bluuebunny/crossref_metadata_2025 huggingface dataset:
Translational Vestibulo‐Ocular Reflex Evoked by a “Head Heave” Stimulus
Authors: RAMAT STEFANO , ZEE DAVID S. , MINOR LLOYD B.
Published: July 2010

Abstract: The gain and symmetry of vestibulo‐ocular reflexes for high‐frequency, high‐acceleration movements of the head are altered following unilateral vestibular lesions. These changes have been well characterized for rotational head movements (thrusts), and provide reliable markers of dysfunction in individual semicircular canals. Alterations in the vestibulo‐ocular reflex (VOR) evoked by lateral, whole‐body translations have also been observed. In an approach directed at the development of a bedside test of otolith function, we have recorded (scleral search coil) the VOR evoked by brief, high‐acceleration lateral translations (heaves). We delivered these stimuli manually and also developed a “head sled” device that minimizes any rotational contaminating component of the stimulus. Our geometrical analysis of the stimuli enables us to take into account the translational and rotational components of the movement, and to calculate an ideal response required for stabilization of images on the fovea at different fixation distances. We observed a tracking response (visually assisted VOR) that was close to ideal for image stabilization when these methods were used to analyze responses to slow, low‐amplitude lateral translations of the head. When applied to rapid, high‐acceleration (0.5 g) translations, the VOR was found to be less than compensatory in subjects with normal vestibular function. In a patient with unilateral vestibular hypofunction following intratympanic gentamicin injections, both the rotational and the translational VOR were asymmetric. Responses for translations toward the treated side had lower gain than those for translations toward the normal side. These findings provide a basis for further development of this technique as a clinical test and as a method for quantitative evaluation of otolith function.
Benign Paroxysmal Positional Vertigo
Authors: OAS JOHN G.
Published: July 2010

Abstract: The pathogenesis and epidemiology of benign paroxysmal positional vertigo are still not well defined. Treatment protocols have emerged along with complementary hypotheses regarding pathogenesis. Ultrastructural studies suggest a multistep process of otoconia metabolism responsible for forming the otolith membrane. A defect in otoconia metabolism leads to an excess of otoconia within the utricular sac (utriculolithiasis). Gravitational forces cause the entrapment of otoconia within the semicircular canal system (canalolithiasis). Localization of these otoconia within the semicircular canal system is deducted by combining the gravitational orientation of the canal involved with the vestibular neurophysiology of the resulting nystagmus recorded during testing and throughout treatment. New terminology is required to differentiate short‐arm from long‐arm canalolithiasis. Evidence from digital videonystagmography recordings of nystagmus is coupled with principles of gravitational fluid mechanics to explain the mechanisms of disease development and treatment. These observations lead to important questions that define future directions in research.
Changes in the Vestibulo‐Ocular Reflex after Plugging of the Semicircular Canals
Authors: YAKUSHIN SERGEI B. , DAI MINGJIA , RAPHAN THEODORE , SUZUKI JUN‐ICHI , ARAI YASUKO , COHEN BERNARD
Published: July 2010

Abstract: The gain of the angular vestibulo‐ocular reflex (aVOR) was determined in monkeys by rotation about a spatial vertical axis while upright or statically tilted forward and backward. Horizontal, vertical, and roll gains were determined at each head orientation and plotted as a function of head tilt. Before canal plugging, animals had maximal (spatial) horizontal gains when upright (spatial phase 0°) and maximal roll gains when tilted forward or backward 90°. Plugging caused striking changes in the characteristics of the aVOR gains at low frequencies. After plugging of the vertical canals, maximal horizontal and roll gains both occurred at head tilts of ∼30° forward. When the lateral canals were plugged, maximal horizontal and roll responses occurred when the head was tilted back ∼50°. The aVOR gains of the canal‐plugged animals were also affected by stimulus frequency. In every instance, as stimulus frequency increased, the spatial phases shifted toward the normal response, that is, the response before plugging. This normalization effect was observed even in the animals with all six semicircular canals plugged, indicating that normalization was not due to spatial adaptation. A three‐dimensional dynamic and kinematic model of the aVOR was able to account for all types of canal plugging by a simple change in the dominant time constant of the plugged canals from 3 s to 5 s to ∼0.07 s. The model accurately predicted responses of the normal and canal‐plugged animals at all frequencies. These data show that the central vestibular system does not spatially adapt to losses resulting from canal plugging.
Human Experience with Canal Plugging
Authors: AGRAWAL SUMIT K. , PARNES LORNE S.
Published: July 2010

Abstract: Canal occlusion/plugging is a very effective technique with a low risk to hearing. The series of posterior semicircular canal occlusions described in this review now becomes the largest in the literature to date. It continues to support this procedure as the treatment of choice for intractable benign paroxysmal positional vertigo (BPPV). All 44 operated ears were relieved of BPPV, with one patient having an atypical late recurrence. Of the 40 ears with normal preoperative hearing, one had a delayed (3‐month) sudden and permanent profound loss, while one other had a mild (20 dB) loss. Six patients had protracted courses of imbalance and motion sensitivity.

Canal plugging has led to several new and innovative developments including the partial labyrinthectomy for difficult‐to‐access skull base lesions and superior semicircular canal plugging for dehiscence. These new procedures and their development are reviewed in this paper.
Gonadotropin‐Releasing Hormone Antagonists
Authors: GORDON KEITH
Published: July 2010

Until recently, gonadotropin‐releasing hormone (GnRH) agonists were the only choice available to physicians for prevention of premature luteinizing hormone (LH) surges in women undergoing controlled ovarian stimulation. The recent FDA approval of GnRH antagonists for this indication gives clinicians some new options. Results of clinical trials to date suggest that, with GnRH antagonists, much shorter treatment regimens with fewer injections and possibly less gonadotropin can achieve good clinical results. In most of the trials performed to date, however, the GnRH antagonist regimens have been associated with a slightly lower pregnancy and implantation rate than the established GnRH agonist protocols. This remains the biggest hurdle to their more general acceptance. Herein, the possible contributing factors are discussed, and the proposal made that differences in serum estradiol patterns preceding oocyte retrieval are the most likely contributing factor.
Understanding Preterm Labor
Authors: CHALLIS JOHN R. G. , LYE STEPHEN J. , GIBB WILLIAM , WHITTLE WENDY , PATEL FALGUNI , ALFAIDY NADIA
Published: July 2010

Increased uterine contractility at term and preterm results from activation and then stimulation of the myometrium. Activation can be provoked by mechanical stretch of the uterus and by an endocrine pathway resulting from increased activity of the fetal hypothalamic‐pituitary‐adrenal (HPA) axis. In fetal sheep, increased cortisol output during pregnancy regulates prostaglandin H synthase type 2 (PGHS2) expression in the placenta in an estrogen‐independent manner, resulting in increased levels of PGE2 in the fetal circulation. Later increases in maternal uterine expresssion of PGHS2 require elevations of estrogen and lead to increased concentrations of PGF2α in the maternal circulation. Thus, regulation of PGHS2 at term is differentially controlled in fetal (trophoblast) and maternal (uterine epithelium) tissue. This difference may reflect expression of the glucocorticoid receptor (GR), but not estrogen receptor (ER), in placental trophoblast cells. In women, cortisol also contributes to increased PG production in fetal tissues through upregulation of PGHS2 (amnion and chorion) and downregulation of 15‐OH PG dehydrogenase (chorion trophoblasts). The effect of cortisol on chorion expression of PGDH reverses a tonic stimulatory effect of progesterone, potentially through a paracrine or autocrine action. We have interpreted this interaction as a reflection of “progesterone withdrawal” in the primate, in relation to birth. Other agents, such as proinflammatory cytokines, similarly upregulate PGHS2 and decrease expression of PGDH, indicating the presence of several mechanisms by which labor at term or preterm may be initiated. These different mechanisms need to be considered in the development of strategies for the detection and management of the patient in preterm labor.
A Critical Birth Weight and Other Determinants of Survival for Infants with Severe Intrauterine Growth Restriction
Authors: LEE MEN‐JEAN , CONNER ELLEN L. , CHARAFEDDINE LAMA , WOODS JAMES R. , Del PRIORE GIUSEPPE
Published: July 2010

Our objective was to assess the perinatal management and neonatal outcomes of premature, severely intrauterine growth‐restricted (IUGR) neonates. A cohort of neonates <1000 grams, ≤ first percentile for weight, and <37 weeks' gestation was identified and matched 2:1 to two control sets of premature, appropriate‐for‐gestational age (AGA) infants—one with similar gestational age (AGA‐GA group) and the other with similar birth weight (AGA‐BW group) to determine the effect of IUGR on the outcome of the premature infant. The IUGR group was then examined in detail for descriptive statistics. Data were analyzed by t‐tests and Chi‐square analyses where appropriate. The IUGR infants had worse outcomes than AGA‐GA controls but had somewhat better results than the AGA‐BW controls. In the IUGR group, a birth weight less than 550 grams was significantly associated with neonatal death (p < 0.001). However, increasing gestational age was not associated with neonatal survival (p= 0.661) if birthweight remained below 550 grams. Classical cesarean delivery was associated with neonatal death (p= 0.003). Neonatal variables associated with poor outcome included patent ductus arteriosus (p= 0.034), feeding intolerance (p= 0.046), and failure to thrive (p= 0.05). Overall, neonatal survival was 73%. Of the surviving neonates, 69% had evidence of neurodevelopmental delay when tested at 6 and 12 months. Premature, growth‐restricted neonates with birth weights of <550 grams versus those of >550 grams have dismal outcomes despite a gestational age that is compatible with survival.
The Embryo Question
Authors: FLAMIGNI CARLO
Published: July 2010

In Italy, the seat of the Vatican, the problem of the “rights of the embryo” has been particularly felt and has caused bitter debate between laymen and clergy. The disagreement has focused primarily on the definition of “person,”“individual,” and the “beginning of life.” Catholics, for the most part, have contested the concept of the “pre‐embryo” and have tried to have a law passed that would impede the production and freezing of supernumerary embryos (according to the hypothesis of the “simple case”). In the same way, Catholics have strongly opposed the possible manipulation of embryos, including pre‐implant genetic investigations. In addition to Catholic teachings, the National Committee for Bioethics has also declared itself favorable to the protection of the “waking life.” It published a special document on the theme in a period in which the Committee was composed only of strict Catholics, following action taken by the then Prime Minister, Berlusconi, who believed it necessary to exclude and remove all lay members from the Committee. The document of the National Committee for Bioethics, which distinguishes itself for having declared that “the embryo is one of us,” has been the cause of a transversal political aggregation that has gathered Catholic parliamentarians from different political parties and that has begun a campaign to acknowledge the prerogatives and rights of the embryo which Italian law attributes only to the baby after its birth. An intense debate continues on all these themes, and in back of all this is the warning from the Church to re‐examine the Italian law dealing with the voluntary interruption of pregnancy.
Historical Perspectives of Hybrid Hepatic Assist Devices
Authors: NOSÉ YUKIHIKO
Published: July 2010

Abstract: A hybrid hepatic assist device using canine liver tissues was developed and clinically applied 38 years ago. However, for many years practical hybrid hepatic assist devices were not clinically introduced owing to the many difficulties encountered in employing cultured hepatocytes. These problems include: (1) maintenance of viable cultured cells, (2) maintenance of normal hepatocyte function with these cells, (3) elimination of toxic substances generated by non‐viable cultured and/or stored cells, (4) elimination of immunological factors generated by cultured cells and by the patient, and (5) difficulties of the biocompatible immunological barrier for cells against the patient. Fortunately, recent progress in apheresis and biomaterial technologies enable us to isolate cultured cells immunologically and yet maintain effective metabolic functions for the patient. These technologies generate an immunological barrier of a hybrid hepatic assist device for the patients. Proper adsorption columns developed for apheresis procedures enable us to remove the toxic substances released by non‐viable cells. Recent development of oxygen‐carrying macromolecules enable us to provide sufficient oxygen supply to the cultured cells and to maintain their normal cellular function, not only during the cultured period of time, but also during their actual clinical application. Together with the advancement of cell culture technologies, including the proper cultured environments and cellular seeding environments, these technologies, primarily developed for therapeutic apheresis, should be able to provide more effective and safe hybrid artificial organs.
Use of Small Animal Models for Screening Immunoisolation Approaches to Cellular Transplantation
Authors: GILL RONALD G.
Published: July 2010

Abstract: It has been recognized for many years that immunoisolation strategies form an attractive approach to preventing the rejection of cellular allografts and xenografts. Although immunoisolation has proven dramatically successful in some cases, the results have tended to be somewhat variable. Although many advances have been made in the development of biocompatible materials for separating host immune cells from the transplanted tissues, much of the experimentation in this area has been outcome driven. That is, the nature of host reactivity and/or biomaterial design resulting in the failure of some immunoisolation strategies has mostly been undefined. A first premise of this discussion is that immunoisolation is primarily cell isolation and not antigen isolation, per se. That is, although varied membrane barriers are designed to prevent cell‐cell contact between host and donor cells, soluble antigens derived from the transplant are likely to gain access to the host immune system. A key question centers on the degree and consequence of this type of antigen presentation in the host to the immunoisolated transplant. To address this and related concerns, this overview presents a simple paradigm for using defined rodent (mouse) models for systematically screening the efficacy of immunoisolated cellular transplants. The proposition is made that understanding the basis of graft failure will aid in the design of future immunoisolation technologies.
NMR Spectroscopy in β Cell Engineering and Islet Transplantation
Authors: PAPAS KLEARCHOS K. , COLTON CLARK K. , GOUNARIDES JOHN S. , ROOS ERIC S. , JAREMA MARY ANN C. , SHAPIRO MICHAEL J. , CHENG LEO L. , CLINE GARY W. , SHULMAN GERALD I. , WU HAIYAN , BONNER‐WEIR SUSAN , WEIR GORDON C.
Published: July 2010

Abstract: Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to β cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c‐myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl‐2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy. (2) Overnight incubation of islets and β cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR‐detectable markers using conventional 13C and 31P NMR spectroscopy of extracts. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high‐resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused β cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy.
Soft, Porous Poly(d,l‐lactide‐co‐glycotide) Microcarriers Designed for Ex Vivo Studies and for Transplantation of Adherent Cell Types Including Progenitors
Authors: XU ARRON S.L. , REID LOLA M.
Published: July 2010

Abstract: Our laboratory is undertaking tissue engineering of liver using enriched liver progenitor cells. We report here our ongoing study to design biodegradable and biocompatible three‐dimensional substratum supports of both natural and synthetic polymeric materials suitable to the adhesion, growth, and differentiation of adult and progenitor liver cells for their transplantation, and for the development of a bioartificial liver assist device. Porous biocompatible and biodegradable microcarriers of diameter 20–40 μm and 100–300 μm were prepared from (α‐hydroxy) acid family of polymers. Human hepatoma cell line HepG2 and adult rodent liver cells were found to attach to collagen‐coated surface of poly(d,l‐lactide‐co‐glycotide) microcarriers. HepG2 cells attached to the degradable microcarriers remained viable and underwent growth expansion, forming three‐dimensional cell‐degradable microcarrier colonies in culture. These cell‐degradable microcarrier colonies may undergo further growth expansion, thus providing a viable approach for three‐dimensional organogenesis of tissue.
Purification of Polymeric Biomaterials
Authors: WANDREY CHRISTINE , VIDAL DIANELYS SAINZ
Published: July 2010

Abstract: Employing a combined filtration and precipitation method, the endotoxin concentration in sodium alginate (SA) and sodium cellulose sulfate (SCS) was reduced to a value of 200 EU/g polymer. This is one tenth of the regulatory threshold calculated, for example, for an appropriate bioartificial pancreas that consists of approximately 420,000 encapsulated islets of Langerhans. The low endotoxin (ET) levels were maintained below this threshold during a six‐month storage period. The purification procedure of the polymers did not negatively influence the final microcapsule properties. The mechanical stability of microcapsules from purified material is even slightly higher than that of microcapsules from the original polymers. A second approach to avoid endotoxin release from the device is its direct complexation during the bead or capsule formation process. The durability of endotoxin binding in binary, ternary, and quaternary complexes could be demonstrated for storage in culture medium and saline. Very low total endotoxin release from the complexes was detected after three months in culture medium and five months in saline. This complexation is primarily based on electrostatic interactions with the participating cationic components and provides additional security for the final bioartificial organ or delivery device.
The Role of a Bioengineered Artificial Kidney in Renal Failure
Authors: FISSELL WILLIAM H. , KIMBALL JASON , MACKAY SHERRILL M. , FUNKE ANGELA , HUMES H. DAVID
Published: July 2010

Abstract: Renal failure continues to carry substantial burden of morbidity and mortality in both acute and chronic forms, despite advances in transplantation and dialysis. There is evidence to suggest that the kidney has metabolic, endocrine, and immune effects transcending its filtration functions, even beyond secretion of renin and erythropoietin. Our laboratory has developed experience in the tissue culture of renal parenchymal cells, and has now been able to demonstrate the metabolic activity of these cells in an extracorporeal circuit recapitulating glomerulotubular anatomy. We have observed active transport of sodium, glucose, and glutathione. We describe the design and initial preclinical testing of the bioartificial kidney, as well as future directions of our research.
Effect of Flow Configuration and Membrane Characteristics on Membrane Fouling in a Novel Multicoaxial Hollow‐Fiber Bioartificial Liver
Authors: MACDONALD JEFFREY M. , WOLFE STEPHEN P. , ROY‐CHOWDHURY INDRAJIT , KUBOTA HIROSHI , REID LOLA M.
Published: July 2010

Abstract: A novel “multicoaxial hollow fiber bioreactor” has been developed consisting of four concentric tubes, the two innermost tubes are called hollow fibers. Bioartificial livers are created by culturing liver progenitors in the space between the two innermost hollow fibers and with culture media contained in the two compartments (intracapillary and extracapillary) sandwiching the cell compartment. The outermost compartment is used for gas exchange. A hydrodynamic model has recently been established to predict the optimum hydraulic permeability and bioreactor operational parameters to create the physicochemical environment found in the liver acinus.1 However, perfusion with serum‐free hormonally‐defined media and inoculation of cells introduces membrane fouling into the equation, and this parameter must be incorporated into the model. Using commercially available semipermeable hollow fibers (1 mm [0.65 μm pores] and 3 mm [0.1 μm pores] outer diameters [o.d]), the primary cause of resistance is the middle hollow fiber. Preliminary studies using bioreactors inoculated with isolated rat hepatocytes and perfused with serum‐containing culture media demonstrated that the middle hollow fiber is the primary site of fouling, and this fouling ultimately causes cell mortality by blocking the transfer of nutrients. Experiments were performed to determine the best commercially available middle hollow fiber for construction of bioreactors and two 3‐mm outer‐diameter middle hollow fibers were compared: polypropylene and polysulfone, with 0.2 μm and 0.1 μm pore sizes, respectively. Dead‐ended and cross flow configurations were compared for their effectiveness at reducing membrane fouling in the middle hollow fiber by determining the change in resistance with time. The results demonstrate that the 0.2‐μm pore size polypropylene hollow fiber is the best choice for construction of the multicoaxial hollow‐fiber bioreactor, and that cross flow results in two orders of magnitude lower resistance than dead‐ended flow after 36 h.
β Cell Replacement for the Treatment of Diabetes
Authors: OBERHOLZER JOSÉ , TOSO CHRISTIAN , RIS FRÉDÉRIC , BUCHER PASCAL , TRIPONEZ FRÉDÉRIC , DEMIRAG ALP , LOU JINNING , MOREL PHILIPPE
Published: July 2010

Abstract:The replacement of insulin‐producing β cells by islet transplantation can efficiently reverse diabetes. The recent improvements in clinical results were made possible by transplanting higher islet masses and through the introduction of new immunosuppressive protocols that avoid diabetogenicity. The need for alternatives to continuous immunosuppression, and an unlimited source of glucose‐sensitive, insulin‐secreting tissue, is emerging. In this review we discuss the various key steps in islet transplantation and offer perspectives for future developments in the replacement of insulin‐producing β cells for the treatment of type I diabetes.
Standards and Guidelines for Biopolymers in Tissue‐Engineered Medical Products
Authors: DORNISH MICHAEL , KAPLAN DAVID , SKAUGRUD ØYVIND
Published: July 2010

Abstract: The American Society for Testing and Materials (ASTM) is making a concerted effort to establish standards and guidelines for the entire field of tissue‐engineered medical products (TEMPS). Safety, consistency, and functionality of biomaterials used as matrices, scaffolds, and immobilizing agents in TEMPS are a concern. Therefore, the ASTM has established a number of task groups to produce standards and guidelines for such biomaterials. Alginate is a naturally occurring biomaterial used for immobilizing living cells to form an artificial organ, such as encapsulated pancreatic islets. In order to aid in successful clinical applications and to help expedite regulatory approval, the alginate used must be fully documented. The ASTM alginate guide gives information on selection of testing methodologies and safety criteria. Critical parameters such as monomer content, molecular weight, and viscosity, in addition to more general parameters, such as dry matter content, heavy metal content, bioburden, and endotoxin content are described in the ASTM document. In a like manner, the characterization parameters for chitosan, a bioadhesive polycationic polysaccharide, are described in a separate guide. For chitosan, the degree of deacetylation is of critical importance. Control of protein content and, hence, potential for hypersensitivity, endotoxin content, and total bioburden are important in chitosan preparations for TEMPS. Together these two guides represent part of the effort on behalf of the ASTM and other interested parties to ensure quality and standardization in TEMPS.
A Framework for the Molecular Classification of Circulating Tumor Markers
Authors: JOHNSON PHILIP J.
Published: July 2010

Abstract: New molecular biological technologies, especially polymerase chain reaction (PCR) and mass spectroscopy, have expanded the pool of molecular targets for cancer diagnosis, monitoring, and prognosis using plasma or serum as the substrate. In this review, a framework is described following the “life history” of a protein—starting with DNA [endogenous (nuclear or mitochondrial) or exogenous (viral)], followed by RNA [endogenous (cell‐based or cell‐free) or exogenous (viral)], and culminating in protein (either the native protein or the glycan portion of glycoproteins). Each of these levels provides unique opportunities to achieve specificity for cancer diagnosis.
Epigenetic Tumor Markers in Plasma and Serum
Authors: WONG IVY H. N. , LO Y. M. DENNIS , JOHNSON PHILIP J.
Published: July 2010

Abstract: Circulating tumor DNA in plasma and serum has been demonstrated to reflect the biological characteristics of tumors, including the rates of apoptosis and necrosis. Aberrant promoter methylation has increasingly emerged as a fundamental molecular abnormality associated with loss of critical gene functions during carcinogenesis. This epigenetic inheritance has significant biological implications for early tumor initiation and cancer progression or metastasis formation. The promoter‐region methylation is crucial in transcriptional silencing of tumor suppressor genes, DNA repair genes, and metastasis inhibitor genes, and is linked to the predisposition of genetic alterations of other cancer‐associated genes. Of clinical relevance, epigenetic markers in plasma and serum have recently been established as specific and sensitive biomarkers for early and noninvasive screening, risk assessment, and monitoring of neoplastic diseases. A panel of epigenetic markers may possibly allow the detection of circulating tumor DNA in virtually all patients with different cancer types. Furthermore, the prognostic value of aberrant DNA methylation and therapeutic implications of demethylation of methylated genes could further improve the management of patients with different kinds of cancer.
Proteomic Approaches within the NCI Early Detection Research Network for the Discovery and Identification of Cancer Biomarkers
Authors: VERMA MUKESH , WRIGHT GEORGE L. , HANASH SAMIR M. , GOPAL‐SRIVASTAVA RASHMI , SRIVASTAVA SUDHIR
Published: July 2010

Abstract: In the postgenome era, proteomics provides a powerful approach for the analysis of normal and transformed cell functions, for the identification of disease‐specific targets, and for uncovering novel endpoints for the evaluation of chemoprevention agents and drug toxicity. Unfortunately, the genomic information that has greatly expounded the genetic basis of cancer does not allow an accurate prediction of what is actually occurring at the protein level within a given cell type at any given time. The gene expression program of a given cell is affected by numerous factors in the in vivo environment resulting from tissue complexity and organ system orchestration, with cells acting in concert with each other and responding to changes in their microenvironment. Repositories of genomic information can be considered master “inventory lists” of genes and their maps, which need to be supplemented with protein‐derived information. The National Cancer Institute's Early Detection Research Network is employing proteomics, or “protein walking”, in the discovery and evaluation of biomarkers for cancer detection and for the identification of high‐risk subjects. Armed with microdissection techniques, including the use of Laser Capture Microdissection (LCM) to procure pure populations of cells directly from human tissue, the Network is facilitating the development of technologies that can overcome the problem of tissue heterogeneity and address the need to identify markers in easily accessible biological fluids. Proteomic approaches complement plasma‐based assays of circulating DNA for cancer detection and risk assessment. LCM, coupled with downstream proteomics applications, such as two‐dimensional polyacrylamide gel electrophoresis and SELDI (surface enhanced laser desorption ionization) separation followed by mass spectrometry (MS) analysis, may greatly facilitate the characterization and identification of protein expression changes that track normal and disease phenotypes. We highlight recent work from Network investigators to demonstrate the potential of proteomics to identify proteins present in cancer tissues and body fluids that are relevant for cancer screening.
Detection of Circulating CEA Molecules in Human Sera and Leukopheresis of Peripheral Blood Stem Cells with E. coli Expressed Bispecific CEAScFv‐Streptavidin Fusion Protein‐Based Immuno‐PCR Technique
Authors: REN JUN , GE LE , LI YONGQI , BAI JUN , LIU WEN CHAO , SI XIAO MING
Published: July 2010

Abstract: In this study, a method called bispecific CEAScFv‐streptavidin fusion protein‐based immuno‐PCR technique will be tested experimentally. The application of the bispecific fusion protein‐based immuno‐PCR technique has the significant advantage that it can be readily applied in the clinical setting, as well as tested as a potential screening tool in high‐risk populations of certain types of cancer.
Circulating RNA as a Tumor Marker
Authors: KOPRESKI MICHAEL S. , BENKO FLOYD A. , GOCKE CHRISTOPHER D.
Published: July 2010

Abstract: Cancer is commonly associated with the inappropriate mRNA expression of nonmutated genes. Recently, several tumor‐associated RNA species, including tyrosinase mRNA and telomerase RNA, have been demonstrated in plasma and serum. The presence of tumor RNA in plasma and serum affords the opportunity to diagnose or stratify patients with cancer when tissue is not readily available. To exemplify the potential for pharmacogenomic and phenotypic stratification of the cancer patient, we evaluated serum for 5T4 mRNA. 5T4 is a trophoblast glycoprotein frequently overexpressed in epithelial malignancies that provides a potential target for cancer therapeutics. Serum was collected from 19 patients with advanced breast cancer (5 patients) or non‐small‐cell lung cancer (14 patients), and from 25 normal control volunteers having amplifiable RNA. RNA extracted from the serum was RT‐PCR amplified using heminested, two‐stage reactions, with products detected by gel electrophoresis. 5T4 mRNA was reproducibly detected in 8/19 (42%) cancer patient sera, including 2/5 breast cancer patient sera and 6/14 lung cancer patient sera, but in only 3/25 (12%) normal control sera (p= 0.035). The potential for circulating mRNA to identify patients who might benefit from a 5T4‐directed therapy offers an example of the utility of circulating RNA as a tumor marker.
Detection of Amplifiable Messenger RNA in the Serum of Patients with Lung Cancer
Authors: FLEISCHHACKER MICHAEL , BEINERT THOMAS , ERMITSCH MICHAEL , SEFERI DZEMILE , POSSINGER KURT , ENGELMANN CLAUS , JANDRIG BURKHARD
Published: July 2010

Abstract: Recently, in addition to the detection of circulating tumor cells in peripheral blood of patients with solid tumors, the presence of free circulating nucleic acids in the plasma and serum has also been described. We have focused on the possibility of isolating and amplifying intact extracellular, tumor‐related mRNA from the plasma/serum of patients with lung cancer. For this purpose, we established several RT‐PCR‐based amplification systems for the detection of a panel of five different genes. The expression of these genes was either shown to be restricted to lung tissue or associated with malignancy. We examined two small groups of 18 patients with lung cancer before and during chemotherapy, respectively. The message for β‐actin (control for integrity of the RNA) was detected in all of the analyzed sera from the control group and patients with lung cancer. Analysis of CK‐19 expression was positive in the majority of tumor patients, but positive results were also shown in all of the control sera. The expression of MAGE‐2 and TTF‐1 genes was not observed in any of the patients in either the lymphocyte preparations or serum samples. Expression of the PGP 9.5 gene was observed in the cells of all 18 patients, but mRNA in the serum was only detectable in one case. The hnRNP‐B1 mRNA was detectable in 14/18 sera, and Her2/neu‐specific mRNA could be amplified from the serum of 7/18 patients. Combining the last two markers, we were able to detect all patients with a malignant lung tumor.
Low‐Molecular‐Weight DNA in Blood Plasma as an Index of the Influence of Ionizing Radiation
Authors: VASILYEVA IRINA N.
Published: July 2010

Abstract: Low‐molecular‐weight DNA (180 bp) appears in blood plasma of irradiated rats. Its amount correlates directly with the irradiation dose. The cloned low‐molecular‐weight DNA is enriched with G/C pairs (48% compared with 40% in the rodent genome). Computer search against EMBL data showed that the sequence of these clones is homologous to various genomic repetitive elements. The program for identification of nucleosome‐positioning sequences in eukaryotic nuclear DNA has shown that low‐molecular‐weight DNA fragments are able to form rather sustainable nucleosomes and have some peculiarity. The difference in the composition of low‐molecular‐weight DNA isolated from blood plasma of rats after irradiation at doses of 8 and 100 Gy consisted of the change in the levels of two dinucleotides (CpG and CpT). The DNA in question originates from different types of cells.
Postradiation DNA Polymorphism
Authors: SHERLINA SOPHIA S. , VLADIMIROV VIKTOR G.
Published: July 2010

Abstract: The presence of DNA molecules with tumor‐specific genetic alterations has been demonstrated in the plasma of cancer patients. Such plasma DNA fragments may be immunogenic and provide immunologic support to the malignant cells. Further, the tumor‐specific plasma DNA may participate in the adaptive processes of the tumor cells in the cancer host. The mechanisms of the latter are unclear. However, from the standpoint of the resistance problem, understanding of DNA transformation after exposure of extreme factors is of great interest.
Circulating Nucleic Acids and Apoptosis
Authors: LICHTENSTEIN ANATOLY V. , MELKONYAN HOVSEP S. , TOMEI L. DAVID , UMANSKY SAMUIL R.
Published: July 2010

Abstract: It is well documented that plasma contains DNA from tissues throughout the body, including developing fetuses, and tumors. A portion of this DNA crosses the kidney barrier and appears in urine (i.e., transrenal DNA). However, molecular, cellular, and physiological mechanisms of the circulating DNA phenomenon and renal clearance are in an early phase of investigation. Here, we discuss possible forms of circulating DNA, factors affecting representation of different tissues and genomic sequences in plasma DNA, possible mechanisms of renal DNA clearance, and technical problems encountered in DNA isolation from urine. We suggest that apoptotic cells are an important source of DNA in both plasma and urine. Further analysis of the data has led us to propose that a significant portion of circulating DNA can be represented in apoptotic bodies.
Cell‐Free DNA in Urine
Authors: ZHONG XIAO YAN , HAHN DEIRDRÉ , TROEGER CAROLYN , KLEMM ANDRÉ , STEIN GÜNTER , THOMSON PETER , HOLZGREVE WOLFGANG , HAHN SINUHE
Published: July 2010

Abstract: Intrigued by the rapid clearance of free fetal DNA from the maternal circulation, we have investigated whether this fetal genetic material could be cleared via the kidney. For this purpose, we examined for the presence of Y chromosome‐specific DNA sequences in urine samples obtained from 8 women pregnant with male fetuses. No male‐specific sequences could be detected, despite the use of a very sensitive nested PCR assay nor a highly reproducible real‐time PCR assay. We did, however, detect maternal DNA sequences. To determine if this cell‐free DNA was derived from the kidney or another source, we next examined urine from female kidney transplant patients who had received male kidneys. Y chromosome‐specific sequences were indeed detectable by both nested and real‐time PCR in these samples, thereby confirming a recent report describing urinary DNA microchimerism. Quantitative analysis of serially obtained samples furthermore suggests that transplant‐derived sequences are elevated during periods of graft rejection. These results imply that the measurement of graft‐derived urinary DNA may serve as a new marker for kidney graft tolerance.
Possible Origin of Extrachromosomal DNA from Human Lymphocytes Following In Vitro Treatment by Phytohemagglutinin
Authors: CHEN JOHN S. , MENESINI CHEN MARIA G.
Published: July 2010

Abstract: Lymphocytes respond to chemical or pathological challenges by synthesizing episomes or extrachromosomal DNA (eDNA). Phytohemagglutinin (PHA) is a mitogen for T lymphocytes and can stimulate the increased production of episomes as well as the release of eDNA, interleukin‐2, and other factors. However, the origin of this eDNA is largely unknown. To confirm the observation on the localization of an antibody directed against a 23‐kb segment of bull acrosomal DNA in cultured human lymphocytes, we performed analyses by fluorescence in situ hybridization (FISH) using the same DNA as a molecular probe.
Epidemiology of Cardiovascular Involvement in HIV Disease and AIDS
Authors: FISHER STACY D. , LIPSHULTZ STEVEN E.
Published: July 2010

Abstract: The epidemiology of cardiac complications related to HIV including cardiomyopathy, increased left ventricular mass, myocarditis, pericardial effusion, endocarditis, and malignancy are discussed. A large number of HIV‐infected individuals will present with cardiac complications in the next decade as chronic viral infection, co‐infections, drug therapy, and immunosuppression affect the heart. Understanding the nature and course of cardiac illness related to HIV infection will allow appropriate monitoring, early intervention and therapy, and will provide a baseline to evaluate the effects of new therapeutic regimens such as highly active antiretroviral therapy on the cardiovascular system.
Cell Death in HIV Pathogenesis and Its Modulation by Retinoids
Authors: SZONDY ZSUZSA , TÓTH RÉKA , SZEGEZDI ÉVA , REICHERT UWE , ANCIAN PHILIPPE , FÉSÜS LÁSZLÓ
Published: July 2010

Abstract: Patients infected with the human immunodeficiency virus exhibit a progressive decline in the CD4 T‐cell number, resulting in immunodeficiency and increased susceptibility to opportunistic infections and malignancies. Although CD4 T cell production is impaired in patients infected with HIV, there is now increasing evidence that the primary basis of T cell depletion is accelerated apoptosis of CD4 and CD8 T cells. The rate of lymphocyte apoptosis in HIV infection correlates inversely with the progression of the disease: it is low in long‐term progressors and in patients undergoing highly active antiretroviral therapy. Interestingly, only a minor fraction of apoptotic lymphocytes are infected by HIV, indicating that the enhanced apoptosis does not necessarily always serve to remove the HIV+ cells and results from mechanisms other than direct infection. Thus, understanding and influencing the mechanisms of HIV‐associated lymphocyte apoptosis may lead to new therapies for HIV disease. In this paper the potential effects of retinoids on CD4 T cell apoptosis is discussed.
HIV Inhibitors: Problems and Reality
Authors: TÖZSÉR JÓZSEF
Published: July 2010

Abstract: The human immunodeficiency virus encodes three replication enzymes, which are required for a productive life‐cycle. Currently, several anti‐retroviral drugs are available for clinical use, and they are inhibitors of either the reverse transcriptase or the viral protease. The introduction of combination anti‐retroviral therapy (HAART) changed the prognosis of HIV infection. However, current therapy is not able to eradicate the virus, only suppress it; therefore, long‐term use of the drugs is required to keep the viral load under control. Most of the problems associated with the HIV therapy are the consequence of the necessarily long‐term use of the drugs. The long‐term effectiveness of current inhibitors as therapeutic agents is limited by the rapid development of drug‐resistant variants. Furthermore, various side effects have been reported. These side effects include hypersensitivity, mitochondrial toxicity, lypodystrophy syndrome, insulin resistance and cardiovascular disorders. Further drug development is necessary to design new compounds that have efficacy similar to the currently used drugs in the management of HIV infection and that are potent against the resistant viruses but do not exhibit unwanted metabolic side effects.
EGFR Transactivation in the Regulation of SMC Function
Authors: KALMES ANDREAS , DAUM GÜNTER , CLOWES ALEXANDER W.
Published: July 2010

Abstract: Vascular smooth muscle cells (SMCs) are the principal cellular component of the normal artery and intimal lesions that develop in response to arterial injury. Several growth factors and their receptors participate in SMC activation, including the tyrosine kinase receptors for platelet‐derived growth factor (PDGF) and basic fibroblast growth factor as well as the G‐protein‐coupled receptors (GPCRs) for thrombin and angiotensin II. During the last couple of years, it has become evident that GPCRs transactivate receptor tyrosine kinases, particularly the epidermal growth factor receptor (EGFR). The EGFR is not well characterized in terms of its role in vascular biology, but recent findings indicate that GPCRs induce EGFR transactivation in cultured vascular SMCs, perhaps by intracellular and extracellular pathways. Studies from our laboratory as well as two other groups have demonstrated that EGFR transactivation by different GPCR agonists and in different cell types, including SMCs, is mediated by heparin‐binding EGF‐like growth factor (HB‐EGF). HB‐EGF‐dependent EGFR activation is blocked by heparin, a growth inhibitor of SMCs in vitro and in vivo. These data suggest that the EGFR may be important in the regulation of SMC function. The complexity of the GPCR‐EGFR crosstalk, involving several different cell surface molecules and an inside‐out signaling step, may provide novel targets for the control of SMC growth and intimal hyperplasia in the arterial injury response.
Polymorphisms in the Platelet‐Endothelial Cell Adhesion Molecule‐1 (PECAM‐1) Gene, Asn563Ser and Gly670Arg, Associated with Myocardial Infarction in the Japanese
Authors: SASAOKA TAISHI , KIMURA AKINORI , HOHTA SHIGERU , FUKUDA NAOTO , KUROSAWA TOSHIRO , IZUMI TOHRU
Published: July 2010

Abstract: We examined three missense polymorphisms of platelet‐endothelial cell adhesion molecule‐1 (PECAM‐1), Val125Leu, Asn563Ser, and Gly670Arg, in 136 Japanese patients with myocardial infarction and 235 healthy Japanese controls. We found that these polymorphisms were in linkage disequilibrium with each other and that frequencies of 125Leu, 563Ser, and 670Arg alleles were significantly increased in patients compared with controls (0.522 vs 0.447, p= 0.048; 0.585 vs 0.502, p= 0.030; and 0.577 vs 0.492, p= 0.032, respectively). The frequencies of homozygotes for 563Ser and 670Arg alleles were also significantly increased in the patients (33.1% vs 23.4%, odds risk [OR] = 1.62, p= 0.040, 95% confidence interval [95%CI] = 1.01‐2.58; and 32.4% vs 23.0%, OR = 1.60, p= 0.048, 95%CI = 1.00‐2.57, respectively). These observations suggest that the 563Ser/Ser genotype and 670Arg/Arg genotype of PECAM‐1 are novel genetic risk factors of myocardial infarction in Japanese. Stratification analysis of the patients showed that the associations of these PECAM‐1 genotypes with myocardial infarction were preferentially found in male and younger patients (age of onset of myocardial infarction less than 60 years). In addition, the associations were stronger in patients with three‐vessel disease than in the others and appeared independent of conventional risk factors including smoking, hypertension, diabetes mellitus, hyperlipidemia, and obesity.
Vascular Gene Discovery Using Laser Capture Microdissection of Human Blood Vessels and Quantitative PCR
Authors: STAGLIANO NANCY E. , CARPINO ALAN J. , ROSS JEFFREY S. , DONOVAN MICHAEL
Published: July 2010

Abstract: Recent approaches to candidate gene identification and cellular localization have included RNA derived from complex whole tissue profiling on cDNA microarrays followed by in situ hybridization with riboprobes. In this study, the Arcturus PixCell II™ laser capture microdissection (LCM) system, an argon‐based laser‐assisted method for the isolation of specific cell types from heterogeneous tissue samples, was used to microdissect the tunica media from normal human arteries and veins (n= 5 in each group). Total RNA was extracted from the sum of 10,000 shots for each blood vessel using the Strataprep MicroKit. RNA was reverse‐transcribed, and the resulting cDNA was analyzed using the Applied Biosystems 7700 quantitative PCR system (Q‐PCR). Control genes, such as the L‐type calcium channel, PECAM (CD‐31), and beta‐2 microglobulin, were used to assess sample quality and purity. Of 10 laser‐captured media samples, five (50%) showed a gene profile that indicated high‐ quality RNA (abundance of housekeeping genes) and smooth muscle cell enrichment (low levels of PECAM and high levels of the L‐type calcium channel). We conclude that the application of the LCM technique to collect smooth muscle cell RNA from the tunica media of human blood vessels can assist in the validation of gene expression and potentially expedite the identification of novel, regulated genes present within vascular smooth muscle.