Relevant Human Model Research Articles

In Vitro Efficacy and Release Study with Anti-Inflammatory Drugs Incorporated in Adhesive Transdermal Drug Delivery Systems

<h2>ABSTRACT</h2> The topical application of two different anti-inflammatory extracts incorporated in adhesive transdermal drug delivery systems (TDDSs) was investigated. Therefore, anti-inflammatory properties and percutaneous absorption behavior of adhesive TDDSs were characterized <i>in vitro</i> conducting experiments with a dermatologically relevant human skin model. Anti-inflammatory efficacy against UV irradiation of both TDDSs was determined <i>in vitro</i> with EpiDerm™. The reduction of the release of proinflammatory cytokines by topically applied TDDSs was compared with the reduction during the presence of the specific cyclooxygenase inhibitor diclofenac in the culture medium. A similar anti-inflammatory efficacy of the topically applied TDDSs in comparison with the use of diclofenac in the culture medium should be achieved. Furthermore, percutaneous absorption in efficacy tests was compared with percutaneous absorption in diffusion studies with porcine cadaver skin. Both the topically applied TDDSs showed a significant anti-inflammatory activity. Permeation coefficients through the stratum corneum and the epidermis gained from the release studies on porcine cadaver skin (<i>Magnolia</i>: 2.23·10⁻⁵ cm/h, licorice: 4.68·10⁻⁶ cm/h) were approximately five times lower than the permeation coefficients obtained with the EpiDerm™ skin model (<i>Magnolia</i>: 9.48·10⁻⁵ cm/h, licorice: 24.0·10⁻⁶ cm/h). Therefore, an adjustment of drug doses during experiments with the EpiDerm™ skin model because of weaker skin barrier properties should be considered. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1142–1148, 2014

Journal of Pharmaceutical Sciences

The topical application of two different anti-inflammatory extracts incorporated in adhesive transdermal drug delivery systems (TDDSs) was investigated. Therefore, anti-inflammatory properties and percutaneous absorption behavior of adhesive TDDSs were characterized in vitro conducting experiments with a dermatologically relevant human skin model. Anti-inflammatory efficacy against UV irradiation of both TDDSs was determined in vitro with EpiDerm™. The reduction of the release of proinflammatory cytokines by topically applied TDDSs was compared with the reduction during the presence of the specific cyclooxygenase inhibitor diclofenac in the culture medium. A similar anti-inflammatory efficacy of the topically applied TDDSs in comparison with the use of diclofenac in the culture medium should be achieved. Furthermore, percutaneous absorption in efficacy tests was compared with percutaneous absorption in diffusion studies with porcine cadaver skin. Both the topically applied TDDSs showed a significant anti-inflammatory activity. Permeation coefficients through the stratum corneum and the epidermis gained from the release studies on porcine cadaver skin (Magnolia: 2.23·10(-5) cm/h, licorice: 4.68·10(-6) cm/h) were approximately five times lower than the permeation coefficients obtained with the EpiDerm™ skin model (Magnolia: 9.48·10(-5) cm/h, licorice: 24.0·10(-6) cm/h). Therefore, an adjustment of drug doses during experiments with the EpiDerm™ skin model because of weaker skin barrier properties should be considered.

1H-NMR spectroscopy for human 3D neural stem cell cultures metabolic profiling

Background The current lack of predictable central nervous system (CNS) models in pharmaceutical industry early stage development strongly contributes for the high attrition rates registered for new therapeutics [1]. Thus, there is an increasing need for a paradigm shift towards more human relevant cell models, which can closely recapitulate the in vivo cell-cell interactions, presenting higher physiological relevance by bridging the gap between animal models and human clinical trials. In this context, human 3D in vitro models are promising tools with great potential for preclinical research, as they can mimic some of the main features of tissues, such as cell-cell and cell-extracellular matrix (ECM) interactions [2,3]. Moreover these complex cell models are suitable for high-throughput screening (HTS) platforms, essential in drug discovery pipelines by reducing both costs and time in clinical trials [2,4]. However, despite important advances in the last years and the increasing clinical and biological relevance, the full establishment of human 3D in vitro models in pre-clinical research requires a significant increase in the power of the available analytical methodologies towards more robust and comprehensive readouts [4]. With the emergence of systems biology field and several “-omics” technologies, such as metabolomics, it became possible to have a more mechanistic approach in the understanding of cellular programs. H-nuclear magnetic resonance (H-NMR) spectroscopy is a powerful and widely accepted high resolution methodology for a number of applications, including metabolic profiling [5]. Despite the low sensitivity when compared with mass spectrometry (MS), H-NMR profiling presents several advantages, enabling a non-invasive and non-destructive quantitative analysis requiring only minimal sample preparation [5]. In this work we present the development of a robust and optimized workflow for the exometabolome profiling of 3D in vitro cultures of human midbrain-derived neural progenitor cells (hmNPC).

Towards a three-dimensional microfluidic liver platform for predicting drug efficacy and toxicity in humans

Although the process of drug development requires efficacy and toxicity testing in animals prior to human testing, animal models have limited ability to accurately predict human responses to xenobiotics and other insults. Societal pressures are also focusing on reduction of and, ultimately, replacement of animal testing. However, a variety of in vitro models, explored over the last decade, have not been powerful enough to replace animal models. New initiatives sponsored by several US federal agencies seek to address this problem by funding the development of physiologically relevant human organ models on microscopic chips. The eventual goal is to simulate a human-on-a-chip, by interconnecting the organ models, thereby replacing animal testing in drug discovery and development. As part of this initiative, we aim to build a three-dimensional human liver chip that mimics the acinus, the smallest functional unit of the liver, including its oxygen gradient. Our liver-on-a-chip platform will deliver a microfluidic three-dimensional co-culture environment with stable synthetic and enzymatic function for at least 4 weeks. Sentinel cells that contain fluorescent biosensors will be integrated into the chip to provide multiplexed, real-time readouts of key liver functions and pathology. We are also developing a database to manage experimental data and harness external information to interpret the multimodal data and create a predictive platform.

Carfilzomib, a Second-Generation Proteasome Inhibitor, In Combination With The Btk Inhibitor Ibrutinib As a Novel Therapeutic Regimen For Treating Double Hit DLBCL

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults; in the United States alone 30,000 new cases are diagnosed every year. Approximately 5% of DLBCLs are double-hit lymphomas (DHL), a recently discovered subtype of lymphoma defined by a chromosomal translocation and subsequent breakpoint at the MYC/8q24 loci in combination with another recurrent breakpoint, usually BCL2/18q21 or BCL6/3q27. DHL is resistant to virtually all therapeutic drugs and chemotherapy, showing extremely poor prognosis even with an R-CHOP regimen. Currently, there is no known therapeutic strategy that can effectively treat double-hit lymphomas. Lack of clinically relevant human experimental models for understanding the molecular biologic and genetic basis of DHL continues to hamper identification of valid therapeutic targets for development of effective therapy. However, we have developed two unique DH-DLBCL cell lines (CJ and RC), harboring both MYC and BCL2 translocations. These cell lines display the morphologic, immunophenotypic, genotypic, and biologic characteristics of the lymphoma cells present in the original diagnostic specimen. We have also obtained another BCL2/MYC DL-DLBCL cell line, U-2973, recently established from another research institution (Uppsala University, Sweden). Together, these DHL cell lines will be useful experimental models for in vitro and in vivo studies of DH-DLBCL pathogenesis and therapeutics. Recent clinical findings have demonstrated that the novel second-generation proteasome inhibitor, carfilzomib (CFZ), and the Btk inhibitor, ibruitinib (IBR), as single agents have shown promising results in relapsed/refractory hematologic malignancies. In our initial experiments, we examined the efficacy of using CFZ and IBR in combination in three DH-DLBCL cell lines using proliferation MTT assays. CJ and U-2973 cells were moderately resistant to IBR (IC50>20 uM) and were highly sensitive to CFZ (IC50=2.5-10 nM) in comparison to RC cells that were sensitive to both IBR (5-10 uM) and CFZ (5-10 nM). When combined, these drugs exhibited strong synergism, particularly in CJ cells, with combination indexes <0.3, as demonstrated using the Chou-Talalay method. In contrast, CFZ/IBR regimens displayed minimal toxicity toward normal peripheral blood mononuclear lymphocytes. In terms of cell death, low drug concentrations of CFZ (2.5 nM) alone caused 30% cell death but drastically increased the apoptotic effect of IBR (10 uM) from 10% to 80% in CJ cells. This apoptotic effect correlated with BCL2 protein cleaving and the induction of BAX protein, leading to caspase 3 activation and PARP cleavage. CFZ and IBR also interacted synergistically to induce DNA damage through the activation of p53 and phosphorylated histone 2AX, molecular markers for DNA damage. Due to the synergistic efficacy of CFZ and IBR on DHL cell lines, these drugs have therapeutic potential in treating patients with DHL. Disclosures:Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership.

Highly optimized DNA vaccine targeting human telomerase reverse transcriptase stimulates potent antitumor immunity.

High levels of human telomerase reverse transcriptase (hTERT) are detected in more than 85% of human cancers. Immunologic analysis supports that hTERT is a widely applicable target recognized by T cells and can be potentially studied as a broad cancer immunotherapeutic, or a unique line of defense against tumor recurrence. There remains an urgent need to develop more potent hTERT vaccines. Here, a synthetic highly optimized full-length hTERT DNA vaccine (phTERT) was designed and the induced immunity was examined in mice and non-human primates (NHP). When delivered by electroporation, phTERT elicited strong, broad hTERT-specific CD8 T-cell responses including induction of T cells expressing CD107a, IFN-γ, and TNF-α in mice. The ability of phTERT to overcome tolerance was evaluated in an NHP model, whose TERT is 96% homologous to that of hTERT. Immunized monkeys exhibited robust [average 1,834 spot forming unit (SFU)/10(6) peripheral blood mononuclear cells (PBMC)], diverse (multiple immunodominant epitopes) IFN-γ responses and antigen-specific perforin release (average 332 SFU/10(6) PBMCs), suggesting that phTERT breaks tolerance and induces potent cytotoxic responses in this human-relevant model. Moreover, in an HPV16-associated tumor model, vaccination of phTERT slows tumor growth and improves survival rate in both prophylactic and therapeutic studies. Finally, in vivo cytotoxicity assay confirmed that phTERT-induced CD8 T cells exhibited specific cytotoxic T lymphocyte (CTL) activity, capable of eliminating hTERT-pulsed target cells. These findings support that this synthetic electroporation-delivered DNA phTERT may have a role as a broad therapeutic cancer vaccine candidate.

Transrectal robotic natural orifice translumenal endoscopic surgery (NOTES) applied to intestinal anastomosis in a porcine intestine model

Natural orifice translumenal endoscopic surgery (NOTES) is a minimally invasive operation using devices such as flexible endoscopes and linear or circular staplers. Nevertheless, hand-sewn anastomosis in NOTES remains challenging. This study aimed to investigate the feasibility of transrectal robotic NOTES requiring intracorporeal small intestinal anastomosis and closure of the rectal anterior wall incision in a relevant human model. The authors developed a flexible rectal proctoscope with a diameter of 43 mm for transrectal robotic NOTES. Small intestinal anastomosis was performed in a porcine intestinal transrectal NOTES model using two robotic arms and a camera inserted through the proctoscope and a rectal anterior wall incision. The quality of transrectal small intestinal anastomosis using the da Vinci surgical system (transrectal robotic NOTES group) was compared with that of transabdominal anastomosis using the da Vinci surgical system (transabdominal robot-assisted surgery group) and transrectal anastomosis using traditional transanal endoscopic microsurgery (TEM) instruments (TEM NOTES group). The quality of transrectal rectal anterior wall suturing in the transrectal robotic NOTES group was compared with that of the TEM NOTES group and the open surgical instruments group (open group). Robotic intracorporeal suturing was performed successfully in the porcine intestine model. During small intestinal anastomosis, burst pressure in the transrectal robotic NOTES group (67.7 ± 29.3 mmHg) was similar to that in the transabdominal robot-assisted surgery group (73.3 ± 18.2 mmHg) but significantly higher than in the TEM NOTES group (20.3 ± 24.0 mmHg; p < 0.01). During rectal anterior wall suturing, the burst pressure did not differ significantly between the transrectal robotic NOTES group (149.9 ± 81.1 mmHg) and the open group (195.0 ± 60.5 mmHg). The preliminary safety and efficacy of transrectal robotic NOTES was established. Further studies are required to determine the practical feasibility of this procedure.

Active Targeting Using HER‐2‐Affibody‐Conjugated Nanoparticles Enabled Sensitive and Specific Imaging of Orthotopic HER‐2 Positive Ovarian Tumors

Despite advances in cancer diagnosis and treatment, ovarian cancer remains one of the most fatal cancer types. The development of targeted nanoparticle imaging probes and therapeutics offers promising approaches for early detection and effective treatment of ovarian cancer. In this study, HER-2 targeted magnetic iron oxide nanoparticles (IONPs) are developed by conjugating a high affinity and small size HER-2 affibody that is labeled with a unique near infrared dye (NIR-830) to the nanoparticles. Using a clinically relevant orthotopic human ovarian tumor xenograft model, it is shown that HER-2 targeted IONPs are selectively delivered into both primary and disseminated ovarian tumors, enabling non-invasive optical and MR imaging of the tumors as small as 1 mm in the peritoneal cavity. It is determined that HER-2 targeted delivery of the IONPs is essential for specific and sensitive imaging of the HER-2 positive tumor since we are unable to detect the imaging signal in the tumors following systemic delivery of non-targeted IONPs into the mice bearing HER-2 positive SKOV3 tumors. Furthermore, imaging signals and the IONPs are not detected in HER-2 low expressing OVCAR3 tumors after systemic delivery of HER-2 targeted-IONPs. Since HER-2 is expressed in a high percentage of ovarian cancers, the HER-2 targeted dual imaging modality IONPs have potential for the development of novel targeted imaging and therapeutic nanoparticles for ovarian cancer detection, targeted drug delivery, and image-guided therapy and surgery.

Gene replacement therapy for retinal CNG channelopathies

Visual phototransduction relies on the function of cyclic nucleotide-gated channels in the rod and cone photoreceptor outer segment plasma membranes. The role of these ion channels is to translate light-triggered changes in the second messenger cyclic guanosine 3'-5'-monophosphate levels into an electrical signal that is further processed within the retinal network and then sent to higher visual centers. Rod and cone photoreceptors express distinct CNG channels. The rod photoreceptor CNG channel is composed of one CNGB1 and three CNGA1 subunits, whereas the cone channel is formed by one CNGB3 and three CNGA3 subunits. Mutations in any of these channel subunits result in severe and currently untreatable retinal degenerative diseases like retinitis pigmentosa or achromatopsia. In this review, we provide an overview of the human diseases and relevant animal models of CNG channelopathies. Furthermore, we summarize recent results from preclinical gene therapy studies using adeno-associated viral vectors and discuss the efficacy and translational potential of these gene therapeutic approaches.

S07.1 Insights into Maternal Gonorrhoea: Human Primary Cervical and Amniochorionic Epithelial Cell Responses to Neisseria Gonorrhoeae Infection

Bacterial infection is widely recognised as a factor contributing to adverse pregnancy outcomes (APOs).Neisseria gonorrhoeaeinfections continue to be a universal and intractable problem. In this regard, maternal gonorrhoea increases a woman’s risk for APO by 6.5-fold. Bacterial infection is thought to trigger a pro-inflammatory response that initiates those processes involved in (preterm) human parturition. The ability of gonococci to invade and transcytose amniotic sac tissues,in vivo, is inferred from the ability to isolate gonococci from these tissues and from amniotic fluid. However, there are currently no data to indicate how gonococcal infection can result in APO, and a physiologically relevant human model of pregnancy that is amendable to scientific analyses has hindered elucidation of factors contributing to APO. Thereby, an understanding of gonococcal infection as it relates to human pregnancy, usinghumancell models of disease, could provide new insights into the pathophysiology of gonococcal disease and of APO as they likely occurin vivo. To this end, we investigated gonococcal infection under conditions reflecting normal pregnancy by using primary epithelial cells derived from the human cervix (i. e. pex cells) and amniochorionic membranes (i. e. pace cells) and by altering the combined concentrations of pertinent steroid hormones. Comparative, quantitative, infection assays indicated that gonococci adhere to and invade amniochorionic cells and tissue, which was further observed to occur by a complement receptor-mediated mechanism. We demonstrate thatN. gonorrhoeaeinfection of pex and pace cells elicits the differential production of nitric oxide and complement proteins, as well as the specific matrix metalloproteases, prostaglandins, and cytokines thought to participate in triggering the onset of human parturition. Hence, we provide the first direct evidence to indicate a potential link between gonococcal infection and the induction of APOs.

Cardiolipin deficiency affects respiratory chain function and organization in an induced pluripotent stem cell model of Barth syndrome

Barth syndrome (BTHS) patients carrying mutations in tafazzin (TAZ1), which is involved in the final maturation of cardiolipin, present with dilated cardiomyopathy, skeletal myopathy, growth retardation and neutropenia. To study how mitochondrial function is impaired in BTHS patients, we generated induced pluripotent stem cells (iPSCs) to develop a novel and relevant human model system for BTHS. BTHS–iPSCs generated from dermal fibroblasts of three patients with different mutations in TAZ1 expressed pluripotency markers, and were able to differentiate into cells derived from all three germ layers both in vitro and in vivo. We used these cells to study the impact of tafazzin deficiency on mitochondrial oxidative phosphorylation. We found an impaired remodeling of cardiolipin, a dramatic decrease in basal oxygen consumption rate and in the maximal respiratory capacity in BTHS–iPSCs. Simultaneous measurement of extra-cellular acidification rate allowed us a thorough assessment of the metabolic deficiency in BTHS patients. Blue native gel analyses revealed that decreased respiration coincided with dramatic structural changes in respiratory chain supercomplexes leading to a massive increase in generation of reactive oxygen species. Our data demonstrate that BTHS–iPSCs are capable of modeling BTHS by recapitulating the disease phenotype and thus are important tools for studying the disease mechanism.

Use of ex vivo and in vitro cultures of the human respiratory tract to study the tropism and host responses of highly pathogenic avian influenza A (H5N1) and other influenza viruses

The tropism of influenza viruses for the human respiratory tract is a key determinant of host-range, and consequently, of pathogenesis and transmission. Insights can be obtained from clinical and autopsy studies of human disease and relevant animal models. Ex vivo cultures of the human respiratory tract and in vitro cultures of primary human cells can provide complementary information provided they are physiologically comparable in relevant characteristics to human tissues in vivo, e.g. virus receptor distribution, state of differentiation. We review different experimental models for their physiological relevance and summarize available data using these cultures in relation to highly pathogenic avian influenza H5N1, in comparison where relevant, with other influenza viruses. Transformed continuous cell-lines often differ in important ways to the corresponding tissues in vivo.The state of differentiation of primary human cells (respiratory epithelium, macrophages) can markedly affect virus tropism and host responses. Ex vivo cultures of human respiratory tissues provide a close resemblance to tissues in vivo and may be used to risk assess animal viruses for pandemic threat. Physiological factors (age, inflammation) can markedly affect virus receptor expression and virus tropism.Taken together with data from clinical studies on infected humans and relevant animal models, data from ex vivo and in vitro cultures of human tissues and cells can provide insights into virus transmission and pathogenesis and may provide understanding that leads to novel therapeutic interventions.

Abstract 4423: Implications for cardiotoxicity with targeted therapies.

Abstract Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen that was not wholly predicted by current FDA mandated pre-clinical tests. Thus a more robust tool to predict the cardiotoxic potential of these compounds is warranted. For this purpose, we developed a panel of in vitro tests in a clinically relevant human cardiomyocyte model that assesses drug impact on overall cardiac health (cell viability, apoptosis, and morphology), mitochondria and metabolic intactness (reactive oxygen species (ROS) generation, AMPK activity, and lipid accumulation), and electrophysiological function (impedance (beat rate) and ion channel blockage). To demonstrate the utility of our panel, we examined the effect of 4 FDA-approved TKi with known clinical outcome on cardiac health. The 3 drugs with known cardiac adverse events (crizotinib, sunitinib, and nilotinib) all proved to be cardiotoxic by our panel although each showed distinct mechanisms of toxicity. However erlotinib, a cardiac-safe drug that targets EGFR and is used primarily in non-small cell lung cancer (NSCLC), did not show any indications of toxicity. Surprisingly, the most cardiotoxic drug by our panel was crizotinib, an ALK/ MET inhibitor that has revolutionized treatment for ALK+ NSCLC patients. Crizotinib potently impaired overall cardiac health by increasing ROS and apoptosis but also impeded electrophysiological function by inducing ion channel blockage and reducing cardiac beat rate. Interestingly crizotinib also induced lipid and cholesterol accumulation which was shown to be correlated with increased expression of the sterol-regulatory binding protein (SREBP) pathway by transcriptome analysis. The multi-targeted TKi sunitinib (used in renal cell carcinoma and gastrointestinal stromal tumors) and nilotinib (used in chronic myelogenous leukemia) also showed unique cardiotoxicity profiles. In conclusion, our studies showed a distinct cardiotoxicity profile for each TKi that correlates with clinical outcome. These results suggest that caution should be taken with these TKi especially when used long-term or in the adjuvant setting. Furthermore, these effects may be exacerbated in patients with adverse cardiac genetic predispositions and studies are ongoing in our lab on patient-derived cardiac cell lines to investigate this possibility. This multi-parameter screening approach allows for a more complete assessment of the potential for drug-induced cardiotoxicity and may allow for earlier detection in the drug development process. In addition, understanding the mechanisms of cardiac toxicities may lead to the discovery of novel mitigation strategies and combination therapies to improve cardiac cell health during treatment with oncology therapies. Citation Format: Kimberly R. Doherty, Robert L. Wappel, Dominique R. Talbert, Patricia B. Trusk, Scott A. Shell, Diarmuid M. Moran, James W. Kramer, Arthur Brown, Sarah Bacus. Implications for cardiotoxicity with targeted therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4423. doi:10.1158/1538-7445.AM2013-4423

Bioengineered 3D Human Kidney Tissue, a Platform for the Determination of Nephrotoxicity

The staggering cost of bringing a drug to market coupled with the extremely high failure rate of prospective compounds in early phase clinical trials due to unexpected human toxicity makes it imperative that more relevant human models be developed to better predict drug toxicity. Drug–induced nephrotoxicity remains especially difficult to predict in both pre-clinical and clinical settings and is often undetected until patient hospitalization. Current pre-clinical methods of determining renal toxicity include 2D cell cultures and animal models, both of which are incapable of fully recapitulating the in vivo human response to drugs, contributing to the high failure rate upon clinical trials. We have bioengineered a 3D kidney tissue model using immortalized human renal cortical epithelial cells with kidney functions similar to that found in vivo. These 3D tissues were compared to 2D cells in terms of both acute (3 days) and chronic (2 weeks) toxicity induced by Cisplatin, Gentamicin, and Doxorubicin using both traditional LDH secretion and the pre-clinical biomarkers Kim-1 and NGAL as assessments of toxicity. The 3D tissues were more sensitive to drug-induced toxicity and, unlike the 2D cells, were capable of being used to monitor chronic toxicity due to repeat dosing. The inclusion of this tissue model in drug testing prior to the initiation of phase I clinical trials would allow for better prediction of the nephrotoxic effects of new drugs.

Sustainable Three-Dimensional Tissue Model of Human Adipose Tissue

The need for physiologically relevant sustainable human adipose tissue models is crucial for understanding tissue development, disease progression, in vitro drug development and soft tissue regeneration. The coculture of adipocytes differentiated from human adipose-derived stem cells, with endothelial cells, on porous silk protein matrices for at least 6 months is reported, while maintaining adipose-like outcomes. Cultures were assessed for structure and morphology (Oil Red O content and CD31 expression), metabolic functions (leptin, glycerol production, gene expression for GLUT4, and PPARγ) and cell replication (DNA content). The cocultures maintained size and shape over this extended period in static cultures, while increasing in diameter by 12.5% in spinner flask culture. Spinner flask cultures yielded improved adipose tissue outcomes overall, based on structure and function, when compared to the static cultures. This work establishes a tissue model system that can be applied to the development of chronic metabolic dysfunction systems associated with human adipose tissue, such as obesity and diabetes, due to the long term sustainable functions demonstrated here.

A simple meningococcal sepsis prognostic score: focusing on the human animal

A simple cheap meningococcal sepsis prognostic score based on readily available, rapid, objective laboratory base excess and platelet count was developed and validated retrospectively. This BEP score should facilitate sepsis clinical trials, allowing study of the relevant human animal model.

Culture models to study leukocyte trafficking across the choroid plexus

BackgroundA critical point during the course of central nervous system infection is the influx of leukocytes from the blood into the brain across the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). However, experimental in vitro models to investigate leukocyte transmigration across cultured choroid plexus epithelial cells have been lacking so far.MethodsWe have developed a porcine and human “inverted” culture insert system that enables leukocyte transmigration specifically from the physiologically relevant basolateral side. The models use primary porcine choroid plexus epithelial cells (PCPEC) and human choroid plexus papilloma cells (HIBCPP). As a prerequisite for a functional barrier, we optimized culture conditions in which cells are maintained in serum-containing medium until high barrier function is reached. Leukocyte transmigration through the plexus epithelial cells is analysed by three-dimensional Apotome®-imaging and electron microscopy, and the route of transmigration through the plexus epithelial cells, i.e. transcellular as well as paracellular, can be determined.DiscussionAs a functionally relevant porcine and human BCSFB model, PCPEC and HIBCPP respectively, offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens. Moreover, our in vitro models facilitate the investigation of leukocyte entry into the CNS via the blood-CSF barrier.

Abstract A36: Resveratrol decreases DNMTs in a human relevant animal model of breast cancer

Abstract Background: We previously reported that trans-resveratrol (resveratrol) downregulates the expression DNA methyltransferases (DNMTs) 1 and 3b in vitro and demethylates the tumor suppressor gene RASSF-1a in the breasts of women who are at increased breast cancer risk. Effects increased with increasing dose/circulating level of the agent, respectively. In the current report, we evaluated the effect of increasing doses of resveratrol on mammary tumor formation in the ACI rat, which develops estrogen dependent mammary carcinoma. Methods: 91 female rats were treated with a slow release estradiol (E2) pellet plus one of the following: 5 mg/kg/day (low dose) resveratrol (n=23), 25 mg/kg/day (high dose) resveratrol (n=22), 0.1mg/kg/week 5-azacytadine (aza) (n=22), an inhibitor of DNMTs which is used clinically, or control (n=24). The animals were examined weekly to assess for the presence of tumor formation. After 5.5 months of treatment, the animals were sacrificed and the mammary glands harvested, with half of each gland snap frozen and the other half formalin fixed. Specimens were evaluated by a board certified pathologist to confirm histology as benign or malignant. DNMT1 and 3b were measured by qRT-PCR. Results: Tumor formation was lowest (18% of rats) in the high dose resveratrol and 5-aza groups, intermediate in the low dose resveratrol group (26%) and highest in control (33%). DMNT1 expression was significantly (p&amp;lt;0.0001) influenced by treatment, while DNMT3b was not. On the other hand, DNMT3b expression was significantly (p&amp;lt;0.0001) influenced by histology (benign vs. malignant), whereas DNMT1 was not. Conclusion: Treatment with resveratrol and 5-aza significantly influence DNMT expression, which may be important in the prevention/delay of mammary tumor formation. This mechanism of action of resveratrol warrants further investigation. Citation Format: Wenyi Qin, Weizhu Zhu, Ke Zhang, Kaitlin Clarke, Edward R. Sauter. Resveratrol decreases DNMTs in a human relevant animal model of breast cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A36.

Marked Hyperglycemia Attenuates Anesthetic Preconditioning in Human-induced Pluripotent Stem Cell-derived Cardiomyocytes

Anesthetic preconditioning protects cardiomyocytes from oxidative stress-induced injury, but it is ineffective in patients with diabetes mellitus. To address the role of hyperglycemia in the inability of diabetic individuals to be preconditioned, we used human cardiomyocytes differentiated from induced pluripotent stem cells generated from patients with or without type 2 diabetes mellitus (DM-iPSC- and N-iPSC-CMs, respectively) to investigate the efficacy of preconditioning in varying glucose conditions (5, 11, and 25 mM). Induced pluripotent stem cells were induced to generate cardiomyocytes by directed differentiation. For subsequent studies, cardiomyocytes were identified by genetic labeling with enhanced green fluorescent protein driven by a cardiac-specific promoter. Cell viability was analyzed by lactate dehydrogenase assay. Confocal microscopy was utilized to measure opening of the mitochondrial permeability transition pore and the mitochondrial adenosine 5'-triphosphate-sensitive potassium channels. Isoflurane (0.5 mM) preconditioning protected N-iPSC- and DM-iPSC-CMs from oxidative stress-induced lactate dehydrogenase release and mitochondrial permeability transition pore opening in 5 mM and 11 mM glucose. Isoflurane triggered mitochondrial adenosine-5'-triphosphate-sensitive potassium channel opening in N-iPSC-CMs in 5 mM and 11 mM glucose and in DM-iPSC-CMs in 5 mM glucose; 25 mM glucose disrupted anesthetic preconditioning-mediated protection in DM-iPSC- and N-iPSC-CMs. The opening of mitochondrial adenosine 5'-triphosphate-sensitive potassium channels are disrupted in DM-iPSC-CMs in 11 mM and 25 mM glucose and in N-iPSC-CMs in 25 mM glucose. Cardiomyocytes derived from healthy donors and patients with a specific disease, such as diabetes in this study, open possibilities in studying genotype- and phenotype-related pathologies in a human-relevant model.

Polar Invasion and Translocation of Neisseria meningitidis and Streptococcus suis in a Novel Human Model of the Blood-Cerebrospinal Fluid Barrier

Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.