1 INTRACELLULAR PROCESSING OF MUC1 IN 201T AND CALU-3 CELLS Ryan Anthony and Joseph M. Pilewski Department of Medicine/Cell Biology University of Pittsburgh Cystic fibrosis (CF) is an autosomal recessive genetic disease characterized by the hypersecretion of mucus by specialized epithelial cells in the lungs. Mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) have been previously determined to cause CF. CFTR functions as an apical membrane chloride channel that may also regulate membrane trafficking and post-translational processing of glycoconjugates. The role of mucins, high molecular weight glycoproteins that comprise the mucus, in the pathophysiology of cystic fibrosis is currently being investigated. The transmembrane mucin MUC1 co- localizes with CFTR. Our hypothesis is that CFTR affects the intracellular trafficking and membrane release of MUC1. In order to examine the endocytosis and release of MUC1, two drugs, brefeldin A and monensin, were used to disrupt MUC1 biosynthesis. Both brefeldin A and monensin disrupt glycoprotein processing by inhibiting protein translocation from the endoplasmic reticulum to the Golgi apparatus. 201T and CALU-3 airway tumor cells were grown to confluency in cell culture plates and Transwell (Costar) filters, respectively, at 37°C in Dulbecco’s Modified Eagle’s medium with 15% fetal bovine serum. Brefeldin A (5μM) was applied for 8, 24, or 48 hours, whereas monensin (1 or 5μM) exposures were restricted to 24 hours. Following incubation, collected supernatants and protein extracts were separated on 6% SDS-polyacrylamide gels. Western blot analysis of treated CALU-3 cells using an antibody against the MUC1 ectodomain suggested that MUC1 expression decreased with exposure to brefeldin A and monensin. Flow cytometry similarly demonstrated a time-dependent decrease in expression in response to these drugs. To evaluate the effects of brefeldin A on MUC1 membrane release, a competitive radioimmunoassay (TruQuant BR) was utilized. CALU-3 cells were treated with brefeldin A for 48 hours, and apical secretions assayed for MUC1. Brefeldin A displayed little effect on MUC1 release at 48 hours; however, MUC1 in apical supernatants was remarkably decreased at each of three collection times following the brefeldin A incubation. Collectively, the effects of brefeldin A are consistent with a model in which MUC1 surface expression is dependent upon both protein synthesis and membrane release. Interestingly, stimulation of CALU-3 cells with forskolin, a cAMP agonist, slightly increased MUC1 surface expression and decreased MUC1 release. This suggests that both CFTR and MUC1 are regulated by cAMP and is consistent with CFTR regulation of MUC1 processing. In future studies, CFTR expression will be down-regulated in CALU-3 cells, and primary cultures of CF and non-CF human airway cells will be used to define the role of CFTR in the intracellular processing of MUC1. 2 EFFECT OF HYPOXIA ON EXPRESSION OF METALLOPROTEINASES AND ORP-150 BY MONOCYTE-DERIVED MACROPHAGES IN VITRO. Hameed Aziz and David Vorp Vascular Surgery and Biomechanics Lab University of Pittsburgh Medical Center Abdominal Aortic Aneurysm (AAA) is the localized dilation of the arterial wall that will result in aortic rupture if left untreated. This pathological state is characterized by destruction of the medial elastic lamellae and a chronic immune/inflammatory response in the diseased aortic wall characterized by significant macrophage infiltration. Mechanical stability of the normal aorta is dependent on the integrity of matrix proteins (elastin and collagen). Aneurysm dilation, gradual expansion, and consequent rupture result from the mechanical failure of these normal aorta extracellular proteins. Increasing evidence within the past few years indicates that enzymatic degradation of matrix proteins by metalloproteinases contributes significantly to the pathogenesis of AAA. Macrophages play an important role in mediating and accelerating this process. Furthermore ORP-150, a novel protein that is expressed by macrophages under ischemic conditions, is likely to be critical in enabling these cells to redirect their biosynthetic activities in hypoxia.
Previous work in our laboratory has shown that the intraluminal thrombus (ILT) present in most AAA induces a hypoxic milieu. Our lab has also identified numerous macrophages within ILT. We therefore set out to investigate the implication of the noted hypoxic conditions on macrophage metabolism. Specifically, the purpose of this project was to investigate the regulation of these proteins by monocyte-derived macrophages in vitro, mimicking the hypoxic conditions of the AAA thrombus. Using freshly drawn human blood, monocytes were isolated using Ficoll-Paque™ sterile solution via density-gradient centrifugation. After further separation and centrifugation techniques using Nycoprep 1.068™, cells were counted by the Trypan Blue Exclusion Test, plated, and cultured for six days. Experimental groups were exposed to a 1% 02 hypoxic chamber and then total RNA or protein was isolated from the macrophages. PCR was used to detect the expression of MMP-2, -9, and ORP150. Western Blot was used to detect presence of ORP-150. This scientific project is still in progress and will continue after the conclusion of my internship with PTEI. 3 REDUCING THE FIBROSIS AFTER MUSCLE INJURY Neil Badlani, Kazumasa Fukishima and Johnny Huard Growth and Development Laboratory, Children’s Hospital of Pittsburgh and University of Pittsburgh Muscle injuries are a challenging problem in traumatology and the most frequently occurring injuries in sports medicine. Even though muscles retain their ability to regenerate after injury, the healing process of muscles after such injuries has been found to be slow and often leads to an incomplete muscle recovery because the regeneration is blocked by the formation of scar tissue. Previously, successful treatments have been developed using growth factors and gene therapy to enhance myoblast proliferation thereby improving muscle healing. However, this healing process cannot be completed because fibrosis occurs at the injured site, blocking the regeneration of skeletal muscle tissue. In ths study, we attempted to reduce this formation of fibrosis in order to allow muscle regeneration to occur more completely. It has been found that the formation of fibrosis is initiated by the release of the growth factor TGF-, which acts as a signaling molecule causing fibroblast cells to proliferate into the injured area and develop scar tissue. Therefore, we tested the effects of a TGF- inhibitor known as decorin, which is a small proteoglycan that has been found to block the activity of TGF- . In vitro, we found that decorin was capable of slowing the growth and proliferation of myo- fibroblasts. In vivo, we used an animal model of muscle laceration to induce the natural formation of fibrosis and then injected various concentrations of decorin into the injured site and found that we could reduce the formation of fibrosis and enhance the healing of the muscle. 4 TRACKING THE FATE OF MUSCLE CELLS USING A Y-CHROMOSOME SPECIFIC PROBE Neil Badlani, Jim Cummins and Johnny Huard Growth and Development Laboratory, Children’s Hospital of Pittsburgh and University of Pittsburgh Duchenne Muscular Dystrophy (DMD) is an x-linked recessive condition thatis caused by a lack of the protein dystrophin. A possible treatment for this condition has been the transplantation of myoblasts from normal muscle tissue which contain the protein dystrophin to DMD muscles. The injection of myoblasts into muscle tissue has also been used in attempts to enhance muscle healing after injuries. However, one drawback of these treatments has been an inability to accurately tag these injected myoblasts in order to track their survival in the new tissue. In this experiment, we have used a y-chromosome specific DNA probe to label our injected cells. In vitro, this probe has shown success in staining specifically male cells. This will be the first attempt to use the probe in vivo. Through a muscle biopsy, normal muscle cells from male mice were isolated and injected into female MDX mice (an animal model for muscular dystrophy). The injected muscle was then harvested five days post-injection and a series of fluoresence in-situ hybridization stains were conducted to test for the presence of our injected male cells and for the expression of the dystrophin protein. 5 SEX DIFFERENCES IN THE EFFECTS OF PHYSOSTIGMINE ON THE RAT HYPOTHALAMO- PITUITARY-ADRENAL AXIS Elena M. Balestreire, Michael E. Rhodes, R. Kenneth Czambel, Stephanie L. Wright and Robert T. Rubin Center for Neurosciences Research Allegheny General Hospital
There are sex differences in the hypothalamo-pituitary-adrenal (HPA) axis response to stimulation by cholinergic agents in rates. Physostigmine (PHYSO), an acetylcholinesterase inhibitor, was administered as a salicylate or hemi-sulfate salt to male and female rats (100ug/kg free base; IP) to determine whether these salts would cause differential responses when injected with equal amounts of free base PHYSO. Salicylate (50 ug/kg salicylic acid; IP) and saline (1 ml/kg: IP) also were administered as controls. Responses were quantified by measuring plasma arginine vasopressin (AVP), adrenocorticotropic hormone (ACTH), and corticosterone concentrations. Blood samples were collected 5 minutes before, and at 20 and 40 minutes following drug administration. PHYSO was without effect on AVP concentration; however, male rats appeared to respond significantly to salicylic acid 20 minutes after injection. PHYSO hemi-sulfate administration resulted in sex differences in plasma ACTH, reflected by a sustained response in males. PHYSO hemi-sulfate was more effective in stimulation of plasma ACTH in males, and corticosterone concentrations in both sexes. Female corticosterone was significantly higher than in males following both salts. These results support existing evidence suggesting sex differences in responsiveness of the HPA axis to PHYSO. Understanding sex differences in cholinergic responses may provide insight into the neurotransmitter activity and hormonal milieu of the central nervous system. The hormonal environment may affect an organism’s acceptance of engineered neural and other tissues. (Supported by an NIMH grant to RTR.) 6 BIOMECHANICAL STRAIN OF THE TEMPOROPARIETAL SUTURE UPON LOADING. Ilian Bandaranayake, Michael Tassick, and Jeremy J. Mao Biomechanics Laboratory University of Pittsburgh School of Dental Medicine In general, the effect of biomechanical loading upon calvarial bones is poorly understood. The objective of the present study was to investigate the magnitude and mode of mechanical strain experienced at various locations across the temporoparietal suture. Three element strain rosettes (WK-06-060WR-350, Measurements Group, Raleigh, NC) were implanted two in a series and each about 3 mm across the temporoparietal suture in three pairs at the anterior, middle and posterior portions of the parietal and temporal bones in four dry adult human skulls. Continuous, posteriorly directed ramp forces up to 90 Newtons were applied to the maxillary first molars using an Instron testing machine (Chatillon Model UTSM, New York, NY): first both parallel to the dental occlusion plane and 30o inferior to the plane and then, ipsilateral and contralateral to the side of rosette placement. In the great majority of cases, the maximum principal bone strain differed significantly across the suture between each pair of rosettes (p < 0.05 to p < 0.001). The peak maximum principal strain was generally small but reached 120 µ in rare cases. One-way ANOVA revealed that the peak maximum principal strain differed significantly between the anterior, middle and posterior portions of both the temporal and parietal bones. The direction of maximum bone strain was variable. These findings suggest that biomechanical stresses of various portions of temporal and parietal bones are different and should be considered accordingly when tissue-engineering reconstruction of these calvarial bones is performed. (Supported by PTEI grants to JJM.) 7 THE STUDY OF THE GROWTH AND ADHESION OF OSTEOBLASTS ON CAPROTITE™ AND THE ENCAPSULATION OF PROTEINS INTO CAPROTITE™ Jonathan Bickel and Kacey G. Marra Institute for Complex Engineered Systems Carnegie Mellon University Caprotite™, a polymer scaffold made from the biodegradable polymers poly(caprolactone) (PCL) and poly(D,L- lactic-co-glycolicacid) (PLGA) and the ceramic hydroxyapatite (HA) is currently undergoing testing for applications in bone tissue engineering. Studies were conducted to determine the osteoconductivity of Caprotite™ with the human osteogenic sarcoma cell line MG63. Cell proliferation studies were conducted with the MG63's physically in contact with Caprotite™. Further study into a novel encapsulation technique using
Caprotite™ was also investigated. Preliminary results show a difference in the osteoconductivity of PCL verses Caprotite™ and that Caprotite™ can be augmented with growth factors to improve its osteoconductivity. 8 IMMORTALIZED CELL LINES AS MODELS FOR GRANULOSA CELL STEROID PRODUCTION Davekumar Chandrasekaran and Jeffrey Clemens Department of Biological Sciences Duquesne University In a developing follicle, the immature egg (oocyte) is surrounded by a layer of granulosa cells, which in turn is encompassed by an outer layer of Theca cells. Granulosa cells are steroidogenic cells that form a complex 3- dimentional structure around the oocyte. These cells form gap junctions with one another, enabling both enhanced message transmission as well as substrate transfer from capillaries outside the follicle across the granulosa cell layer to the developing oocyte. The cells also express the receptor for the gonadotropin Follicle- stimulating Hormone (FSH), a large glycoprotein hormone which activates a cyclic AMP (cAMP) pathway. This pathway induces the expression of p450 Aromatase (ARM) and p450 Side Chain Cleavage (SCC), enzymes involved in the steroid biosynthetic pathway. Through their steroidogenic activity, granulosa cells help regulate follicular development during the ovarian cycle. The specific cell organization and extracellular environment are thought to play influential roles in granulosa cell steroid synthesis and secretion, and thus may be indirectly responsible for the development of the oocyte. Most studies done using granulosa cells, however, have been as monolayers on tissue culture plastic. Granulosa cells in culture may lose much of their in vivo functional activity because the cells are not able to orient themselves in their complex 3-dimentional structure in their normal extracellular matrix. Immortalized granulosa cell lines have recently been used as a model of granulosa cell study. It has been shown in primary cultures of both human and rat granulosa cells that luteinization (high levels of progesterone production) can be induced using various hormonal stimulants. The focus of my research was whether the PO GS 5 and PO GRS 1 immortalized granulosa cell lines (Amsterdam et al.) are inducible for progesterone production upon stimulation with forskolin (an activator of the cAMP pathway). I also examined the effects of extracellular matrix and growth serum on forkolin induction in an attempt to better mimic the more complex in vivo conditions of granulosa cell steroid biosynthesis. Forskolin addition did in fact induce luteinization in both cell lines, with a nearly 10-fold greater response in the PO GRS 1 cells. Further studies with the PO GRS 1 cell line showed that both Fetal Bovine Serum and extracellular matrix (in the form of an EHS gel) do affect granulosa cell growth, morphology, and progesterone production. The EHS gel enhanced the cells’ ability to attach to the plastic, while FBS accelerated cell growth. Both led to increased luteinization, similar to the results seen in primary cultures of rat granulosa cells. 9 GENE THERAPY USING HUMAN LIPO-DERIVED STROMAL VASCULAR FRACTION CELLS William Chong and Aaron Mason Division of Plastic and Reconstructive Surgery University of Pittsburgh Gene therapy is a promising therapy for many patients suffering from genetic and other diseases. New delivery vehicles for engineered cells are sought. One encouraging vehicle is human adipose stromal vascular fraction cells (SVF cells). SVC cells can be obtained in large numbers from liposuctions. The main question facing the use of SVF cells in gene therapy is whether the cells will accept the specific gene and produce the desired product. To determine the usefulness of SVF cells in gene therapy, the cells were transfected with retroviruses engineered to express a bioactive factor. The cells were then grown and supernatant was collected. Once confluent, the cells were trypsinized and injected into mice. On days 3, 7 and 14 blood was collected. An ELISA assay was then performed on the samples of supernatant and blood to measure levels of expressed factor. From this, it was found that elevated levels of the bioactive factor were produced. 10 GEOMETRIC ANALYSIS OF THE HEART VALVE CUSP
Michael DeRenzo and Michael Sacks Department of Bioengineering University of Pittsburgh Knowledge of the biomechanics of the natural valve during the cardiac cycle are essential to establishing guidelines for developing tissue engineering cardiac valve replacements. In particular, translation of micro- mechanical changes to the valvular level requires quantification of the deformation history (bending and stretching). In the present work, we utilized a finite element based surface fitting method for geometric analysis of a deforming heart valve cusp. The biquintic finite element program utilizes a fifth order hermite interpolation scheme and generates a surface of C2 continuity, allowing computations of the surface curvature as well as the in-plane strains. In addition, we developed a smoothing process that enables us to minimize the effects of data noise. Results were displayed using Tecplot (Bellevue, Washington) to make animations of the heart valve cusp opening and closing. These animations enabled us to see how the values of the curvature and strain rates were distributed along the surface of the cusp throughout the cycle of opening and closing. This method was validated by computing the principal curvature distributions for phantoms of known curvatures. In our analysis, the strain rates as well as the principal stretch values increased throughout the cycle of the cusp closing. In addition, we found that the area located directly above the central belly region experienced the greatest amount of curvature, which correlated to the regions that had the greatest structural damage on the cusp. We were able to deal with noisy data and use the biquintic finite element method to successfully compute the strain rates and curvature of the heart valve cusp. 11 CHARACTERIZATION AND IDENTIFICATION OF MUSCLE-DERIVED STEM CELLS Charley Gates, Shari Rogal, Ryan Pruchnic and Johnny Huard Growth and Development Laboratory, Children’s Hospital of Pittsburgh and University of Pittsburgh Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. Although this technique can deliver genes to skeletal muscle, it is hindered by the poor survival of the injected cells. We have recently identified a population of muscle-derived cells which entirely survive post-injection (J. Cell Biology) and which differentiate into other lineages, suggesting their pluripotentiality. In this study, we have investigated the effects of different nutrient mediums and growth factors on the proliferation of these muscle-derived cells in vitro. More importantly, we have found that these cells originate from skeletal muscle. In a section of gastrocnemius muscle taken from an adult mouse, we identified cells lying entirely within the basal lamina and plasma membrane that were positive for BCl-2, CD34, and desmin, three proteins which are crucial markers for stem cells. In all, these findings suggest the identification of a population of muscle-derived stem cells which reside within skeletal muscle. The isolation of such a cell population will be important for muscle-based gene therapy and tissue engineering for the musculo- skeletal system. 12 DEVELOPMENT OF A CONJOINING FLUORESCENT BASED ASSAY TO STUDY DIFFUSION OF GROWTH FACTORS WITH COMPLEX INTERSTITIAL INTERACTIONS Beth Ann Kaminski, Fred Lanni, Ratnakar Mujumdar and Phil Campbell Institute for Complex Engineered Systems Carnegie Mellon University Within tissues, macromolecules including growth factors, are made available to cells via interstitial transport. Transport mechanisms include diffusion through complex interactive 3-dimensional matrices. Furthermore, growth factors, such as insulin-like growth factor-I (IGF-I) do not occur in a free bioactive state but are associated with both fluid- and solid-phase specific binding proteins (IGFBPs). A basic understanding of these processes are required for any rational development of therapies using localized delivery of growth factors. As such, the primary purpose of this project was to develop a unique conjoining assay system to determine the diffusion of IGF-I, as our paradigm growth factor, through a simulated 3-D extracellular matrix. Critical components of this project consisted of fluorescently labeling IGF-I, while maintaining bioactivity, and overcoming the technical aspects of the physical assay itself. The diffusion assay involves the conjoining of two small, sealed, capillary tubes, one filled with a fluorescently labeled growth factor and the second containing a
non-labeled buffer. The fluorescence can be visually recorded and measured as a gradient is created while diffusion of the growth factor occurs. Future work will include the optimization of the conjoining assay and determining more specific details of growth factor diffusion into complex matrices. 13 AN AMINO ACID BASED POLYURETHANE FOR BONE REGENERATION Eric Krauland, Scott Beaver, Eric Beckman and Sudha Agarwal School of Dental Medicine University of Pittsburgh Our purpose is to develop a polymer that acts as a scaffold for osteoblasts and aids in the regeneration of damaged bone tissue. The goal is a nontoxic biodegradable polyurethane with less physical limitations and acidic by-products than existing FDA-approved polylactides. The polyurethanes are lysine diisocyanate (LDI) based, which is synthesized by transforming the two primary amine groups of lysine ethyl ester into isocyanates. LDI is readily combined with alcohol groups from various diols, polyols and other amino acids to form a urethane bond between the substrates. Finally, solidified foam was produced by reacting water with excess isocyanate groups, producing carbon dioxide gas. The polymers are placed in an incubated culture medium with osteoblasts taken from rabbit femur marrow. Polymers that supported cell growth were composites of LDI with glycerol, glycerol and tyrosine, glucose and collagen. Ongoing experiments with pH and UV spectrometry are tracing the degradation of these polymers. 14 LABELING IGF-1 FOR NON-INVASIVE IMAGING Julie Mackey, Byron Ballou and Daniel Farkas Center for Light Microscope Imaging and Biotechnology Carnegie Mellon University Sites of growth factor receptor expression are of much interest for tissue engineering applications. We have elected to study insulin-like growth factor- 1 (IGF-1), a 6300 MW polypeptide, because of local extensive experience with this molecule. We have established a sufficient database using radiolabeled IGF-1 for fruitful comparision with the fluorescently labeled molecule. Historically and up to present date, insulin-like growth factor has been radiolabeled for use in biochemical and physiological studies. However, there are strong limitations on the use of radiolabels, including safety, long-term storage, and the long time required for autoradiographic imaging. Fluorescence technology circumvents all of these problems and offers the possibility of imaging a few or single molecules in situ. In addition, real-time monitoring of multiple labeled components is feasible. We selected Cy5, a far red fluorochrome, for its high brightness, photostability, low fluorescence background, and visibility in highly scattering media. Initially we used traditional solution phase labeling techniques, but obtained several labeled species of IGF-1 that required extensive purification. We then developed a new technique of labeling the molecule bound to solid phase. The adsorption simultaneously purified and protected the binding site. Preliminary testing of the labeled components indicated that binding capacity was retained. Bead technology with camera-based quantitation provided a ready means of assessing specific binding using very small amounts of material. 15 FLOW SUBTRACTION PROJECT Kimberly Madia and Albert D. Donnenberg University of Pittsburgh Cancer Institute
First proposed in 1934 as an effective method for counting and sizing particles, flow cytometry has evolved into a sophisticated tool for quantitating the chemical and physical properties of cells and cell particles. In practice a laser beam is directed toward a stream of cells. Based on the dyes used to stain the cells and the wavelength of the laser beam, a myriad of colors is emitted at specific angles. Each cell, also referred to as an event, is associated with a set of data points indicating its behavior resulting from the laser beam. These statistics, along with other useful information (i.e. time), are associated with parameters. The parameters are easily compared and analyzed through graphical techniques. Graphical representations are generated using modern software
supported in the windows environment. The software, written in the Visual C++ programming language, adheres to specific guidelines for representing flow files as dictated by Data File Standard Committee of the Society for Analytical Cytology. Some of the most practical, valuable, and widely used options available with this software are the gating capabilities. Gating allows for the ability to analyze very clustered cell populations in greater detail.
The flow subtraction project attempts to compare two different flow files, with similar cell populations, in an effort to determine differences and similarities between the distribution of events in multi-parameter space. Four output files result from the comparison of two files. Two files list events unique to the respective input files, and two files list events that are common to the input files. Using several mathematical equations, the data from the original two files are collapsed into several bin values. This binning is dependent on the aggregate distribution of the data in the input files. After the binning process, the new data sets are compared using commercial analytical software. The ability to easily compare the n-dimensional data distribution of two flow cytometry files finds numerous applications especially in the study of leukocyte cell populations.
16 A NOVEL STRATEGY FOR INFLUENZA VACCINATION Anuj Patel, Hongmei Liang and Joseph M. Ahearn Arthritis Institute University of Pittsburgh
Influenza virus is recognized as a health problem in all regions of the world. Epidemic and pandemic outbreaks of the disease have caused severe morbidity and mortality rates throughout mankind’s history. Influenza is still the sixth most important cause of death in the United States, especially among young children and the elderly.
The threat of influenza can be reduced by the use of currently available vaccines. Research is being conducted, however, to develop safer, more effective vaccination techniques. One such method being pursued is the manipulation of the complement system, which serves as a link between the innate and acquired immune responses.
The influenza virus envelope is spiked by surface glycoproteins. The protein hemagglutinin(HA) is most responsible for triggering antibody responses. Current research focuses on using the complement system to assist in reaction to the HA antigen. A fusion protein derived from HA and a complement ligand is used to target the HA to complement receptors on B cells and follicular dendritic cells. These cells can be found in the germinal centers of secondary lymphoid organs, the cell regions responsible for antibody production during an immune response.
While experiments have shown an ability to enhance antibody response through this complement manipulation, the physical mechanism behind this response is not fully understood. We hope to begin to reveal this mechanism by studying the histology of the immune response. Spleen tissue sections of mice immunized with different antigens were immunostained to identify germinal centers, B-cells, and HA-specific cells. These results allow us to associate physical processes with the antibody response detected in the sera of these mice. By further analyzing and advancing these findings, we may be able to further enhance the efficacy of this novel strategy of vaccination.
17 THE EFFICACY OF TACROLIMUS / STEROIDS VS TACROLIMUS / MYCOPHENOLATE MOFETIL / STEROIDS IN POST-RENAL TRANSPLANT IMMUNOSUPPRESSION Monica Rivera and Mark L. Jordan Division of Urological Surgery University of Pittsburgh Medical Center Background: Each year almost 12,000 kidney transplants are performed in the United States alone. Despite advances made in transplantation, rejection remains a major problem. Therefore, we investigated the efficacy of two drug combinations in an attempt to reduce rejection and, in turn, increase patient and graft survival rates.
Methods: A total of 106 cadaveric renal transplant recipients were randomized to the Tacrolimus (FK)/Steroids Study and 104 to the Tacrolimus (FK)/Mycophenolate Mofetil (MMF)/Steroids Study. Rejections were determined by needle biopsies and graded according to Banff Schema (97). The average follow-up was 708 ± 354 days. Demographic data: FK/Steroids ¾ age 52.4 ± 13.4 years, 61.3% males, 38.7% females, 81.1% Caucasians, 12.3% African-Americans, 6.6% others. FK/MMF/Steroids¾ age 48.4 ± 13.6 years, 54.8% males, 45.2% females, 85.6% Caucasians, 11.5% African-Americans, 2.9% others. Risk factors: FK + Steroids¾ 86.8% primary transplants, 13.2% secondary transplants, 13.2% en bloc transplants, 18.9% with delayed graft function, cold ischemia time 29:05 ± 8:41. FK + MMF + Steroids¾ 82.7% primary transplants, 17.3% secondary, 9.6% en bloc transplants, 20.2% with delayed graft function, cold ischemia time 32:11 ± 9:26. Results: The patient and graft survival rates for FK/Steroids were 87.7% and 79.2% while the FK/MMF/Steroids rates were improved to 90.4% and 81.7%, respectively. The average time to graft failure was prolonged in the FK/MMF/Steroids with a value of 297.1 ± 359.2 days over the double drug therapy value of 249.5 ± 291.4 days. The incidence of rejection was decreased in the triple drug therapy with 51.9% of the patients in the study having acute rejection and 6.7% having vascular rejection, while its double therapy counterpart had results of 59.4% and 19.8% respectively. In addition, the severity of the rejection episodes in the FK/MMF/Steroids study was less, with 33.3% of the episodes having a grade of borderline, 53.7% of them having a grade of one, 9.3% having a grade of two, and 3.7% having a grade of three. The rejection episodes of FK/Steroids were divided accordingly with results of 22.2%, 44.4%, 33.3% and 0%. Finally, the triple therapy decreased the need for OKT3 and high doses of Solu-Medrol from 7.6% and 40.6% to 2.9% and 28.8%. Conclusions: The immunosuppressive regimen of Tacrolimus/Mycophenolate Mofetil/Steroids 1) provides improved long term patient and graft survival rates, 2) decreases the incidence and severity of acute rejection episodes and 3) decreases the need for OKT3 and high doses of Solu-Medrol. 18 CONSTRUCTION OF PLASMID VECTORS FOR GENE EXPRESSION IN BACTERIA AND INSECT CELLS Cynthia Schmid, Laura Sheahan and Saleem Khan Department of Molecular Genetics and Biochemistry University of Pittsburgh School of Medicine The goal of our lab is to understand Human Papillomavirus (HPV) replication. Replication of HPV DNA requires the viral proteins E1 and E2. E1 is involved in initiating viral DNA replication, and E2 both activates and represses viral transcription and replication. Further investigation of the E1 and E2 proteins could reveal how the HPV DNA integrates into the genome to produce malignancy. To carry out these studies, we have constructed vectors for gene therapy and protein expression. If a viral vector could be designed to repress replication in maglignant cells, then a new treatment could be developed for cancer, specifically cervical cancer. Also, viral vector development could lead to treatment of other diseases associated with the skin. In our attempt to understand the structure and function of the E1 protein, a hybrid vector has been constucted. This vector, or donor plasmid, serves as a tool for generation of recombinant baculoviruses which will be used to infect insect cells and allow for viral protein expression. This plasmid’s components consist of the pFASTBAC plasmid, a 71 base paid (bp) oligonucleotide, and a ahybrid E1 gene, composed of various fragments of the HPV-1a E1 and HPV-18 E1. The plasmid, pFASTBAC, was chosen because of its ability to be expressed in an eukaryotic cell, such as an insect sell. The 71 bp oligonucleotide includes a double “flag-tag”. The “flag-tag” serves as a marker, or epitope, for an antibody to recognize the E1 protein, and allow for purification of the E1 protein from the insect cells. The hybrid E1 gene, once cloned into pFASTBAC-Mod (pFASTBAC with oligonucleotide), will be expressed using the baculovirus system. These studies will further our understanding of the biochemical properties of the E1 protein and HPV replication.
TRANSFECTION OF MAMMALIAN CELLS TO EXPRESS GREEN FLUORESCENT PROTEIN- TUBULIN (GFP-TUB) AND-ACTIN(GFP-ACTIN Lisa Raenae Sproul and Melissa Melan Department of Biological Sciences Duquesne University Melatonin has been shown to produce neurite-like outgrowths in mammalian cells that express the human mt1 receptor. To better view these changes, Chinese Hamster Ovary cells expressing the human mt1 receptor (mt1- CHO) have been transfected with the pEGFP-Tub or pEGFP-Actin to express one of two green fluorescent fusion proteins, either tubulin or actin. The pEGFP vectors were purchased from Clontech. They were then successfully transformed into E.coli cells. DNA from the kanamycin resistant colonies were then cut with several restriction enzymes to ensure the success of cloning the correct vector. Once the clonings were verified, we purified large amounts of DNA because several micrograms of DNA were required for each transfection. The purified DNA was then quantified, either according to a UV/vis spec reading or gel quantification. These DNA’s were then used to transfect the mt1 CHO cell line. We then assayed the success of the transfections using fluorescence microscopy both prior to and post treatment of the cells with 1 micromolar melatonin for five hours. The purpose of this research was to transfect the cells with the GFP-fusion to directly visualize the cytoskeleton and better enable study of the cells undergoing change due to melatonin. This research may eventually lead to studies of treatments that promote recovery from CNS trauma or stroke. 20 WOUNDS THAT HEAL WITHOUT SCARS - AN INVESTIGATION WITH DIFFERENTIAL DISPLAY Michael Sweeney, Jay D. Hayes and Garth D. Ehrlich Center for Genomic Sciences Allegheny General Hospital The formation of scar tissue after injury or operation can lead to serious health problems and cosmetic concerns. Unlike limb regeneration in some amphibians and invertebrates, scarring in adult humans produces tissue that is structurally different and weaker than normal skin. However, research has shown that the wounds of a mammalian fetus heal without scars in a much shorter period. The goal of the current work is the identification of which, if any, genes are responsible for this phenomenon. Using a rabbit model, we apply a technique called "differential display" to visualize the genetic expression in fetal and adult wound tissue and screen for unique signals - genes potentially nvolved in scarless wound healing. Differential display is based on the polymerase chain reaction (PCR) and electrophoretic gels. Messenger RNA (mRNA), the immediate product of genetic transcription, is extracted from the tissue sample. Using an enzyme from retroviruses, the mRNA is copied into complementary DNA (cDNA) through reverse transcriptase PCR (RT-PCR). The copy number of each cDNA is greatly increased in an amplifying PCR that incorporates fluorescent primers into the cDNA. The products of these reactions are run on an electrophoretic gel, yielding a fingerprint pattern of bands that represents the pool of mRNA in the original tissue. This process was performed on four types of tissue: wounded and normal (control) adult skin and wounded and control fetal skin. Comparison between lanes shows that some treatments contain bands not found in the others - differential display. This band corresponds to an RNA transcript, and therefore genetic activity, exclusive to that treatment. Ongoing work is looking for genes either upregulated or downregulated in the fetal wound skin. Differentially expressed bands will be isolated, cloned, sequenced, and identified through comparison with known genes. If the gene or genes associated with scarless wound healing can be identified, clinical applications may eventually be devised to benefit people recovering from injury or operation. 21 MUC-1 IN NON-CANCEROUS LUNG LYMPH NODES Monica Jo Tomaszewski and Jill Siegfried Department of Thoracic Surgery University of Pittsburgh School of Medicine
MUC-1 is an underglycosylated mucin found in tumors. Because of its difference from normal epithelial mucin, mutated MUC-1 is seen as a gateway to diagnose the spread of cancer. It is thought that when the cancer begins to spread, MUC-1 would be the indication of the spread of cancer even before the sample would be histolocally positive. This project investigates the use of MUC-1 as a precursor to cancer mobility. Lymph nodes were taken from non-cancerous patients and analyzed for MUC-1 content by RT-PCR using a nested primer system. The resulting gels containing a band at 248 base pairs were then examined. The band at 248 was removed for future sequencing to positively confirm the presence of MUC-1. Because of the amount of benign lymph nodes that contain MUC-1, it was concluded that MUC-1 identification is not a useful diagnostic procedure for determination of cancer diffusion. 22 ALTERNATIVES FOR ISOLATION OF HEMATOPOIETIC STEM CELLS Chad Zatezalo, Sara Cole and Nadir Askenasy Institute for Cellular Therapies Carnegie Mellon University In an attempt to improve the approach to bone marrow transplantation (BMT), alternatives were analyzed for Fluorescent Activated Cell Sorting (FACS) to isolate hematopoietic stem cells (HSC). The FACS procedure is time consuming, sorting only 2,500 cell per hour. Selectively depleting various cell populations contained in the bone marrow, aided in enriching the donor inoculum with HSC for BMT. Immunomagnetic positive HSC isolation provedto be an effective and rapid method for enlarging the concentration of HSC in the bone marrow. Other techniques used were less successful in enriching the HSC population and may to be too time consuming for utilization.
Zyklen, Präparate und Stabilisatoren Als chemotherapeutischen Zyklus bezeichnet man den Zeitraum einer oder mehrerer kurzfristig aufeinander folgender Verabreichungen von chemotherapeutischen Präparaten. Jeder Zyklus kann sich über einige Minuten, Stunden oder auch Tage erstrecken. Erfolgt die Medikation beispielsweise einmal wöchentlich für Minuten oder Stunden, so stellt diese in s
Topic: DRUD EFFECTS ON THYROID FUNCTION Title: Metformin may inhibit thyrotropin (TSH) secretion in patients with hypothyroidism treated with thyroxine. Authors: Vigersky RA, Filmore-Nassar A, Glass AR (Washington DC, USA) Reference: Journal of Clinical Endocrinology and Metabolism: 91: 225-227, 2006 Context: Drug-drug interactions are common but often are discovered only lo