Retreat 2007 M.D./Ph.D. Student Abstracts
DETERMINANTS OF REPRODUCTIVE HEALTH IN RURAL MEXICAN-AMERICAN WOMEN
PSTP Student: Teresa Barcellos
Mentor: Dr Marc Schenker
PSTP Student: Jolene M. Chang
Mentor: Michael Ferns
Collaborator: Jacinthe Gingras
Synapse formation is important for formation, regeneration, and plasticity of the nervous system, and is regulated by bidirectional signalling between pre and postsynaptic neurons which in turn induce pre and postsynaptic differentiations to align together to mediate efficient synaptic transmission. Synaptogenesis has been studied extensively in the neuromuscular junction, where an extracellular matrix protein called agrin is deposited by the presynaptic motoneuron into the basal lamina of the muscle cell, where it signals through MuSK receptor tyrosine kinase to cluster acetylcholine receptors at the postsynaptic site. Outside the neuromuscular junction, agrin has also been studied in the superior cervical ganglion (SCG). Interneuronal synapses in the SCG system shares some similarities to the neuromuscular junction – it is also a fast cholinergic synapse. Agrin knockout mice have fewer synapses in SCG and impaired synaptic transmission. Thus, agrin regulates synaptic differentiation in the peripheral nervous system as well as at the neuromuscular junction. The mechanism through which agrin acts to stabilize and maintain synapses in interneuronal cholinergic synapses is unknown. Most importantly, the receptor for agrin in neurons has not yet been identified. My project is to characterize and identify the agrin receptor in neurons. One approach is to use recombinant agrin fragments to to map agrin’s interaction domain with the receptor, in order to find an existing receptor or a novel receptor using biochemistry and/or expression cloning. Another approach is to take a candidate receptor, MuSK, and examine for similar synaptic defects to agrin knockouts. Preliminary results show that there is a qualitative decrease in the overlap of pre and postsynaptic staining in vivo in MuSK knockout SCG. MuSK knockout SCG also displayed an unexpected upregulation of acetylcholine receptor expression as displayed through immunostaining. The decreased colocalization of pre and postsynaptic markers mirrors the agrin knockout SCG phenotype, whereas the upregulation of acetylcholine receptor in MuSK knockout SCG mirrors the MuSK knockout phenotype in muscle, where acetylcholine receptor levels are also upregulated. These two findings suggest that MuSK is possibly the receptor for agrin in the SCG. Future studies will have to show a direct correlation of agrin activation of MuSK, which can be shown by looking at agrin effects of MuSK tyrosine phosphorylation of neurons in vitro.
PSTP Student: Yu-Ming Chang
Mentors: Drs. Christopher P. Evans and Hsing-Jien Kung
Introduction and Objectives: Prostate cancer (CaP) is the most frequently diagnosed cancer in American men. Src is the first proto-oncogene discovered. Previous studies demonstrate that Src is involved in CaP growth, migration, angiogenesis, and apoptosis.
Methods: Src basal activation and expression levels were profiled in commonly used CaP cell lines CWR22Rv1, DU145, LNCaP, and PC3. CaP cell lines were treated with nano- to micromolar doses of AZD0530, a novel Src kinase inhibitor in MTT cell proliferation and Boyden Chamber cell migration assays. Flow cytometry was used to determine cell cycling changes in DU145 cells following two day treatment of 1µM of AZD0530. Signaling studies were conducted in DU145 cells by Western Blotting using various doses of AZD0530 at 30 minutes and 1µM of AZD0530 at multiple time points. Mice orthotopically implanted with 2x10^6 DU145 cells were treated with 25mg of AZD0530 per kg of mouse weight for seven weeks and four days.
Results: Src is expressed and activated in all CaP cell lines examined. AZD0530 inhibits Src in a dose-dependent fashion with near complete inhibition achieved at 1µM and within five minutes of treatment. CaP cell lines treated with AZD0530 demonstrate dose-dependent cell proliferation inhibition with IC50s in the single-digit micromolar range. Treatment with AZD0530 increased G0/G1 and decreased S phase cell populations. ERK1/2, a kinase linked to Src important in cell proliferation is inhibited by AZD0530. Treatment with AZD0530 lead to decreased c-Myc, a transcription factor involved in cell cycling. IC50s for cell migration inhibition in PC3 and DU145 cells by AZD0530 are 62.5nM and 500nM, respectively. Cell motility factors FAK, p130CAS, and paxillin activation are inhibited by AZD0530. In DU145-implanted mice, control and AZD0530-treated mice developed primary tumors with mean tumor mass of 2.01g (n=10) and 1.107g (n=10), respectively. No metastasis were found in either control or AZD053-treated mice.
Conclusions: We conclude that Src is a mediator of cell proliferation and migration in CaP. We further conclude that AZD0530-inhibited cell proliferation is mediated by G0/G1 cell cycle arrest and likely through c-Myc down-regulation. Thus, AZD0530 may be an effective therapy in the treatment of CaP.
PSTP Student: William Frederick
Mentor: Dr. Jonathan P. Heritage
We wish to test the hypothesis that monochromatic x-ray radiation just above the K-edge of various heavy metals within neoplastic cells is more effective than conventional radiation therapy for killing neoplastic cells in vitro. This project is part of the Compton Light Source (CLS) project at the Stanford Linear Accelerator Center (SLAC) Klystron Department to develop a tunable, compact source of quasi-monoenergetic x-rays for use in cancer detection and treatment. While the CLS is under development, we use both filtered conventional x-ray sources and beamlines at several synchrotron sources (The Stanford Synchrotron Radiation Lab, the Argonne Advanced Photon Source, and The Berkeley Advanced Light Source) for studying dose enhancement in x-ray phototherapy by irradiating established cell lines labeled with high-Z atoms. Cellular uptake and perinuclear localization of such metals will increase probability of secondary radiation products to induce raidation damage. Dose enhancement is assessed through dose-response curves generated both by traditional colony counting and by flow cytometric analysis of immunolabeled DNA double-stranded breaks. Current radiosensitizing agents under consideration include proteins and metalloporphyrins containing iodine, tin, platinum, and gold. Use of metalloporphyrins to which the cell membrane is permeable should allow increased DNA damage by secondary ionization products, thus increasing dose enhancement. Samples are irradiated at energies 0-30 keV above the K-edges of the radiosensitizer metals. The high energy, monochromatic fluxes available at synchrotron sources enable use of higher Z metals for which radiosensitization should be more pronounced. Furthermore, the monochromatic nature of such sources further minimizes radiation damage to normal tissues.
PSTP Student: Philbert Huang
Mentor: Sarigul-Klijn, Nesrin
Collaborators: Gupta M., and Hazelwood S.
Both bone graft and metallic implants have been used in combination with the necessary anterior rod or plate instrumentation to fill the voids left by vertebral body removal, with the ultimate goal of restoring stability. One type of device that has recently been introduced is an expandable titanium telescoping cage that is designed to be used as a strut implant to fill corpectomy defects. The use of these devices has met varying success. Acceptance by surgeons and spine biomechanicians has been limited by clinical failure with subsequent loss of reduction and increase in kyphosis. In order to further improve patient care, it is critical to evaluate the use of these implants through biomechanical as well as other modes of testing.
The purpose of this study is to compare and contrast the spinal fusion outcome of using allograft bone versus the expandable vertebral body replacement titanium implant in a lumbar corpectomy procedure. A controlled biomechanical study of lumbar spine fusion was performed using bone graft and the expandable cage in an in vivo bovine model following a 4-month postoperative healing period. Calves aged 4-6 months with L3 removed were used to create a simulated lumbar corpectomy defect. The calves were randomly allocated to groups for corpectomy defect repair with 1) allograft metatarsal bone and Thoracolumbar Spine Locking Plate, n=6, or 2) Expandable vertebral body replacement device and Thoracolumbar Spine Locking Plate, n=6. Following a 4-month postoperative period, the animals were sacrificed and lumbar spines harvested for biomechanical testing. Strength of fusion was assessed by stiffness of ex vivo spine specimens in flexion-extension, lateral bending, and torsion obtained from biomechanical testing. Uniaxial strain at various positions on the surface of the anterior plate was measured during loading as an additional stability parameter. Loading tests were repeated following removal of the anterior instrumentation (plate and screws).
At 4 months postoperative, the stiffness of the calf spines repaired by the bone graft and locking plate was significantly greater than that of the spines repaired by the expandable cage and locking plate. This finding was true for all three directions of loading. Greater strain values were observed from the gauges on the locking plate of the spines utilizing the expandable cage than the spines utilizing allograft bone. This finding held for all gauge positions (anterior edge, anterior face, posterior edge, and posterior face of the plate). Following locking plate removal and a repeat of the loading tests, a decrease in stiffness of the construct was observed for both the allograft and cage groups. These data may indicate that the use of allograft bone for corpectomy defect repair in the lumbar spine contributes to a stiffer and perhaps more stable spine segment compared to using the expandable cage device for such a repair following a 4-month healing period in this in vivo calf model.
PSTP Student: Brian A. Jonas
Mentor: Dr. Martin L. Privalsky
SMRT and N-CoR are corepressor paralogs that play a critical role in mammalian development, reproduction, and homeostasis by partnering with and mediating transcriptional repression by a wide variety of metazoan transcription factors, including nuclear hormone receptors. Although encoded by distinct genetic loci, SMRT and N-CoR share substantial sequence interrelatedness and can form analogous assemblies with histone deacetylases and auxiliary factors, interact with similar sets of transcription factor partners, and exert overlapping functions. Notably, MAP kinase (MAPK) cascades operating downstream of growth factor and stress signaling pathways can phosphorylate SMRT, inducing its release from its transcription factor partners, export from nucleus to cytoplasm, and derepression of target gene expression. We previously reported that although SMRT is regulated by these MAPK cascades, the otherwise closely related N-CoR is not. Subsequently it was demonstrated that SMRT and N-CoR are expressed as a series of alternatively-spliced protein variants that differ in their structure and function. We therefore characterized the impact of these alternative mRNA splicing events on the corepressor response to MAPK signaling. Whereas the SMRTa, SMRTt and SMRTsp2 splice variants are released from their nuclear receptor partners in response to MAPK activation, the SMRTsp18 variant, which resembles N-CoR in its overall molecular architecture, is largely refractory to this kinase-induced release. Alternative splicing of N-CoR, in contrast, had only minimal effects on the resistance of this corepressor to MAPK inhibition. In addition, while all of the SMRT splice variants examined redistributed from the nucleus to cytoplasm in response to MAPK cascade signaling, none of N-CoR splice variants did so. Different tiers of the MAPK cascade hierarchy contributed to these aspects of corepressor regulation, with MEKK1 and MEK1 regulating subcellular redistribution, and ERK2 regulating nuclear receptor-corepressor interaction. We conclude that cells can customize their transcriptional response to MAPK cascade signaling by selective expression of the SMRT or N-CoR locus, utilization of specific corepressor splice variants, and exploitation of specific MAPK cascade tiers.
PSTP Student: Randie Kim
Mentor: Dr. Richard Bold and Dr. Hsing-Jien Kung
Arginine is a semi-essential amino acid synthesized from citrulline by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Many hepatocellular carcinomas and melanomas are deficient in ASS, making arginine essential. Arginine deiminase (ADI) degrades arginine into citrulline, and in a pegylated form, is a promising clinical agent for cancers lacking ASS. Some prostate cancers are sensitive to arginine depletion, leading us to hypothesize that prostate cancer can be inhibited with ADI. In this study, we demonstrate ASS expression in different prostate cancer lines correlate with cell survival after ADI. In particular, CWR22Rv1 exhibits reduced ASS mRNA and protein expression and is most sensitive to ADI. FACS analysis shows ADI-induced apoptosis in CWR22Rv1 is synergistically enhanced in combination with paclitaxel. Interestingly, autophagy, a process of organellar self-digestion that can ultimately lead to cell death, is detectable in PC3 by pEGFP-LC3 translocation after ADI treatment under fluorescence microscopy. Arginine deprivation possibly triggers autophagy to prolong cell survival under metabolic stress. Autophagic defects have been associated with tumor progression and noted in prostate cancer. We hope to identify any autophagic defects and correlate it to ADI response.
PSTP Student: Claudia M. Krispel
Mentor: Dr. Marie
Signaling through heterotrimeric G proteins is a ubiquitous mechanism in cell biology. Despite the intense study of processes that regulate G protein pathways (e.g. G protein receptor kinases, arrestins, RGSs), it is unknown which regulatory steps determine the overall duration of the resulting intracellular signals. Even in the well-studied G protein cascade of vertebrate rod photoreceptors, the identity of the biochemical step that dictates the time course of the response under normal conditions has been unclear. In rods, timely recovery of the photoresponse requires efficient deactivation of both the G protein coupled receptor, rhodopsin, and the G protein/effector complex. The slowest of these two steps determines the overall rate of response recovery (Nikonov et al., 1998). While some experiments have suggested that rhodopsin lifetime is the slowest, or rate-limiting, step in recovery, other studies have argued that the rate-limiting step is deactivation of the G-protein, transducin.
A principal difficulty in settling this controversy has been that most experimental approaches that have investigated these mechanisms have abnormally prolonged deactivation, either by diluting endogenous regulators in biochemical experiments or by slowing the time course of physiological responses through pharmacological or genetic loss-of-function manipulations. Because lengthening the lifetime of rhodopsin or transducin/PDE does not address which of these two species normally rate-limit recovery, settlement of this controversy demands a manipulation to directly speed the rate-limiting step in intact cells.
We have addressed this question by utilizing mice which over-express (and thus have increased total activity of) rhodopsin kinase and RGS9-1, the regulators that control rhodopsin and transducin/PDE deactivation, respectively. Using suction electrodes, we recorded flash responses from rods of RGS9-over expressing mice and rods of RK-over expressing mice, and compared the time courses of their flash responses. We report that increasing the expression of RGS9-1, accelerates recovery of the light response in mammalian rods, while over expression of RK has no effect on the time course of flash responses. This resolves a long-standing controversy by demonstrating that deactivation of many G protein molecules, rather than the shutoff of long-lived photoexcited rhodopsin, determines the duration of the rod's response to light. Furthermore, these results indicate that RGSs are not merely negative regulators of G protein activity, but can precisely tune the duration of cellular responses.
Nikonov S, Engheta N, Pugh EN Jr. (1998). Kinetics of recovery of the dark-adapted salamander rod response. J Gen Physiol 111(1):7-37.
The nervous systems of vertebrates are characterized by a complex array of intricate connections. However, early in development these connections are in excess and are less precise. Proper nervous system development requires the removal of inappropriate connections while functionally relevant ones are retained. One mechanism for removing excess connections is through the process of stereotyped axon pruning. In stereotyped axon pruning, transient long axon collaterals are predictably pruned away from their targets. We have previously addressed the importance of plexin signaling on the stereotyped pruning of long axon collaterals in the vertebrate CNS; however, a cellular basis for plexins on stereotyped pruning has not been determined. Using quantitative electron microscopy and immunocytochemistry, we found that infrapyramidal mossy fiber axon collaterals form transient synaptic complexes with basal dendrites of CA3 pyramidal cells in the early postnatal mouse hippocampus. At later postnatal ages, these synaptic complexes stop maturing and are removed before stereotyped pruning by a mechanism that does not involve axon degeneration and glial cell engulfment. In knock-out mice that lack plexin-A3 signaling, the synaptic complexes continue to mature, and, as a result, the collaterals are not pruned. Thus, our results suggest that intact plexin-A3 signaling contributes to synaptic complex elimination, which is associated with stereotyped axon pruning.
Investigating the Cancer Stem Cell Hypothesis in Human Glioblastomas: Co-localization of ATF5 and CD133 Identifies a Subpopulation of Progenitor-like Cells
The cancer stem cell hypothesis suggests that cells with progenitor qualities may play a role in the origin and maintenance of neoplastic clones. The ependymal cells lining the ventricles, as well as the subependymal area have been identified as a source of neurocortical progenitor cells which supply the normal brain with additional differentiated cells. The cancer stem cell hypothesis explores the possibility that mutations in progenitor cells may possibly be the origin of tumorigenesis. ATF5 is a transcription factor that plays an important role in suppressing differentiation while stimulating proliferation of neuroprogenitor cells into three main possible terminal cell fates, and thus is a general mediator of progenitor cell cycle control. Downregulation of ATF5 is required for differentiation of progenitor cells into neurons, astrocytes, and oligodendrocytes. Our studies focus on the identification of a subpopulation of progenitor cells in human glioblastoma. To study the cancer stem cell hypothesis in human brain glioblastomas, immunohistochemical staining of tumor sections were performed to determine the presence and distribution of adult stem cell markers CD133, GFAP, and their colocalization with ATF5. CD133+ cells were localized to the ventricular zone in patient tumor tissues and in non-tumor contralateral tissues at coronal tumor section. Absence of CD133+ cells were noted in sections more rostral to the tumor section, while robust staining of CD133+ cells were identified in ipsilateral tumor sections. In comparison, non-tumor contralateral tissue at the same coronal section demonstrated minimal staining for CD133. At sections more rostral to the tumor, ATF5 staining is minimal compared to tumor ventricular zone. Our preliminary results in human tissue support previous findings demonstrating the presence of a progenitor cell population in the ventricular area in murine models, and may be the first to demonstrate the co-existence of a putative stem cell population in human glioblastoma pathogenesis. Our study is also the first to confirm the specificity of CD133+ as a useful marker in identifying cancer stem cells in human glioblastoma. Further studies proving or disproving whether gliomas originate from subjacent stems cells remain to be investigated.
INTRAVENOUS, INTRAPERITONEAL, AND SUBCUTANEOUS ROUTES OF ADMINISTERING 64CU-DOTA-HB22.7 DISPLAY EQUIVALENT TUMOR TARGETING ABILITY
Mentor: Joseph Tuscano
Collaborators: Joseph M. Tuscano, Robert T. O’Donnell, Hayes McKnight, David L. Kukis, and Julie.L.Sutcliffe.
BACKGROUND: Chemotherapy for Non-Hodgkin’s Lymphoma (NHL) is initially effective, but often limited by toxicity and resistance. New agents for the treatment of NHL are needed. HB22.7 is a monoclonal antibody that binds CD22 and has previously been shown to reduce human lymphoma xenograft volume in nude mice. OBJECTIVES: (1) Develop a 64Cu-DOTA-HB22.7 antibody, for in vivo imaging and potential therapy of NHL. (2) Determine if tumor targeting of 64Cu-DOTA-HB22.7 is equivalent between intravenous (IV), intraperitoneal (IP), and subcutaneous (SQ) routes of administration. METHODS: DOTA-HB22.7’s ability to bind CD22+ cells was assessed by flow cytometry. 64Cu-DOTA-HB22.7 was injected IV, IP, or SQ into xenograft-bearing nude mice and tumor targeting assessed by positron emission tomography (PET) and biodistribution assay. Pharmacokinetics were determined by measuring radioactivity of blood samples. Serum was analyzed by radio-TLC. RESULTS: DOTA conjugation does not affect HB22.7’s ability to bind CD22. 64Cu-DOTA-HB22.7 demonstrates specific tumor targeting at 24 and 48 hrs. Targeting is equivalent regardless of route of administration. Pharmacokinetics demonstrate that 64Cu-DOTA-HB22.7 can access the bloodstream (and thus, target tumor) in IP or SQ injections. By 48 hrs, blood 64Cu levels are (a) equivalent, regardless of injection route and (b) below peak levels, indicating clearance from the circulation. Serum analysis shows that right-shifted TLC peaks, possibly 64Cu-DOTA-HB22.7 metabolites, are present only in the IV injected group at 48 hrs. CONCLUSION: These findings establish 64Cu-DOTA-HB22.7 as an NHL-specific imaging agent and indicate its potential for use in radioimmunotherapy. These findings provide evidence that more accessible routes of administration can achieve equivalent targeting results, and may lead to more efficient and accurate administration of antibody-based therapeutics in mice.
PSTP Student: Noah M. Merin
Mentor: Sally J. Rogers
Autism is a neurodevelopmental disorder that profoundly affects social development and language. Genetic factors are thought to play an important role in the risk of developing the disorder; twin-concordance studies have estimated heritability at 60-90%, one of the highest for any psychiatric disorder. It is presently diagnosed using a structured clinical observation of young toddlers who show signs of abnormal development. This clinical observation assesses behaviors that do not emerge until the second year of life (such as language, peer interaction, pointing, etc.). Therefore, autism cannot currently be diagnosed until after these behaviors typically appear. If we could identify the presence of behavioral markers of the disorder at an earlier age, there would be more time to intervene before the onset of symptoms. Knowing the age of onset also has theoretical importance—it would help in the search for environmental triggers and focus attention on specific neurodevelopmental processes.
Infant gaze behavior, particularly during reciprocal social interaction, may be a useful behavioral marker for autism in young infants.
In the present study, we tested 31 six-month-old infants at elevated risk for developing autism, and 24 typically-developing controls. We recorded eye-tracking data during a live reciprocal interaction between the infant and the mother. In the at-risk group, we found 10 infants who made poor eye contact, and focused visual attention predominantly on the mother’s mouth. Only one infant in the control group showed this pattern of gaze. Ongoing longitudinal work will determine if the subgroup of at-risk infants who do not look at their mother’s eyes later develop autism or autism-related disorders.
PSTP Student: Karen J. Mu
Mentors: Ebenezer Yamoah, Tsung-Yu Chen
Collaborators:_Liping Nie, Wei-Nan Lian, Wei-Ping Yu, Ana
Barttin is an auxiliary subunit of the ClC-K channel and its functional importance in sound transduction is underlined by the fact that mutations result in Bartter Syndrome type IV, of which deafness is a component. Barttin has been identified as a necessary subunit of the ClC-K channel, crucial for translocation and function. However, the stoichiometry of the assembly between Barttin and the ClC-K dimer is not known.
We employed Fluoresence Resonance Energy Transfer
A unique property of ClC-K1 channels within the ClC family is the potentiation of current upon increased extracellular calcium, and we have observed this phenomenon in macroscopic recordings from Xenopus laevis oocytes expressing ClC-K and Barttin. Upon examination of this effect on HEK cells co-expressing the fusion protein constructs using 1 mM EGTA and 5 and 20 mM of extracellular calcium, we observed that the increase of extracellular calcium has an effect on FRET efficiency, implicating that the observed potentiation of current upon increasing extracellular calcium may be due to modifiable physical interactions between the ClC-K and Barttin subunits. These findings provide a working model to help us better understand the structure and function of ClC-K channels.
PSTP Student: Ekama Onofiok
Mentor: Kit S. Lam
Cellular interaction with functionalized macro-molecular scaffolds is being studied for further investigation of cell adhesion, signaling, protease-mediated extracellular matrix degradation, and migratory processes involved in tumor cell metastasis. Herein, we report the use of both modified agarose and polyvinyl alcohol (PVA) scaffolds for these purposes. The macromolecular scaffolds were first ketone or azido-derivatized for further conjugation to various compounds of interest, such as cell adhesion peptides and small molecules, other extracellular matrix components, and finally, substrates of certain proteases found to be overexpressed in several malignancies. The functionalized scaffolds were subsequently physically crosslinked, evaluated and optimized for adherence and proliferation of a lymphoid cancer cell line. Using urokinase (uPA) substrate-functionalized scaffolds, we also report studies aimed at the development of an in-situ releasable protease assay, in an effort to identify and characterize small molecule inhibitors of uPA from a one-bead one-compound combinatorial library of novel compounds.
MICROVASCULAR CHANGES FOLLOWING HEMORRHAGE AND RESUSCITATION WITH POLYETHYLENE GLYCOL-ALBUMIN IN A RABBIT HEMORRHAGIC SHOCK MODEL
PSTP Student: Patricia L. To
: Dr. Anthony T. Cheung Mentor
Concerns associated with the use of allogeneic blood transfusion as treatment for acute blood loss have led to the development of various blood substitutes with emphasis placed on restoration of systemic function, biochemistry, and oxygenation variables. However, studies have suggested that maintaining a uniformly distributed and normalized microcirculation may be more effective than substitution of red blood cells in the treatment of hemorrhagic shock. PEGylated plasma expanders, in which polyethylene glycol (PEG) is covalently bound to proteins such as albumin to produce a high colloid osmotic pressure, may restore and maintain the microcirculation during hemorrhagic shock. A rabbit hemorrhagic shock model was used to study the effects of resuscitation with PEGylated bovine serum albumin (PEG-BSA). Animals were anesthetized and instrumented for monitoring of systemic function and oxygenation characteristics. Following an equilibration period, animals were hemorrhaged to and maintained at a mean arterial pressure (MAP) of 35-40 mmHg (~40% blood loss) for one hour to allow for the establishment of hemorrhagic shock. Animals were then resuscitated with normal saline (NS), bovine serum albumin (BSA), or PEG-BSA. Computer-assisted intravital microscopy was used to videotape and quantify microvascular changes in the conjunctival microcirculation continuously throughout the study to correlate with systemic function and oxygenation changes. Pre-hemorrhagic MAP (72.3±7.8 mmHg), cardiac output (CO) (735±198 ml/min), and microvascular characteristics were similar in all animals. All animals showed similar post-hemorrhagic changes, including decreases in venular diameter and variability in red-cell velocity along with significant reductions in MAP (35.0±4.4 mmHg) and CO (232±137 ml/min). Resuscitation with a large volume of NS resulted in improvement of MAP and microvascular characteristics but not to pre-hemorrhagic levels and without improvement in CO. Resuscitation with a small volume of BSA (25% removed blood volume) did not restore MAP but restored microvascular characteristics and CO to some extent. Resuscitation with a small volume (25% removed blood volume) of PEG-BSA did not improve MAP or microvascular characteristics but restored CO close to pre-hemorrhagic values. This unexpected result may be due to the fact that a greater volume of blood was removed compared to the other animals to achieve a MAP of 35-40 mmHg.
ABATACEPT (CTLA4-Ig), ALEFACEPT (LFA3-Ig), WHOLE BLOOD VENOUS DONOR SPECIFIC TRANSFUSION, AND SIROLIMUS PROLONG RENAL ALLOGRAFT SURVIVAL IN NON-HUMAN PRIMATES
PSTP Student: Tim Weaver
Collaborator: Allan D. Kirk M.D., Ph.D.
Abatacept is a fusion protein CTLA4-Ig, and binds CD80 (B7.1) CD86 (B7.2) on Antigen Presenting Cells (APCs), blocking the binding of costimulatory ligand CD28 on T-cells. Alefacept is also human fusion protein consisting of the extracellular domain of LFA-3 and the Fc portion of IgG1. It binds to CD2 on T-cells thereby preventing LFA-3 ligation (on APCs). Multiple studies have shown that alefacept blocks T-cell activation, particularly memory T cell activation. Furthermore, it is well know that Abatacept works significantly works on naïve T-cell populations, inhibiting activation, and inducing a state of T-cell anergy. Donor specific transfusion (DST) helps to drive the initial T-cell receptor binding to antigen (signal 1). This will then up-regulates costimulatory molecules on both T-cells and APCs. Various murine models and large animal models of organ transplant have shown that if costimulation is inhibited during signal 1 activation, the T-cell will not be fully activated and acceptance or tolerance against that antigen can be induced. In transplant this has shown to be an effective way of inducing allograft tolerance. However, some subsets of memory T-cells do not need co-stimulation to become fully activated, thus eventually causing an immune rejection response against the allograft. This is the first time these two immunomodulatory fusion protiens have been used together. We hypothesized that synergy between these two therapies will inhibit the full T-cell repertoire (naive and memory) from activating a full immune response during the time of organ transplant. As a result, creating an initial state of T-cell anergy which will allow acceptance / tolerance of the allograft. Taking either one of these therapies by themselves in the same transplant model, will get allograft rejection within 20 – 25 days. Currently, we have three nonhuman primates post renal transplant without allograft rejection, 98, 33, and 33 days. One more nonhuman primate rejected at 56 days post transplant. Outbred MHC non-identical rhesus macaques were used as donor-recipient pairs for renal transplant studies performed on day 0. Animals received DST 7ml/kg whole blood, abatacept 10mg/kg IV, alefacept 0.3 mg/kg IV, and sirolimus 1mg/kg PO on day -1, 0, 3, 7, then weekly for two months, at which time all medications are stopped. These animals were followed serially by 7-color flow cytometry to determine the relative and absolute number of CD4 and CD8 naive and memory cells. Thus far we have seen a slight deletion of memory T-cells. Naïve T-cells have increased to retain T-cell homeostasis, however their activation potential is decreased, making them hyporesponsive to donor antigen as shown in MLR assays. Mixed lymphocyte reactions (MLR) assays are used to monitor recipient immune response, proliferation, when stimulated by donor antigen in vitro. Overall, we are definitely having a positive synergism between immunomodulatory therapies abatacept and alefacept by increased non-rejection times in nonhuman primate renal allograft transplantation and a hyporesponsiveness in T-cell response to donor antigen. Whether this will lead to a strong therapy for operational tolerance induction remains to be determined.