Efficacy Screening Research Articles

Curtailment: A method to reduce the length of self-report questionnaires while maintaining diagnostic accuracy

Minimizing the respondent burden and maximizing the classification accuracy of tests is essential for efficacious screening for common mental health disorders. In previous studies, curtailment of tests has been shown to reduce average test length considerably, without loss of accuracy. In the current study, we simulate Deterministic (DC) and Stochastic (SC) Curtailment for three self-report questionnaires for common mental health disorders, to study the potential gains in efficiency that can be obtained in screening for these disorders. The curtailment algorithms were applied in an existing dataset of item scores of 502 help-seeking participants. Results indicate that DC reduces test length by up to 37%, and SC reduces test length by up to 46%, with only very slight decreases in diagnostic accuracy. Compared to an item response theory based adaptive test with similar test length, SC provided better diagnostic accuracy. Consequently, curtailment may be useful in improving the efficiency of mental health self-report questionnaires.

Challenges in clinical trial design for recurrent glioblastoma

Glioblastomas are rare tumors but aggressive and often incurable. Since clinical trial enrollment is limited by the relative rarity of the disease, clinical trials should be well designed so that advances are made efficiently. The field has been dominated by smaller Phase II studies since they provide the initial screening of drug efficacy. These studies are prone to issues of selection bias, inappropriate historic controls and confounding clinical variables. There are also issues specific to glioblastoma, including the difficulty in interpreting radiographic responses in the setting of treatment effects and agents that affect vascular permeability and the difficulty of performing pharmacokinetic and pharmacodynamic studies given the relative inaccessibility of the CNS compartment. These barriers have also hindered the development of radiographic and molecular biomarkers. As we move into the era of personalized medicine, it is increasingly important to address these issues in the design of clinical trials.

How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria‐targeted drugs

Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here, we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, including ubiquinone mimetics idebenone and decylubiquinone, and the antimalarial atovaquone. Zebrafish (Danio rerio) embryos were chronically and acutely exposed to mitochondrial inhibitors and quinone analogues. Concentration-response curves, developmental and cardiovascular phenotyping were performed together with sequence analysis of inhibitor-binding mitochondrial subunits in zebrafish versus mouse, human and parasites. Phenotype rescuing was assessed in co-exposure assays. Complex I and II inhibitors induced developmental abnormalities, but their submaximal toxicity was not additive, suggesting active alternative pathways for complex III feeding. Complex III inhibitors evoked a direct normal-to-dead transition. ATP synthase inhibition arrested gastrulation. Menadione induced hypochromic anaemia when transiently present following primitive erythropoiesis. Atovaquone was over 1000-fold less lethal in zebrafish than reported for Plasmodium falciparum, and its toxicity partly rescued by the ubiquinone precursor 4-hydroxybenzoate. Idebenone and decylubiquinone delayed rotenone- but not myxothiazol- or antimycin-evoked cardiac dysfunction. This study characterizes pharmacologically induced mitochondrial dysfunction phenotypes in zebrafish, laying the foundation for comparison with future studies addressing mitochondrial dysfunction in this model organism. It has relevant implications for interpreting zebrafish disease models linked to complex I/II inhibition. Further, it evidences zebrafish's potential for in vivo efficacy or toxicity screening of ubiquinone analogues or antiparasitic mitochondria-targeted drugs.

Multicenter phase I/II trial of topotecan (T) and ifosfamide (I) combination as second-line therapy for pediatric solid cancer: Phase II results.

10050 Background: Both of T and I are key drugs for recurrent pediatric solid malignancies but rarely used in combination. Results of the phase I portion of the combination of T and I chemotherapy (TI) which is dose-finding study by CRM method in ASCO 2010. We hereby report the results of efficacy and safety screening of TI conducted as a phase II portion. Methods: Recurrent pediatric solid malignancies with history of chemotherapy no more than 20 cycles (Cs) are eligible. Combination of I (1.2g/m2/day) and T (0.6 mg/m2/day) were administered day 1 through 5 up to six Cs. In the phase II portion, a Simon optimal two-stage design was adopted using treatment compliance with 4 Cs of TI as primary endpoint. Unacceptable compliance was set at 20% and promising one at 40%. If more than two patients (pts) among initial 13-15 pts successfully complete 4 Cs, patients’ enrollment is allowed to continue until a total of 33 pts in the phase II portion. Complete or Partial Response (CR+PR) was evaluated according to RECIST 1.0 criteria and toxicity, CTCAE v3.0. Results: 35 pts, median age 12 years (range 1-28) has been registered in both phase I and II between March 2008 and January 2013 in 8 centers: 7 neuroblastoma (NB), 8 rhabdomyosarcoma (RMS), 8 osteosarcoma (OS), 6 Ewing’s sarcoma (ES), 2 hepatoblastoma (HB), and 4 others. 13 pts out of total 34 pts (38%) completed 6 Cs. 13 pts out of 28 pts (36%, the phaseII portion) received more than 4 Cs without meeting stopping criteria of protocol treatment. This has reached the predefined efficient level. No Grade 4 non-hematological toxicity occurred. Grade 2-4 fatigue, anorexia, and nausea were 20% or less. Grade 3 or 4 neutropenia was frequent (105/127 Cs, 83%), though only 9 % (12 Cs) developed febrile neutropenia. 2 CR (HB, ES) and 2 PR (RMS, OS) were observed at best response among 22 pts with measurable disease, leading to an estimated OR rate of 18% (95%CI 5-40%). 14 patients had disease stabilization. One year progression-free survival rate and overall survival rate are 21% and 64%. Conclusions: TI achieved very good compliance and favorable toxicity as well as good tumor control rate. This combination is a new promising 2nd line therapy for pediatric solid malignancies. Clinical trial information: UMIN000001037.

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

On being the right size: scaling effects in designing a human-on-a-chip

Developing a human-on-a-chip by connecting multiple model organ systems would provide an intermediate screen for therapeutic efficacy and toxic side effects of drugs prior to conducting expensive clinical trials. However, correctly designing individual organs and scaling them relative to each other to make a functional microscale human analog is challenging, and a generalized approach has yet to be identified. In this work, we demonstrate the importance of rational design of both the individual organ and its relationship with other organs, using a simple two-compartment system simulating insulin-dependent glucose uptake in adipose tissues. We demonstrate that inter-organ scaling laws depend on both the number of cells and the spatial arrangement of those cells within the microfabricated construct. We then propose a simple and novel inter-organ 'metabolically supported functional scaling' approach predicated on maintaining in vivo cellular basal metabolic rates by limiting resources available to cells on the chip. This approach leverages findings from allometric scaling models in mammals that limited resources in vivo prompt cells to behave differently than in resource-rich in vitro cultures. Although applying scaling laws directly to tissues can result in systems that would be quite challenging to implement, engineering workarounds may be used to circumvent these scaling issues. Specific workarounds discussed include the limited oxygen carrying capacity of cell culture media when used as a blood substitute and the ability to engineer non-physiological structures to augment organ function, to create the transport-accessible, yet resource-limited environment necessary for cells to mimic in vivo functionality. Furthermore, designing the structure of individual tissues in each organ compartment may be a useful strategy to bypass scaling concerns at the inter-organ level.

RNA Interference Targeting Connective Tissue Growth Factor Inhibits the Transforming Growth Factor-β2Induced Proliferation in Human Tenon Capsule Fibroblasts

Purpose. This study was to determine the effect of CTGF-small interfering RNA (siRNA) on TGF-β 2-induced proliferation in human Tenon capsule fibroblasts (HTFs). Methods. HTFs were transfected with four of CTGF-siRNAs separately for screening of gene silencing efficacy that was determined by transcript level measured by quantitative real-time PCR (qRT-PCR). Recombinant TGF-β 2 was added into the culture to stimulate the proliferation of HTFs. The gene silencing efficacy of the siRNAs was evaluated by qRT-PCR and immunofluorescence of CTGF transcript and protein levels. The viability of HTFs was determined by cell counting kit-8 (CCK-8). FCM was used to assess cell cycle after CTGF-siRNA transfection. Results. The expression of CTGF and proliferation of HTFs were increased significantly by TGF-β 2 stimulation. The transfection of CTGF-siRNA abolished the upregulation of CTGF and cell proliferation induced by TGF-β 2. The analysis of cell cycle indicated that CTGF-siRNA treatment stimulated cells from S phase to G0/G1 phase in comparison with the inverse physiologic function of TGF-β 2. Conclusion. CTGF targeting siRNA could effectively suppress the expression of CTGF and attenuate the proliferation of HTFs. The siRNA approach may provide a therapeutic option for eliminating filtration bleb scarring after glaucoma filtration surgery (GFS).

Oophorectomy

Ovarian cancer remains the fifth deadliest cancer among women because of its early asymptomatic nature and lack of efficacious screening methods, leading to frequent late-stage diagnosis. Elective oophorectomy is an option for women undergoing benign hysterectomy as a means of reducing their ovarian cancer risk. Benefits also include reduced risk of repeat surgical operation due to adnexal masses and reduced anxiety related to perceived risk of ovarian and breast cancer. The potential negative side effects of elective oophorectomy, such as decreased cognition and sexual function and increased risk of osteoporosis and cardiac mortality, offer support for ovarian conservation. The implications of this elective procedure and the possible consequences without it require physicians to review the pros and cons with patients in light of the patient's individual circumstances and ovarian cancer risk.

Multimodality Imaging of Tumor and Bone Response in a Mouse Model of Bony Metastasis

Cancer drug development generally performs in vivo evaluation of treatment effects that have traditionally relied on detection of morphologic changes. The emergence of new targeted therapies, which may not result in gross morphologic changes, has spurred investigation into more specific imaging methods to quantify response, such as targeted fluorescent probes and bioluminescent cells. The present study investigated tissue response to docetaxel or zoledronic acid (ZA) in a mouse model of bony metastasis. Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored throughout this study using several modalities: molecular resonance imaging (MRI) tumor volume and apparent diffusion coefficient (ADC), micro-computed tomography (µCT) bone volume, bioluminescence imaging (BLI) reporting cancer cell apoptosis, and fluorescence using Osteosense 800 and CatK 680-FAST. Docetaxel treatment resulted in tumor cell kill reflected by ADC and BLI increases and tumor volume reduction, with delayed bone recovery seen in µCT prefaced by increased osteoblastic activity (Osteosense 800). In contrast, the ZA treatment group produced similar values in MRI, BLI, and Osteosense 800 fluorescence imaging readouts when compared to controls. However, µCT bone volume increased significantly by the first week post-treatment and the CatK 680-FAST signal was slightly diminished by 4 weeks following ZA treatment. Multimodality imaging provides a more comprehensive tool for new drug evaluation and efficacy screening through identification of morphology as well as function and apoptotic signaling.

R&D Outsourcing: AstraZeneca teams up with two major research contractors

The British drugmaker AstraZeneca has established two research outsourcing agreements as part of the restructuring of its R&D organization. In Beijing, the Chinese pharmaceutical R&D contractor Pharmaron will set up a dedicated research center for AstraZeneca that can accommodate “several hundred” scientists, according to Pharmaron. Under a multiyear agreement, the facility will provide AstraZeneca discovery services in chemistry, drug metabolism and pharmacokinetics, and efficacy screening. It will be located within a campus where Pharmaron already operates an R&D center for Merck Serono. AstraZeneca also selected Charles River Laboratories, with headquarters in Wilmington, Mass., as its preferred partner for regulated safety assessment and development drug metabolism and pharmacokinetics. The firms say the deal will increase Charles River’s annual sales, which were $1.1 billion last year, by about 1%. AstraZeneca is in the midst of a major restructuring of its R&D organization. Early this year, it annou...

Abstract PL05-04: Introducing HPV screening into low-resource settings: Opportunities and challenges.

Abstract Despite the promise of a highly efficacious prophylactic human papillomavirus (HPV) vaccination, screening and treatment of precancerous lesions will be needed for generations to come. In its current form, HPV vaccination will not prevent approximately 30% of invasive cervical cancer, does not treat pre-existing infections and precancerous lesions, and remains expensive and not yet feasible for many countries. Even if vaccines were made widely available at a greatly reduced cost and widespread vaccination could be rapidly implemented globally, the benefits of HPV vaccination will not be realized in 30 years or more. With adequate screening and treatment, the vast majority of cervical cancer can be prevented during the typical 10-15 year precancerous period. Unfortunately, successful screening programs in low resource settings have been difficult to achieve because of insensitive screening tests, low population coverage, and inadequate follow-up of screen positives. Cervical screening for carcinogenic HPV infection is being considered for low income countries because HPV testing provides excellent risk stratification and is amenable to vaginal self-collection. Fortunately, tests are being developed for low-resource settings. The natural history of HPV and cervical cancer informs the relative advantage of HPV screening compared to other screening tests that aim to detect precancerous lesions. In most populations, the age-specific prevalence pattern of HPV infections observed in sexually active women resembles that of a typical sexually transmissible infection. Incidence and prevalence peak at young ages soon after the start of sexual activity, with a subsequent decline as women age because their infections clear and fewer incident infections are acquired. The high sensitivity of HPV testing suggests that one or two screens in a lifetime, starting around 30 years of age and ending by menopause, before invasive cervical cancer arises, would be sufficient to substantially reduce cervical cancer mortality. This superior performance was observed in a large community randomized trial from India where HPV screening reduced mortality in just 7 years. The high sensitivity of HPV testing means that most women (80%–90%) who test HPV negative will have reassurance for at least 10 years that they are at low risk of cervical cancer. Numerous studies have demonstrated the long-term negative predictive value of HPV testing. Even when an affordable HPV test is available for low resource settings, challenges remain for successful implementation of HPV screening. Although vaginal sampling allows increased population coverage, it has been associated with loss of sensitivity. Women who screen HPV-positive must be managed adequately and various approaches with unique limitations have been considered. Finally, not all populations experience the same epidemiologic HPV-age trends where prevalence declines in older women. Some population-based HPV studies conducted in Sub-Saharan Africa observed elevated HPV prevalence in older women. In addition, the relationship between HPV and cervical cancer is further complicated in some settings by HIV infection. Compared to HIV-negative women, HIV-positive women are at an increased risk of HPV infection. Once infected, they also have a greater risk of persistent infection and cervical precancer (even with access to HIV antiretroviral therapies). This talk will review HPV epidemiologic research pertaining to the incorporation of HPV testing into cervical cancer prevention initiatives for traditionally underserved communities and low-resource settings. Citation Format: Julia C. Gage. Introducing HPV screening into low-resource settings: Opportunities and challenges. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL05-04.

Genetically Engineered Cancer Models, But Not Xenografts, Faithfully Predict Anticancer Drug Exposure in Melanoma Tumors

Rodent studies are a vital step in the development of novel anticancer therapeutics and are used in pharmacokinetic (PK), toxicology, and efficacy studies. Traditionally, anticancer drug development has relied on xenograft implantation of human cancer cell lines in immunocompromised mice for efficacy screening of a candidate compound. The usefulness of xenograft models for efficacy testing, however, has been questioned, whereas genetically engineered mouse models (GEMMs) and orthotopic syngeneic transplants (OSTs) may offer some advantages for efficacy assessment. A critical factor influencing the predictability of rodent tumor models is drug PKs, but a comprehensive comparison of plasma and tumor PK parameters among xenograft models, OSTs, GEMMs, and human patients has not been performed. In this work, we evaluated the plasma and tumor dispositions of an antimelanoma agent, carboplatin, in patients with cutaneous melanoma compared with four different murine melanoma models (one GEMM, one human cell line xenograft, and two OSTs). Using microdialysis to sample carboplatin tumor disposition, we found that OSTs and xenografts were poor predictors of drug exposure in human tumors, whereas the GEMM model exhibited PK parameters similar to those seen in human tumors. The tumor PKs of carboplatin in a GEMM of melanoma more closely resembles the tumor disposition in patients with melanoma than transplanted tumor models. GEMMs show promise in becoming an improved prediction model for intratumoral PKs and response in patients with solid tumors.

Targeted Provision of Oral Iron: The Evolution of a Practical Screening Option

AbstractUniversal oral iron supplementation, undertaken according to 1998 WHO guidelines, produced adverse consequences among some children in malaria-endemic areas. Prompted by the Pemba trial, which revealed excessive hospitalizations and deaths, WHO advised that iron supplementation in such regions be accompanied by previous screening for iron deficiency. This agenda, however, poses issues of cost, benefit, acceptability, technical feasibility, and reliability of such screening. The cost of equipment and personnel is balanced against savings from iron supplements spared and treatment for morbidity averted. Costs aside, the most efficacious acceptable screening approach for avoiding hospitalization and deaths must be fielded. Screening before supplementation can be used to assess hematological, iron, and possible inflammatory status to differentiate the source of decreased hemoglobin concentration. Iron deficiency has often been inferred from hematological status markers. The need for extraction of blood, albeit capillary in origin, and high assay costs limit the use of validated methods in screening. Noninvasive methods, i.e., not requiring the extraction of blood, provide the most acceptable and potentially least expensive approach for determining hematological or iron status. Although a noninvasive technique for iron and inflammatory status would be the ideal, it is unattained. Field-friendly, skin-probe hemoglobin devices, derived from instruments for clinical settings, are being developed and tested for eventual rollout in malarial areas. Given a firm grounding for the theoretical requirements needed to advance the screening agenda, evaluation and monitoring of the performance of screening devices can proceed hand in hand.

O2‐05‐06: Mechanistic investigation of nitrate chimeras for Alzheimer's disease

Alzheimer's Disease is multifactorial and elicits diverse neurological and psychiatric abnormalities, and therefore presents a unique challenge in drug development. The earliest event in Alzheimer's Disease (AD) is synaptic failure, making the disease fundamentally a disorder of impaired cognition and memory. A drug that targets synaptic failure and secondarily prevents neuronal loss would likely prove beneficial to AD patients, and recent efforts showing modification of neuropathology after increased synaptic plasticity suggest these effects would be disease modifying. Nomethiazoles are chimeric compounds that combine the neuroprotective GABA-potentiating activity of the clinically used compound, chlormethiazole (CMZ), and add the ability to activate CREB via NO-mimetic actions. The transcription factor cAMP responsive element-binding protein (CREB) has been shown to be necessary for synaptic plasticity and long-term memory formation, and can be activated through second messenger cascades initiated by NO. In AD, decreased activity of pCREB has been observed in post-mortem AD brains, and molecular network analysis converges on aberrant CREB-mediated gene regulation in the AD hippocampus. Protection from insults in primary neuronal culture, reversal of deficits of long-term potentiation in aged transgenic mice, and long-term in vivo studies with behavioral and biochemical endpoints were used both for efficacy screening and investigation into mechanism of action for all novel compounds. In primary neuronal culture models of excitotoxicity and amyloid-induced dysfunction, neuroprotection in some novel compounds correlated to the GABA-potentiating ability of methiazole backbones, while other compounds worked through an unknown mechanism. In electrophysiological investigation of synaptic plasticity in hippocampal slices, reversal of deficits in LTP in 3xTg mice were found to be mediated through an NO/cGMP mechanism. Additionally, in long-term in vivo studies in 3xTg models of Alzheimer's disease, procognitive effect was demonstrated in STPA, a behavioral model of memory, which neatly paralleled increases in levels of pCREB. Finally, these neurorestorative effects in vivo also led to a decrease in levels of Aβ, tau and the neuroinflammatory/apoptotic marker TNF-Îα. The above investigation resulted in four optimized novel nomethiazoles slated for pre-IND submission in 2013, with a defined mechanism of action through GABA potentiation and pCREB activation.

Early Stage Efficacy and Toxicology Screening for Antibiotics and Enzyme Inhibitors

The rise in organisms resistant to existing drugs has added urgency to the search for new antimicrobial agents. Aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in an essential microbial pathway that is absent in mammals. Our laboratory is using fragment library screening to identify efficient and selective ASADH inhibitors. These preliminary agents are then tested to identify compounds with desired antimicrobial properties for further refinement. Toward this end, we have established a microplate-based, dual-assay approach using a single reagent to evaluate antibiotic activity and mammalian cell toxicity during early stage development. The bacterial assay uses nonpathogenic bacteria to allow efficacy testing without a dedicated microbial laboratory. Toxicity assays are performed with a panel of mammalian cells derived from representative susceptible tissues. These assays can be adapted to target other microbial systems, such as fungi and biofilms, and additional mammalian cell lines can be added as needed. Application of this screening approach to antibiotic standards demonstrates the ability of these assays to identify bacterial selectivity and potential toxicity issues. Tests with selected agents from the ASADH inhibitor fragment library show some compounds with antibiotic activity, but as expected, most of these early agents display higher than desired mammalian cell toxicity.

Metabolism of ibuprofen in zebrafish larvae

The application of zebrafish (Danio rerio) larvae to drug discovery assays and toxicity testing, and the occurrence of pharmaceuticals in the environment, has resulted in a need to understand the extent of the metabolic capabilities in the early life stages of this species.The aims of this study were to determine if zebrafish larvae absorbed, metabolized and excreted the model pharmaceutical, ibuprofen. Zebrafish larvae (72 h post fertilization) were exposed to ibuprofen (100 µg/L), 14C-ibuprofen (100 µg/L) or a solvent control (ethanol) for ≤24 h. Water samples and larval extracts were assessed for metabolites of ibuprofen using liquid chromatography mass spectrometry (LC–MS–MS). Fractions from the separation of the samples treated with 14C-ibuprofen were collected after chromatography and analysed for 14C content by scintillation counting.Assessment of larval extracts and water samples by LC–MS–MS at 24 h resulted in the identification of hydroxy-ibuprofen in both water samples and larval extracts (8.2 and 0.08% of the total detected 14C, respectively). A second putative hydroxy-ibuprofen moiety was also observed in water samples at trace levels, and a third minor unknown metabolite was detected in larval extracts only by scintillation counting (0.02% of the total 14C detected).This study provides evidence that zebrafish larvae can metabolize and excrete ibuprofen in a manner known to be cytochrome P450-dependent in mammals, and the similarity to the mammalian pathway supports the use of this system as a surrogate in toxicity and efficacy screening.

Effects of applying glutaraldehyde-containing desensitizer formulations on reducing dentin permeability

Background/purposeThe efficacy of dentin-desensitizing agents is commonly evaluated in clinical studies by measuring patients’ pain response upon stimulation. Although indispensable, such trials are time-consuming, and the results depend on an individual’s subjective pain rating. Therefore, in vitro efficacy screening prior to clinical testing is highly desirable. The objective of this study was to investigate in vitro dentin permeability of two glutaraldehyde-containing desensitizer formulations after different modes and times of application. Materials and methodsCoronal tooth slices, 1.3mm thick, were dissected from 60 freshly extracted third molars. Specimens were treated with EDTA to remove the cutting smear. The dentin disks were clamped in a split chamber device to determine the baseline permeability under a liquid pressure of 2.5kPa for 2 minutes and 13kPa for 1 minute, to record liquid flow through the dentin using a photochemical method. Slices were soaked in a 2% albumin solution and reevaluated under the same pressure cycles prior to active or passive application for 15, 30, or 60 seconds of either Gluma Desensitizer or Gluma PowerGel (GDP) (Heraeus Kulzer, Hanau, Germany) and then reevaluated. Dentin-disk permeability was determined as the area under the photo signal output voltage line during the pressurizing period (mV s). The statistical data analysis used a Kruskal-Wallis ANOVA and Mann-Whitney post-hoc test with the significance level set to 5%. ResultsPermeability at the baseline and after albumin soaking did not significantly differ. For both desensitizing compounds, 30 and 60 seconds of active and passive applications resulted in significantly reduced dentin permeability. After the 15 second application, only the actively treated samples with GDP showed a significant reduction in permeability. ConclusionsThe liquid and the gel desensitizing agents both significantly reduced dentin permeability. The obvious advantage of a gel formulation is the controlled application, limited to the hypersensitive tooth area, thus avoiding inadvertent contact with adjacent gingival tissues.

A transient expression assay for the in planta efficacy screening of an antimicrobial peptide against grapevine bacterial pathogens

Natural and synthetic antimicrobial peptides (AMPs) are of increasing interest as potential resistance conferring elements in plants against pathogen infection. The efficacy of AMPs against pathogens is prescreened by in vitro assays, and promising AMP candidates are introduced as transgenes into plants. As in vitro and in planta environments differ, a prescreening procedure of the AMP efficacy in the plant environment is desired. Here, we report the efficacy of the purified synthetic peptide D4E1 against the grapevine-infecting bacterial pathogens Agrobacterium vitis and Xylophilus ampelinus in vitro and describe for the first time an in planta prescreening procedure based on transiently expressed D4E1. The antimicrobial effect of D4E1 against Ag. vitis and X. ampelinus was shown by a reduction in colony-forming units in vitro in a traditional plate-based assay and by a reduction in bacterial titres in planta as measured by quantitative real-time PCR (qPCR) in grapevine leaves transiently expressing D4E1. A statistically significant reduction in titre was shown for X. ampelinus, but for Ag. vitis, a significant reduction in titre was only observed in a subset of plants. The titres of both grapevine-infecting bacterial pathogens were reduced in an in vitro assay and for X. ampelinus in an in planta assay by D4E1 application. This widens the applicability of D4E1 as a potential resistance-enhancing element to additional pathogens and in a novel plant species. D4E1 is a promising candidate to confer enhanced resistance against the two tested grapevine bacterial pathogens, and the applied transient expression system proved to be a valuable tool for prescreening of D4E1 efficacy in an in planta environment. The described prescreening procedure can be used for other AMPs and might be adapted to other plant species and pathogens before the expensive and tedious development of stably transgenic lines is started.

Abstract 4070: Development of circulating tumor cell capture from whole blood using beads and microfluidics

Abstract Capture of circulating tumor cells (CTCs) from blood shows promise as a relatively non-invasive screen for early stage metastasis, treatment efficacy, and disease progression. Capture rates exceeding 80% of CTCs from whole blood have been demonstrated in microfluidic chips, with good specificity and throughput at approximately 1 mL/h. The most common approach is to design a high surface-area microdevice and functionalize the surface with anti-EpCAM to capture epithelial tumor cells. As an alternative to this monolithic design, we have developed a CTC capture strategy based on functionalized beads for analyzing small numbers of CTCs from whole blood. Beads for the device were commercially available in a variety of sizes and surface chemistries or could be synthesized by multiple techniques. We utilized avidin-functionalized beads to enable capture using biotinylated antibodies (Ab) for surface-expressed proteins. Critically, batches of beads were assayed in bulk and then used for multiple experiments, avoiding characterization of each device. Beads packed well as columns in microdevices, ensuring multiple surface interactions as cells flow through. We simulated CTC-carrying blood by spiking human breast cancer adenocarcinoma (MFC7) cells (prestained with a membrane dye) into whole human blood between 1000 cells/mL and 105 cells/mL. Samples were delivered through the packed bed of beads using a syringe pump. We found that adding Ab to whole blood samples provided superior results to pre-coating the beads with the Ab. This approach was tested for multiple bead compositions and enabled (1) use of less Ab, and (2) higher capture rates, perhaps due to the rapid biotin:avidin binding. Significant cell capture was observed when as few as 300 cells were introduced into the device. At all cell densities, the majority of captured cells bound to the first few rows of beads, with decreasing capture frequency along the length of the column. Quantification of the absolute number of captured cells was a challenge in whole blood samples due to the large numbers of cells captured and optical distortion effects. We estimated capture by observing the initial capture rates of the same cells at the same densities in serum-spiked phosphate-buffered saline. This indicated that during the initial stages of flow, over 80% of the Ab-labeled cells were selectively captured. We conclude that the device can handle higher flowrates and still capture the majority of labeled cells. We are exploring the feasibility of this approach for capturing even lower CTC concentrations (1 cell/ml to 100 cells/mL range). Our bead/microfluidic approach simplifies CTC capture by enabling bulk surface functionalization for multiple experiments and allowing commercial sourcing of all functional elements. Further, there is flexibility in antibody selection, such that alternate Ab could rapidly be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4070. doi:1538-7445.AM2012-4070

Translating heart failure biomarkers from animal models to humans

The generation of a self‐evident paradigm where chemokines play a role in the remodeling of the heart is consistent with the idea that inflammatory molecules are responsible of the chemotactic recruitment and of a specific “remodeling oriented” inflammatory expression. In our in‐vivo experiments with animal models for heart failure (HF) such as transverse aortic constricted mice and myocardial infarcted rats we observed a common increased level of 4 novel (i.e. never investigated in this setting) circulating chemokines. The blood levels of >40 analytes were assessed semi‐quantitatively using a membrane‐bound antibody technique, called Proteome Profiler™(enhanced with fluorescence). The potential of the identified biomarkers in clinical studies, monitoring either new treatments or new strategies of clinical management, is capitalizing on recent advances in technology, bearing the promise of expediting the transition from “bench to bedside”, early diagnosis, individualized therapy and, as a consequence, better efficacy screening. In order to bridge the results obtained in the animal models with the clinic we proved the feasibility of the study using the blood of a small cohort of patients with heterogeneous HF. ELISA measurements plotted versus the membrane technique reached a linear correlation of R=0.93. These findings prompted us to begin a study in a larger cohort with established HF.