ITI PUBLICATIONS – April 2012 (77)

1)Nat Protoc. 2012 Apr 5;7(5):829-38. doi: 10.1038/nprot.2012.021.Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns.Sanchez-Freire V, Ebert AD, Kalisky T, Quake SR, Wu JC.

1] Department of Medicine, Stanford University School of Medicine, Stanford, California, USA. [2] Department of Radiology, Stanford University School of Medicine, Stanford, California, USA. [3].

Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.PMID: 22481529 [PubMed - in process]--2)Trends Immunol. 2012 Apr 2. [Epub ahead of print]A deep profiler's guide to cytometry.Bendall SC, Nolan GP, Roederer M, Chattopadhyay PK.

Baxter Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA.

In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.Published by Elsevier Ltd.PMID: 22476049 [PubMed - as supplied by publisher]--3)Pediatr Transplant. 2012 Apr 4. doi: 10.1111/j.1399-3046.2012.01688.x. [Epub ahead of print]Steroid-free immunosuppression in teenagers: Living without a safety net.Grimm PC, Concepcion W.

Departments of Pediatrics Surgery, Pediatric Kidney Transplant Program, Stanford University and Lucile Packard Children's Hospital at Stanford, G306, 300 Pasteur Drive MC 5208, Stanford, CA 94305-5208 E-mail: pgrimm@stanford.edu.

PMID: 22471858 [PubMed - as supplied by publisher]--4)Cell Mol Life Sci. 2012 Apr 1. [Epub ahead of print]Immune aging and autoimmunity.Goronzy JJ, Weyand CM.

Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, CCSR Building Room 2215, Mail Code 5166, 269 Campus Drive West, Stanford, CA, 94305-5166, USA, jgoronzy@stanford.edu.

Age is an important risk for autoimmunity, and many autoimmune diseases preferentially occur in the second half of adulthood when immune competence has declined and thymic T cell generation has ceased. Many tolerance checkpoints have to fail for an autoimmune disease to develop, and several of those are susceptible to the immune aging process. Homeostatic T cell proliferation which is mainly responsible for T cell replenishment during adulthood can lead to the selection of T cells with increased affinity to self- or neoantigens and enhanced growth and survival properties. These cells can acquire a memory-like phenotype, in particular under lymphopenic conditions. Accumulation of end-differentiated effector T cells, either specific for self-antigen or for latent viruses, have a low activation threshold due to the expression of signaling and regulatory molecules and generate an inflammatory environment with their ability to be cytotoxic and to produce excessive amounts of cytokines and thereby inducing or amplifying autoimmune responses.PMID: 22466672 [PubMed - as supplied by publisher]--5)J Neurosci Methods. 2012 Mar 28. [Epub ahead of print]Distal hypoxic stroke: A new mouse model of stroke with high throughput, low variability and a quantifiable functional deficit.Doyle KP, Fathali N, Siddiqui MR, Buckwalter MS.

Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305-5489, United States; Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305-5489, United States.

C57BL/6J are the most commonly used strain of mouse for stroke experiments but vascular anatomy of the Circle of Willis within this strain is extremely variable and the cortex has extensive collateralization. This causes large variability in stroke models that target the middle cerebral artery proximally and confers resistance to ischemia in those that target it distally. We tested the hypothesis that by combining distal middle cerebral artery occlusion with 1h of hypoxia, we could generate a large lesion that causes a behavioral deficit with low variability. We found that this new distal hypoxic (DH) model of stroke generates a lesion with a volume of 25% of the ipsilateral hemisphere, extends to the motor cortex and causes a behavioral deficit. It also has a very clear border, exceptionally low variability, and can be performed by a single surgeon on up to 30 animals a day. Moreover, survivability is 100% in young adult animals, the model can be performed on old animals, and therapeutic intervention can reduce infarct volume. Therefore DH stroke is an excellent complement to existing stroke models and could be used for preclinical studies in C57BL/6J mice.Copyright © 2012. Published by Elsevier B.V.

PMID: 22465679 [PubMed - as supplied by publisher]--6)Pediatr Transplant. 2012 Mar 30. doi: 10.1111/j.1399-3046.2012.01677.x. [Epub ahead of print]The impact of hepatic portoenterostomy on liver transplantation for the treatment of biliary atresia: Early failure adversely affects outcome.Alexopoulos SP, Merrill M, Kin C, Matsuoka L, Dorey F, Concepcion W, Esquivel C, Bonham A.

Department of Surgery, University of Southern California, Los Angeles Department of Surgery, Stanford University, Palo Alto Department of Biostatistics, Children's Hospital of Los Angeles, Los Angeles, CA, USA.

Alexopoulos SP, Merrill M, Kin C, Matsuoka L, Dorey F, Concepcion W, Esquivel C, Bonham A. The impact of hepatic portoenterostomy on liver transplantation for the treatment of biliary atresia: Early failure adversely affects outcome. Abstract:  The most common indication for pediatric LTx is biliary atresia with failed HPE, yet the effect of previous HPE on the outcome after LTx has not been well characterized. We retrospectively reviewed a single-center experience with 134 consecutive pediatric liver transplants for the treatment of biliary atresia from 1 May 1995 to 28 April 2008. Of 134 patients, 22 underwent LTx without prior HPE (NPE), while 112 patients underwent HPE first. HPE patients were grouped into EF, defined as need for LTx within the first year of life, and LF, defined as need for LTx beyond the first year of life. NPE and EF groups differed significantly from the LF group in age, weight, PELD, and ICU status (p < 0.05) with NPE having the highest PELD and ICU status. Patients who underwent salvage LTx after EF following HPE had a significantly higher incidence of post-operative bacteremia and septicemia (p < 0.05), and subsequently lower survival rates. One-year patient survival and graft survival were as follows: NPE 100%, EF 81%, and LF 96% (p < 0.05); and NPE 96%, EF 79%, and LF 96% (p < 0.05). Further investigation into the optimal treatment of biliary atresia should focus on identifying patients at high risk of EF who may benefit from proceeding directly to LTx given the increased risk of post-LTx bacteremia, sepsis, and death after failed HPE.© 2012 John Wiley & Sons A/S.PMID: 22463739 [PubMed - as supplied by publisher]--7)J Biomed Opt. 2012 Feb;17(2):021102.In vivo near-infrared dual-axis confocal microendoscopy in the human lower gastrointestinal tract.Piyawattanametha W, Ra H, Qiu Z, Friedland S, Liu JT, Loewke K, Kino GS, Solgaard O, Wang TD, Mandella MJ, Contag CH.

Stanford University, James H. Clark Center for Biomedical Engineering & Sciences, Departments of Pediatrics, Radiology and Microbiology & Immunology, Molecular Imaging Program. Stanford, California 94305.

Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope

with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5  frames/sec with a field of view of 362×212  μm(2) and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.PMID: 22463020 [PubMed - in process]--8)Physiol Genomics. 2012 Mar 27. [Epub ahead of print]Dynamic microRNA Expression During the Transition from Right Ventricular Hypertrophy to Failure.Reddy S, Zhao M, Hu DQ, Fajardo GA, Hu S, Ghosh Z, Rajagopalan V, Wu JC, Bernstein D.

Stanford University.

Background: MicroRNAs (miRs) are small, non-coding RNAs that are emerging as crucial regulators of cardiac remodeling in left ventricular hypertrophy (LVH) and failure (LVF). However, there is no data on their role in right ventricular hypertrophy (RVH) and failure (RVF). This is a critical question given that the RV is uniquely at risk in patients with congenital right-sided obstructive lesions and in those with systemic RVs. Methods and Results: We have developed a murine model of RVH and RVF using pulmonary artery constriction (PAC). miR microarray analysis of RV from PAC versus control demonstrates altered miR expression with gene targets associated with cardiomyocyte survival and growth during hypertrophy (miR 199a-3p) and reactivation of the fetal gene program during heart failure (miR-208b). The transition from hypertrophy to heart failure is characterized by apoptosis and fibrosis (miRs-34, 21, 1). Most are similar to LVH/LVF. However, there are several key differences between RV and LV: 4 miRs (34a, 28, 148a and 93) were upregulated in RVH/RVF that are downregulated or unchanged in LVH/LVF. Furthermore, there is a corresponding downregulation of their putative target genes involving cell survival, proliferation, metabolism, ECM turnover and impaired proteosomal function. Conclusions: The current study demonstrates, for the first time, alterations in miRs during the process of RV remodeling and the gene regulatory pathways leading to RVH and RVF. Many of these alterations are similar to those in the afterload stressed LV. miRs differentially regulated between the RV and LV may contribute to the RVs increased susceptibility to heart failure.PMID: 22454450 [PubMed - as supplied by publisher]--9)Proc Natl Acad Sci USA. 2012 Mar 26. [Epub ahead of print]The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors.Willingham SB, Volkmer JP, Gentles AJ, Sahoo D, Dalerba P, Mitra SS, Wang J, Contreras-Trujillo H, Martin R, Cohen JD, Lovelace P, Scheeren FA, Chao MP, Weiskopf K, Tang C, Volkmer AK, Naik TJ, Storm TA, Mosley AR, Edris B, Schmid SM, Sun CK, Chua MS, Murillo O, Rajendran P, Cha AC, Chin RK, Kim D, Adorno M, Raveh T, Tseng D, Jaiswal S, Enger PO,

Steinberg GK, Li G, So SK, Majeti R, Harsh GR, van de Rijn M, Teng NN, Sunwoo JB, Alizadeh AA, Clarke MF, Weissman IL.

Institute for Stem Cell Biology and Regenerative Medicine and the Ludwig Cancer Center, Department of Urology, Stanford Research Initiative for Systems Biology of Cancer, Department of Internal Medicine, Division of Oncology, Asian Liver Center, Department of Radiation Oncology, Department of Neurosurgery, Department of Internal Medicine, Division of Hematology, Department of Otolaryngology, Head and Neck Surgery, Department of Pathology, and Department of Obstetrics and Gynecology, Stanford University Medical Center, Stanford, CA 94305.

CD47, a "don't eat me" signal for phagocytic cells, is expressed on the surface of all human solid tumor cells. Analysis of patient tumor and matched adjacent normal (nontumor) tissue revealed that CD47 is overexpressed on cancer cells. CD47 mRNA expression levels correlated with a decreased probability of survival for multiple types of cancer. CD47 is a ligand for SIRPα, a protein expressed on macrophages and dendritic cells. In vitro, blockade of CD47 signaling using targeted monoclonal antibodies enabled macrophage phagocytosis of tumor cells that were otherwise protected. Administration of anti-CD47 antibodies inhibited tumor growth in orthotopic immunodeficient mouse xenotransplantation models established with patient tumor cells and increased the survival of the mice over time. Anti-CD47 antibody therapy initiated on larger tumors inhibited tumor growth and prevented or treated metastasis, but initiation of the therapy on smaller tumors was potentially curative. The safety and efficacy of targeting CD47 was further tested and validated in immune competent hosts using an orthotopic mouse breast cancer model. These results suggest all human solid tumor cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination. These data, taken together with similar findings with other human neoplasms, show that CD47 is a commonly expressed molecule on all cancers, its function to block phagocytosis is known, and blockade of its function leads to tumor cell phagocytosis and elimination. CD47 is therefore a validated target for cancer therapies.PMID: 22451913 [PubMed - as supplied by publisher]--10)Ann Neurol. 2012 Mar;71(3):287-8. doi: 10.1002/ana.23538.Re-engineering of pathogenic aquaporin 4-specific antibodies as molecular decoys to treat neuromyelitis optica.Steinman L, Zamvil SS.

Department of Neurology and Neurological Sciences Stanford University Palo Alto, CA.

PMID: 22451198 [PubMed - in process]--11)Nature. 2012 Mar 25. doi: 10.1038/nature10975. [Epub ahead of print]Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'Levin AM, Bates DL, Ring AM, Krieg C, Lin JT, Su L, Moraga I, Raeber ME, Bowman GR, Novick P, Pande VS, Fathman CG, Boyman O, Garcia KC.

1] Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305, USA [2].

The immunostimulatory cytokine interleukin-2 (IL-2) is a growth factor for a wide range of leukocytes, including T cells and natural killer (NK) cells. Considerable effort has been invested in using IL-2 as a therapeutic agent for a variety of immune disorders ranging from AIDS to cancer. However, adverse effects have limited its use in the clinic. On activated T cells, IL-2 signals through a quaternary 'high affinity' receptor complex consisting of IL-2, IL-2Rα (termed CD25), IL-2Rβ and IL-2Rγ. Naive T cells express only a low density of IL-2Rβ and IL-2Rγ, and are therefore relatively insensitive to IL-2, but acquire sensitivity after CD25 expression, which captures the cytokine and presents it to IL-2Rβ and IL-2Rγ. Here, using in vitro evolution, we eliminated the functional requirement of IL-2 for CD25 expression by engineering an IL-2 'superkine' (also called super-2) with increased binding affinity for IL-2Rβ. Crystal structures of the IL-2 superkine in free and receptor-bound forms showed that the evolved mutations are principally in the core of the cytokine, and molecular dynamics simulations indicated that the evolved mutations stabilized IL-2, reducing the flexibility of a helix in the IL-2Rβ binding site, into an optimized receptor-binding conformation resembling that when bound to CD25. The evolved mutations in the IL-2 superkine recapitulated the functional role of CD25 by eliciting potent phosphorylation of STAT5 and vigorous proliferation of T cells irrespective of CD25 expression. Compared to IL-2, the IL-2 superkine induced superior expansion of cytotoxic T cells, leading to improved antitumour responses in vivo, and elicited proportionally less expansion of T regulatory cells and reduced pulmonary oedema. Collectively, we show that in vitro evolution has mimicked the functional role of CD25 in enhancing IL-2 potency and regulating target cell specificity, which has implications for immunotherapy.PMID: 22446627 [PubMed - as supplied by publisher]--12)Mol Immunol. 2012 Mar 23. [Epub ahead of print]Cd14 SNPs regulate the innate immune response.Liu HH, Hu Y, Zheng M, Suhoski MM, Engleman EG, Dill DL, Hudnall M, Wang J, Spolski R, Leonard WJ, Peltz G.

Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305, USA.

CD14 is a monocytic differentiation antigen that regulates innate immune responses to pathogens. Here, we show that murine Cd14 SNPs regulate the length of Cd14 mRNA and CD14 protein translation efficiency, and consequently the basal level of soluble CD14 (sCD14) and type I IFN production by murine macrophages. This has substantial downstream consequences for the innate immune response; the level of expression of at least 40 IFN-responsive murine genes was altered by this mechanism. We also observed that there was substantial variation in the length of human CD14 mRNAs and in their translation efficiency. sCD14 increased cytokine production by human dendritic cells (DCs), and sCD14-primed DCs augmented human CD4T cell proliferation. These findings may provide a mechanism for exploring the complex relationship between CD14 SNPs, serum sCD14 levels, and susceptibility to human infectious and allergic diseases.Copyright © 2012 Elsevier Ltd. All rights reserved.PMID: 22445606 [PubMed - as supplied by publisher]--13)Neuron. 2012 Mar 22;73(6):1100-7. Epub 2012 Mar 21.Neuroprotection from Stroke in the Absence of MHCI or PirB.

Adelson JD, Barreto GE, Xu L, Kim T, Brott BK, Ouyang YB, Naserke T, Djurisic M, Xiong X, Shatz CJ, Giffard RG.

Department of Biology and Neurobiology, Stanford University, Stanford, CA 94305-5437, USA.

Recovery from stroke engages mechanisms of neural plasticity. Here we examine a role for MHC class I (MHCI) H2-Kb and H2-Db, as well as PirB receptor. These molecules restrict synaptic plasticity and motor learning in the healthy brain. Stroke elevates neuronal expression not only of H2-Kb and H2-Db, but also of PirB and downstream signaling. KbDb knockout (KO) or PirB KO mice have smaller infarcts and enhanced motor recovery. KO hippocampal organotypic slices, which lack an intact peripheral immune response, have less cell death after in vitro ischemia. In PirB KO mice, corticospinal projections from the motor cortex are enhanced, and the reactive astrocytic response is dampened after MCAO. Thus, molecules that function in the immune system act not only to limit synaptic plasticity in healthy neurons, but also to exacerbate brain injury after ischemia. These results suggest therapies for stroke by targeting MHCI and PirB.Copyright © 2012 Elsevier Inc. All rights reserved.PMID: 22445338 [PubMed - in process] PMCID: PMC3314229 [Available on 2013/3/22]--14)Immunity. 2012 Mar 23;36(3):438-50.Plasmacytoid dendritic cells transport peripheral antigens to the thymus to promote central tolerance.Hadeiba H, Lahl K, Edalati A, Oderup C, Habtezion A, Pachynski R, Nguyen L, Ghodsi A, Adler S, Butcher EC.

Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA; The Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA.

Central tolerance can be mediated by peripheral dendritic cells (DCs) that transport innocuous antigens (Ags) to the thymus for presentation to developing T cells, but the responsible DC subsets remained poorly defined. Immature plasmacytoid DCs (pDCs) express CCR9, a chemokine receptor involved in migration of T cell precursors to the thymus. We show here that CCR9 mediated efficient thymic entry of endogenous or i.v. transfused pDCs. pDCs activated by Toll-like receptor (TLR) ligands downregulated CCR9 and lost their ability to home to the thymus. Moreover, endogenous pDCs took up subcutaneously injected fluorescent Ag and, in the absence of TLR signals, transported Ag to the thymus in a CCR9-dependent fashion. Injected, Ag-loaded pDCs effectively deleted Ag-specific thymocytes, and this thymic clonal deletion required CCR9-mediated homing and was prevented by infectious signals. Thus, peripheral pDCs can contribute to immune tolerance through CCR9-dependent transport of peripheral Ags and subsequent deletion of Ag-reactive thymocytes.Copyright © 2012 Elsevier Inc. All rights reserved.PMID: 22444632 [PubMed - in process] PMCID: PMC3315699 [Available on 2013/3/23]--15)Chem Biol. 2012 Mar 23;19(3):340-52.An optimized activity-based probe for the study of caspase-6 activation.

Edgington LE, van Raam BJ, Verdoes M, Wierschem C, Salvesen GS, Bogyo M.

Cancer Biology Program, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5324, USA; Department of Pathology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5324, USA.

Although significant efforts have been made to understand the mechanisms of caspase activation during apoptosis, many questions remain regarding how and when executioner caspases get activated. We describe the design and synthesis of an activity-based probe that labels caspase-3/-6/-7, allowing direct monitoring of all executioner caspases simultaneously. This probe has enhanced in vivo properties and reduced cross-reactivity compared to our previously reported probe, AB50. Using this probe, we find that caspase-6 undergoes a conformational change and can bind substrates even in the absence of cleavage of the proenzyme. We also demonstrate that caspase-6 activation does not require active caspase-3/-7, suggesting that it may autoactivate or be cleaved by other proteases. Together, our results suggest that caspase-6 activation proceeds through a unique mechanism that may be important for its diverse biological functions.Copyright © 2012 Elsevier Ltd. All rights reserved.PMID: 22444589 [PubMed - in process] PMCID: PMC3314226 [Available on 2013/3/23]--16)Pain. 2012 Mar 21. [Epub ahead of print]Pain sensitivity and opioid analgesia: a pharmacogenomic twin study.Angst MS, Phillips NG, Drover DR, Tingle M, Ray A, Swan GE, Lazzeroni LC, Clark JD.

Department of Anesthesia, Stanford University School of Medicine, Stanford, CA 94305, USA.

Opioids are the cornerstone medication for the management of moderate to severe pain. Unfortunately, vast inter-individual differences in dose requirements complicate their effective and safe clinical use. Mechanisms underlying such differences are incompletely understood, are likely multifactorial, and include genetic and environmental contributions. While accumulating evidence suggests that variants of several genes account for some of the observed response variance, the relative contribution of these factors remains unknown. This study used a twin paradigm to provide a global estimate of the genetic and environmental contributions to inter-individual differences in pain sensitivity and analgesic opioid effects. Eighty one monozygotic and 31 dizygotic twin pairs successfully underwent a computer-controlled infusion with the μ-opioid agonist alfentanil in a single occasion, randomized, double-blind and placebo-controlled study design. Pain sensitivity and analgesic effects were assessed with experimental heat and cold pressor pain models along with important covariates including demographic factors, depression, anxiety, and sleep quality. Significant heritability was detected for cold pressor pain tolerance and opioid-mediated elevations in heat and cold pressor pain thresholds. Genetic effects accounted for 12-60% of the observed response variance. Significant familial effects accounting for 24-32% of observed variance were detected for heat and cold pressor pain thresholds and opioid-mediated elevation in cold pressor pain tolerance. Significant covariates included age, gender, race, education, and anxiety. Results provide a strong rationale for more detailed molecular genetic studies to elucidate mechanisms underlying inter-individual differences in pain sensitivity and analgesic opioid responses. Such studies will require careful consideration of the studied pain phenotype.

Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.PMID: 22444188 [PubMed - as supplied by publisher]--17)J Burn Care Res. 2012 Mar;33(2):e49-54.In Vivo Molecular Imaging of Murine Embryonic Stem Cells Delivered to a Burn Wound Surface via Integra® Scaffolding.Hamrahi VF, Goverman J, Jung W, Wu JC, Fischman AJ, Tompkins RG, Yu YY, Fagan SP, Carter EA.

From the Departments of *Pediatrics and †Surgery, Massachusetts General Hospital; ‡Shriners Hospitals for Children; and §Harvard Medical School, Boston, Massachusetts; and ‖Department of Radiology, Stanford University, Stanford, California.

It has been demonstrated that restoration of function to compromised tissue can be accomplished by transplantation of bone marrow stem cells and/or embryonic stem cells (ESCs). One limitation to this approach has been the lack of noninvasive techniques to longitudinally monitor stem cell attachment and proliferation. Recently, murine ESC lines that express green fluorescent protein (GFP), luciferase (LV), and herpes simplex thymidine kinase (HVTK) were developed for detection of actively growing cells in vivo by imaging. In this study, the authors investigated the use of these ESC lines in a burned mouse model using Integra® as a delivery scaffolding/matrix. Two different cell lines were used: one expressing GFP and LV and the other expressing GFP, LV, and HVTK. Burn wounds were produced by application of a brass block (2 × 2 cm kept in boiling water before application) to the dorsal surface of SV129 mice for 10 seconds. Twenty-four hours after injury, Integra® with adherent stem cells was engrafted onto a burn wound immediately after excision of eschar. The stem cells were monitored in vivo by measuring bioluminescence with a charge-coupled device camera and immunocytochemistry of excised tissue. Bioluminescence progressively increased in intensity over the time course of the study, and GFP-positive cells growing into the Integra® were detected. These studies demonstrate the feasibility of using Integra® as a scaffolding, or matrix, for the delivery of stem cells to burn wounds as well as the utility of bioluminescence for monitoring in vivo cellular tracking of stably transfected ESC cells.PMID: 22441149 [PubMed - in process] PMCID: PMC3311992 [Available on 2013/3/1]--18)Proc Natl Acad Sci USA. 2012 Apr 10;109(15):5820-5. Epub 2012 Mar 22.Co-transplantation of pure blood stem cells with antigen-specific but not bulk T cells augments functional immunity.Müller AM, Shashidhar S, Küpper NJ, Kohrt HE, Florek M, Negrin RS, Brown JM, Shizuru JA.

Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University, Stanford, CA 94305.

Impaired immunity is a fundamental obstacle to successful allogeneic hematopoietic cell transplantation. Mature graft T cells are thought to provide protection from infections early after transplantation, but can cause life-threatening graft-vs.-host disease. Human CMV is a major pathogen after transplantation. We studied reactivity against the mouse homologue, murine

CMV (MCMV), in lethally irradiated mice given allogeneic purified hematopoietic stem cells (HSCs) or HSCs supplemented with T cells or T-cell subsets. Unexpectedly, recipients of purified HSCs mounted superior antiviral responses compared with recipients of HSC plus unselected bulk T cells. Furthermore, supplementation of purified HSC grafts with CD8(+) memory or MCMV-specific T cells resulted in enhanced antiviral reactivity. Posttransplantation lymphopenia promoted massive expansion of MCMV-specific T cells when no competing donor T cells were present. In recipients of pure HSCs, naive and memory T cells and innate lymphoid cell populations developed. In contrast, the lymphoid pool in recipients of bulk T cells was dominated by effector memory cells. These studies show that pure HSC transplantations allow superior protective immunity against a viral pathogen compared with unselected mature T cells. This reductionist transplant model reveals the impact of graft composition on regeneration of host, newly generated, and mature transferred T cells, and underscores the deleterious effects of bulk donor T cells. Our findings lead us to conclude that grafts composed of purified HSCs provide an optimal platform for in vivo expansion of selected antigen-specific cells while allowing the reconstitution of a naive T-cell pool.PMID: 22440752 [PubMed - in process]--19)Lymphat Res Biol. 2012 Mar;10(1):1.Reflections on a decade of lymphatic research and biology.Rockson SG.

Falk Cardiovascular Research Center, Stanford University School of Medicine , Stanford, California.

PMID: 22439852 [PubMed - in process]--20)Proc Natl Acad Sci USA. 2012 Apr 10;109(15):E879-88. Epub 2012 Mar 20.Signal inhibition by the dual-specific phosphatase 4 impairs T cell-dependent B-cell responses with age.Yu M, Li G, Lee WW, Yuan M, Cui D, Weyand CM, Goronzy JJ.

Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305.

T cell-dependent B-cell responses decline with age, suggesting defective CD4 T-cell function. CD4 memory T cells from individuals older than 65 y displayed increased and sustained transcription of the dual-specific phosphatase 4 (DUSP4) that shortened expression of CD40-ligand (CD40L) and inducible T-cell costimulator (ICOS) (both P < 0.001) and decreased production of IL-4, IL-17A, and IL-21 (all P < 0.001) after in vitro activation. In vivo after influenza vaccination, activated CD4 T cells from elderly individuals had increased DUSP4 transcription (P = 0.002), which inversely correlated with the expression of CD40L (r = 0.65, P = 0.002), ICOS (r = 0.57, P = 0.008), and IL-4 (r = 0.66, P = 0.001). In CD4 KO mice reconstituted with DUSP4 OT-II T cells, DUSP4 had a negative effect on the expansion of antigen-specific B cells (P = 0.003) and the production of ova-specific antibodies (P = 0.03) after immunization. Silencing of DUSP4 in memory CD4 T cells improved CD40L (P < 0.001), IL-4 (P = 0.007), and IL-21 (P = 0.04) expression significantly more in the elderly than young adults. Consequently, the ability of CD4 memory T cells to support B-cell differentiation that was impaired in the

elderly (P = 0.004) was restored. Our data suggest that increased DUSP4 expression in activated T cells in the elderly in part accounts for defective adaptive immune responses.PMID: 22434910 [PubMed - in process]--21)Proc Natl Acad Sci USA. 2012 Apr 3;109(14):5475-80. Epub 2012 Mar 19.Prokineticin 2 is an endangering mediator of cerebral ischemic injury.Cheng MY, Lee AG, Culbertson C, Sun G, Talati RK, Manley NC, Li X, Zhao H, Lyons DM, Zhou QY, Steinberg GK, Sapolsky RM.

Departments of Biological Sciences, Neurosurgery, and Psychiatry, Stanford University, Stanford, CA 94305.

Stroke causes brain dysfunction and neuron death, and the lack of effective therapies heightens the need for new therapeutic targets. Here we identify prokineticin 2 (PK2) as a mediator for cerebral ischemic injury. PK2 is a bioactive peptide initially discovered as a regulator of gastrointestinal motility. Multiple biological roles for PK2 have been discovered, including circadian rhythms, angiogenesis, and neurogenesis. However, the role of PK2 in neuropathology is unknown. Using primary cortical cultures, we found that PK2 mRNA is up-regulated by several pathological stressors, including hypoxia, reactive oxygen species, and excitotoxic glutamate. Glutamate-induced PK2 expression is dependent on NMDA receptor activation and extracellular calcium. Enriched neuronal culture studies revealed that neurons are the principal source of glutamate-induced PK2. Using in vivo models of stroke, we found that PK2 mRNA is induced in the ischemic cortex and striatum. Central delivery of PK2 worsens infarct volume, whereas PK2 receptor antagonist decreases infarct volume and central inflammation while improving functional outcome. Direct central inhibition of PK2 using RNAi also reduces infarct volume. These findings indicate that PK2 can be activated by pathological stimuli such as hypoxia-ischemia and excitotoxic glutamate and identify PK2 as a deleterious mediator for cerebral ischemia.PMID: 22431614 [PubMed - in process]--22)J Am Chem Soc. 2012 Apr 4;134(13):5734-7. Epub 2012 Mar 23.Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics.Chen J, Tsai A, Petrov A, Puglisi JD.

Department of Applied Physics, Stanford University , Stanford, California 94305-4090, United States.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor

to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.PMID: 22428667 [PubMed - in process]--23)Cell. 2012 Mar 16;148(6):1293-307.Personal omics profiling reveals dynamic molecular and medical phenotypes.Chen R, Mias GI, Li-Pook-Than J, Jiang L, Lam HY, Chen R, Miriami E, Karczewski KJ, Hariharan M, Dewey FE, Cheng Y, Clark MJ, Im H, Habegger L, Balasubramanian S, O'Huallachain M, Dudley JT, Hillenmeyer S, Haraksingh R, Sharon D, Euskirchen G, Lacroute P, Bettinger K, Boyle AP, Kasowski M, Grubert F, Seki S, Garcia M, Whirl-Carrillo M, Gallardo M, Blasco MA, Greenberg PL, Snyder P, Klein TE, Altman RB, Butte AJ, Ashley EA, Gerstein M, Nadeau KC, Tang H, Snyder M.Source

Department of Genetics, Stanford University School of Medicine, Stanford University, Stanford, CA 94305, USA.

Personalized medicine is expected to benefit from combining genomic information with regular monitoring of physiological states by multiple high-throughput methods. Here, we present an integrative personal omics profile (iPOP), an analysis that combines genomic, transcriptomic, proteomic, metabolomic, and autoantibody profiles from a single individual over a 14 month period. Our iPOP analysis revealed various medical risks, including type 2 diabetes. It also uncovered extensive, dynamic changes in diverse molecular components and biological pathways across healthy and diseased conditions. Extremely high-coverage genomic and transcriptomic data, which provide the basis of our iPOP, revealed extensive heteroallelic changes during healthy and diseased states and an unexpected RNA editing mechanism. This study demonstrates that longitudinal iPOP can be used to interpret healthy and diseased states by connecting genomic information with additional dynamic omics activity.Copyright © 2012 Elsevier Inc. All rights reserved.PMID: 22424236 [PubMed - in process]--24)Int J Epidemiol. 2012 Mar 15. [Epub ahead of print]Systematic evaluation of environmental factors: persistent pollutants and nutrients correlated with serum lipid levels.Patel CJ, Cullen MR, Ioannidis JP, Butte AJ.

Department of Pediatrics, Division of Systems Medicine, Stanford University School of Medicine, Stanford, CA, USA, Lucile Packard Children's Hospital, Palo Alto, CA, USA, Department of Medicine, Division of General Medical Disciplines, Stanford University School of Medicine, Stanford, CA, USA, Department of Medicine, Stanford Prevention Research Center, Stanford University School of Medicine, Stanford, CA, USA, Department of Health Research and Policy, Stanford University School of Medicine, Stanford, CA, USA and Department of Statistics, Stanford University School of Humanities and Sciences, Stanford, CA USA.BACKGROUND:Both genetic and environmental factors contribute to triglyceride, low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein-cholesterol (HDL-C) levels.

Although genome-wide association studies are currently testing the genetic factors systematically, testing and reporting one or a few factors at a time can lead to fragmented literature for environmental chemical factors. We screened for correlation between environmental factors and lipid levels, utilizing four independent surveys with information on 188 environmental factors from the Centers of Disease Control, National Health and Nutrition Examination Survey, collected between 1999 and 2006.METHODS:We used linear regression to correlate each environmental chemical factor to triglycerides, LDL-C and HDL-C adjusting for age, age(2), sex, ethnicity, socio-economic status and body mass index. Final estimates were adjusted for waist circumference, diabetes status, blood pressure and survey. Multiple comparisons were controlled for by estimating the false discovery rate and significant findings were tentatively validated in an independent survey.RESULTS:We identified and validated 29, 9 and 17 environmental factors correlated with triglycerides, LDL-C and HDL-C levels, respectively. Findings include hydrocarbons and nicotine associated with lower HDL-C and vitamin E (γ-tocopherol) associated with unfavourable lipid levels. Higher triglycerides and lower HDL-C were correlated with higher levels of fat-soluble contaminants (e.g. polychlorinated biphenyls and dibenzofurans). Nutrients and vitamin markers (e.g. vitamins B, D and carotenes), were associated with favourable triglyceride and HDL-C levels.CONCLUSIONS:Our systematic association study has enabled us to postulate about broad environmental correlation to lipid levels. Although subject to confounding and reverse causality bias, these findings merit evaluation in additional cohorts.PMID: 22421054 [PubMed - as supplied by publisher]--25)Transfusion. 2012 Mar 13. doi: 10.1111/j.1537-2995.2012.03596.x. [Epub ahead of print]How we treat: risk mitigation for ABO-incompatible plasma in plateletpheresis products.Fontaine MJ, Mills AM, Weiss S, Hong WJ, Viele M, Goodnough LT.

From the Department of Pathology, Stanford Hospital and Clinics, and the Department of Medicine/Division of Hematology, Stanford University, Stanford, California.

PMID: 22414003 [PubMed - as supplied by publisher]--26)PLoS Genet. 2012 Mar;8(3):e1002565. Epub 2012 Mar 8.Tcf7 is an important regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line.Wu JQ, Seay M, Schulz VP, Hariharan M, Tuck D, Lian J, Du J, Shi M, Ye Z, Gerstein M, Snyder MP, Weissman S.

Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America.

A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-

renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34- cells, using RNA-Seq. We subsequently identified the target genes bound by TCF7, using ChIP-Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34- state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems.PMID: 22412390 [PubMed - in process] PMCID: PMC3297581--27)Am J Transplant. 2012 Mar 8. doi: 10.1111/j.1600-6143.2012.03992.x. [Epub ahead of print]Tolerance and Withdrawal of Immunosuppressive Drugs in Patients Given Kidney and Hematopoietic Cell Transplants.Scandling JD, Busque S, Dejbakhsh-Jones S, Benike C, Sarwal M, Millan MT, Shizuru JA, Lowsky R, Engleman EG, Strober S.

Departments of Medicine (Nephrology), Stanford University School of Medicine, Stanford, CA Departments of Surgery (Abdominal Transplantation), Stanford University School of Medicine, Stanford, CA Departments of Medicine (Immunology and Rheumatology), Stanford University School of Medicine, Stanford, CA Departments of Pathology, Stanford University School of Medicine, Stanford, CA Departments of Pediatrics (Nephrology), Stanford University School of Medicine, Stanford, CA Departments of Medicine (Blood and Marrow Transplantation), Stanford University School of Medicine, Stanford, CA.

Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.PMID: 22405058 [PubMed - as supplied by publisher]--28)Aging Dis. 2011 Dec;2(6):524-37. Epub 2011 Dec 2.Telomere dysfunction, autoimmunity and aging.Hohensinner PJ, Goronzy JJ, Weyand CM.

Department of Medicine: Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California and Palo Alto Department of Veterans Affairs Health Care System, Palo Alto, California, USA.

Immune aging is associated with loss of critical immune functions, such as host protection from infection and malignancy. Unexpectedly, immunosenescence also renders the host susceptible to inflammation, which may translate into tissue-damaging disease as the senescent immune system loses its ability to maximize inflammatory protection while minimizing inflammatory injury. On the other hand, chronic inflammation associated with immune-mediated disease represents a profound stress factor for the immune system, affecting cellular turn-over, replication and exhaustion. Immune cell longevity is tightly connected to the functional integrity of telomeres which are regulated by cell multiplication, exposure to oxidative stress and DNA repair mechanisms. Lymphocytes are amongst the few cell types that can actively elongate telomeres through the action of telomerase. In patients with the autoimmune disease rheumatoid arthritis (RA), telomerase deficiency is associated with prematurity of immune aging. Patients with RA have other defects in DNA repair mechanisms, including the kinase Ataxia telangiectasia mutated (ATM), critically involved in the repair of DNA double strand breaks. ATM deficiency in RA shortens lymphocyte survival. Dynamics of telomeric length and structure are beginning to be understood and have distinct patterns in different autoimmune diseases, suggesting a multitude of molecular mechanisms defining the interface between chronic immune stimulation and progressive aging of the immune system.PMID: 22396899 [PubMed - in process] PMCID: PMC3295061--29)Aging Dis. 2011 Oct;2(5):398-413. Epub 2011 Oct 28.Finding Balance: T cell Regulatory Receptor Expression during Aging.Cavanagh MM, Qi Q, Weyand CM, Goronzy JJ.

Department of Medicine: Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305 and Palo Alto Department of Veterans Affairs Health Care System, Palo Alto, CA 94306, USA.

Aging is associated with a variety of changes to immune responsiveness. Reduced protection against infection, reduced responses to vaccination and increased risk of autoimmunity are all hallmarks of advanced age. Here we consider how changes in the expression of regulatory receptors on the T cell surface contribute to altered immunity during aging.PMID: 22396890 [PubMed - in process] PMCID: PMC3295076--30)Cold Spring Harb Perspect Med. 2012 Mar;2(3):a007724.

Connecting Type 1 and Type 2 Diabetes through Innate Immunity.Odegaard JI, Chawla A.

Department of Pathology, Stanford University School of Medicine, Stanford, California 94305.

The escalating epidemic of obesity has driven the prevalence of both type 1 and 2 diabetes mellitus to historically high levels. Chronic low-grade inflammation, which is present in both type 1 and type 2 diabetics, contributes to the pathogenesis of insulin resistance. The accumulation of activated innate immune cells in metabolic tissues results in release of inflammatory mediators, in particular, IL-1β and TNFα, which promote systemic insulin resistance and β-cell damage. In this article, we discuss the central role of innate immunity and, in particular, the macrophage in insulin sensitivity and resistance, β-cell damage, and autoimmune insulitis. We conclude with a discussion of the therapeutic implications of this integrated understanding of diabetic pathology.PMID: 22393536 [PubMed - in process] PMCID: PMC3282495--31)J Immunol. 2012 Apr 1;188(7):3513-21. Epub 2012 Mar 5.Inhibition of epidermal growth factor receptor tyrosine kinase ameliorates collagen-induced arthritis.Swanson CD, Akama-Garren EH, Stein EA, Petralia JD, Ruiz PJ, Edalati A, Lindstrom TM, Robinson WH.

Division of Immunology and Rheumatology, Department of Medicine, Stanford University, Stanford, CA 94305.

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.PMID: 22393153 [PubMed - in process] PMCID: PMC3311775 [Available on 2013/4/1]--32)FASEB J. 2012 Mar 5. [Epub ahead of print]

Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB.Banfi A, von Degenfeld G, Gianni-Barrera R, Reginato S, Merchant MJ, McDonald DM, Blau HM.

*Baxter Laboratory for Stem Cell Biology, Institute for Regenerative Medicine and Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, California, USA;

Therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, yet clinical trials have thus far failed to show efficacy. As a result, limb amputation remains a common outcome for muscle ischemia due to severe atherosclerotic disease, with an overall incidence of 100 per million people in the United States per year. A challenge has been that the angiogenic master regulator vascular endothelial growth factor (VEGF) induces dysfunctional vessels, if expressed, outside of a narrow dosage window. We tested the hypothesis that codelivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, could induce normal angiogenesis in skeletal muscle irrespective of VEGF levels. Coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Notably, in an ischemic hindlimb model, single-vector expression led to efficient growth of collateral arteries, revascularization, increased blood flow, and reduced tissue damage. Furthermore, these results were confirmed in a clinically applicable gene therapy approach by adenoviral-mediated delivery of the bicistronic vector. We conclude that coordinated expression of VEGF and PDGF-BB via a single vector constitutes a novel strategy for harnessing the potency of VEGF to induce safe and efficacious angiogenesis.-Banfi, A., von Degenfeld, G., Gianni-Barrera, R., Reginato, S., Merchant, M. J., McDonald, D. M., Blau, H. M. Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB.PMID: 22391130 [PubMed - as supplied by publisher]--33)Mol Biol Evol. 2012 Mar 1. [Epub ahead of print]Evolutionary meta-analysis of association studies reveals ancient constraints affecting disease marker discovery.Dudley JT, Chen R, Sanderford M, Butte AJ, Kumar S.

Program in Biomedical Informatics, Stanford University School of Medicine, Stanford, California, United States of America.

Genome-wide disease association studies contrast genetic variation between disease cohorts and healthy populations to discover single nucleotide polymorphisms (SNPs) and other genetic markers revealing underlying genetic architectures of human diseases. Despite many efforts over the past decade, many reproducible genetic variants that explain substantial proportions of the heritable risk of common human diseases remain undiscovered. We have conducted a multispecies genomic analysis of 5,831 putative human risk variants for more than 230 disease phenotypes reported in 2,021 studies. We show that the current approaches show a propensity for discovering disease associated SNPs (dSNPs) at conserved genomic positions, because the effect

size (odds ratio) and allelic P-value of genetic association of a SNP relates strongly to the evolutionary conservation of their genomic position. We propose a new measure for ranking SNPs that integrates evolutionary conservation scores and the P-value (E-rank). Using published data from a large case-control study, we demonstrate that E-rank method prioritizes SNPs with a greater likelihood of bona fide and reproducible genetic disease associations, many of which may explain greater proportions of genetic variance. Therefore, long-term evolutionary histories of genomic positions offer key practical utility in reassessing data from existing disease association studies, and in the design and analysis of future studies aimed at revealing the genetic basis of common human diseases.PMID: 22389448 [PubMed - as supplied by publisher]--34)Circulation. 2012 Feb 21;125(7):931-44.DNA sequencing: clinical applications of new DNA sequencing technologies.Dewey FE, Pan S, Wheeler MT, Quake SR, Ashley EA.

Center for Inherited Cardiovascular Disease, Division of Cardiovascular Medicine, Stanford University School of Medicine, Falk CVRB, 300 Pasteur Dr, Stanford, CA 94305, USA.

PMID: 22354974 [PubMed - indexed for MEDLINE]--35)Proc Natl Acad Sci USA. 2012 Feb 7;109(6):2078-83. Epub 2012 Jan 19.Three differentiation states risk-stratify bladder cancer into distinct subtypes.Volkmer JP, Sahoo D, Chin RK, Ho PL, Tang C, Kurtova AV, Willingham SB, Pazhanisamy SK, Contreras-Trujillo H, Storm TA, Lotan Y, Beck AH, Chung BI, Alizadeh AA, Godoy G, Lerner SP, van de Rijn M, Shortliffe LD, Weissman IL, Chan KS.

Institute of Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA. jvolkmer@stanford.eduErratum in

* Proc Natl Acad Sci U S A. 2012 Feb 28;109(9):3600.

Current clinical judgment in bladder cancer (BC) relies primarily on pathological stage and grade. We investigated whether a molecular classification of tumor cell differentiation, based on a developmental biology approach, can provide additional prognostic information. Exploiting large preexisting gene-expression databases, we developed a biologically supervised computational model to predict markers that correspond with BC differentiation. To provide mechanistic insight, we assessed relative tumorigenicity and differentiation potential via xenotransplantation. We then correlated the prognostic utility of the identified markers to outcomes within gene expression and formalin-fixed paraffin-embedded (FFPE) tissue datasets. Our data indicate that BC can be subclassified into three subtypes, on the basis of their differentiation states: basal, intermediate, and differentiated, where only the most primitive tumor cell subpopulation within each subtype is capable of generating xenograft tumors and recapitulating downstream populations. We found that keratin 14 (KRT14) marks the most primitive differentiation state that precedes KRT5 and KRT20 expression. Furthermore, KRT14 expression is consistently associated with worse prognosis in both univariate and multivariate

analyses. We identify here three distinct BC subtypes on the basis of their differentiation states, each harboring a unique tumor-initiating population.PMID: 22308455 [PubMed - indexed for MEDLINE] PMCID: PMC3277552--36)Proc Natl Acad Sci USA. 2012 Jan 31;109(5):1613-8. Epub 2012 Jan 17.Complementary costimulation of human T-cell subpopulations by cluster of differentiation 28 (CD28) and CD81.Sagi Y, Landrigan A, Levy R, Levy S.

Division of Oncology, Department of Medicine, and Division of Immunology and Rheumatology, Stanford University Medical Center, Stanford, CA 94305, USA.

Cluster of differentiation 81 (CD81) is a widely expressed tetraspanin molecule that physically associates with CD4 and CD8 on the surface of human T cells. Coengagement of CD81 and CD3 results in the activation and proliferation of T cells. CD81 also costimulated mouse T cells that lack CD28, suggesting either a redundant or a different mechanism of action. Here we show that CD81 and CD28 have a preference for different subsets of T cells: Primary human naïve T cells are better costimulated by CD81, whereas the memory T-cell subsets and Tregs are better costimulated by CD28. The more efficient activation of naïve T cells by CD81 was due to prolonged signal transduction compared with that by CD28. We found that IL-6 played a role in the activation of the naïve T-cell subset by CD81. Combined costimulation through both CD28 and CD81 resulted in an additive effect on T-cell activation. Thus, these two costimulatory molecules complement each other both in the strength of signal transduction and in T-cell subset inclusions. Costimulation via CD81 might be useful for expansion of T cells for adoptive immunotherapy to allow the inclusion of naïve T cells with their broad repertoire.PMID: 22307619 [PubMed - indexed for MEDLINE]PMCID: PMC3277132 [Available on 2012/7/31]--37) 2012 Jan 27;36(1):142-52.Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes.Newell EW, Sigal N, Bendall SC, Nolan GP, Davis MM.

Department of Microbiology and Immunology, Stanford University, CA 94305, USA.

Cytotoxic CD8(+) T lymphocytes directly kill infected or aberrant cells and secrete proinflammatory cytokines. By using metal-labeled probes and mass spectrometric analysis (cytometry by time-of-flight, or CyTOF) of human CD8(+) T cells, we analyzed the expression of many more proteins than previously possible with fluorescent labels, including surface markers, cytokines, and antigen specificity with modified peptide-MHC tetramers. With 3-dimensional principal component analysis (3D-PCA) to display phenotypic diversity, we observed a relatively uniform pattern of variation in all subjects tested, highlighting the interrelatedness of previously described subsets and the continuous nature of CD8(+) T cell differentiation. These data also showed much greater complexity in the CD8(+) T cell compartment than previously appreciated, including a nearly combinatorial pattern of cytokine expression, with distinct niches occupied by virus-specific cells. This large degree of functional

diversity even between cells with the same specificity gives CD8(+) T cells a remarkable degree of flexibility in responding to pathogens.Comment in

* Immunity. 2012 Jan 27;36(1):10-2.

PMID: 22265676 [PubMed - indexed for MEDLINE]--38)Nat Med. 2012 Jan 6;18(1):59-65. doi: 10.1038/nm.2625.Optimization of current and future therapy for autoimmune diseases.Steinman L, Merrill JT, McInnes IB, Peakman M.

Department of Neurology and Neurological Sciences, Stanford University, Stanford, California, USA. steinman@stanford.edu

PMID: 22227674 [PubMed - indexed for MEDLINE]--39)Ann NY Acad Sci. 2012 Jan;1247:69-82. doi: 10.1111/j.1749-6632.2011.06297.x. Epub 2012 Jan 6.Mechanisms of immunosenescence: lessons from models of accelerated immune aging.Le Saux S, Weyand CM, Goronzy JJ.

Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, California, USA.

With increasing age, the ability of the adaptive immune system to respond to vaccines and to protect from infection declines. In parallel, the production of inflammatory mediators increases. While cross-sectional studies have been successful in defining age-dependent immunological phenotypes, studies of accelerated immune aging in human subpopulations have been instrumental in obtaining mechanistic insights. The immune system depends on its regenerative capacity; however, the T cell repertoire, once established, is relatively robust to aging and only decompensates when additionally stressed. Such stressors include chronic infections such as CMV and HIV, even when viral replication is controlled, and autoimmune diseases. Reduced regenerative capacity, chronic immune activation in the absence of cell exhaustion, T cell memory inflation, and accumulation of highly potent effector T cells in these patients synergize to develop an immune phenotype that is characteristic of the elderly. Studies of accelerated immune aging in autoimmune diseases have identified an unexpected link to chronic DNA damage responses that are known to be important in aging, but so far had not been implicated in immune aging.© 2012 New York Academy of Sciences.PMID: 22224726 [PubMed - indexed for MEDLINE]--40)Nat Struct Mol Biol. 2012 Jan 5;19(1):9-16. doi: 10.1038/nsmb.2203.New approaches for dissecting protease functions to improve probe development and drug discovery.Deu E, Verdoes M, Bogyo M.

Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.

Proteases are well-established targets for pharmaceutical development because of their known enzymatic mechanism and their regulatory roles in many pathologies. However, many potent clinical lead compounds have been unsuccessful either because of a lack of specificity or because of our limited understanding of the biological roles of the targeted protease. In order to successfully develop protease inhibitors as drugs, it is necessary to understand protease functions and to expand the platform of inhibitor development beyond active site-directed design and in vitro optimization. Several newly developed technologies will enhance assessment of drug selectivity in living cells and animal models, allowing researchers to focus on compounds with high specificity and minimal side effects in vivo. In this review, we highlight advances in the development of chemical probes, proteomic methods and screening tools that we feel will help facilitate this paradigm shift in drug discovery.PMID: 22218294 [PubMed - indexed for MEDLINE]--41)PLoS Genet. Dec;7(12):e1002410. doi: 10.1371/journal.pgen.1002410. Epub 2011 Dec 15.Ancestral components of admixed genomes in a Mexican cohort.Johnson NA, Coram MA, Shriver MD, Romieu I, Barsh GS, London SJ, Tang H.

Department of Statistics, Stanford University, Stanford, California, United States of America.

For most of the world, human genome structure at a population level is shaped by interplay between ancient geographic isolation and more recent demographic shifts, factors that are captured by the concepts of biogeographic ancestry and admixture, respectively. The ancestry of non-admixed individuals can often be traced to a specific population in a precise region, but current approaches for studying admixed individuals generally yield coarse information in which genome ancestry proportions are identified according to continent of origin. Here we introduce a new analytic strategy for this problem that allows fine-grained characterization of admixed individuals with respect to both geographic and genomic coordinates. Ancestry segments from different continents, identified with a probabilistic model, are used to construct and study "virtual genomes" of admixed individuals. We apply this approach to a cohort of 492 parent-offspring trios from Mexico City. The relative contributions from the three continental-level ancestral populations-Africa, Europe, and America-vary substantially between individuals, and the distribution of haplotype block length suggests an admixing time of 10-15 generations. The European and Indigenous American virtual genomes of each Mexican individual can be traced to precise regions within each continent, and they reveal a gradient of Amerindian ancestry between indigenous people of southwestern Mexico and Mayans of the Yucatan Peninsula. This contrasts sharply with the African roots of African Americans, which have been characterized by a uniform mixing of multiple West African populations. We also use the virtual European and Indigenous American genomes to search for the signatures of selection in the ancestral populations, and we identify previously known targets of selection in other populations, as well as new candidate loci. The ability to infer precise ancestral components of admixed genomes will facilitate studies of disease-related phenotypes and will allow new insight into the adaptive and demographic history of indigenous people.PMID: 22194699 [PubMed - indexed for MEDLINE] PMCID: PMC324059942)Proc Natl Acad Sci USA. 2012 Jan 10;109(2):466-71. Epub 2011 Dec 21.

The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.Yan KS, Chia LA, Li X, Ootani A, Su J, Lee JY, Su N, Luo Y, Heilshorn SC, Amieva MR, Sangiorgi E, Capecchi MR, Kuo CJ.

Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.PMID: 22190486 [PubMed - indexed for MEDLINE] PMCID: PMC3258636

---43)Proc Natl Acad Sci USA. 2012 Jan 10;109(2):600-5. Epub 2011 Dec 21.Signal transducer and activator of transcription 3 (STAT3) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis.Sen N, Che X, Rajamani J, Zerboni L, Sung P, Ptacek J, Arvin AM.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA. nandinis@stanford.edu

Varicella-zoster virus (VZV) is a human α-herpesvirus that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation. Like other viruses, VZV must subvert the intrinsic antiviral defenses of differentiated human cells to produce progeny virions. Accordingly, VZV inhibits the activation of the cellular transcription factors IFN regulatory factor 3 (IRF3) and signal transducers and activators of transcription 1 (STAT1), thereby downregulating antiviral factors, including IFNs. Conversely, in this study, we found that VZV triggers STAT3 phosphorylation in cells infected in vitro and in human skin xenografts in SCID mice in vivo and that STAT3 activation induces the anti-apoptotic protein survivin. Small-molecule inhibitors of STAT3 phosphorylation and survivin restrict VZV replication in vitro, and VZV infection of skin xenografts in vivo is markedly impaired by the administration of the phospho-STAT3 inhibitor S3I-201. STAT3 and survivin are required for malignant transformation caused by γ-herpesviruses, such as Kaposi's sarcoma virus. We show that STAT3

activation is also critical for VZV, a nononcogenic herpesvirus, via a survivin-dependent mechanism. Furthermore, STAT3 activation is critical for the life cycle of the virus because VZV skin infection is necessary for viral transmission and persistence in the human population. Therefore, we conclude that takeover of this major cell-signaling pathway is necessary, independent of cell transformation, for herpesvirus pathogenesis and that STAT3 activation and up-regulation of survivin is a common mechanism important for the pathogenesis of lytic as well as tumorigenic herpesviruses.PMID: 22190485 [PubMed - indexed for MEDLINE] PMCID: PMC3258638 [Available on 2012/7/10]--44)Blood. 2012 Feb 9;119(6):1581-9. Epub 2011 Dec 15.Interactions between NKT cells and Tregs are required for tolerance to combined bone marrow and organ transplants.Hongo D, Tang X, Dutt S, Nador RG, Strober S.

Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, 296 Campus Drive, Stanford, CA 94305, USA.

We used a model of combined bone marrow and heart transplantation, in which tolerance and stable chimerism is induced after conditioning with fractionated irradiation of the lymphoid tissues and anti-T-cell antibodies. Graft acceptance and chimerism required host CD4(+)CD25(+) Treg production of IL-10 that was in-turn enhanced by host invariant natural killer (NK) T-cell production of IL-4. Up-regulation of PD-1 on host Tregs, CD4(+)CD25(-) conventional T (Tcon) cells, and CD8(+) T cells was also enhanced by NKT cell production of IL-4. Up-regulated PD-1 expression on Tregs was linked to IL-10 secretion, on CD8(+) T cells was linked to Tim-3 expression, and on CD4(+) Tcon cells was associated with reduced IFNγ secretion. Changes in the expression of PD-1 were induced by the conditioning regimen, and declined after bone marrow transplantation. In conclusion, NKT cells in this model promoted changes in expression of negative costimulatory receptors and anti-inflammatory cytokines by Tregs and other T-cell subsets in an IL-4-dependent manner that resulted in tolerance to the bone marrow and organ grafts.PMID: 22174155 [PubMed - indexed for MEDLINE] PMCID: PMC3286219 [Available on 2013/2/9]--45)J Histochem Cytochem. 2012 Feb;60(2):97-109. Epub 2011 Dec 1.Three-dimensional microstructural changes in murine abdominal aortic aneurysms quantified using immunofluorescent array tomography.Saatchi S, Azuma J, Wanchoo N, Smith SJ, Yock PG, Taylor CA, Tsao PS.

Department of Bioengineering, Stanford University, Stanford, California 94305, USA. sanazsaatchi@gmail.com

This study investigated the spatial and temporal remodeling of blood vessel wall microarchitecture and cellular morphology during abdominal aortic aneurysm (AAA) development using immunofluorescent array tomography (IAT), a high-resolution three-dimensional (3D) microscopy technology, in the murine model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7, and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified volume fractions (VF) of elastin, smooth muscle cell

(SMC) actin, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, non-adventitial wall thickness, and nuclei amount. The 3D renderings depicted elastin and collagen type I degradation and SMC morphological changes. Elastin VF decreased 37.5% (p<0.01), thickness decreased 48.9%, and fragmentation increased 449.7% (p<0.001) over 28 days. SMC actin VF decreased 78.3% (p<0.001) from days 0 to 7 and increased 139.7% (p<0.05) from days 7 to 28. Non-adventitial wall thickness increased 61.1%, medial nuclei amount increased 159.1% (p<0.01), and adventitial collagen type I VF decreased 64.1% (p<0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the dynamics of vascular remodeling during AAA development.PMID: 22140132 [PubMed - indexed for MEDLINE]--46)PLoS One. 2011;6(11):e28029. Epub 2011 Nov 21.Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe.Ren G, Blum G, Verdoes M, Liu H, Syed S, Edgington LE, Gheysens O, Miao Z, Jiang H, Gambhir SS, Bogyo M, Cheng Z.

Molecular Imaging Program at Stanford, Stanford University, Stanford, California, United States of America.

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.PMID: 22132198 [PubMed - indexed for MEDLINE] PMCID: PMC3221694--47)PLoS One. 2011;6(11):e27881. Epub 2011 Nov 21.Astrocyte proliferation following stroke in the mouse depends on distance from the infarct.Barreto GE, Sun X, Xu L, Giffard RG.

Department of Anesthesia, Stanford University School of Medicine, Stanford, California, United States of America.

Reactive gliosis is a hallmark of brain pathology and the injury response, yet the extent to which astrocytes proliferate, and whether this is central to astrogliosis is still controversial. We determined the fraction of mature astrocytes that proliferate in a mouse stroke model using unbiased stereology as a function of distance from the infarct edge. Cumulatively 11.1±1.2% of Aldh1l1(+) astrocytes within 400 µm in the cortical penumbra incorporate BrdU in the first week following stroke, while the overall number of astrocytes does not change. The number of astrocytes proliferating fell sharply with distance with more than half of all proliferating astrocytes found within 100 µm of the edge of the infarct. Despite extensive cell proliferation primarily of microglia and neutrophils/monocytes in the week following stroke, few mature astrocytes re-enter cell cycle, and these are concentrated close to the infarct boundary.PMID: 22132159 [PubMed - indexed for MEDLINE] PMCID: PMC3221692--48)Circ Res. 2012 Jan 20;110(2):312-24. Epub 2011 Nov 23.miR-29b participates in early aneurysm development in Marfan syndrome.Merk DR, Chin JT, Dake BA, Maegdefessel L, Miller MO, Kimura N, Tsao PS, Iosef C, Berry GJ, Mohr FW, Spin JM, Alvira CM, Robbins RC, Fischbein MP.

Department of Cardiothoracic Surgery, 300 Pasteur Drive, Falk Cardiovascular Research Building, Stanford University, Stanford, CA 94305, USA.

RATIONALE:Marfan syndrome (MFS) is a systemic connective tissue disorder notable for the development of aortic root aneurysms and the subsequent life-threatening complications of aortic dissection and rupture. Underlying fibrillin-1 gene mutations cause increased transforming growth factor-β (TGF-β) signaling. Although TGF-β blockade prevents aneurysms in MFS mouse models, the mechanisms through which excessive TGF-β causes aneurysms remain ill-defined.OBJECTIVE:We investigated the role of microRNA-29b (miR-29b) in aneurysm formation in MFS.METHODS AND RESULTS:Using quantitative polymerase chain reaction, we discovered that miR-29b, a microRNA regulating apoptosis and extracellular matrix synthesis/deposition genes, is increased in the ascending aorta of Marfan (Fbn1(C1039G/+)) mice. Increased apoptosis, assessed by increased cleaved caspase-3 and caspase-9, enhanced caspase-3 activity, and decreased levels of the antiapoptotic proteins, Mcl-1 and Bcl-2, were found in the Fbn1(C1039G/+) aorta. Histological evidence of decreased and fragmented elastin was observed exclusively in the Fbn1(C1039G/+) ascending aorta in association with repressed elastin mRNA and increased matrix metalloproteinase-2 expression and activity, both targets of miR-29b. Evidence of decreased activation of nuclear factor κB, a repressor of miR-29b, and a factor suppressed by TGF-β, was also observed in Fbn1(C1039G/+) aorta. Furthermore, administration of a nuclear factor κB inhibitor increased miR-29b levels, whereas TGF-β blockade or losartan effectively decreased miR-29b levels in Fbn1(C1039G/+) mice. Finally, miR-29b blockade by locked nucleic acid antisense oligonucleotides prevented early aneurysm development, aortic wall apoptosis, and extracellular matrix deficiencies.CONCLUSIONS:We identify increased miR-29b expression as key to the pathogenesis of early aneurysm development in MFS by regulating aortic wall apoptosis and extracellular matrix abnormalities.PMID: 22116819 [PubMed - indexed for MEDLINE]49)Br J Anaesth. 2012 Feb;108(2):271-7. Epub 2011 Nov 23.

Non-invasive haemoglobin measurement in patients undergoing elective Caesarean section.Butwick A, Hilton G, Carvalho B.

Department of Anaesthesia, Stanford University School of Medicine, Stanford CA 94305, USA. ajbut@stanford.edu

BACKGROUND:The ability to measure haemoglobin (Hb) real-time and non-invasively offers important clinical value in the assessment of acute changes in maternal Hb during the peripartum period. This study evaluates the Masimo Rainbow SET(®) Radical-7 Pulse CO-Oximeter in a pregnant population undergoing Caesarean section (CS).METHODS:Fifty patients undergoing elective CS were enrolled in this prospective, controlled study and followed for 48 h after surgery. Non-invasive Masimo Hb (SpHb) values were compared with laboratory Hb values from venous blood samples drawn at baseline, immediately post-CS, and 24 h post-CS using the Bland-Altman plots. Longitudinal analysis of SpHb changes over time was performed using mixed-effects regression modelling.RESULTS:For the comparison between SpHb and laboratory Hb, SpHb displayed a significant positive bias at baseline {1.22 g dl(-1) [95% confidence interval (CI): 0.89-1.54]} and at 24 h post-CS [1.36 g dl(-1) (95% CI: 1.04-1.68)]. The bias immediately post-CS was 0.14 g dl(-1) (95% CI: -0.18 to 0.46). The limits of agreement at baseline, immediately post-CS, and at 24 h post-CS were: -0.9 and 3.33, -2.35 and 2.56, and -0.55 and 3.27 g dl(-1), respectively. The mean decrease in SpHb from baseline to 48 h post-CS was ∼1 g dl(-1).CONCLUSIONS:The variability in bias and limits of agreements of the Rainbow SET(®) Radical-7 Pulse CO-Oximeter SpHb may limit its clinical utility for assessing Hb concentration in patients undergoing elective CS. Modifications are needed in the calibration of the device to improve accuracy and precision in an obstetric setting. The study was registered at clinicaltrials.gov (NCT01108471) before participant enrolment: URL=http://clinicaltrials.gov/ct2/show/NCT01108471?term=butwick&rank=1.PMID: 22116296 [PubMed - indexed for MEDLINE]--50)Biomark Med. 2011 Dec;5(6):731-44.Endothelial progenitor cells in cardiovascular disease and chronic inflammation: from biomarker to therapeutic agent.Grisar JC, Haddad F, Gomari FA, Wu JC.

Department of Medicine, Division of Immunology & Rheumatology, Stanford School of Medicine, 265 Campus Drive, Stanford, CA 94305-5454, USA

The discovery of endothelial progenitor cells in the 1990s challenged the paradigm of angiogenesis by showing that cells derived from hematopoietic stem cells are capable of forming new blood vessels even in the absence of a pre-existing vessel network, a process termed vasculogenesis. Since then, the majority of studies in the field have found a strong association between circulating endothelial progenitor cells and cardiovascular risk. Several studies have also reported that inflammation influences the mobilization and differentiation of endothelial progenitor cells. In this review, we discuss the emerging role of endothelial progenitor cells as biomarkers of cardiovascular disease as well as the interplay between inflammation and endothelial progenitor cell biology. We will also review the challenges in the field of endothelial

progenitor cell-based therapy.PMID: 22103609 [PubMed - indexed for MEDLINE] PMCID: PMC3285378 [Available on 2012/10/1]--51)Curr Opin Chem Biol. 2011 Dec;15(6):798-805. Epub 2011 Nov 16.Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes.Edgington LE, Verdoes M, Bogyo M.

Cancer Biology Program, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5324, USA.

Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few 'druggable' classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.Copyright © 2011 Elsevier Ltd. All rights reserved.PMID: 22098719 [PubMed - indexed for MEDLINE] PMCID: PMC3237724 [Available on 2012/12/1]--52)Expert Rev Proteomics. 2011 Dec;8(6):705-15.Recent advances in biomarker discovery in solid organ transplant by proteomics.Sigdel TK, Sarwal MM.

Department of Pediatrics, Stanford University Medical School, Stanford University, Stanford, CA 94305, USA.

The identification and clinical use of more sensitive and specific biomarkers in the field of solid organ transplantation is an urgent need in medicine. Solid organ transplantation has seen improvements in the short-term survival of transplanted organs due to recent advancements in immunosuppressive therapy. However, the currently available methods of allograft monitoring are not optimal. Recent advancements in assaying methods for biomolecules such as genes, mRNA and proteins have helped to identify surrogate biomarkers that can be used to monitor the transplanted organ. These high-throughput 'omic' methods can help researchers to significantly speed up the identification and the validation steps, which are crucial factors for biomarker discovery efforts. Still, the progress towards identifying more sensitive and specific biomarkers

remains a great deal slower than expected. In this article, we have evaluated the current status of biomarker discovery using proteomics tools in different solid organ transplants in recent years. This article summarizes recent reports and current status, along with the hurdles in efficient biomarker discovery of protein biomarkers using proteomics approaches. Finally, we will touch upon personalized medicine as a future direction for better management of transplanted organs, and provide what we think could be a recipe for success in this field.© 2011 Expert Reviews LtdPMID: 22087656 [PubMed - indexed for MEDLINE] PMCID: PMC3282122 [Available on 2012/10/1]--53)Vaccine. 2012 Jan 5;30(2):247-53. Epub 2011 Nov 12.Lack of association between childhood immunizations and encephalitis in California, 1998-2008.Pahud BA, Rowhani-Rahbar A, Glaser C, Gavali S, Salibay CJ, Fireman B, Dekker CL.

Stanford University, School of Medicine, 300 Pasteur Drive, Stanford, CA 94305-5208, USA. bapahud@cmh.edu

OBJECTIVE:A number of new and combination vaccines have been introduced for children in the past two decades. Encephalitis cases occurring within defined time windows following administration of pertussis- or measles-containing vaccines are eligible for compensation by the Vaccine Injury Compensation Program. Due to increased parental concerns about vaccine safety and potential neurologic adverse events following immunization with new and multiple vaccines administered at the same visit, our aim was to determine whether immunizations are associated with an increased risk of encephalitis within defined risk windows.METHODS:We reviewed immunization records from 246 pediatric encephalitis cases referred to the California Encephalitis Project between July 1998 and December 2008. We included data on 110 cases who had been immunized in the year prior to the onset of encephalitis (observation period) and had complete immunization records. We used the case-centered method to test whether cases were more likely to have developed encephalitis in defined risk windows-42, 30 and 21 days after any vaccination, 3 days after pertussis-containing vaccines and 5-15 days after measles-virus containing vaccines-compared with the rest of the observation period.RESULTS:All vaccines recommended in the current immunization schedule were represented in our sample. No increased risk of encephalitis was seen following administration of pertussis-containing vaccines, measles-containing vaccines or any number of vaccines administered in a single visit (vaccine episode); the odds ratios and 95% confidence intervals for encephalitis after a vaccine episode were: 1.0 (0.6-1.8) in a 42-day risk window, 0.9 (0.5-1.6) in a 30-day risk window and 1.2 (0.7-2.2) in a 21-day risk window.CONCLUSION:No association between receipt of currently recommended immunizations and subsequent development of encephalitis was observed in this study.Copyright © 2011 Elsevier Ltd. All rights reserved.PMID: 22080172 [PubMed - indexed for MEDLINE]--54)PLoS One. 2011;6(10):e26846. Epub 2011 Oct 31.Clinical validation of integrated nucleic acid and protein detection on an electrochemical biosensor array for urinary tract infection diagnosis.Mohan R, Mach KE, Bercovici M, Pan Y, Dhulipala L, Wong PK, Liao JC.

Department of Urology and Bio-X Program, Stanford University School of Medicine, Stanford, California, United States of America.

BACKGROUND:Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.METHODOLOGY/PRINCIPAL FINDINGS:The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both).CONCLUSION/SIGNIFICANCE:We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.PMID: 22066011 [PubMed - indexed for MEDLINE] PMCID: PMC3204982--55)Blood. 2012 Jan 12;119(2):355-63. Epub 2011 Nov 1.In situ vaccination against mycosis fungoides by intratumoral injection of a TLR9 agonist combined with radiation: a phase 1/2 study.Kim YH, Gratzinger D, Harrison C, Brody JD, Czerwinski DK, Ai WZ, Morales A, Abdulla F, Xing L, Navi D, Tibshirani RJ, Advani RH, Lingala B, Shah S, Hoppe RT, Levy R.

Department of Dermatology, Stanford University School of Medicine, CA, USA. younkim@stanford.edu

We have developed and previously reported on a therapeutic vaccination strategy for indolent B-cell lymphoma that combines local radiation to enhance tumor immunogenicity with the injection into the tumor of a TLR9 agonist. As a result, antitumor CD8(+) T cells are induced, and systemic tumor regression was documented. Because the vaccination occurs in situ, there is no need to manufacture a vaccine product. We have now explored this strategy in a second disease: mycosis fungoides (MF). We treated 15 patients. Clinical responses were assessed at the distant, untreated sites as a measure of systemic antitumor activity. Five clinically meaningful

responses were observed. The procedure was well tolerated and adverse effects consisted mostly of mild and transient injection site or flu-like symptoms. The immunized sites showed a significant reduction of CD25(+), Foxp3(+) T cells that could be either MF cells or tissue regulatory T cells and a similar reduction in S100(+), CD1a(+) dendritic cells. There was a trend toward greater reduction of CD25(+) T cells and skin dendritic cells in clinical responders versus nonresponders. Our in situ vaccination strategy is feasible also in MF and the clinical responses that occurred in a subset of patients warrant further study with modifications to augment these therapeutic effects. This study is registered at www.clinicaltrials.gov as NCT00226993.Comment in

* Blood. 2012 Jan 12;119(2):321-2.

PMID: 22045986 [PubMed - indexed for MEDLINE] PMCID: PMC3257006 [Available on 2013/1/12]--56)Eukaryot Cell. 2011 Dec;10(12):1637-47. Epub 2011 Oct 21.Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii bradyzoites.Buchholz KR, Fritz HM, Chen X, Durbin-Johnson B, Rocke DM, Ferguson DJ, Conrad PA, Boothroyd JC.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124, USA.

The Toxoplasma gondii bradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days postinfection, in vitro bradyzoites 4 days postinfection, and in vivo bradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall.PMID: 22021236 [PubMed - indexed for MEDLINE] PMCID: PMC3232729 [Available on 2012/6/1]--57)Transplantation. 2011 Dec 27;92(12):1335-41.Validity of surrogate measures for functional nephron mass.Tan JC, Paik J, Chertow GM, Grumet FC, Busque S, Lapasia J, Desai M.

Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA. jane.tan@stanford.edu

BACKGROUND:Transplanted nephron mass is an important determinant of long-term allograft survival, but accurate assessment before organ retrieval is challenging. Newer radiologic imaging techniques allow for better determination of total kidney and cortical volumes.METHODS:Using volume measurements reconstructed from magnetic resonance or computed tomography imaging from living donor candidates, we characterized total kidney (n=312) and cortical volumes (n=236) according to sex, age, weight, height, body mass index (BMI), and body surface area (BSA).RESULTS:The mean cortical volume was 204 mL (range 105-355 mL) with no significant differences between left and right cortical volumes. The degree to which existing anthropomorphic surrogates predict nephron mass was quantified, and a diligent attempt was made to derive a better surrogate model for nephron mass. Cortical volumes were strongly associated with sex and BSA, but not with weight, height, or BMI. Four prediction models for cortical volume constructed using combinations of age, sex, race, weight, and height were compared with models including either BSA or BMI.CONCLUSIONS:Among existing surrogate measures, BSA was superior to BMI in predicting renal cortical volume. We were able to construct a statistically superior proxy for cortical volume, but whether relevant improvements in predictive accuracy could be gained needs further evaluation in a larger population.PMID: 22011765 [PubMed - indexed for MEDLINE]--58)Blood. 2011 Dec 22;118(26):6930-8. Epub 2011 Oct 14.Reduced mast cell and basophil numbers and function in Cpa3-Cre; Mcl-1fl/fl mice.Lilla JN, Chen CC, Mukai K, BenBarak MJ, Franco CB, Kalesnikoff J, Yu M, Tsai M, Piliponsky AM, Galli SJ.

Department of Pathology, Stanford University School of Medicine, CA 94305-5324, USA.

It has been reported that the intracellular antiapoptotic factor myeloid cell leukemia sequence 1 (Mcl-1) is required for mast cell survival in vitro, and that genetic manipulation of Mcl-1 can be used to delete individual hematopoietic cell populations in vivo. In the present study, we report the generation of C57BL/6 mice in which Cre recombinase is expressed under the control of a segment of the carboxypeptidase A3 (Cpa3) promoter. C57BL/6-Cpa3-Cre; Mcl-1(fl/fl) mice are severely deficient in mast cells (92%-100% reduced in various tissues analyzed) and also have a marked deficiency in basophils (58%-78% reduced in the compartments analyzed), whereas the numbers of other hematopoietic cell populations exhibit little or no changes. Moreover, Cpa3-Cre; Mcl-1(fl/fl) mice exhibited marked reductions in the tissue swelling and leukocyte infiltration that are associated with both mast cell- and IgE-dependent passive cutaneous anaphylaxis (except at sites engrafted with in vitro-derived mast cells) and a basophil- and IgE-dependent model of chronic allergic inflammation, and do not develop IgE-dependent passive systemic anaphylaxis. Our findings support the conclusion that Mcl-1 is required for normal mast cell and basophil development/survival in vivo in mice, and also suggest that Cpa3-Cre; Mcl-1(fl/fl) mice may be useful in analyzing the roles of mast cells and basophils in health and disease.Comment in

* Blood. 2011 Dec 22;118(26):6729-30.

PMID: 22001390 [PubMed - indexed for MEDLINE] PMCID: PMC3245213 [Available on 2012/12/22]--59)Stem Cells. 2011 Dec;29(12):2018-29. doi: 10.1002/stem.757.Nonintegrating knockdown and customized scaffold design enhances human adipose-derived stem cells in skeletal repair.Levi B, Hyun JS, Nelson ER, Li S, Montoro DT, Wan DC, Jia FJ, Glotzbach JC, James AW, Lee M, Huang M, Quarto N, Gurtner GC, Wu JC, Longaker MT.

Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, California 94305-5148, USA.

An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine.Copyright © 2011 AlphaMed Press.PMID: 21997852 [PubMed - indexed for MEDLINE]--60)Aliment Pharmacol Ther. 2011 Nov;34(10):1145-58. doi: 10.1111/j.1365-2036.2011.04869.x. Epub 2011 Oct 7.Review article: current antiviral therapy of chronic hepatitis B.Ayoub WS, Keeffe EB.

Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University Medical Center, 750 Welch Road, Stanford, CA 94304, USA. wayoub@stanford.edu

BACKGROUND:The indications and endpoints for treatment of chronic hepatitis B continue to evolve. The aim of the therapy for chronic hepatitis B is to achieve a long-term continued suppression of the hepatitis B virus (HBV) DNA to prevent disease progression leading to the development of cirrhosis and hepatocellular carcinoma.AIM:To summarise current literature on therapy of chronic hepatitis B, with a focus on indications for therapy, preferred treatment options, and management of resistance and partial responders.METHODS:A systematic review of the literature, with a focus on international guidelines, was performed.RESULTS:Seven drugs are licensed for the treatment of chronic hepatitis B in many countries. The selection of a drug with high potency and low rate of resistance is essential to achieve rapid and long-term viral suppression. The prevention of the sequelae of antiviral drug resistance and

appropriate management of viral breakthrough are major goals of current management. The addition or change to an antiviral agent that is not cross-resistant is critical to restore suppression of viral replication for patients with breakthrough resistance. Patient adherence to medication is essential to achieve adequate HBV DNA suppression.CONCLUSIONS:The current treatment strategy of chronic hepatitis B is now standard: initial selection of entecavir, tenofovir, or peginterferon alfa-2a. Future studies are required to determine if combination therapy using two oral agents or peginterferon with an oral agent with a high genetic barrier to resistance might be superior to standard current monotherapy.© 2011 Blackwell Publishing Ltd.PMID: 21978243 [PubMed - indexed for MEDLINE]--62)Am J Transplant. 2011 Dec;11(12):2675-84. doi: 10.1111/j.1600-6143.2011.03763.x. Epub 2011 Sep 22.The PROMISE study: a phase 2b multicenter study of voclosporin (ISA247) versus tacrolimus in de novo kidney transplantation.Busque S, Cantarovich M, Mulgaonkar S, Gaston R, Gaber AO, Mayo PR, Ling S, Huizinga RB, Meier-Kriesche HU; PROMISE Investigators.Collaborators (35)

Laftavi M, Mateo R, Goggins M, Jevnikar A, Bowers V, Alloway R, Campbell P, Jain A, Kapur S, Magee J, Klintmalm G, Harland R, Stegall M, Bass B, Zaltzman J, Tomlanovich S, Hartman E, Maluf D, Liang L, Nezakatgoo N, Butt K, Baliga P, Cohen A, McKeown JW, Mandelbrot D, Steinberg S, Bunnapradist M, Kuo P, Zhang R, Chan L, Foster C, Leventhal J, Cooper M, Batiuk T, Bloom R.Source

Division of Abdominal Transplantation, Department of Surgery, Stanford University School of Medicine, Stanford, CA, USA. sbusque@stanford.edu

Voclosporin (VCS, ISA247) is a novel calcineurin inhibitor being developed for organ transplantation. PROMISE was a 6-month, multicenter, randomized, open-label study of three ascending concentration-controlled groups of VCS (low, medium and high) compared to tacrolimus (TAC) in 334 low-risk renal transplant recipients. The primary endpoint was demonstration of noninferiority of biopsy proven acute rejection (BPAR) rates. Secondary objectives included renal function, new onset diabetes after transplantation (NODAT), hypertension, hyperlipidemia and pharmacokinetic-pharmacodynamic evaluation. The incidence of BPAR in the VCS groups (10.7%, 9.1% and 2.3%, respectively) was noninferior to TAC (5.8%). The incidence of NODAT for VCS was 1.6%, 5.7% and 17.7% versus 16.4% in TAC (low-dose VCS, p = 0.03). Nankivell estimated glomerular filtration rate was respectively: 71, 72, 68 and 69 mL/min, statistically lower in the high-dose group, p = 0.049. The incidence of hypertension and adverse events was not different between the VCS groups and TAC. VCS demonstrated an excellent correlation between trough and area under the curve (r(2) = 0.97) and no difference in mycophenolic acid exposure compared to TAC. This 6-month study shows VCS to be as efficacious as TAC in preventing acute rejection with similar renal function in the low- and medium-exposure groups, and potentially associated with a reduced incidence of NODAT.

©Copyright 2011 The American Society of Transplantation and the American Society of Transplant Surgeons.PMID: 21943027 [PubMed - indexed for MEDLINE]--63)Plast Reconstr Surg. 2012 Jan;129(1):53-66.Enhancement of human adipose-derived stromal cell angiogenesis through knockdown of a BMP-2 inhibitor.Levi B, Nelson ER, Hyun JS, Glotzbach JP, Li S, Nauta A, Montoro DT, Lee M, Commons GC, Hu S, Wu JC, Gurtner GC, Longaker MT.

Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, Calif 94305-51Abstract

BACKGROUND:Previous studies have demonstrated the role of noggin, a bone morphogenetic protein-2 inhibitor, in vascular development and angiogenesis. The authors hypothesized that noggin suppression in human adipose-derived stromal cells would enhance vascular endothelial growth factor secretion and angiogenesis in vitro and in vivo to a greater extent than bone morphogenetic protein-2 alone.METHODS:Human adipose-derived stromal cells were isolated from human lipoaspirate (n = 6) noggin was knocked down using lentiviral techniques. Knockdown was confirmed and angiogenesis was assessed by tubule formation and quantitative real-time polymerase chain reaction. Cells were seeded onto scaffolds and implanted into a 4-mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluorescence and immunohistochemistry.RESULTS:Human adipose-derived stromal cells with noggin suppression secreted significantly higher amounts of angiogenic proteins, expressed higher levels of angiogenic genes, and formed more tubules in vitro. In vivo, calvarial defects seeded with noggin shRNA human adipose-derived stromal cells exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (p < 0.05). In addition, bone morphogenetic protein-2-releasing scaffolds significantly enhanced vascular signaling in the defect site.CONCLUSIONS:Human adipose-derived stromal cells demonstrate significant increases in angiogenesis in vitro and in vivo with both noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased bone morphogenetic protein-2 signaling, a more angiogenic niche can be created.PMID: 21915082 [PubMed - indexed for MEDLINE] PMCID: PMC3246067 [Available on 2013/1/1]--64)Gastroenterol Clin North Am. 2011 Sep;40(3):495-505.Hepatitis B: modern end points of treatment and the specter of viral resistance.Lee M, Keeffe EB.

Division of Gastroenterology and Hepatology, Stanford University Medical Center, 300 Pasteur Drive, Alway Building M211, Palo Alto, CA 94305-5187, USA.

The goal of antiviral treatment of chronic hepatitis B is to prevent the complications of cirrhosis, hepatic decompensation, HCC, and death. Because these clinical outcomes may take a long

period of time to develop, it is important to use intermediate or surrogate end points to evaluate the efficacy and response to antiviral treatment, and to determine whether treatment can be safely stopped, especially given concern for the development of antiviral resistance with NUC therapy. Although normalization of ALT and suppression of HBV DNA viral replication are associated with favorable outcomes, the durability of their response is low, and these end points are insufficient markers for stopping treatment. HBeAg seroconversion is currently used to discontinue NUC treatment in patients with HBeAg-positive chronic hepatitis B, whereas the stopping rule for HBeAg-negative disease relies on HBsAg loss. However, HBsAg loss occurs very infrequently and is not a practical end point for clinical use, although quantitative HBsAg levels may be useful in identifying patients who could achieve a sustained virologic response to treatment.PMID: 21893270 [PubMed - indexed for MEDLINE]--65)Pediatr Transplant. 2011 Nov;15(7):733-41. doi: 10.1111/j.1399-3046.2011.01563.x. Epub 2011 Aug 23.Genotype, phenotype, and outcomes of nine patients with T-B+NK+ SCID.Yu GP, Nadeau KC, Berk DR, de Saint Basile G, Lambert N, Knapnougel P, Roberts J, Kavanau K, Dunn E, Stiehm ER, Lewis DB, Umetsu DT, Puck JM, Cowan MJ.

Division of Immunology and Allergy, Department of Pediatrics, Stanford University School of Medicine and Lucile Packard Children's Hospital at Stanford, Palo Alto, CA, USA.Abstract

There are few reports of clinical presentation, genotype, and HCT outcomes for patients with T-B+NK+ SCID. Between 1981 and 2007, eight of 84 patients with SCID who received and/or were followed after HCT at UCSF had the T-B+NK+ phenotype. One additional patient with T-B+NK+ SCID was identified as the sibling of a patient treated at UCSF. Chart reviews were performed. Molecular analyses of IL7R, IL2RG, JAK3, and the genes encoding the CD3 T-cell receptor components δ (CD3D), ε (CD3E), and ζ (CD3Z) were carried out. IL7R mutations were documented in four patients and CD3D mutations in two others. Three patients had no defects found. Only two of nine patients had an HLA-matched related HCT donor. Both survived, and neither developed GVHD. Five of seven recipients of haploidentical grafts survived. Although the majority of reported cases of T-B+NK+ SCID are caused by defects in IL7R, CD3 complex defects were also found in this series and should be considered when evaluating patients with T-B+NK+ SCID. Additional genes, mutations in which account for T-B+NK+ SCID, remain to be found. Better approaches to early diagnosis and HCT treatment are needed for patients lacking an HLA-matched related donor.© 2011 John Wiley & Sons A/S.PMID: 21883749 [PubMed - indexed for MEDLINE] PMCID: PMC3196791 [Available on 2012/11/1]--66)Kidney Int. 2011 Dec;80(12):1364-76. doi: 10.1038/ki.2011.245. Epub 2011 Aug 31.Progressive histological damage in renal allografts is associated with expression of innate and adaptive immunity genes.Naesens M, Khatri P, Li L, Sigdel TK, Vitalone MJ, Chen R, Butte AJ, Salvatierra O, Sarwal MM.

Division of Nephrology, Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.

The degree of progressive chronic histological damage is associated with long-term renal allograft survival. In order to identify promising molecular targets for timely intervention, we examined renal allograft protocol and indication biopsies from 120 low-risk pediatric and adolescent recipients by whole-genome microarray expression profiling. In data-driven analysis, we found a highly regulated pattern of adaptive and innate immune gene expression that correlated with established or ongoing histological chronic injury, and also with development of future chronic histological damage, even in histologically pristine kidneys. Hence, histologically unrecognized immunological injury at a molecular level sets the stage for the development of chronic tissue injury, while the same molecular response is accentuated during established and worsening chronic allograft damage. Irrespective of the hypothesized immune or nonimmune trigger for chronic allograft injury, a highly orchestrated regulation of innate and adaptive immune responses was found in the graft at the molecular level. This occurred months before histologic lesions appear, and quantitatively below the diagnostic threshold of classic T-cell or antibody-mediated rejection. Thus, measurement of specific immune gene expression in protocol biopsies may be warranted to predict the development of subsequent chronic injury in histologically quiescent grafts and as a means to titrate immunosuppressive therapy.Comment in

* Kidney Int. 2011 Dec;80(12):1254-5.

PMID: 21881554 [PubMed - indexed for MEDLINE]--67)Arthritis Res Ther. 2011 Aug 2;13(4):231.Giant cell arteritis: immune and vascular aging as disease risk factors.Mohan SV, Liao YJ, Kim JW, Goronzy JJ, Weyand CM.

Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305-5166, USA.

Susceptibility for giant cell arteritis increases with chronological age, in parallel with age-related restructuring of the immune system and age-induced remodeling of the vascular wall. Immunosenescence results in shrinkage of the naïve T-cell pool, contraction of T-cell diversity, and impairment of innate immunity. Aging of immunocompetent cells forces the host to take alternative routes for protective immunity and confers risk for pathogenic immunity that causes chronic inflammatory tissue damage. Dwindling immunocompetence is particularly relevant as the aging host is forced to cope with an ever growing infectious load. Immunosenescence coincides with vascular aging during which the arterial wall undergoes dramatic structural changes and medium and large arteries lose their pliability and elasticity. On the molecular level, elastic fibers deteriorate and matrix proteins accumulate biochemical modifications. Thus, the aging process impacts the two major biologic systems that liaise to promote giant cell arteritis; the immune system and the vessel wall niche.PMID: 21861860 [PubMed - indexed for MEDLINE] PMCID: PMC3239337--68)Nat Biotechnol. 2011 Aug 14;29(9):829-34. doi: 10.1038/nbt.1947.An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells.

Tang C, Lee AS, Volkmer JP, Sahoo D, Nag D, Mosley AR, Inlay MA, Ardehali R, Chavez SL, Pera RR, Behr B, Wu JC, Weissman IL, Drukker M.

Institute of Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.Comment in

* Cell Stem Cell. 2011 Oct 4;9(4):291-2. * Nat Biotechnol. 2011 Sep;29(9):803-5.

PMID: 21841799 [PubMed - indexed for MEDLINE]--69)J Rheumatol. 2011 Aug;38(8):1759-64.The PROMIS of better outcome assessment: responsiveness, floor and ceiling effects, and Internet administration.Fries J, Rose M, Krishnan E.

Department of Medicine, Stanford University School of Medicine, Palo Alto, California, USA. jff@stanford.edu

OBJECTIVE:Use of item response theory (IRT) and, subsequently, computerized adaptive testing (CAT), under the umbrella of the NIH-PROMIS initiative (National Institutes of Health-Patient-Reported Outcomes Measurement Information System), to bring strong new assets to the development of more sensitive, more widely applicable, and more efficiently administered patient-reported outcome (PRO) instruments. We present data on current progress in 3 crucial areas: floor and ceiling effects, responsiveness to change, and interactive computer-based administration over the Internet.METHODS:We examined nearly 1000 patients with rheumatoid arthritis and related diseases in a series of studies including a one-year longitudinal examination of detection of change; compared responsiveness of the Legacy SF-36 and HAQ-DI instruments with IRT-based instruments; performed a randomized head-to-head trial of 4 modes of item administration; and simulated the effect of lack of floor and ceiling items upon statistical power and sample sizes.

RESULTS:IRT-based PROMIS instruments are more sensitive to change, resulting in the potential to reduce sample size requirements substantially by up to a factor of 4. The modes of administration tested did not differ from each other in any instance by more than one-tenth of a standard deviation. Floor and ceiling effects greatly reduce the number of available subjects, particularly at the ceiling.CONCLUSION:Failure to adequately address floor and ceiling effects, which determine the range of an instrument, can result in suboptimal assessment of many patients. Improved items, improved instruments, and computer-based administration improve PRO assessment and represent a fundamental advance in clinical outcomes research.PMID: 21807798 [PubMed - indexed for MEDLINE]--70)Arthritis Res Ther. 2011 Jun 24;13(3):R102.Novel multiplex technology for diagnostic characterization of rheumatoid arthritis.Chandra PE, Sokolove J, Hipp BG, Lindstrom TM, Elder JT, Reveille JD, Eberl H, Klause U, Robinson WH.

Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

INTRODUCTION:The aim of this study was to develop a clinical-grade, automated, multiplex system for the differential diagnosis and molecular stratification of rheumatoid arthritis (RA).METHODS:We profiled autoantibodies, cytokines, and bone-turnover products in sera from 120 patients with a diagnosis of RA of < 6 months' duration, as well as in sera from 27 patients with ankylosing spondylitis, 28 patients with psoriatic arthritis, and 25 healthy individuals. We used a commercial bead assay to measure cytokine levels and developed an array assay based on novel multiplex technology (Immunological Multi-Parameter Chip Technology) to evaluate autoantibody reactivities and bone-turnover markers. Data were analyzed by Significance Analysis of Microarrays and hierarchical clustering software.RESULTS:We developed a highly reproducible, automated, multiplex biomarker assay that can reliably distinguish between RA patients and healthy individuals or patients with other inflammatory arthritides. Identification of distinct biomarker signatures enabled molecular stratification of early-stage RA into clinically relevant subtypes. In this initial study, multiplex measurement of a subset of the differentiating biomarkers provided high sensitivity and specificity in the diagnostic discrimination of RA: Use of 3 biomarkers yielded a sensitivity of 84.2% and a specificity of 93.8%, and use of 4 biomarkers a sensitivity of 59.2% and a specificity of 96.3%.CONCLUSIONS:The multiplex biomarker assay described herein has the potential to diagnose RA with greater sensitivity and specificity than do current clinical tests. Its ability to stratify RA patients in an automated and reproducible manner paves the way for the development of assays that can guide RA therapy.PMID: 21702928 [PubMed - indexed for MEDLINE] PMCID: PMC3218917--71)Obes Surg. 2011 Dec;21(12):1965-70.Potential nutritional conflicts in bariatric and renal transplant patients.Lightner AL, Lau J, Obayashi P, Birge K, Melcher ML.

Department of Surgery, Stanford University School of Medicine, MSLS, 3rd Floor, MC: 5492, 1201 Welch Road, Stanford, CA 94305-5102, USA. amy4@stanford.edu

An increasing number of morbidly obese patients with end stage renal disease (ESRD) are sequentially undergoing bariatric surgery followed by renal transplantation. Discrepancies between the nutritional recommendations for obesity and chronic kidney disease (CKD) are often confusing for the obese patient in renal failure. However, when recommendations are structured according to stage and treatment of disease, a consistent plan can be clearly communicated to the patient. Therefore, to optimize patient and graft outcomes we present nutritional recommendations tailored to three patient populations: obese patients with ESRD, patients post Roux-en-Y gastric bypass (RYGBP) with ESRD, and patients post RYGBP and post renal transplantation.PMID: 21526378 [PubMed - indexed for MEDLINE]--72)Cent Nerv Syst Agents Chem. 2011 Jun 1;11(2):164-73.Astrocytes: targets for neuroprotection in stroke.Barreto G, White RE, Ouyang Y, Xu L, Giffard RG.

Department of Anesthesia, Stanford University School of Medicine, S272, Stanford, CA 94305, USA.

In the past two decades, over 1000 clinical trials have failed to demonstrate a benefit in treating stroke, with the exception of thrombolytics. Although many targets have been pursued, including antioxidants, calcium channel blockers, glutamate receptor blockers, and neurotrophic factors, often the focus has been on neuronal mechanisms of injury. Broader attention to loss and dysfunction of non-neuronal cell types is now required to increase the chance of success. Of the several glial cell types, this review will focus on astrocytes. Astrocytes are the most abundant cell type in the higher mammalian nervous system, and they play key roles in normal CNS physiology and in central nervous system injury and pathology. In the setting of ischemia astrocytes perform multiple functions, some beneficial and some potentially detrimental, making them excellent candidates as therapeutic targets to improve outcome following stroke and in other central nervous system injuries. The older neurocentric view of the central nervous system has changed radically with the growing understanding of the many essential functions of astrocytes. These include K+ buffering, glutamate clearance, brain antioxidant defense, close metabolic coupling with neurons, and modulation of neuronal excitability. In this review, we will focus on those functions of astrocytes that can both protect and endanger neurons, and discuss how manipulating these functions provides a novel and important strategy to enhance neuronal survival and improve outcome following cerebral ischemia.PMID: 21521168 [PubMed - indexed for MEDLINE] PMCID: PMC3167939--73)ACS Chem Biol. 2011 Jun 17;6(6):563-72. Epub 2011 Feb 28.Development of activity-based probes for cathepsin X.Paulick MG, Bogyo M.

Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5324, United States.

Cathepsin X is a lysosomal cysteine protease that functions as a carboxypeptidase with broad substrate specificity. Cathepsin X was discovered only recently, and its physiological roles are still not well understood. A number of studies suggest that cathepsin X may be involved in a variety of biological processes, including cancer, aging and degenerative conditions of the brain, inflammation, and cellular communication. Here we present the synthesis and characterization of several activity-based probes (ABPs) that target active cathepsin X. These ABPs were used to label cathepsin X in complex lysates, whole cells, and in vivo. Furthermore, we have developed a method for selectively labeling and visualizing active cathepsin X in vitro and in vivo. Overall, the probes developed in this study are valuable tools for the study of cathepsin X function.PMID: 21322635 [PubMed - indexed for MEDLINE] PMCID: PMC3117957 [Available on 2012/6/17]--74)Mol Imaging Biol. 2011 Dec;13(6):1061-6.In vivo bioluminescence imaging of inducible nitric oxide synthase gene expression in vascular inflammation.Terashima M, Ehara S, Yang E, Kosuge H, Tsao PS, Quertermous T, Contag CH, McConnell MV.

Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA 94305-5233, USA.

PURPOSE:Inflammation plays a critical role in atherosclerosis and is associated with upregulation of inducible nitric oxide synthase (iNOS). We studied bioluminescence imaging (BLI) to track iNOS gene expression in a murine model of vascular inflammation.PROCEDURES:Macrophage-rich vascular lesions were induced by carotid ligation plus high-fat diet and streptozotocin-induced diabetes in 18 iNOS-luc reporter mice. In vivo iNOS expression was imaged serially by BLI over 14 days, followed by in situ BLI and histology.RESULTS:BLI signal from ligated carotids increased over 14 days (9.7 ± 4.4 × 10(3 ) vs. 4.4 ± 1.7 × 10(3) photons/s/cm(2)/sr at baseline, p < 0.001 vs. baseline, p < 0.05 vs. sham controls). Histology confirmed substantial macrophage infiltration, with iNOS and luciferase expression, only in ligated left carotid arteries and not controls.CONCLUSIONS:BLI allows in vivo detection of iNOS expression in murine carotid lesions and may provide a valuable approach for monitoring vascular gene expression and inflammation in small animal models.PMID: 21057879 [PubMed - indexed for MEDLINE] PMCID: PMC3102789 [Available on 2012/12/1]--75)Photosynth Res. 2011 Jan;107(1):37-57. Epub 2010 Sep 7.The evolutionary consequences of oxygenic photosynthesis: a body size perspective.Payne JL, McClain CR, Boyer AG, Brown JH, Finnegan S, Kowalewski M, Krause RA Jr, Lyons SK, McShea DW, Novack-Gottshall PM, Smith FA, Spaeth P, Stempien JA, Wang SC.

Department of Geological and Environmental Sciences, Stanford University, 450 Serra Mall, Bldg. 320, Stanford, CA 94305, USA. jlpayne@stanford.edu

The high concentration of molecular oxygen in Earth's atmosphere is arguably the most conspicuous and geologically important signature of life. Earth's early atmosphere lacked

oxygen; accumulation began after the evolution of oxygenic photosynthesis in cyanobacteria around 3.0-2.5 billion years ago (Gya). Concentrations of oxygen have since varied, first reaching near-modern values ~600 million years ago (Mya). These fluctuations have been hypothesized to constrain many biological patterns, among them the evolution of body size. Here, we review the state of knowledge relating oxygen availability to body size. Laboratory studies increasingly illuminate the mechanisms by which organisms can adapt physiologically to the variation in oxygen availability, but the extent to which these findings can be extrapolated to evolutionary timescales remains poorly understood. Experiments confirm that animal size is limited by experimental hypoxia, but show that plant vegetative growth is enhanced due to reduced photorespiration at lower O(2):CO(2). Field studies of size distributions across extant higher taxa and individual species in the modern provide qualitative support for a correlation between animal and protist size and oxygen availability, but few allow prediction of maximum or mean size from oxygen concentrations in unstudied regions. There is qualitative support for a link between oxygen availability and body size from the fossil record of protists and animals, but there have been few quantitative analyses confirming or refuting this impression. As oxygen transport limits the thickness or volume-to-surface area ratio-rather than mass or volume-predictions of maximum possible size cannot be constructed simply from metabolic rate and oxygen availability. Thus, it remains difficult to confirm that the largest representatives of fossil or living taxa are limited by oxygen transport rather than other factors. Despite the challenges of integrating findings from experiments on model organisms, comparative observations across living species, and fossil specimens spanning millions to billions of years, numerous tractable avenues of research could greatly improve quantitative constraints on the role of oxygen in the macroevolutionary history of organismal size.PMID: 20821265 [PubMed - indexed for MEDLINE]--76)Adv Mater. 2011 Oct 18;23(39):4509-15. doi: 10.1002/adma.201102371. Epub 2011 Sep 8.Nanogel star polymer architectures: a nanoparticle platform for modular programmable macromolecular self-assembly, intercellular transport, and dual-mode cargo delivery.Lee VY, Havenstrite K, Tjio M, McNeil M, Blau HM, Miller RD, Sly J.

IBM Almaden Research Center, San Jose, CA 95120, USA.

PMID: 21901765 [PubMed - indexed for MEDLINE] PMCID: PMC3324179 [Available on 2012/10/18]--77)FASEB J. 2012 Mar 5. [Epub ahead of print]Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB.Banfi A, von Degenfeld G, Gianni-Barrera R, Reginato S, Merchant MJ, McDonald DM, Blau HM.

*Baxter Laboratory for Stem Cell Biology, Institute for Regenerative Medicine and Stem Cell Biology, Department of Microbiology and Immunology, Stanford University, Stanford, California, USA;

Therapeutic angiogenesis by delivery of vascular growth factors is an attractive strategy for treating debilitating occlusive vascular diseases, yet clinical trials have thus far failed to show

efficacy. As a result, limb amputation remains a common outcome for muscle ischemia due to severe atherosclerotic disease, with an overall incidence of 100 per million people in the United States per year. A challenge has been that the angiogenic master regulator vascular endothelial growth factor (VEGF) induces dysfunctional vessels, if expressed, outside of a narrow dosage window. We tested the hypothesis that codelivery of platelet-derived growth factor-BB (PDGF-BB), which recruits pericytes, could induce normal angiogenesis in skeletal muscle irrespective of VEGF levels. Coexpression of VEGF and PDGF-BB encoded by separate vectors in different cells or in the same cells only partially corrected aberrant angiogenesis. In marked contrast, coexpression of both factors in every cell at a fixed relative level via a single bicistronic vector led to robust, uniformly normal angiogenesis, even when VEGF expression was high and heterogeneous. Notably, in an ischemic hindlimb model, single-vector expression led to efficient growth of collateral arteries, revascularization, increased blood flow, and reduced tissue damage. Furthermore, these results were confirmed in a clinically applicable gene therapy approach by adenoviral-mediated delivery of the bicistronic vector. We conclude that coordinated expression of VEGF and PDGF-BB via a single vector constitutes a novel strategy for harnessing the potency of VEGF to induce safe and efficacious angiogenesis.-Banfi, A., von Degenfeld, G., Gianni-Barrera, R., Reginato, S., Merchant, M. J., McDonald, D. M., Blau, H. M. Therapeutic angiogenesis due to balanced single-vector delivery of VEGF and PDGF-BB.PMID: 22391130 [PubMed - as supplied by publisher]