Evaluation Of Novel Therapeutic Agents Research Articles

Role of Animal Models in Parkinson's Disease (PD): What Role They Play in Preclinical Translational Research.

Animal models for drug discovery and development in Parkinson 's disease have played an important role in the characterization of the pathophysiology of diseases and associated mechanisms of injury, drug target identification, and evaluation of novel therapeutic agents for toxicity/ safety, pharmacokinetics, pharmacodynamics, and efficacy. The review is intended to reform the scope, advantages, and limitations of various Parkinson's Disease models and their scope in translational research. The lack of a gold standard for PD animal models presents a major challenge in devising a validation system. This review is an attempt to provide a way to adopt the validation approach for PD animal model for research. Because underlying disease mechanisms are so similar across species, it is possible to extrapolate results from Parkinson's disease studies using animal models. Furthermore, behavioural tests used to access the neurobehavioral test with its limitations were explored for rodents, non-human primates, lower-order animals, and invertebrates. The role of gender selectivity and non-selectivity is the one major concern in PD model validation that is addressed in the review. The rigorous validation has been done on animal models for Parkinson's disease (PD) based on comparisons to the human state. Regarding toxicological and safety investigations in PD, non-animal options must be thoroughly validated. There are both advantages and disadvantages to using animal models of Parkinson's disease as proof-of-concept research. The specific animal model selected for a given drug to be tested and developed depends on the goal of the specific study.

Experimental animal models for gastric ulcer / peptic ulcer: An overview

In the present study we have discussed around sixteen different animal models used worldwide for the scientific research and new drug discovery. The main aim of the using experimental animal models in drug discovery is to establish and provide evidence for non-clinical 'proof-of-concept' for the safety, efficacy, and target of interest for specific drug molecules. Experimental preparations developed in one species for the purpose of studying phenomena occurring in another species. The use of experimental animal models serves to better understand the origins, pathology, and the overall nature of comparable diseases of humans being. Similarly, animal models perform duties for in the development of safe and effective treatments and cures of such diseases and/or associated symptoms. Experimental animal models for drug discovery and development have played a major role in the characterization of the pathophysiology of diseases and associated mechanisms of injury, drug target identification, and evaluation of novel therapeutic agents for toxicity, pharmacokinetics and pharmacodynamics activity. Through animal model researchers can perform experiments that would be impractical or ethically prohibited with humans. There are various animal models used for screening of uncountable therapeutic activities, in this review our main focus is animal models used for peptic ulcer. Peptic ulcer is one of the worldwide diseases where 10% of adults are affected by peptic ulcer once in their lifetime. The antiulcer models for drug development against gastric and duodenal ulcer studies are limited in number that has hindered the progress of targeted therapy in this field. Therefore, it is necessary to review the literature on experimental animal models that are used to screen agents with potential anti-gastric ulcer activity and describe their biochemical basis in order to facilitate their use in the development of new preventive and curative antiulcer drugs. There are many models used to induce ulcer such as pylorus ligation or it can be chemically induced by ethanol, NSAIDs (e.g. indomethacin) or many more. In this review paper, current in-vivo animal models of ulcers and the pathophysiological mechanisms underlying their induction, their drawbacks, as well as the challenges associated with their use have been discussed.

Unilateral Nephrectomized SHR/NDmcr-cp Rat Shows a Progressive Decline of Glomerular Filtration With Tubular Interstitial Lesions

In patients with diabetic kidney disease (DKD), the estimated glomerular filtration rate (eGFR) or creatinine clearance rate (Ccr) is always used as an index of decline in renal function. However, there are few animal models of DKD that could be used to evaluate renal function based on GFR or Ccr. For this reason, it is desirable to develop animal models to assess renal function, which could also be used for the evaluation of novel therapeutic agents for DKD. Therefore, we aimed to develop such animal model of DKD by using spontaneously hypertensive rat (SHR)/NDmcr-cp (cp/cp) rats with the characteristics of obese type 2 diabetes and metabolic syndrome. As a result, we have found that unilateral nephrectomy (UNx) caused a chronic Ccr decline, development of glomerular sclerosis, tubular lesions, and tubulointerstitial fibrosis, accompanied by renal anemia. Moreover, losartan-mixed diet suppressed the Ccr decline in UNx-performed SHR/NDmcr-cp rats (UNx-SHR/cp rats), with improvement in renal anemia and histopathological changes. These results suggest that UNx-SHR/cp rats could be used as a DKD model for evaluating the efficacy of therapeutic agents based on suppression of renal function decline.

Salt loading with unilateral nephrectomy accelerates decline in glomerular filtration rate in the hypertensive, obese, type 2 diabetic SDT fatty rat model of diabetic kidney disease.

For the evaluation of novel therapeutic agents for diabetic kidney disease (DKD), it is desirable to examine their efficacy in animal models by using the glomerular filtration rate (GFR) as an index. For this purpose, animal models that demonstrate a short-term GFR decline because of disease progression are required. Therefore, we aimed to develop such an animal model of DKD by using obese type 2 diabetic spontaneously diabetic Torii (SDT) fatty rats treated with salt loading by drinking water containing sodium chloride with or without unilateral nephrectomy. As a result, we have found that 0.3% salt loading with unilateral nephrectomy or 0.8% salt loading alone caused a rapid GFR decline, hypertension and rapid development of tubulointerstitial fibrosis. Moreover, the addition of losartan to a mixed diet suppressed the GFR decline in SDT fatty rats treated with 0.3% salt loading with unilateral nephrectomy. These results suggest that the model of SDT fatty rats treated with 0.3% salt loading and unilateral nephrectomy could be used as a hypertensive DKD model for evaluating therapeutic agents based on suppression of GFR decline.

Abstract 3003: In vitro and in vivo model for evaluation of novel therapeutic agents in patient -derived acute B-lymphoblastic leukemia (B-ALL)

Abstract B-cell Acute Lymphoblastic Leukemia is a hematological malignancy with malignant transformation and proliferation of B-Cell progenitors. Young patients fare well with common therapeutic agents and hematopoietic stem cell transplant, but a subpopulation is chemo-resistant and has high relapse rate. Besides, prognosis in adult patient is worse than younger individuals. Thus, there is an increase need for development of novel and targeted therapeutics. Lack of suitable in vitro culture conditions for primary B-ALL cells severely impairs the evaluation of new therapeutics. We have established a short-term culture system that supports survival of primary B-ALL cells ex vivo for screening of candidate therapeutic agents. Different culture conditions were evaluated, and cell growth/viability was assessed using Cell titer-Glo assay. Out of 13 primary B-ALL models studied 5 models proliferated and 5 remain stable and viable till day 3. Flow cytometry study showed that all the models stably expressed CD19 and CD20 throughout the assay. The models were tested for sensitivity to chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. Heterogeneous dose dependent responses were observed across the models (IC50 17nM - 0.3 µM doxorubicin), (IC50 3 - 79 nM vincristine) indicating a range of relative sensitivity to B-ALL models. Interestingly, BCR-ABL-ve models were resistant to imatinib and BCR-ABL+ve sensitive. Next ,to develop patient derived xenograft preclinical B-ALL model, two million PBMC from B-ALL patients (n=14) were intravenously transplanted into sub-lethally irradiated NCG mice. 100% engraftment(n=5,P1) was observed in all the models, but latency of engraftment(>3%) in peripheral blood varied from 15 days to 3.5 months. The time of passage varied from 40 to 120 days. Flow cytometry analyses showed 30-95% of the bone marrow cells was hCD45+, hCD19+ cells. Splenomegaly was observed and infiltration of 40-98% of B-ALL cells was detected in spleen. Engrafted cells matched the immunophenotype of the corresponding patient. On serial transplantation (n=15) these models showed similar engraftment and immunophenotype. Thus, at Champions we have established a robust in vitro platform for screening new drugs for B-ALL and a passagable patient derived preclinical xenograft model for in vivo drug efficacy study. Citation Format: Bandana Ajay Vishwakarma, Bhavna Verma, Amy Wesa. In vitro and in vivo model for evaluation of novel therapeutic agents in patient -derived acute B-lymphoblastic leukemia (B-ALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3003.

Patient-derived orthotopic xenograft models of medulloblastoma lack a functional blood-brain barrier.

Novel targeted therapies for children diagnosed with medulloblastoma (MB), the most common malignant pediatric brain tumor, are urgently required. A major hurdle in the development of effective therapies is the impaired delivery of systemic therapies to tumor cells due to a specialized endothelial blood-brain barrier (BBB). Accordingly, the integrity of the BBB is an essential consideration in any preclinical model used for assessing novel therapeutics. This study sought to assess the functional integrity of the BBB in several preclinical mouse models of MB. Dynamic contrast enhancement magnetic resonance imaging (MRI) was used to evaluate blood-brain-tumor barrier (BBTB) permeability in a murine genetically engineered mouse model (GEMM) of Sonic Hedgehog (SHH) MB, patient-derived orthotopic xenograft models of MB (SHH and Gp3), and orthotopic transplantation of GEMM tumor cells, enabling a comparison of the direct effects of transplantation on the integrity of the BBTB. Immunofluorescence analysis was performed to compare the structural and subcellular features of tumor-associated vasculature in all models. Contrast enhancement was observed in all transplantation models of MB. No contrast enhancement was observed in the GEMM despite significant tumor burden. Cellular analysis of BBTB integrity revealed aberrancies in all transplantation models, correlating to the varying levels of BBTB permeability observed by MRI in these models. These results highlight functional differences in the integrity of the BBTB and tumor vessel phenotype between commonly utilized preclinical models of MB, with important implications for the preclinical evaluation of novel therapeutic agents for MB.

Establishment of a Patient-Derived Acute B Lymphoblastic Leukemia (B-ALL)Ex Vivo and In Vivoplatform for Evaluation of Novel Therapeutic Agents

B-cell Acute Lymphoblastic Leukemia (B-ALL)is a hematological malignancy with malignant transformation and proliferation of B-Cell progenitors in bone marrow, blood and extramedullary sites. It is more prevalent in children and adolescent and is one of the top leading cause of death related to malignancies in this population. Majority of young patients fare well with common therapeutic agents and hematopoietic stem cell transplant, but a subpopulation is chemo-resistant and has a high relapse rate. Besides, prognosis in adult B-ALL patient is worse than younger individuals. Thus, there is an increasing need for development of novel and targeted therapeutic strategies to improve outcome in elderly and resistant individual. Lack of suitablein vitroculture conditions for primary B-ALL cells severely impairs their experimental accessibility and evaluation of new therapeutics. To corroborate theex vivostudies in a more natural microenvironment there is an unmet necessity of a robust preclinical patient derived xenograft model for B-ALL. We have established and optimized a short-term culture system that supports survival of primary B-ALL cellsex vivofor evaluation and/or screening of candidate agents. Our B-ALL bank comprise of patient-derived specimens with complete cytogenetics information including Philadelphia translocation positive and negative samples and surface marker expression. Different medias, supplements and culture conditions were evaluated to select conditions that support the survival and maintenance of primary B-ALL specimens. Cell growth/viability was assessed using Cell titer-Glo assay. Out of 13 primary B-ALL models studied 5 models proliferated in the optimized media and 5 remain stable and viable till day 3. Phenotypic characterization by flow cytometry showed that all the models stably expressed CD19 and CD20 throughout the assay. To verify the applicability of the 3-day ex vivo system for drug testing, models were tested for sensitivity towards chemotherapeutic drugs doxorubicin, vincristine, imatinib and cytarabine. Heterogeneous dose dependent responses were observed across the models (IC50 17nM to 0.3 µM doxorubicin), (IC50 3 nM to 79 nM vincristine) indicating a range of relative sensitivity to B-ALL models. Interestingly, BCR-ABL-ve models were resistant to imatinib and BCR-ABL+ve sensitive, when treated with small molecule inhibitor imatinib. Next, to develop patient derived xenograft preclinical B-ALL models for drug efficacy study , sub lethally irradiated NCG mice were transplanted intravenously with two million PBMC from B-ALL patients(n=14). 100% engraftment(n=5/model P1) was observed in all the models, but the latency of engraftment(>3%) in peripheral blood varied from 15 days to 3.5 months depending on the aggressiveness of the models. The time of passage varied from 40 to 120 days at P1. Mice were sacrificed when 10-20% of hCD45+ve cells were observed in peripheral blood by flow cytometry. 30-95% of the bone marrow was occupied by hCD45+, hCD19+ cells. Splenomegaly was observed and infiltration of 40-98% of B-ALL cells was detected in spleen. Engrafted cells were lymphoid-restricted and matched the immunophenotype of the corresponding patient. On serial transplantation in secondary NCG mice (n=15/model P2,P3), these models showed similar engraftment pattern and immunophenotype characteristic. Thus , here we show that theex vivoculture condition optimized at Champions supports growth and proliferation of patient derived B-ALL specimen and is a good platform for screening new therapeutic drugs for B-ALL . We also demonstrate that NCG mice supports the engraftment and development of B-ALL disease initiating and leukemic cells in vivo, allowing to create a robust passagable patient derived preclinical xenograft model that recapitulates the disease and probably could be used for drug efficacy study. Disclosures No relevant conflicts of interest to declare.

A Comprehensive Review on Preclinical Diabetic Models.

Preclinical experimental models historically play a critical role in the exploration and characterization of disease pathophysiology. Further, these in-vivo and in-vitro preclinical experiments help in target identification, evaluation of novel therapeutic agents and validation of treatments. Diabetes mellitus (DM) is a multifaceted metabolic disorder of multidimensional aetiologies with the cardinal feature of chronic hyperglycemia. To avoid or minimize late complications of diabetes and related costs, primary prevention and early treatment are therefore necessary. Due to its chronic manifestations, new treatment strategies need to be developed, because of the limited effectiveness of the current therapies. The study included electronic databases such as Pubmed, Web of Science and Scopus. The datasets were searched for entries of studies up to June, 2018. A large number of in-vivo and in-vitro models have been presented for evaluating the mechanism of anti-hyperglycaemic effect of drugs in hormone-, chemically-, pathogen-induced animal models of diabetes mellitus. The advantages and limitations of each model have also been addressed in this review. This review encompasses the wide pathophysiological and molecular mechanisms associated with diabetes, particularly focusing on the challenges associated with the evaluation and predictive validation of these models as ideal animal models for preclinical assessments and discovering new drugs and therapeutic agents for translational application in humans. This review may further contribute to discover a novel drug to treat diabetes more efficaciously with minimum or no side effects. Furthermore, it also highlights ongoing research and considers the future perspectives in the field of diabetes.

Isoquinolinamine FX-9 Exhibits Anti-Mitotic Activity in Human and Canine Prostate Carcinoma Cell Lines.

Current therapies are insufficient for metastatic prostate cancer (PCa) in men and dogs. As human castrate-resistant PCa shares several characteristics with the canine disease, comparative evaluation of novel therapeutic agents is of considerable value for both species. Novel isoquinolinamine FX-9 exhibits antiproliferative activity in acute lymphoblastic leukemia cell lines but has not been tested yet on any solid neoplasia type. In this study, FX-9′s mediated effects were characterized on two human (PC-3, LNCaP) and two canine (CT1258, 0846) PCa cell lines, as well as benign solid tissue cells. FX-9 significantly inhibited cell viability and induced apoptosis with concentrations in the low micromolar range. Mediated effects were highly comparable between the PCa cell lines of both species, but less pronounced on non-malignant chondrocytes and fibroblasts. Interestingly, FX-9 exposure also leads to the formation and survival of enlarged multinucleated cells through mitotic slippage. Based on the results, FX-9 acts as an anti-mitotic agent with reduced cytotoxic activity in benign cells. The characterization of FX-9-induced effects on PCa cells provides a basis for in vivo studies with the potential of valuable transferable findings to the benefit of men and dogs.

Abstract 3710: Establishment of a patient-derived acute myeloid leukemia (AML) ex vivo platform for evaluation of novel therapeutic agents

Abstract Acute myelogenous leukemia (AML) is the most common acute leukemia in adults. A hematologic cancer, the disease is highly heterogeneous, with multiple subtypes. Despite advances in treatment, the long-term survival for AML remains poor and the development of novel treatments is an unmet need. Due to the highly divergent subtypes and mutation profiles in AML, the use of patient-derived models may improve drug discovery and development. To address this, we have established a short-term culture system that supports the growth of primary AML cells ex vivo to permit the evaluation and/or screening of candidate agents. Our AML bank is comprised of patient-derived specimens across a range of subtypes (which includes M1, M2, M4, M5, and others), and includes models with common mutations in FLT3 (ITD), IDH1/IDH2 and NPM. Primary AML specimens were characterized for common mutations by TruSight sequencing and for surface marker expression by flow cytometry. The ex vivo assay system was evaluated for the ability to support the survival and expansion of 22 primary AML specimens. Among these, 15 had evidence of proliferation, 5 had no net expansion, and 2 failed to survive. Extension of the culture period for up to 14 days was feasible, with most models having equal or increased cell numbers by the end of the culture period (8 of 10 evaluated). All models stably expressed CD33 throughout the assay. To verify the applicability of this system for drug testing, a standard of care agent cytarabine (ara-C) was assessed for each of the AML models. Cell growth/viability was assessed using Celltiter-Glo assay. Concentration-dependent responses to ara-C were observed across multiple models (IC50 10 nM to 150 nM), indicating a range of relatively sensitive to resistant AML models. A cohort of models were evaluated in vivo for sensitivity to ara-C. Engraftment of the systemic patient-derived xenograft AML models (into NOG or NOG-EXL mice) was evaluated in circulation by flow cytometry. When engrafted, AML-bearing mice were randomized into ara-C or vehicle control groups. Two weeks later, mice were evaluated for the presence of human CD45+CD33+ AML cells. Models that were relatively sensitive to Ara C ex vivo (IC50 < 30 nM) showed a greater in vivo response as evidenced by a 60-80% reduction in the mean circulating AML cells versus models with >100 nM IC50 values, that had little response. In conclusion, Champions offers over 30 well characterized models of AML for study in ex vivo culture and in vivo systemic xenograft models. The diversity in these AML models is reflective of patient diversity, enhancing their utility in the evaluation of novel therapeutic candidates. These data indicate the feasibility of utilization of these primary models, ex vivo as well as in vivo, for drug discovery for AML, from screening to preclinical efficacy modeling. Citation Format: Bhavna Verma, Swetha Tati, Bruce Ruggeri, Amy Wesa. Establishment of a patient-derived acute myeloid leukemia (AML) ex vivo platform for evaluation of novel therapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3710.

Experimental Diabetes Mellitus in Different Animal Models.

Animal models have historically played a critical role in the exploration and characterization of disease pathophysiology and target identification and in the evaluation of novel therapeutic agents and treatments in vivo. Diabetes mellitus disease, commonly known as diabetes, is a group of metabolic disorders characterized by high blood glucose levels for a prolonged time. To avoid late complications of diabetes and related costs, primary prevention and early treatment are therefore necessary. Due to its chronic symptoms, new treatment strategies need to be developed, because of the limited effectiveness of the current therapies. We overviewed the pathophysiological features of diabetes in relation to its complications in type 1 and type 2 mice along with rat models, including Zucker Diabetic Fatty (ZDF) rats, BB rats, LEW 1AR1/-iddm rats, Goto-Kakizaki rats, chemically induced diabetic models, and Nonobese Diabetic mouse, and Akita mice model. The advantages and disadvantages that these models comprise were also addressed in this review. This paper briefly reviews the wide pathophysiological and molecular mechanisms associated with type 1 and type 2 diabetes, particularly focusing on the challenges associated with the evaluation and predictive validation of these models as ideal animal models for preclinical assessments and discovering new drugs and therapeutic agents for translational application in humans.

Generation of Noninvasive, Quantifiable, Orthotopic Animal Models for NF2-Associated Schwannoma and Meningioma.

Schwannomas and meningiomas are nervous system tumors that can occur sporadically or in patients with neurofibromatosis type 2 (NF2). Mutations of the Neurofibromatosis 2 (NF2) gene are frequently observed in these tumors. Schwannomas and meningiomas cause significant morbidities, and an FDA-approved medical therapy is currently not available. The development of preclinical animal models that accurately capture the clinical characteristics of these tumors will facilitate the evaluation of novel therapeutic agents for the treatment of these tumors, ultimately leading to more productive clinical trials. Here, we describe the generation of luciferase-expressing NF2-deficient schwannoma and meningioma cells and the use of these cells to establish orthotopic tumor models in immunodeficient mice. The growth of these tumors and their response to treatment can be measured effectively by bioluminescence imaging (BLI) and confirmed by small-animal magnetic resonance imaging (MRI). These and other animal models, such as genetically-engineered models, should substantially advance the investigation of promising therapies for schwannomas and meningiomas.

Integration of PKPD relationships into benefit-risk analysis.

Despite the continuous endeavour to achieve high standards in medical care through effectiveness measures, a quantitative framework for the assessment of the benefit-risk balance of new medicines is lacking prior to regulatory approval. The aim of this short review is to summarise the approaches currently available for benefit-risk assessment. In addition, we propose the use of pharmacokinetic-pharmacodynamic (PKPD) modelling as the pharmacological basis for evidence synthesis and evaluation of novel therapeutic agents. A comprehensive literature search has been performed using MESH terms in PubMed, in which articles describing benefit-risk assessment and modelling and simulation were identified. In parallel, a critical review of multi-criteria decision analysis (MCDA) is presented as a tool for characterising a drug's safety and efficacy profile. A definition of benefits and risks has been proposed by the European Medicines Agency (EMA), in which qualitative and quantitative elements are included. However, in spite of the value of MCDA as a quantitative method, decisions about benefit-risk balance continue to rely on subjective expert opinion. By contrast, a model-informed approach offers the opportunity for a more comprehensive evaluation of benefit-risk balance before extensive evidence is generated in clinical practice. Benefit-risk balance should be an integral part of the risk management plan and as such considered before marketing authorisation. Modelling and simulation can be incorporated into MCDA to support the evidence synthesis as well evidence generation taking into account the underlying correlations between favourable and unfavourable effects. In addition, it represents a valuable tool for the optimization of protocol design in effectiveness trials.

Real-time imaging of glioblastoma using bioluminescence in a U-87 MG xenograft model mouse

Glioblastoma multiforme (GBM), the most common malignant brain tumor, is characterized by aggressive proliferation and invasive potential. Xenograft animal models of GBM have critically contributed to evaluation of novel therapeutic agents, drug delivery system, and diagnostic tools. To mimic intrinsic behavior of GBM, orthotopic transplantation of cancer cells and continuous observation of cell growth should be conducted in animal study. Here, we generated xenograft model mouse of GBM in which U-87 MG human glioblastoma cells were intracranially implanted for live imaging. Introducing luciferase gene into U-87 MG cell line enabled real-time observation and quantification of tumor survival and propagation by detecting photon emission derived from luciferase. Our GBM model mouse has potentials to bring great advantages in pharmacological and mechanistic investigation on brain tumors.

Development of a mouse model of osteoarthritis of knee by induction of obesity and bipedal walking

Purpose: Animal models of osteoarthritis(OA) are used extensively in research of its pathogenesis and in search of potential disease modifying anti-OA drugs. However, whether current animal models of OA properly represent human OA is a critical question. In devising an animal model which can be extrapolated into human disease, 2 factors, obesity and bipedal walking inherent in human locomotion, have been under-represented. In this study, we sought to investigate the influence of obligatory bipedal walking on OA development in C57BL6 mice. In addition, by inducing obesity with high fat diet, we observed whether excess body weight acts synergistically with bipedal walking in the development of OA. Methods: Seventy-two 31 week-old C57BL6 mice were divided into 2 groups and one group was fed with 30% fat diet and another group with control diet for 2 months. After induction of obesity, mice from each group were again divided into 2 groups and obligatory bipedal exercise was induced with specially designed treadmill for 1-4 hours daily in each group, resulting in 4 experimental groups of mice (control, control bipedal, obese, obese bipedal). After 8,10 and 12 weeks of bipedal walking, animals are sacrificed. Knee joints were obtained and graded microscopically according to scoring system recommended by OARSI histopathology initiative and modified Mankin score. Serum levels of cartilage oligomeric matrix protein(COMP) and type II collagen degradation product were measured with ELISA. Pain behavior was observed with von Frey fiber test and regularity index measured by catwalk test. Results: High fat diet for 2 months induced significant weight gain in C57BL6 mouse without any obvious signs of physical illness. Typical findings of human OA cartilage, including surface fibrillation, proteoglycan matrix depletion and chondrocyte loss began to appear after 8 weeks of exercise in bipedal groups and progressed as the duration of exercise increased. At 12 weeks, vertical erosion to calficifed cartilage a nd total loss of cartilage typical of mouse OA was observed in none of control and obese groups, and in 66.6 and 100% of control bipedal and obese bipedal animals. Cartilage grading was significantly higher in bipedal groups compared to obese and control groups, and significantly higher in obese bipedal group compared to control bipedal group. OA change in bipedal groups was accompanied by synovitis and subchondroal bone thickening while osteophyte formation was not significantly increased compared to control group. There was no significant difference between obese and control groups. Serum levels of COMP were significantly increased in bipedal groups compared to control or obese group at 8 and 10 weeks, while the level of serum type II collage degradation product was not significantly different between the 4 groups. Threshold for von Frey fiber test decreased significantly in bipedal groups compared to control group while it significantly increased in obese group. Abnormal gait pattern measured by regularity index in catwalk test tended to increase in obese bipedal group compared to other groups. Conclusions: By induction of obesity and bipedal exercise, natural OA mimicking human OA was induced in C57BL7 mouse. The regulation of relevant molecular markers and signaling mechanism during progression of OA in this model would contribute to the understanding of pathogenetic mechanism of human OA and efficient evaluation of novel therapeutic agents.

Measurement of apolipoprotein E and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling

BackgroundAbnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain.ResultsCohorts of mice were pulse labeled with 13 C6-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid β (Aβ). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and Aβ clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of Aβ deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of Aβ, suggesting that ABCA1 does not regulate Aβ metabolism in the brain.ConclusionsOur SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents.

Chemically Detachable Polyelectrolyte Multilayer Platform for Cell Sheet Engineering

Human gingival fibroblasts (HGFs) cell sheets have a potential use for in vivo wound healing due to the ability of HGFs to adopt a contractile phenotype which is typically expressed during extracellular matrix tissue remodeling. For this purpose, we developed a chemically detachable platform based on poly(allylamine hydrochloride)/poly(styrene sulfonate) multilayer film built on a sacrificial precursor film which served as a substrate for HGF cell layer formation. The sacrificial precursor film, based on disulfide-containing polycation and polyanion, is degradable under mild conditions compatible for cell sheet detachment. Cellular viability and cell phenotype analysis of HGF show that the designed platform promotes cell phenotype switch into contractile phenotype, maintained after cell sheet lift-off. This contractile phenotype is acquired by fibroblasts during in vivo wound healing and tissue remodeling. HGFs cell sheet fragments, obtained by this detachment process, could be cultured later on showing a good retention of the typical spindle-shape of differentiated cells after 10 days of culture. HGFs cell sheets have great potential applications as autologous substrates for tissue repair and cellular synthetic platforms for research on connective tissue diseases or evaluation of novel therapeutic agents.

Response Assessment Challenges in Clinical Trials of Gliomas

Accurate, reproducible criteria for determining tumor response and progression after therapy are critical for optimal patient care and effective evaluation of novel therapeutic agents. Currently, the most widely used criteria for determining treatment response in gliomas is based on two-dimensional tumor measurements using neuroimaging studies (Macdonald criteria). In recent years, the limitation of these criteria, which only address the contrast-enhancing component of the tumor, have become increasingly apparent. This review discusses challenges that have emerged in assessing response in patients with gliomas and approaches being introduced to address them.

Synergistic effects of Curcumin and Garcinol on human pancreatic cancer cells

Pancreatic cancer (PaCa) is the fourth leading cause of cancer related death worldwide with a low five year survival rate of <1% due to its aggressive local invasion and early metastases. Thus early detection and evaluation of novel therapeutic agents is crucial for a favorable prognosis. Curcumin and Garcinol from the plants, Curcuma longa and Garcinia indica respectively have proven anti‐inflammatory, antioxidant, and anticarcinogenic activity in several cancer types. The objective of this study was to investigate the synergistic effect of these two chemopreventive dietary agents in PaCa cells. For this, PaCa cells (BxPC‐3 and Panc‐1: K‐ras wild and mutant) were treated with Curcumin (0‐50µM) and Garcinol (0 ‐ 40µM) and in combination for 24, 48 and 72hrs to examine viability, antiproliferative and apoptotic effects using MTT, Trypan Blue, cytoplasmic histone‐DNA fragment quantification and DAPI staining. The molecular mechanism was investigated by Western immunobloting, targeting different apoptotic signaling moieties. Synergistic effect of these compounds was tested using Isobologram analysis. Cell cycle phase analysis was done by Flow Cytometry. Both compounds individually and in combination significantly enhanced the anti‐proliferative and apoptotic effect in these cells relative to untreated control cells. This is first report on therapeutic effect of Garcinol on PaCa cells.Grant Funding SourceGraduate Teaching Assistantship

Combining surgery and chemotherapy for invasive bladder cancer: current and future directions

More than 13,000 patients died from invasive bladder cancer in 2005 alone. Radical cystectomy is the most commonly prescribed treatment for patients with muscle-invasive bladder cancer, or for those with a nonmuscle-invasive disease that is refractory to intravesical therapy. Despite advances in surgical technique and improved understanding of the role of pelvic lymphadenectomy, 5-year survival probabilities suggest that improvements in treatment are necessary. The maturation of several randomized clinical trials on perioperative chemotherapy, and particularly neoadjuvant chemotherapy, clearly suggest that an integrated treatment program of systemic chemotherapy and definitive locoregional therapy may improve the outcome for bladder cancer patients. The next frontier is the molecular characterization of this spectrum of diseases that make up invasive bladder cancer and targeted therapeutics. Prospective validation of molecular markers and evaluation of novel therapeutic agents, alone or in combination with established cytotoxic agents, provide hope of better outcomes for bladder cancer patients.