeloma (MM): Opportunities for Studying MM In Its Natural Environment and for Preclinical Testing

AbstractAbstract 981Here we report the development of a unique mouse model to study the pathobiology of multiple myeloma (MM) by implementing a technology for creating a natural human bone environment in the immune deficient RAG2γ-/-c-/- mouse. To this end we combined a procedure to culture-expand human BM-derived mesenchymal stromal cells (MSC) that were seeded on biphasic calcium phosphate (BCP) particles and subsequently implanted subcutaneously in RAG2γ-/-c-/- mice. Within 6 weeks this leads to the formation of so-called ossicles that contain substantial amounts of human bone, while the remaining open spaces are filled with mouse hematopoietic cells and blood vessels, creating an environment that strongly resembles the human bone marrow. A striking finding was that this humanized environment in the mouse acts as a 3-D natural “niche” for patient MM (pMM) cells. Intrascaffold injection of pMM cells resulted in engraftment and outgrowth of tumor cells in close contact with the human bone layer in the ossicles. In addition, intracardial injection, revealed that these primary tumor cells were also capable of homing to the implanted artificial BM-niches, while no tumor cells were detected in the mouse BM. The outgrowth of pMM in this model is accompanied by an increase in osteoclast number on the bone surface, indicating the presence of bone resorption, one of the most important clinical sequelae of MM. Interestingly, by gene-marking pMM cells with luciferase and using bioluminescent imaging, we were able to follow myeloma outgrowth in time, and visualize the effect of treatment. Hence, this novel humanized mouse model provides the first opportunity to investigate patient-derived MM plasma cells in their natural environment, which may lead to better insights in the pathogenesis of this disease. Furthermore, it could serve as a model for preclinical testing of new therapeutic approaches for the treatment of MM patients. Disclosures:de Weers:Genmab bv: Employment.

The Humanized Multiple Myeloma Mouse Model: Opportunities for Studying the Pathogenesis of MM in Its Natural Environment.

Abstract 1847Poster Board I-873Multiple myeloma (MM), one of the most common hematological malignancies in adults, is a neoplasm of terminally differentiated B cells, i.e. plasma cells. The transition of a plasma cell to a fully transformed, aggressive myeloma is a multistep process, which requires the acquisition of mutations in multiple genes. Most of this evolution takes place in the bone marrow (BM). Studying the pathogenesis of MM is seriously hampered by the lack of appropriate conditions for the engraftment of patient derived MM cells (pMM) which, unlike MM cell lines, strongly depend on a human microenvironment to engraft, survive and expand, indicating that the interaction of MM cells with the cellular and extracellular components of the human BM microenvironment plays a crucial role in the growth behavior of MM cells. To date only few animal models are described that facilitate the outgrowth of pMM, however, they depend on the co-implantation of human fetal tissues or prior implantation of rabbit bone. Moreover, these do not represent the human adult BM microenvironment in which MM cells normally exist (e.g. in the SCID-Rab model and in the NOD-SCID mouse model) or there are ethical concerns (e.g. the SCID-Hu model).Here we report the development of a unique mouse model to study the pathobiology of MM by implementing a technology for creating a natural human bone environment in the immune deficient RAG2−/−γc−/− mouse. To this end we combined a procedure to culture-expand human BM-derived mesenchymal stromal cells (MSC) that were seeded on biphasic calcium phosphate (BCP) particles and subsequently implanted subcutaneously in RAG2−/−γc−/− mice. Within 6 to 8 weeks this leads to the formation of so-called ossicles that contain substantial amounts of human bone, while the remaining open structures are filled with hematopoietic cells (of mouse origin) at various stages of erythroid, megakaryocytic and myeloid differentiation, creating an environment that strongly resembles the human bone marrow. A stricking finding was that this humanized environment in the mouse proved to be capable to not only support human GFP-luciferase gene-marked MM cell lines but also pMM cells. Intravenous or intracardial injection of MM cell lines or pMM cells showed homing to, engraftment and outgrowth of tumor cells in connection with the human bone layer in the ossicles. Hence, this novel humanized mouse model will provide the first opportunity to investigate patient derived MM plasma cells in their natural environment which may lead to better insights in the pathogenesis of this disease and serve as a model for preclinical testing of new therapeutic approaches for the treatment of Multiple Myeloma patients. Disclosures:de Weers:Genmab: Employment.

Transgenic model of cardiac rhabdomyosarcoma formation

Cardiac rhabdomyosarcomas are rare, and the pathogenesis of this detrimental disease is widely unknown. Most data are obtained from case reports or small series, and models for systematic pathogenetic studies are lacking. We aimed to establish a transgenic mouse model of cardiac rhabdomyosarcoma formation. Standard techniques were used to construct a minigene comprised of the 5' region of the 1.4-kb SM22alpha gene (expressed in embryonic cardiac muscle) and the 2.7-kb SV40 T antigen early region. This T antigen fragment includes the coding sequences for the binding sites of p53 and the proteins of the pRb family. Genotyping of transgenic mice was performed by means of polymerase chain reaction, and phenotypic expression was evaluated by means of immunohistochemistry. Transgenic mice were studied at the age of approximately 8 to 12 weeks. Cardiac tumors were found of variable size in the left or right sides of the heart and were associated with T antigen expression. Histologic analysis revealed a 3.1-fold enhanced cell density, enlarged cell nuclei, and a 3.4-fold enhanced DNA content. Phenotypic characterization of cardiac tumors resulted in positive staining for desmin, smooth muscle alpha-actin, troponin C, and Myo D1, which met the criteria for rhabdomyosarcomas. To the best of our knowledge, the present study is the first description of a mouse model of cardiac rhabdomyosarcoma formation based on genetic modulation. Our model will be a valuable tool for illuminating the pathogenesis of cardiac rhabdomyosarcomas and will allow the testing of new therapeutic approaches to fight this dreadful disease.

Conditionally inducible tau mice – designing a better mouse model of neurodegenerative diseases

Neuronal cell death underlies the majority of age-related human neurodegenerative disorders that culminate with salient and severe cognitive decline affecting patients' quality of life, identity and eventually leading to death. The identification of disease-causing genes in familial forms of neurodegenerative diseases enabled the development of genetic models closely replicating pathologies found in human central nervous system. These models dramatically precipitated our understanding of molecular events leading to neuronal death in many neurodegenerative disorders. Today's large range of cellular and animal models generate rapidly accumulating biochemical and neuropathological data on changes induced by mutated or dysfunctional proteins implicated in neuronal loss. Most of these models are complementary, although all have intrinsic limitations as well as specific advantages. Development of conditional transgenic mouse models in which a deleterious effect of a transgene can be regulated in a controlled way created new possibilities of addressing the basic mechanisms of neurodegeneration and provided a new angle for the development and testing of new therapeutic approaches.