Experimental Biology and Medicine

This symposium addresses careers in drug development in industry; the performance of translational research by academia, industry, and both; and numerous factors pertinent to alliances essential to drug discovery and development. Drug development is a complex process that regularly involves effective collaborations between academic and physician scientists and industry. There are specific occupational factors affecting recruitment of scientists and physicians in drug development programs in industry; ideal backgrounds for successful applicants for positions in industry in drug development; ethical and regulatory considerations particularly germane to the performance of scientists and physicians in drug development programs in industry and at universities; and particular gratifications available to scientists in Industry working on drug development. Both similarities and differences characterize the performance of translational research in industry compared with academia. In industry, logistic, operational, and scientific oversight is complex, especially because It often Involves relationships with clinical enterprises outside of the corporation. The process is long and arduous from formulation of a good Idea in discovery to acceptance of a novel drug in the marketplace. Collaborations and partnerships by industry often involving academia and confrontation of multiple Issues are pivotal.

A career in industry has become a widely accepted alternative for those of us trained in medicine and/or science who have traditionally focused on careers in academia. Like any career decision, consideration of a position in industry should include asking yourself a series of fundamental questions. A few of the key questions should include: 1) What kind of work environment do you find most enjoyable (e.g., patient care setting, basic research lab, team-oriented setting)?; 2) What are you focused on accomplishing in your career (basic research discoveries, contributions to clinical medicine, compensation)?; 3) Are you team oriented in your interactions or are you more of an Individual contributor? A successful career in any endeavor, including industry, starts with a careful and honest examination of what you are best suited for and inspired to do.

Research that bridges between scientific insights and clinical application is one of the most active and exciting areas of current biomedical activity. Much of this translational work occurs through collaborations between academic and industrial institutions, taking advantage of the respective strengths and resources of the two sectors. However, such collaborations sometimes can be challenging due to differences between the cultures and priorities of the two parties. This article discusses the nature of translational research, with a focus on the academia-industry interface, analyzes the factors important for effective collaborations, and describes specific examples of successful translational research programs.

This manuscript briefly addresses the drug discovery and development process. It is a long road from the formulation of a good discovery idea to the acceptance of a new drug in the marketplace, and there are many challenges faced along the way to the patient. Collaborations and partnerships are an important part of this process. There are a variety of partnering opportunities, ranging from the discovery of novel technologies and drug targets to lead discovery, compound gifts, and external sourcing. These partnerships help increase confidence and improve decision making on issues of safety and efficacy preclinically, which can reduce attrition and expedite the provision of new quality drugs to patients more quickly and at lower costs. Collaborations involve addressing multiple issues that include infrastructure, safety, regulatory matters, intellectual property, technical and personnel considerations, source document capture and data analysis Issues, and legal and strategic alliances. A number of success factors are Identified as important for quality collaborations in the drug development process.

Preterm birth is associated with immature digestive function that may require the use of total parenteral nutrition and special oral feeding regimens. Little is known about the responses to oral food in the preterm neonate and how enteral nutrients affect the immature gastrointestinal tract (GIT). In vivo studies are difficult to perform in laboratory rodents because of their small body size and that of immature organs at birth, and this makes the large farm animals (e.g., pigs, cattle, sheep) more attractive models in this field. In these species, preterm delivery at 88%–95% gestation is associated clinical complications and degrees of GIT immaturity similar to those in infants born at 70%–90% gestation. Studies in both animals and infants indicate that the immature GIT responds to the first enteral food with rapid increases in gut mass and surface area, blood flow, motility, digestive capacity, and nutrient absorption. To a large extent, the enteral food responses are birth independent, and can be elicited also In utero, at least during late gestation. Nevertheless, preterm neonates show compromised GIT structure, function, and immunology, particularly when delivered by caesarean section and fed diets other than mother's milk. Formula-fed preterm infants are thus at increased risk of developing diseases such as necrotizing enterocolitis, unless special care is taken to avoid excessive nutrient fermentation and bacterial overgrowth. The extent to which results obtained in preterm animals (most notably the pig) can be used to reflect similar conditions in preterm infants is discussed.

Bovine pericardium (BPC) and polytetrafluoroethylene (PTFE) have been widely used to reinforce staple lines in lung resection. Since limited Information regarding the calcification of these biomaterials is available, we undertook an In vitro study to evaluate their calcification potential. Commercially available BPC and PTFE biomaterials were evaluated and compared with custom-prepared BPC tissue. In vitro calcification was performed via submersion in supersaturated solution In a double-walled glass reactor at 37.0°C ± 0.1°C, pH 7.4 ± 0.1, mimicking most ion concentrations of human blood plasma. In processing of calcification, the pH decrease of the solution simulated the addition of consumed H⁺, Ca²⁺, and PO₄^3– ions from titrant solutions, the concentrations of which were based on the stolchiometry of octacalcium phosphate. The molar ion addition with time was recorded, and the initial slope of the curve was computed for each experiment. The rate of calcification developed (molar calcium phosphate ion addition rate per time and total surface area) (R) was computed after that with respect to the relative supersaturation (σ) used in each experiment. R for custom-prepared BPC tissues was found to be in the range of 0.19 ± 0.08 to 0.52 ± 0.19 (n = 17) in σ range of 0.72 to 1.42. Commercial BPC was found to be 0.016 to 0.052 (n = 4), and PTFE was 0.005 to 0.05 (n = 8) in the same σ range. Both clinically applied biomaterials, BPC and PTFE, seemed to be calcified with rates of at least one order of magnitude lower than the custom-prepared BPC tissue. This data suggested that BPC and PTFE biomaterials showed a similar, relatively very low tendency for calcification compared with custom-prepared BPC tissue. Although further studies are necessary, staple line reinforcement by these two biomaterials should be considered safe from the calcification point of view.

To further develop the hen as a model of ovarian adenocarcinoma, we have studied normal and neoplastic ovaries as well as cultured cells from the ovarian surface epithelium (OSE). We characterized the OSE layer of the hen for specific histologic markers and evaluated these markers on tumor tissue. We also isolated and characterized the epithelial cells that are the likely source of the ovarian tumors of the hen. The surface epithelium of normal ovaries demonstrated positive staining for cytokeratin, proliferating cell nuclear antigen (PCNA), progesterone receptor (PR), and negative staining for vimentin. Ovarian tumors demonstrated positive cytokeratin, PCNA, PR, and weak vimentin staining in the gland-like areas. Epithelial cell cultures were obtained by an explant method utilizing small and large yellow follicles. These cells were positive for cytokeratin and negative for vimentin on Days 1 and 3. By Day 10, cytokeratin protein expression was less for some cells, and vimentin expression was weakly present in some cells. Expression of PCNA was observed at Days 1 and 3, but was rarely seen in cells cultured for 10 days. Expression of PR was observed on Day 10 after 24-hr estrogen treatment. Epithelial cells grew slowly in culture, and were susceptible to trypsin or other dissociation treatments.

There is increasing evidence showing dual functions of antioxidant enzymes in coping with reactive oxygen species (ROS) versus reactive nitrogen species (RNS). The objective of this study was to compare the impacts of knockout of Cu, Zn-superoxide dismutase (SOD1) and Se-dependent glutathione peroxidase-1 (GPX1) on cell death and related signaling mediated by acetaminophen (APAP), a RNS inducer in liver. Two groups of young adult knockout mice (SOD1^−-/– and GPX1^−-/–), along with their wild types (WT), were killed 5 hrs after an ip injection of saline or APAP (300 mg/kg body wt). While the WT mice showed more hepatic necrosis and DNA breakage than the GPX1^−-/– mice, the SOD1^−-/– mice had essentially no positive response compared with their saline-injected controls. The APAP treatment activated liver c-jun N-terminal kinase (JNK) in the WT and GPX1^−-/– mice, but not in the SOD1^−-/– mice. The APAP-induced changes in other cell death-related signal proteins such as p21, caspase-3, and poly(ADP-ribose) polymerase (PARP) also were obviated in the SOD1^−-/– mice. In conclusion, knockout of GPX1 did not potentiate APAP-induced cell death and related signaling, whereas the SOD1 null blocked APAP-induced hepatic JNK phosphorylation and cell death.

Fruits and vegetables are the major sources of biologically active compounds, and carotenoids and tocopherols constitute important groups in human diets. Bioavailability is a critical feature in the assessment of the role of micronutrients in human health, and the approaches to this issue include in vitro and in vivo methods. Our aim was to evaluate the bioavailability of carotenoids and tocopherols present in broccoli and to compare in vitro and in vivo approaches. Fourteen apparently healthy volunteers consumed 200 g broccoli once a day for seven days. Blood samples were drawn at baseline and after intervention to determine changes in lutein, β-carotene, and Α- and γ-tocopherol as relevant phytochemicals provided with this vegetable. Broccoli also was subjected to simulated gastrointestinal digestion to assess changes related to preabsorptive processes. Analytes in serum and at each phase of the digestion were assayed by high-performance liquid chromatography. During the intervention, the amounts supplied dally ranged from 2.4 to 3.1 mg lutein, 1.4 to 1.8 mg β-carotene, 4.5 to 6.8 mg Α-tocopherol, and 0.8 to 1.8 mg γ-tocopherol.

Significant changes in serum in both men and women were observed only for lutein, whereas for γ-tocopherol a significant change was detected in women. No changes were observed for Α-tocopherol, β-carotene, retinol, the Α-tocopherol-to-cholesterol ratio, or serum lipids. Using the in vitro model, more than 75% of lutein, β-carotene, γ-tocopherol, and Α-tocopherol remained at the duodenal phase, whereas Incorporation Into the supernatants accounted for <20% of the initial content in food. Regular consumption of broccoli at dietary levels increased serum concentrations of lutein and γ-tocopherol without affecting Α-tocopherol or β-carotene status in serum. The behavior of these phytochemicals under in vitro gastrointestinal conditions does not fully explain the changes observed in vivo.

A number of traditional medicine plants are hepatotoxic. Thus, while the traditional uses of Atuna racemosa suggest little indication for toxicity, it is nonetheless important to examine the potential for this extract to target the liver. Using Jurkat T cells and HepG2 hepatocytes as a model, the potential hepatotoxicity of this extract was evaluated. The results of a conditioned media experiment suggest that A. racemosa extract would likely be detoxified by the liver. These results provide the necessary background to Initiate an in vivo toxicology investigation.

In this study we examined the expression of cytochrome P450 (CYP) 2C and CYP2J Isoforms in renal proximal tubules and microvessels isolated from rats at different stages of pregnancy. We also selectively inhibited epoxyeicosatrienoic acid (EET) production by the administration of N-methanesulfonyl-6-(2-proparyloxyphenyl)hexanamide (MSPPOH 20 mg/kg/day iv) to rats during Days 14–17 of gestation and to age-matched virgin rats and determined the consequent effects on renal function. Western blot analysis showed that CYP2C11, CYP2C23, and CYP2J2 expression was significantly increased in the renal microvessels of pregnant rats on Day 12 of gestation. In the proximal tubules, CYP2C23 expression was significantly increased throughout pregnancy, while the expression of CYP2C11 was increased in early and late pregnancy and the expression of CYP2J2 was increased in middle and late pregnancy. MSPPOH treatment significantly Increased pregnant rats’ mean arterial pressure, renal vascular resistance, and sodium balance but significantly decreased renal blood flow, glomerular filtration rate, and urinary sodium excretion, as well as fetal pups’ body weight and length. In contrast, MSPPOH treatment had no effect on renal hemodynamics or urinary sodium excretion in age-matched virgin rats. In pregnant rats, MSPPOH treatment also caused selective inhibition of renal cortical EET production and significantly decreased the expression of CYP2C11, CYP2C23, and CYP2J2 in the renal cortex, renal microvessels, and proximal tubules. These results suggest that upregulation of renal vascular and tubular EETs contributes to the control of blood pressure and renal function during pregnancy.

Human embryonic stem cells (hESCs) can be coaxed to differentiate Into specific cell types, including cardiomyocyte-like cells. These cells express cardiac-specific markers and display functional similarities to their adult counterparts. Based on these properties, hESC-derived cardiomyocytes have the potential to be extremely useful in various in vitro applications and to provide the opportunity for cardiac cell replacement therapies. However, before this can become a reality, the molecular and functional characteristics of these cells need to be Investigated in more detail. In the present study we differentiate hESCs into cardiomyocyte-like cells via embryoid bodies (EBs). The fraction of spontaneously beating clusters obtained from the EBs averaged approximately 30% of the total number of EBs used. These cell clusters were isolated, dissociated into single-cell suspensions, and frozen for long-term storage. The cryopreserved cells could be successfully thawed and subcultured. Using electron microscopy, we observed Z discs and tight Junctions in the hESC-derived cardiomyocytes, and by Immunohistochemical analysis we detected expression of cardiac-specific markers (cTnl and cMHC). Notably, using BrdU labeling we also could demonstrate that some of the hESC-derived cardiomyocytes retain a proliferative capacity. Furthermore, pharmacological stimulation of the cells resulted in responses Indicative of functional adrenergic and muscarinic receptor coupling systems. Taken together, these results lend support to the notion that hESCs can be used as a source for the procurement of cardiomyocytes for in vitro and in vivo applications.