Microfluidic Single-Cell Real-Time PCR for Comparative Analysis of Gene Expression Patterns

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]
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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]
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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: .

PMID: 22471858 [PubMed - as supplied by publisher]
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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, .

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]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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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]
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14)Immunity.2012 Mar 23;36(3):438-50.

Plasmacytoid dendritic cells transport peripheral antigens to the thymus to promote central tolerance.