Cell therapy for intervertebral disc repair: advancing cell therapy from bench to clinics

Intervertebral disc (IVD) degeneration is a major cause of pain and disability; yet therapeutic options are limited and treatment often remains unsatisfactory. In recent years, research activities have intensified in tissue engineering and regenerative medicine, and pre-clinical studies have demonstrated encouraging results. Nonetheless, the translation of new biological therapies into clinical practice faces substantial barriers. During the symposium "Where Science meets Clinics", sponsored by the AO Foundation and held in Davos, Switzerland, from September 5-7, 2013, hurdles for translation were outlined, and ways to overcome them were discussed. With respect to cell therapy for IVD repair, it is obvious that regenerative treatment is indicated at early stages of disc degeneration, before structural changes have occurred. It is envisaged that in the near future, screening techniques and non-invasive imaging methods will be available to detect early degenerative changes. The promises of cell therapy include a sustained effect on matrix synthesis, inflammation control, and prevention of angio- and neuro-genesis. Discogenic pain, originating from "black discs" or annular injury, prevention of adjacent segment disease, and prevention of post-discectomy syndrome were identified as prospective indications for cell therapy. Before such therapy can safely and effectively be introduced into clinics, the identification of the patient population and proper standardisation of diagnostic parameters and outcome measurements are indispensable. Furthermore, open questions regarding the optimal cell type and delivery method need to be resolved in order to overcome the safety concerns implied with certain procedures. Finally, appropriate large animal models and well-designed clinical studies will be required, particularly addressing safety aspects.

Characterization of eosinophilic esophagitis murine models using optical coherence tomography.

Pre-clinical studies using murine models are critical for understanding the pathophysiological mechanisms underlying immune-mediated disorders such as Eosinophilic esophagitis (EoE). In this study, an optical coherence tomography (OCT) system capable of providing three-dimensional images with axial and transverse resolutions of 5 µm and 10 µm, respectively, was utilized to obtain esophageal images from a murine model of EoE-like disease ex vivo. Structural changes in the esophagus of wild-type (Tslpr(+/+) ) and mutant (Tslpr(-/-) ) mice with EoE-like disease were quantitatively evaluated and food impaction sites in the esophagus of diseased mice were monitored using OCT. Here, the capability of OCT as a label-free imaging tool devoid of tissue-processing artifacts to effectively characterize murine EoE-like disease models has been demonstrated.

Quantification of anandamide and 2-arachidonoylglycerol plasma levels to examine potential influences of tetrahydrocannabinol application on the endocannabinoid system in humans

The effects of tetrahydrocannabinol (THC) and endogenous cannabinoids (endocannabinoids, ECs) are both mediated by activation of the cannabinoid receptors CB1 and CB2. Exogenous activation of these receptors by THC could therefore alter EC levels. We tested this hypothesis in healthy volunteers (n = 25) who received a large intravenous dose of THC (0.10 mg/kg). Effects on the EC system were quantified by serial measurements of plasma ECs after THC administration. Eleven blood samples were drawn during the first 5 h after THC administration and two more samples after 24 and 48 h. THC, its metabolites THC-OH (biologically active) and THC-COOH (non-active), and the ECs anandamide and 2-arachidonoylglycerol (2-AG) were quantified by liquid chromatography-mass spectrometry. EC-plasma levels showed a biphasic response after THC injection reaching maximal values at 30 min. Anandamide increased slightly from 0.58 ± 0.21 ng/ml at baseline to 0.64 ± 0.24 ng/ml (p < 0.05) and 2-AG from 7.60 ± 4.30 ng/ml to 9.50 ± 5.90 ng/ml (p < 0.05). After reaching maximal concentrations, EC plasma levels decreased markedly to a nadir of 300 min after THC administration (to 0.32 ± 0.15 ng/ml for anandamide and to 5.50 ± 3.01 ng/ml for 2-AG, p < 0.05). EC plasma concentrations returned to near baseline levels until 48 h after the experiment. THC (0.76 ± 0.16 ng/ml) and THC-OH (0.36 ± 0.17 ng/ml) were still measurable at 24 h and remained detectible until 48 h after THC administration. Although the underlying mechanism is not clear, high doses of intravenous THC appear to influence endogenous cannabinoid concentrations and presumably EC-signalling.

L-mimosine increases the production of vascular endothelial growth factor in human tooth slice organ culture model.

AIM To assess the pro-angiogenic and pro-inflammatory capacity of the dentine-pulp complex in response to the prolyl hydroxylase inhibitor L-mimosine in a tooth slice organ culture model. METHODOLOGY Human teeth were sectioned transversely into 600-μm-thick slices and cultured in medium supplemented with serum and antibiotics. Then, pulps were stimulated for 48 h with L-mimosine. Pulps were subjected to viability measurements based on formazan formation in MTT assays. In addition, histological evaluation of pulps was performed based on haematoxylin and eosin staining. Culture supernatants were subjected to immunoassays for vascular endothelial growth factor (VEGF) to determine the pro-angiogenic capacity and to immunoassays for interleukin (IL)-6 and IL-8 to assess the pro-inflammatory response. Interleukin-1 served as pro-inflammatory control. Echinomycin was used to inhibit hypoxia-inducible factor-1 (HIF-1) alpha activity. Data were analysed using Student's t-test and Mann-Whitney U test. RESULTS Pulps within tooth slices remained vital upon L-mimosine stimulation as indicated by formazan formation and histological evaluation. L-mimosine increased VEGF production when normalized to formazan formation in the pulp tissue of the tooth slices (P < 0.05). This effect on VEGF was reduced by echinomycin (P < 0.01). Changes in normalized IL-6 and IL-8 levels upon treatment with L-mimosine did not reach the level of significance (P > 0.05), whilst treatment with IL-1, which served as positive control, increased IL-6 (P < 0.05) and IL-8 levels (P < 0.05). CONCLUSIONS The prolyl hydroxylase inhibitor L-mimosine increased VEGF production via HIF-1 alpha in the tooth slice organ culture model whilst inducing no prominent increase in IL-6 and IL-8. Pre-clinical studies will reveal if these in vitro effects translate into dental pulp regeneration.

Minimal residual disease in cancer therapy - Small things make all the difference

Minimal residual disease (MRD) is a major hurdle in the eradication of malignant tumors. Despite the high sensitivity of various cancers to treatment, some residual cancer cells persist and lead to tumor recurrence and treatment failure. Obvious reasons for residual disease include mechanisms of secondary therapy resistance, such as the presence of mutant cells that are insensitive to the drugs, or the presence of cells that become drug resistant due to activation of survival pathways. In addition to such unambiguous resistance modalities, several patients with relapsing tumors do not show refractory disease and respond again when the initial therapy is repeated. These cases cannot be explained by the selection of mutant tumor cells, and the precise mechanisms underlying this clinical drug resistance are ill-defined. In the current review, we put special emphasis on cell-intrinsic and -extrinsic mechanisms that may explain mechanisms of MRD that are independent of secondary therapy resistance. In particular, we show that studying genetically engineered mouse models (GEMMs), which highly resemble the disease in humans, provides a complementary approach to understand MRD. In these animal models, specific mechanisms of secondary resistance can be excluded by targeted genetic modifications. This allows a clear distinction between the selection of cells with stable secondary resistance and mechanisms that result in the survival of residual cells but do not provoke secondary drug resistance. Mechanisms that may explain the latter feature include special biochemical defense properties of cancer stem cells, metabolic peculiarities such as the dependence on autophagy, drug-tolerant persisting cells, intratumoral heterogeneity, secreted factors from the microenvironment, tumor vascularization patterns and immunosurveillance-related factors. We propose in the current review that a common feature of these various mechanisms is cancer cell dormancy. Therefore, dormant cancer cells appear to be an important target in the attempt to eradicate residual cancer cells, and eventually cure patients who repeatedly respond to anticancer therapy but lack complete tumor eradication.

Dissecting abdominal aortic aneurysm in Angiotensin II-infused mice: the importance of imaging

INTRODUCTION Since the initial publication in 2000, Angiotensin II-infused mice have become one of the most popular models to study abdominal aortic aneurysm in a pre-clinical setting. We recently used phase contrast X-ray based computed tomography to demonstrate that these animals develop an apparent luminal dilatation and an intramural hematoma, both related to mural ruptures in the tunica media in the vicinity of suprarenal side branches. AIMS The aim of this narrative review was to provide an extensive overview of small animal applicable techniques that have provided relevant insight into the pathogenesis and morphology of dissecting AAA in mice, and to relate findings from these techniques to each other and to our recent PCXTM-based results. Combining insights from recent and consolidated publications we aimed to enhance our understanding of dissecting AAA morphology and anatomy. RESULTS AND CONCLUSION We analyzed in vivo and ex vivo images of aortas obtained from macroscopic anatomy, histology, high-frequency ultrasound, contrast-enhanced micro-CT, micro-MRI and PCXTM. We demonstrate how in almost all publications the aorta has been subdivided into a part in which an intact lumen lies adjacent to a remodeled wall/hematoma, and a part in which elastic lamellae are ruptured and the lumen appears to be dilated. We show how the novel paradigm fits within the existing one, and how 3D images can explain and connect previously published 2D structures. We conclude that PCXTM-based findings are in line with previous results, and all evidence points towards the fact that dissecting AAAs in Angiotensin II-infused mice are actually caused by ruptures of the tunica media in the immediate vicinity of small side branches.

Cellular and molecular effects of the mTOR inhibitor everolimus.

mTOR (mechanistic target of rapamycin) functions as the central regulator for cell proliferation, growth and survival. Up-regulation of proteins regulating mTOR, as well as its downstream targets, has been reported in various cancers. This has promoted the development of anti-cancer therapies targeting mTOR, namely fungal macrolide rapamycin, a naturally occurring mTOR inhibitor, and its analogues (rapalogues). One such rapalogue, everolimus, has been approved in the clinical treatment of renal and breast cancers. Although results have demonstrated that these mTOR inhibitors are effective in attenuating cell growth of cancer cells under in vitro and in vivo conditions, subsequent sporadic response to rapalogues therapy in clinical trials has promoted researchers to look further into the complex understanding of the dynamics of mTOR regulation in the tumour environment. Limitations of these rapalogues include the sensitivity of tumour subsets to mTOR inhibition. Additionally, it is well known that rapamycin and its rapalogues mediate their effects by inhibiting mTORC (mTOR complex) 1, with limited or no effect on mTORC2 activity. The present review summarizes the pre-clinical, clinical and recent discoveries, with emphasis on the cellular and molecular effects of everolimus in cancer therapy.

An interferon-inducible protein, p202, in cancer gene therapy

Interferons (IFNs) have been shown to exert antiviral, cell growth regulatory, and immunomodulatory effects on target cells. Both type I (α and β) and type II (γ) IFNs regulate cellular activities by specifically inducing the expression or activation of endogenous proteins that perform distinct biological functions. p202 is a 52 kDa nuclear phosphoprotein known to be induced by IFNs. p202 interacts with a variety of cellular transcription and growth regulatory factors and affects their functions. ^ In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. Cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. More importantly, p202 expression reduced the tumorigenicity of human prostate cancer cells. p202-expressing cells exhibit an elevated level of hypophosphorylated form of pRb, and reduced level of cyclin B1 and p55CDC. ^ Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype and tumorigenicity of prostate cancer cells. ^ In addition to inhibiting in vitro cell growth, suppressing the tumorigenicity of breast cancer cells in vivo, p202 expression could sensitize breast cancer cells to apoptosis induced by TNF-α treatment. One possible mechanism contributing to this sensitization is the inactivation of NF-κB by its interaction with p202. These results provide a scientific basis for a novel therapeutic strategy that combines p202 and TNF-α treatment against breast cancer. ^ It has been reported that NF-κB is constitutively active in human pancreatic cancer cells. Since p202 interacts with NF-κB and inhibits its activity, we examined a potential p202-mediated anti-tumor activity in pancreatic cancer. We used both ectopic and orthotopic xenograft models and demonstrated that p202 expression is associated with multiple anti-tumor activities that include inhibition of tumor growth, reduced tumorigenicity, prolonged survival, and remarkably, suppression of metastasis and angiogenesis. In vitro invasion assay also showed that p202-expressing pancreatic cancer cells are less invasive than those without p202 expression. That observation was supported by the findings that p202-expressing tumors showed reduced expression of angiogenic factors such as IL-8, and VEGF by inhibiting their transcription, and p202-expressing pancreatic cancer cells have reduced level of MAP-2 activity, a secreted protease activity important for metastasis. Together, our results strongly suggest that p202 expression mediates multiple anti-tumor activities against pancreatic cancer, and that may provide a scientific basis for developing a p202-based gene therapy in pancreatic cancer treatment. ^ Importantly, we demonstrated a treatment efficacy by using p202/SN2 liposome complex in a nude mice orthotopic breast cancer, and an ectopic pancreatic cancer xenograft model, through systemic and intra-tumor injection respectively. These results suggest a feasibility of using p202/SN2 liposome in future pre-clinical gene therapy experiments. ^

The transcription factor nuclear factor kappa B (NFkappaB) maintains TRAIL resistance in human pancreatic cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF family of cytokines that induces apoptosis in a variety of tumor cells while sparing normal cells. However, many human cancer cell lines display resistance to TRAIL-induced apoptosis and the mechanisms contributing to resistance remain controversial. Previous studies have demonstrated that the dimeric transcription factor Nuclear Factor kappa B (NFκB) is constitutively active in a majority of human pancreatic cancer cell lines and primary tumors, and although its role in tumor progression remains unclear it has been suggested that NFκB contributes to TRAIL resistance. Based on this, I examined the effects of NFκB inhibitors on TRAIL sensitivity in a panel of nine pancreatic cancer cell lines. I show here that inhibitors of NFκB, including two inhibitors of the proteasome (bortezomib (Velcade™, PS-341) and NPI-0052), a small molecule inhibitor of IKK (PS1145), and a novel synthetic diterpene NIK inhibitor (NPI-1342) reverse TRAIL resistance in pancreatic cancer cell lines. Further analysis revealed that the expression of the anti-apoptosic proteins BclXL and XIAP was significantly decreased following exposure to these inhibitors alone and in combination with TRAIL. Additionally, treatment with NPI0052 and TRAIL significantly reduced tumor burden relative to the control tumors in an L3.6pl orthotopic pancreatic xenograft model. This was associated with a significant decrease in proliferation and an increase in caspase 3 and 8 cleavage. Combination therapy employing PS1145 or NPI-1342 in combination with TRAIL also resulted in a significant reduction in tumor burden compared to either agent alone in a Panc1 orthotopic xenograft model. My studies show that combination therapy with inhibitors of NFκB alone and TRAIL is effective in pre-clinical models of pancreatic cancer and suggests that the approach should be evaluated in patients. ^

The role of SRC family kinase activation in prostate cancer growth and lymph node metastasis

Aberrant expression and/or activation of Src Family of non-receptor protein tyrosine kinases (SFKs) occur frequently during progressive stages of multiple types of human malignancies, including prostate cancer. Two SFKs, Src and Lyn, are expressed and implicated in prostate cancer progression. Work in this dissertation investigated the specific roles of Src and Lyn in the prostate tumor progression, and the effects of SFK inhibition on prostate tumor growth and lymph node metastasis in pre-clinical mouse models. ^ Firstly, using a pharmacological inhibitor of SFKs in clinical trials, dasatinib, I demonstrated that SFK inhibition affects both cellular migration and proliferation in vitro. Systemic administration of dasatinib reduced primary tumor growth, as well as development of lymph node metastases, in both androgen-sensitive and -resistant orthotopic prostate cancer mouse models. Immunohistochemical analysis of the primary tumors revealed that dasatinib treatment decreased SFK phosphorylation but not expression, resulting in decreased cellular proliferation and increased apoptosis. For this analysis of immunohistochemical stained tissues, I developed a novel method of quantifying immunohistochemical stain intensity that greatly reduced the inherent bias in analyzing staining intensity. ^ To determine if Src and Lyn played overlapping or distinct roles in prostate cancer tumor growth and progression, Src expression alone was inhibited by small-interfering RNA. The resulting stable cell lines were decreased in migration, but not substantially affected in proliferation rates. In contrast, an analogous strategy targeting Lyn led to stable cell lines in which proliferation rates were significantly reduced. ^ Lastly, I tested the efficacy of a novel SFK inhibitor (KX2-391) targeting peptide substrate-binding domain, on prostate cancer growth and lymph node metastasis in vivo. I demonstrated that KX2-391 has similar effects as dasatinib, an ATP-competitive small molecular inhibitor, on both the primary tumor growth and development of lymph node metastasis in vivo, work that contributed to the first-in-man Phase I clinical trial of KX2-391. ^ In summary, studies in this dissertation provide the first demonstration that Src and Lyn activities affect different cellular functions required for prostate tumor growth and metastasis, and SFK inhibitors effectively reduce primary tumor growth and lymph node metastasis. Therefore, I conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer. ^

Evaluation of Knowledge Regarding Diagnostic Strategies for Genetic Diseases in Select Residents

Genetics education for physicians has been a popular publication topic in the United States and in Europe for over 20 years. Decreasing numbers of medical genetics professionals and an increasing volume of genetic information has created a dire need for increased genetics training in medical school and in clinical practice. This study aimed to assess how well pediatrics-focused primary care physicians apply their general genetics knowledge to clinical genetic testing using scenario-based questions. We chose to specifically focus on knowledge of the diagnostic applicability of Chromosomal Microarray (CMA) technology in pediatrics because of its recent recommendation by the International Standard Cytogenomic Array (ISCA) Consortium as a first-tier genetic test for individuals with developmental disabilities and/or congenital anomalies. Proficiency in ordering baseline genetic testing was evaluated for eighty-one respondents from four pediatrics-focused residencies (categorical pediatrics, pediatric neurology, internal medicine/pediatrics, and family practice) at two large residency programs in Houston, Texas. Similar to other studies, we found an overall deficit of genetic testing knowledge, especially among family practice residents. Interestingly, residents who elected to complete a genetics rotation in medical school scored significantly better than expected, as well as better than residents who did not elect to complete a genetics rotation. We suspect that the insufficient knowledge among physicians regarding a baseline genetics work-up is leading to redundant (i.e. concurrent karyotype and CMA) and incorrect (i.e. ordering CMA to detect achondroplasia) genetic testing and is contributing to rising health care costs in the United States. Our results provide specific teaching points upon which medical schools can focus education about clinical genetic testing and suggest that increased collaboration between primary care physicians and genetics professionals could benefit patient health care overall.

A review of the Malaria Chemoprophylaxis Compliance Program at ExxonMobil Corporation

ExxonMobil, a Fortune 500 oil and gas corporation, has a global workforce with employees assigned to projects in areas at risk for infectious diseases, particularly malaria. As such, the corporation has put in place a program to protect the health of workers and ensure their safety in malaria endemic zones. This program is called the Malaria Control Program (MCP). One component of this program is the more specific Malaria Chemoprophylaxis Compliance Program (MCCP), in which employees enroll following consent to random drug testing for compliance with the company's chemoprophylaxis requirements. Each year, data is gathered on the number of employees working in these locations and are selected randomly and tested for chemoprophylaxis compliance. The selection strives to test each eligible worker once per year. Test results that come back positive for the chemoprophylaxis drug are considered "detects" and tests that are negative for the drug and therefore show the worker is non-compliant at risk for severe malaria infection are considered "non-detect". ^ The current practice report used aggregate data to calculate statistics on test results to reflect compliance among both employees and contractors in various malaria-endemic areas. This aggregate, non-individualized data has been compiled and reflects the effectiveness and reach of ExxonMobil's Malaria Chemoprophylaxis Compliance Program. In order to assess compliance, information on the number of non-detect test results was compared to the number of tests completed per year. The data shows that over time, non-detect results have declined in both employee and contractor populations, and vary somewhat by location due to size and scope of the MCCP implemented in-country. Although the data indicate a positive trend for the corporation, some recommendations have been made for future implementation of the program.^

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.

Left, right and interval bivariate censored data: Evaluating screening mammography in the presence of lead -time bias, length bias and over-detection

Of the large clinical trials evaluating screening mammography efficacy, none included women ages 75 and older. Recommendations on an upper age limit at which to discontinue screening are based on indirect evidence and are not consistent. Screening mammography is evaluated using observational data from the SEER-Medicare linked database. Measuring the benefit of screening mammography is difficult due to the impact of lead-time bias, length bias and over-detection. The underlying conceptual model divides the disease into two stages: pre-clinical (T0) and symptomatic (T1) breast cancer. Treating the time in these phases as a pair of dependent bivariate observations, (t0,t1), estimates are derived to describe the distribution of this random vector. To quantify the effect of screening mammography, statistical inference is made about the mammography parameters that correspond to the marginal distribution of the symptomatic phase duration (T1). This shows the hazard ratio of death from breast cancer comparing women with screen-detected tumors to those detected at their symptom onset is 0.36 (0.30, 0.42), indicating a benefit among the screen-detected cases. ^

An Open-Label Investigation of the Pharmacokinetics and Tolerability of Oral Cysteamine in Adults with Cystic Fibrosis

This study was funded by Health Sciences Scotland (West Medical Building, University Avenue, Glasgow G12 8QQ. UK) and the Cystic Fibrosis Trust (One Aldgate, London. EC3N 1RE. UK). The funders did not contribute to study design, data collection, analysis, this report or the decision to publish.