Publications by authors named "Clifford A Lowell"

153 Publications

A CD22-Shp1 phosphatase axis controls integrin β display and B cell function in mucosal immunity.

Nat Immunol 2021 Mar 15;22(3):381-390. Epub 2021 Feb 15.

Palo Alto Veterans Institute for Research, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA, USA.

The integrin αβ selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of αβ surface expression and gut immunity. Shp1 selectively inhibited β endocytosis, enhancing surface αβ display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin β on the cell surface to target intracellular Shp1 to β. Shp1 restrained plasma membrane β phosphorylation and inhibited β endocytosis without affecting β integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface αβ and in homing to GALT. Consistent with the specialized role of αβ in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.
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http://dx.doi.org/10.1038/s41590-021-00862-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116842PMC
March 2021

A Neutrophil Subset Defined by Intracellular Olfactomedin 4 is Associated with Mortality in Sepsis.

Am J Physiol Lung Cell Mol Physiol 2020 Dec 23. Epub 2020 Dec 23.

Departments of Medicine and Anesthesiology and the Cardiovascular Research Institute, University of California, San Francisco, California.

Sepsis is a heterogeneous syndrome clinically and biologically but biomarkers of distinct host response pathways for early prognostic information and testing targeted treatments are lacking. We hypothesized that Olfactomedin 4 (OLFM4), a matrix glycoprotein of neutrophil specific granules defines a distinct neutrophil subset that may be an independent risk factor for poor outcomes in sepsis. In a single-center, prospective cohort study, we enrolled adults admitted to an academic medical center from the Emergency Department (ED) with suspected sepsis (identified by 2 or greater Systemic Inflammatory Response Syndrome [SIRS] criteria and antibiotic receipt) from March 2016 through December 2017, followed by sepsis adjudication according to Sepsis-3. We collected 200mL of whole blood within 24 hours of admission and stained for the neutrophil surface marker CD66b followed by intracellular staining for OLFM4 quantitated by flow cytometry. The predictor for 60-day mortality was the percentage of OLFM4+ neutrophils and at a cut-point of OLFM4+ ≥37.6% determined by the Youden Index. Of 120 enrolled patients with suspected sepsis, 97 had sepsis and 23 had non-sepsis SIRS. The mean percentage of OLFM4+ neutrophils was significantly increased in both sepsis and non-sepsis SIRS patients who died (P ≤ 0.01). Among sepsis patients with elevated OLFM4+(≥37.6%), 56% died compared to 18% with OLFM4+ <37.6% (P=0.001).The association between OLFM4+ and mortality withstood adjustment for demographics, co-morbidities and measures of severity of illness (P<0.03). In sepsis, OLFM4+ neutrophil percentage is independently associated with 60-day mortality and may represent a novel measure of the heterogeneity of host response to sepsis.
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http://dx.doi.org/10.1152/ajplung.00090.2020DOI Listing
December 2020

Shp1 Loss Enhances Macrophage Effector Function and Promotes Anti-Tumor Immunity.

Front Immunol 2020 29;11:576310. Epub 2020 Sep 29.

Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, United States.

Shp1, encoded by the gene , is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells . We also generated a novel mouse model to evaluate the impact of global, inducible deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when is knocked out from birth. This indicates that acute perturbation of Shp1 could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.
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http://dx.doi.org/10.3389/fimmu.2020.576310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550718PMC
September 2020

ORAI1 and ORAI2 modulate murine neutrophil calcium signaling, cellular activation, and host defense.

Proc Natl Acad Sci U S A 2020 09 14;117(39):24403-24414. Epub 2020 Sep 14.

Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110;

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.
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http://dx.doi.org/10.1073/pnas.2008032117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533673PMC
September 2020

Complement activation on endothelium initiates antibody-mediated acute lung injury.

J Clin Invest 2020 11;130(11):5909-5923

Department of Medicine, UCSF, San Francisco, California, USA.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.
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http://dx.doi.org/10.1172/JCI138136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598054PMC
November 2020

The Ubiquitin-Modifying Enzyme A20 Terminates C-Type Lectin Receptor Signals and Is a Suppressor of Host Defense against Systemic Fungal Infection.

Infect Immun 2020 08 19;88(9). Epub 2020 Aug 19.

Department of Immunology, Duke University Medical Center, Durham, North Carolina, USA

C-type lectin receptors (CLRs) play key roles in antifungal defense. CLR-induced NF-κB is central to CLR functions in immunity, and thus, molecules that control the amplitude of CLR-induced NF-κB could profoundly influence host defense against fungal pathogens. However, little is known about the mechanisms that negatively regulate CLR-induced NF-κB, and molecules which act on the CLR family broadly and which directly regulate acute CLR-signaling cascades remain unidentified. Here, we identify the ubiquitin-editing enzyme A20 as a negative regulator of acute NF-κB activation downstream of multiple CLR pathways. Absence of A20 suppression results in exaggerated CLR responses in cells which are A20 deficient and also cells which are A20 haplosufficient, including multiple primary immune cells. Loss of a single allele of A20 results in enhanced defense against systemic infection and prolonged host survival. Thus, A20 restricts CLR-induced innate immune responses and is a suppressor of host defense against systemic fungal infection.
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http://dx.doi.org/10.1128/IAI.00048-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440764PMC
August 2020

Inhibition of the SRC Kinase HCK Impairs STAT3-Dependent Gastric Tumor Growth in Mice.

Cancer Immunol Res 2020 04 28;8(4):428-435. Epub 2020 Jan 28.

Olivia Newton-John Cancer Research Institute and La Trobe University School of Cancer Medicine, Victoria, Australia.

Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow-derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity.
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http://dx.doi.org/10.1158/2326-6066.CIR-19-0623DOI Listing
April 2020

Src family kinase-mediated vesicle trafficking is critical for neutrophil basement membrane penetration.

Haematologica 2020 07 7;105(7):1845-1856. Epub 2019 Nov 7.

Walter-Brendel-Center of Experimental Medicine, Institute of Cardiovascular Physiology and Pathophysiology, Klinikum der Universität, Ludwig-Maximilians-University Munich, Planegg-Martinsried, Germany

Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment using mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.
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http://dx.doi.org/10.3324/haematol.2019.225722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327629PMC
July 2020

Relief of tumor hypoxia unleashes the tumoricidal potential of neutrophils.

J Clin Invest 2020 01;130(1):389-403

Department of Laboratory Medicine, UCSF, San Francisco, California, USA.

Polymorphonuclear neutrophils (PMNs) are increasingly recognized to influence solid tumor development, but why their effects are so context dependent and even frequently divergent remains poorly understood. Using an autochthonous mouse model of uterine cancer and the administration of respiratory hyperoxia as a means to improve tumor oxygenation, we provide in vivo evidence that hypoxia is a potent determinant of tumor-associated PMN phenotypes and direct PMN-tumor cell interactions. Upon relief of tumor hypoxia, PMNs were recruited less intensely to the tumor-bearing uterus, but the recruited cells much more effectively killed tumor cells, an activity our data moreover suggested was mediated via their production of NADPH oxidase-derived reactive oxygen species and MMP-9. Simultaneously, their ability to promote tumor cell proliferation, which appeared to be mediated via their production of neutrophil elastase, was rendered less effective. Relieving tumor hypoxia thus greatly improved net PMN-dependent tumor control, leading to a massive reduction in tumor burden. Remarkably, this outcome was T cell independent. Together, these findings identify key hypoxia-regulated molecular mechanisms through which PMNs directly induce tumor cell death and proliferation in vivo and suggest that the contrasting properties of PMNs in different tumor settings may in part reflect the effects of hypoxia on direct PMN-tumor cell interactions.
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http://dx.doi.org/10.1172/JCI130952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934192PMC
January 2020

CARD9 mediates dendritic cell-induced development of Lyn deficiency-associated autoimmune and inflammatory diseases.

Sci Signal 2019 10 8;12(602). Epub 2019 Oct 8.

Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.

CARD9 is an immune adaptor protein in myeloid cells that is involved in C-type lectin signaling and antifungal immunity. CARD9 is implicated in autoimmune and inflammatory-related diseases, such as rheumatoid arthritis, IgA nephropathy, ankylosing spondylitis, and inflammatory bowel disease (IBD). Given that Lyn-deficient ( mice are susceptible to both autoimmunity and IBD, we investigated the immunological role of CARD9 in the development of these diseases using the mouse model. We found that genetic deletion of CARD9 was sufficient to reduce the development of both spontaneous autoimmune disease as well as DSS- or IL-10 deficiency-associated colitis in mice. Mechanistically, CARD9 was a vital component of the Lyn-mediated regulation of Toll-like receptor (TLR2 and TLR4) signaling in dendritic cells, but not in macrophages. In the absence of Lyn, signaling through a CD11b-Syk-PKCδ-CARD9 pathway was amplified, leading to increased TLR-induced production of inflammatory cytokines. Dendritic cell-specific deletion of CARD9 reversed the development of autoimmune and experimental colitis observed in dendritic cell-specific, Lyn-deficient mice. These findings suggest that targeting CARD9 may suppress the development of colitis and autoimmunity by reducing dendritic cell-driven inflammation.
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http://dx.doi.org/10.1126/scisignal.aao3829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546115PMC
October 2019

L-selectin shedding affects bacterial clearance in the lung: a new regulatory pathway for integrin outside-in signaling.

Blood 2019 10;134(17):1445-1457

Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Muenster, Muenster, Germany.

Pneumonia induced by Gram-negative bacteria is a common and serious disease associated with high morbidity and mortality. Elimination of bacterial pathogens relies on the recruitment and functions of neutrophils. The adhesion molecule L-selectin has recently been implicated in integrin activation in neutrophils (inside-out signaling). However, the molecular mechanism by which L-selectin participates in host defense against Klebsiella pneumoniae-induced pulmonary inflammation is unknown. We demonstrate that L-selectin-deficient mice are prone to pulmonary infection compared with wild-type controls. Mechanistically, L-selectin cleavage from the neutrophil surface triggered by integrin engagement is involved in neutrophil recruitment into the lung and bacterial clearance. Downstream of integrin ligation, the metalloproteinase A disintegrin and metalloproteinase 17 (ADAM17) sheds L-selectin from the neutrophil surface in an IRhom2-dependent manner. L-selectin cleavage enhances integrin-mediated outside-in signaling, resulting in increased neutrophil effector functions. Thus, we identify a novel regulatory mechanism in neutrophils required for an adequate immune response triggered by integrin engagement during K pneumoniae-induced pulmonary inflammation.
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http://dx.doi.org/10.1182/blood.2019000685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839956PMC
October 2019

CRAC channel regulation of innate immune cells in health and disease.

Cell Calcium 2019 03 9;78:56-65. Epub 2019 Jan 9.

Department of Laboratory Medicine, University of California, San Francisco, CA, United States.

Calcium is a major intracellular signaling messenger in innate immune cells. Similar to other immune cell subsets, the majority of calcium entry into innate immune cells is induced by cell surface receptors that stimulate store-operated calcium entry through calcium-release activated calcium (CRAC) channels. Since the molecular description of the STIM family of calcium sensors and the ORAI family of CRAC channel proteins, the majority of studies support a dominant role for these proteins in calcium signaling in innate cells. In reviewing the literature on CRAC channel function in innate cells, several general themes emerge. All innate cells express multiple members of the STIM and ORAI family members, however the ratio and relative contribution of individual isoforms changes depending on the cell type and activation state of the cell. It is evident that study of functional roles for STIM molecules is clearly ahead of studies of specific ORAI family members in all innate cell types, and that studies of CRAC channels in innate cells are not nearly as advanced as studies in lymphocytes. However, taken together, evidence from both STIM calcium sensors and ORAI channels in innate cells indicates that deficiency of STIM and ORAI proteins tends not to affect the development of any innate cell lineage, but certainly affects their function, in particular activation of the neutrophil oxidase and mast cell activation via IgE receptors. Furthermore, there are clearly hints that therapeutic targeting of CRAC channels in innate cells offers a new approach to various inflammatory and allergic diseases.
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http://dx.doi.org/10.1016/j.ceca.2019.01.003DOI Listing
March 2019

Neutrophils: Their Role in Innate and Adaptive Immunity 2017.

J Immunol Res 2017;2017:9748345. Epub 2017 Nov 7.

Facultad de Odontología, Universidad Nacional Autónoma de México, 04510 Ciudad de México, Mexico.

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http://dx.doi.org/10.1155/2017/9748345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5697120PMC
January 2019

PRN473, an inhibitor of Bruton's tyrosine kinase, inhibits neutrophil recruitment via inhibition of macrophage antigen-1 signalling.

Br J Pharmacol 2018 02 22;175(3):429-439. Epub 2017 Dec 22.

Department of Anesthesiology, Intensive Care and Pain Medicine, University Hospital Münster, Münster, Germany.

Background And Purpose: Following inflammatory stimuli, neutrophils are recruited to sites of inflammation and exert effector functions that often have deleterious effects on tissue integrity, which can lead to organ failure. Bruton's tyrosine kinase (Btk) is expressed in neutrophils and constitutes a promising pharmacological target for neutrophil-mediated tissue damage. Here, we evaluate a selective reversible inhibitor of Btk, PRN473, for its ability to dampen neutrophil influx via inhibition of adhesion receptor signalling pathways.

Experimental Approach: In vitro assays were used to assess fMLP receptor 1 (Fpr-1)-mediated binding of ligands to the adhesion receptors macrophage antigen-1 (Mac-1) and lymphocyte function antigen-1. Intravital microscopy of the murine cremaster was used to evaluate post-adhesion strengthening and endoluminal crawling. Finally, neutrophil influx was visualized in a clinically relevant model of sterile liver injury in vivo. Btk knockout animals were used as points of reference for Btk functions.

Key Results: Pharmacological inhibition of Btk by PRN473 reduced fMLP-induced phosphorylation of Btk and Mac-1 activation. Biochemical experiments demonstrated the specificity of the inhibitor. PRN473 (20 mg·kg ) significantly reduced intravascular crawling and neutrophil recruitment into inflamed tissue in a model of sterile liver injury, down to levels seen in Btk-deficient animals. A higher dose did not provide additional reduction of intravascular crawling and neutrophil recruitment.

Conclusions And Implications: PRN473, a highly selective inhibitor of Btk, potently attenuates sterile liver injury by inhibiting the activation of the β -integrin Mac-1 and subsequently neutrophil recruitment into inflamed tissue.
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http://dx.doi.org/10.1111/bph.14090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773957PMC
February 2018

B cell autoimmunity at the extremes.

Nat Immunol 2017 09;18(10):1065-1066

Department of Laboratory Medicine, University of California San Francisco, San Francisco, California, USA.

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http://dx.doi.org/10.1038/ni.3840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334840PMC
September 2017

STIM1 and STIM2 cooperatively regulate mouse neutrophil store-operated calcium entry and cytokine production.

Blood 2017 09 19;130(13):1565-1577. Epub 2017 Jul 19.

Department of Laboratory Medicine, University of California, San Francisco, CA.

Neutrophils are key effector cells of the innate immune system. Calcium-dependent signaling pathways initiated by store-operated calcium entry (SOCE) are known to regulate neutrophil activation; however, the precise mechanism of this process remains unclear. STIM1 and STIM2 are calcium-sensing molecules that link calcium depletion of the endoplasmic reticulum with opening of plasma membrane calcium channels. Although a role for STIM1 in neutrophil SOCE and activation has been established, the function of STIM2 is unknown. Here we use mice with conditional ablation of and/or to investigate the role of STIM2 in neutrophil activation. We demonstrate that loss of STIM2 results in decreased SOCE, particularly at lower doses of agonists. Reactive oxygen species (ROS) production, degranulation, and phagocytosis are normal in the absence of STIM2, suggesting STIM1 is the dominant calcium sensor required for classical short-term neutrophil responses. However, neutrophil cytokine production required STIM2, but not STIM1, at least in part as a result of redox regulation of cytokine gene expression. In vivo loss of STIM2 results in lower cytokine levels and protection from mortality in a mouse model of systemic inflammatory response syndrome. These data, combined with previous studies focusing on STIM1, define distinct but cooperative functions for STIM1 and STIM2 in modulating neutrophil bactericidal and cytokine responses.
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http://dx.doi.org/10.1182/blood-2016-11-751230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620414PMC
September 2017

Role of MyD88 signaling in the imiquimod-induced mouse model of psoriasis: focus on innate myeloid cells.

J Leukoc Biol 2017 09 22;102(3):791-803. Epub 2017 Jun 22.

Department of Medicine, Division of General Pathology, University of Verona, Verona, Italy;

Psoriasis is a chronic skin disease associated with deregulated activation of immune cells and keratinocytes. In this study, we used the imiquimod (IMQ)-induced mouse model of psoriasis to dissect better the contribution of hematopoietic and skin-resident stromal cells to psoriasis development. The comparison of disease development in mice carrying the hematopoietic cell-specific deletion of MyD88 ( mice) with mice carrying the total MyD88 deficiency ( mice), we show that the progression of skin and systemic inflammation, as well as of epidermal thickening, was completely dependent on MyD88 expression in hematopoietic cells. However, both mouse strains developed some degree of epidermal thickening during the initial stages of IMQ-induced psoriasis, even in the absence of hematopoietic cell activation and infiltration into the skin, suggesting a contribution of MyD88-independent mechanisms in skin-resident stromal cells. With the use of conditional knockout mouse strains lacking MyD88 in distinct lineages of myeloid cells ( and mice), we report that MyD88 signaling in monocytes and Mϕ, but not in neutrophils, plays an important role in disease propagation and exacerbation by modulating their ability to sustain γδ T cell effector functions via IL-1β and IL-23 production. Overall, these findings add new insights into the specific contribution of skin-resident stromal vs. hematopoietic cells to disease initiation and progression in the IMQ-induced mouse model of psoriasis and uncover a potential novel pathogenic role for monocytes/Mϕ to psoriasis development.
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http://dx.doi.org/10.1189/jlb.3MA0217-054RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608051PMC
September 2017

Shp1 function in myeloid cells.

J Leukoc Biol 2017 09 12;102(3):657-675. Epub 2017 Jun 12.

Department of Laboratory Medicine and Immunology Program, University of California, San Francisco, California, USA

The mouse was first described in 1975 as a model of systemic inflammation and autoimmunity, as a result of immune system dysregulation. The phenotype was later ascribed to mutations in the cytoplasmic tyrosine phosphatase Shp1. This phosphatase is expressed widely throughout the hematopoietic system and has been shown to impact a multitude of cell signaling pathways. The determination of which cell types contribute to the different aspects of the phenotype caused by global Shp1 loss or mutation and which pathways within these cell types are regulated by Shp1 is important to further our understanding of immune system regulation. In this review, we focus on the role of Shp1 in myeloid cells and how its dysregulation affects immune function, which can impact human disease.
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http://dx.doi.org/10.1189/jlb.2MR0317-105RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557645PMC
September 2017

Differentiating pulmonary transfusion reactions using recipient and transfusion factors.

Transfusion 2017 07 3;57(7):1684-1690. Epub 2017 May 3.

Blood Systems Research Institute.

Background: It is increasingly recognized that recipient risk factors play a prominent role in possible transfusion-related acute lung injury (pTRALI) and transfusion-associated circulatory overload (TACO). We hypothesized that both transfusion and recipient factors including natriuretic peptides could be used to distinguish TRALI from TACO and pTRALI.

Study Design And Methods: We performed a post hoc analysis of a case-control study of pulmonary transfusion reactions conducted at the University of California at San Francisco and Mayo Clinic, Rochester. We evaluated clinical data and brain natriuretic peptides (BNP) levels drawn after transfusion in patients with TRALI (n = 21), pTRALI (n = 26), TACO (n = 22), and controls (n = 24). Logistic regression and receiver operating characteristics curve analyses were used to determine the accuracy of clinical and biomarker predictors in differentiating TRALI from TACO and pTRALI.

Results: We found that pTRALI and TACO were associated with older age, higher fluid balance, and elevated BNP levels relative to those of controls and TRALI. The following variables were useful in distinguishing cases of pTRALI and TACO from TRALI: age more than 70 years, BNP levels more than 1000 pg/mL, 24-hour fluid balance of more than 3 L, and a lower number of transfused blood components. Using the above variables, our logistic model had a 91% negative predictive value in the differential diagnosis of TRALI.

Conclusions: Models incorporating readily available clinical and biomarker data can be used to differentiate transfusion-related respiratory complications. Additional studies examining recipient risk factors and the likelihood of TRALI may be useful in decision making regarding donor white blood cell antibody testing.
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http://dx.doi.org/10.1111/trf.14118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758383PMC
July 2017

SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin.

Nature 2017 04 19;544(7651):493-497. Epub 2017 Apr 19.

Laboratory of Molecular Oncology, Institut de recherches cliniques de Montréal (IRCM), Montréal, Québec H2W 1R7, Canada.

Cancer cells elude anti-tumour immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors. Phagocytosis by macrophages plays a critical role in cancer control. Therapeutic blockade of signal regulatory protein (SIRP)-α, an inhibitory receptor on macrophages, or of its ligand CD47 expressed on tumour cells, improves tumour cell elimination in vitro and in vivo, suggesting that blockade of the SIRPα-CD47 checkpoint could be useful in treating human cancer. However, the pro-phagocytic receptor(s) responsible for tumour cell phagocytosis is(are) largely unknown. Here we find that macrophages are much more efficient at phagocytosis of haematopoietic tumour cells, compared with non-haematopoietic tumour cells, in response to SIRPα-CD47 blockade. Using a mouse lacking the signalling lymphocytic activation molecule (SLAM) family of homotypic haematopoietic cell-specific receptors, we determined that phagocytosis of haematopoietic tumour cells during SIRPα-CD47 blockade was strictly dependent on SLAM family receptors in vitro and in vivo. In both mouse and human cells, this function required a single SLAM family member, SLAMF7 (also known as CRACC, CS1, CD319), expressed on macrophages and tumour cell targets. In contrast to most SLAM receptor functions, SLAMF7-mediated phagocytosis was independent of signalling lymphocyte activation molecule-associated protein (SAP) adaptors. Instead, it depended on the ability of SLAMF7 to interact with integrin Mac-1 (refs 18, 19, 20) and utilize signals involving immunoreceptor tyrosine-based activation motifs. These findings elucidate the mechanism by which macrophages engulf and destroy haematopoietic tumour cells. They also reveal a novel SAP adaptor-independent function for a SLAM receptor. Lastly, they suggest that patients with tumours expressing SLAMF7 are more likely to respond to SIRPα-CD47 blockade therapy.
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http://dx.doi.org/10.1038/nature22076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5565268PMC
April 2017

Efficiency and Specificity of Gene Deletion in Lung Epithelial Doxycycline-Inducible Cre Mice.

Am J Respir Cell Mol Biol 2017 08;57(2):248-257

Department of Laboratory Medicine and Program in Immunology, University of California, San Francisco, California.

The transgenic mouse strains surfactant protein C-reverse tetracycline transactivator (SP-C-rtTA), club cell secretory protein (CCSP)-rtTA, and tetracycline operator (TetO)-Cre have been invaluable for spatiotemporally regulating gene deletion in the pulmonary epithelium. In this study, we measured the efficiency and specificity of gene deletion that can be achieved in these mice using the Rosa26-eYFP reporter. Triple-transgenic mice (tTg or rtTA/TetO-Cre/Rosa-eYFP) were bred and treated with various doxycycline (dox) regimens to induce gene deletion, which was then quantified in various cell populations by flow cytometry. In these crosses, we found that the TetO-Cre transgene must be transmitted through the female parent to avoid germline gene deletion. With dox exposure during lung development, SP-C-tTg mice deleted in ∼65-75% of alveolar epithelial type II (ATII) cells, but in only ∼45-50% of the integrin β4 population, which consisted of club cells and distal lung progenitor cells. In contrast, CCSP-tTg mice deleted in ∼50% of ATII cells and ∼80% of integrin β4 cells. Upon dox treatment of adults, deletion in ATII cells and integrin β4 cells in SP-C-tTg mice dropped significantly to ∼20% and ∼6%, respectively, whereas CCSP-tTg mice deleted in ∼57% of ATII and ∼40% of integrin β4 cells. Interestingly, untreated CCSP-tTg mice also deleted in ∼40% of integrin β4 cells, indicating significant leakiness of CCSP-tTg in β4 cells. In all mouse groups, minimal deletion occurred in mouse tracheal epithelial cells or in mesenchymal or hematopoietic cells. These data provide the first quantitative, side-by-side comparison of the deletion efficiency for these widely used transgenic mouse strains.
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http://dx.doi.org/10.1165/rcmb.2016-0208OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576580PMC
August 2017

Isolation of Highly Pure Primary Mouse Alveolar Epithelial Type II Cells by Flow Cytometric Cell Sorting.

Bio Protoc 2016 Nov;6(22)

Department of Laboratory Medicine and the Program in Immunology, University of California, San Francisco, USA.

In this protocol, we describe the method for isolating highly pure primary alveolar epithelial type II (ATII) cells from lungs of naïve mice. The method combines negative selection for a variety of lineage markers along with positive selection for EpCAM, a pan-epithelial cell marker. This method yields 2-3 × 10 ATII cells per mouse lung. The cell preps are highly pure and viable and can be used for genomic or proteomic analyses or cultured to understand their roles in various biological processes.
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http://dx.doi.org/10.21769/BioProtoc.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293249PMC
November 2016

Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9.

PLoS One 2016 15;11(12):e0167693. Epub 2016 Dec 15.

Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, United States of America.

Excessive type 2 helper T cell responses to environmental antigens can cause immunopathology such as asthma and allergy, but how such immune responses are induced remains unclear. We studied this process in the airways by immunizing mice intranasally with the antigen ovalbumin together with either of two Toll-like receptor (TLR) ligands. We found the TLR5 ligand flagellin promoted a type 2 helper T cell response, whereas, a TLR9 ligand CpG oligodeoxyribonucleotide (ODN) promoted a type 1 helper T cell response. CpG ODN induced mRNA encoding interleukin (IL)-12 p40, whereas, flagellin caused IL-33 secretion and induced mRNAs encoding IL-1 and thymic stromal lymphopoietin (TSLP). By using mice deficient in the TLR and IL-1R signaling molecule, myeloid differentiation primary response 88 (MyD88), in conventional dendritic cells (cDCs) and alveolar macrophages (AMs), and by cell sorting different lung populations after 2 hours of in vivo stimulation, we characterized the cell types that rapidly produced inflammatory cytokines in response to TLR stimulation. CpG ODN was likely recognized by TLR9 on cDCs and AMs, which made mRNA encoding IL-12. IL-12 was necessary for the subsequent innate and adaptive interferon-γ production. In contrast, flagellin stimulated multiple cells of hematopoietic and non-hematopoietic origin, including AMs, DCs, monocytes, and lung epithelial cells. AMs were largely responsible for IL-1α, whereas lung epithelial cells made TSLP. Multiple hematopoietic cells, including AMs, DCs, and monocytes contributed to other cytokines, including IL-1β and TNFα. MyD88-dependent signals, likely through IL-1R and IL-33R, and MyD88-independent signals, likely from TSLP, were necessary in cDCs for promotion of the early IL-4 response by CD4 T cells in the draining lymph node. Thus, the cell types that responded to TLR ligands were a critical determinant of the innate cytokines produced and the character of the resulting adaptive immune response in the airways.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167693PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5157987PMC
July 2017

Leishmania Uses Mincle to Target an Inhibitory ITAM Signaling Pathway in Dendritic Cells that Dampens Adaptive Immunity to Infection.

Immunity 2016 10 11;45(4):788-801. Epub 2016 Oct 11.

Immunobiology Laboratory, Fundación Centro Nacional de Investigaciones Cardiovasculares "Carlos III" (CNIC), Melchor Fernández Almagro 3, Madrid 28029, Spain. Electronic address:

C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection. Mincle deficiency enhanced adaptive immunity against the parasite, correlating with increased activation, migration, and priming by Mincle-deficient dendritic cells (DCs). Leishmania triggered a Mincle-dependent inhibitory axis characterized by SHP1 coupling to the FcRγ chain. Selective loss of SHP1 in CD11c cells phenocopies enhanced adaptive immunity to Leishmania. In conclusion, Leishmania shifts Mincle to an inhibitory ITAM (ITAMi) configuration that impairs DC activation. Thus, ITAMi can be exploited for immune evasion by a pathogen and may represent a paradigm for ITAM-coupled receptors sensing self and non-self.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074365PMC
http://dx.doi.org/10.1016/j.immuni.2016.09.012DOI Listing
October 2016

LYN- and AIRE-mediated tolerance checkpoint defects synergize to trigger organ-specific autoimmunity.

J Clin Invest 2016 10 29;126(10):3758-3771. Epub 2016 Aug 29.

Studies of the genetic factors associated with human autoimmune disease suggest a multigenic origin of susceptibility; however, how these factors interact and through which tolerance pathways they operate generally remain to be defined. One key checkpoint occurs through the activity of the autoimmune regulator AIRE, which promotes central T cell tolerance. Recent reports have described a variety of dominant-negative AIRE mutations that likely contribute to human autoimmunity to a greater extent than previously thought. In families with these mutations, the penetrance of autoimmunity is incomplete, suggesting that other checkpoints play a role in preventing autoimmunity. Here, we tested whether a defect in LYN, an inhibitory protein tyrosine kinase that is implicated in systemic autoimmunity, could combine with an Aire mutation to provoke organ-specific autoimmunity. Indeed, mice with a dominant-negative allele of Aire and deficiency in LYN spontaneously developed organ-specific autoimmunity in the eye. We further determined that a small pool of retinal protein-specific T cells escaped thymic deletion as a result of the hypomorphic Aire function and that these cells also escaped peripheral tolerance in the presence of LYN-deficient dendritic cells, leading to highly destructive autoimmune attack. These findings demonstrate how 2 distinct tolerance pathways can synergize to unleash autoimmunity and have implications for the genetic susceptibility of autoimmune disease.
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http://dx.doi.org/10.1172/JCI84440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087700PMC
October 2016

Downregulation of the Syk Signaling Pathway in Intestinal Dendritic Cells Is Sufficient To Induce Dendritic Cells That Inhibit Colitis.

J Immunol 2016 10 24;197(7):2948-57. Epub 2016 Aug 24.

Division of Gastroenterology-Hepatology, Department of Internal Medicine, Tufts Medical Center, Boston, MA 02111;

Helminthic infections modulate host immunity and may protect people in less-developed countries from developing immunological diseases. In a murine colitis model, the helminth Heligmosomoides polygyrus bakeri prevents colitis via induction of regulatory dendritic cells (DCs). The mechanism driving the development of these regulatory DCs is unexplored. There is decreased expression of the intracellular signaling pathway spleen tyrosine kinase (Syk) in intestinal DCs from H. polygyrus bakeri-infected mice. To explore the importance of this observation, it was shown that intestinal DCs from DC-specific Syk(-/-) mice were powerful inhibitors of murine colitis, suggesting that loss of Syk was sufficient to convert these cells into their regulatory phenotype. DCs sense gut flora and damaged epithelium via expression of C-type lectin receptors, many of which signal through the Syk signaling pathway. It was observed that gut DCs express mRNA encoding for C-type lectin (CLEC) 7A, CLEC9A, CLEC12A, and CLEC4N. H. polygyrus bakeri infection downmodulated CLEC mRNA expression in these cells. Focusing on CLEC7A, which encodes for the dectin-1 receptor, flow analysis showed that H. polygyrus bakeri decreases dectin-1 expression on the intestinal DC subsets that drive Th1/Th17 development. DCs become unresponsive to the dectin-1 agonist curdlan and fail to phosphorylate Syk after agonist stimulation. Soluble worm products can block CLEC7A and Syk mRNA expression in gut DCs from uninfected mice after a brief in vitro exposure. Thus, downmodulation of Syk expression and phosphorylation in intestinal DCs could be important mechanisms through which helminths induce regulatory DCs that limit colitis.
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http://dx.doi.org/10.4049/jimmunol.1600063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026942PMC
October 2016

MyD88 Shapes Vaccine Immunity by Extrinsically Regulating Survival of CD4+ T Cells during the Contraction Phase.

PLoS Pathog 2016 08 19;12(8):e1005787. Epub 2016 Aug 19.

Departments of Pediatrics, University of Wisconsin School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in Myd88-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines.
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http://dx.doi.org/10.1371/journal.ppat.1005787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991787PMC
August 2016

Neutrophils in animal models of autoimmune disease.

Semin Immunol 2016 04 7;28(2):174-86. Epub 2016 Apr 7.

Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA 94143, USA. Electronic address:

Neutrophils have traditionally been thought to play only a peripheral role in the genesis of many autoimmune and inflammatory diseases. However, recent studies in a variety of animal models suggest that these cells are central to the initiation and propagation of autoimmunity. The use of mouse models, which allow either deletion of neutrophils or the targeting of specific neutrophil functions, has revealed the many complex ways these cells contribute to autoimmune/inflammatory processes. This includes generation of self antigens through the process of NETosis, regulation of T-cell and dendritic cell activation, production of cytokines such as BAFF that stimulate self-reactive B-cells, as well as indirect effects on epithelial cell stability. In comparing the many different autoimmune models in which neutrophils have been examined, a number of common underlying themes emerge - such as a role for neutrophils in stimulating vascular permeability in arthritis, encephalitis and colitis. The use of animal models has also stimulated the development of new therapeutics that target neutrophil functions, such as NETosis, that may prove beneficial in human disease. This review will summarize neutrophil contributions in a number of murine autoimmune/inflammatory disease models.
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http://dx.doi.org/10.1016/j.smim.2016.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867286PMC
April 2016

FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection.

PLoS Pathog 2016 Apr 8;12(4):e1005555. Epub 2016 Apr 8.

Division of Pulmonary and Critical Care Medicine, University of California, San Francisco, San Francisco, California.

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.
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http://dx.doi.org/10.1371/journal.ppat.1005555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4825926PMC
April 2016

Neutrophils: Their Role in Innate and Adaptive Immunity.

J Immunol Res 2016 24;2016:1469780. Epub 2016 Feb 24.

Facultad de Odontología, Universidad Nacional Autónoma de México, 04510 Ciudad de México, DF, Mexico.

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http://dx.doi.org/10.1155/2016/1469780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783580PMC
November 2016