Publications by authors named "Mary C Nakamura"

38 Publications

Glucocorticoids and B Cell Depleting Agents Substantially Impair Immunogenicity of mRNA Vaccines to SARS-CoV-2.

medRxiv 2021 Apr 9. Epub 2021 Apr 9.

Background: Individuals with chronic inflammatory diseases (CID) are frequently treated with immunosuppressive medications that can increase their risk of severe COVID-19. While novel mRNA-based SARS-CoV-2 vaccination platforms provide robust protection in immunocompetent individuals, the immunogenicity in CID patients on immunosuppression is not well established. Therefore, determining the effectiveness of SARS-CoV-2 vaccines in the setting of immunosuppression is essential to risk-stratify CID patients with impaired protection and provide clinical guidance regarding medication management.

Methods: We conducted a prospective assessment of mRNA-based vaccine immunogenicity in 133 adults with CIDs and 53 immunocompetent controls. Blood from participants over 18 years of age was collected before initial immunization and 1-2 weeks after the second immunization. Serum anti-SARS-CoV-2 spike (S) IgG binding, neutralizing antibody titers, and circulating S-specific plasmablasts were quantified to assess the magnitude and quality of the humoral response following vaccination.

Results: Compared to immunocompetent controls, a three-fold reduction in anti-S IgG titers (P=0.009) and SARS-CoV-2 neutralization (p<0.0001) were observed in CID patients. B cell depletion and glucocorticoids exerted the strongest effect with a 36- and 10-fold reduction in humoral responses, respectively (p<0.0001). Janus kinase inhibitors and antimetabolites, including methotrexate, also blunted antibody titers in multivariate regression analysis (P<0.0001, P=0.0023, respectively). Other targeted therapies, such as TNF inhibitors, IL-12/23 inhibitors, and integrin inhibitors, had only modest impacts on antibody formation and neutralization.

Conclusions: CID patients treated with immunosuppressive therapies exhibit impaired SARS-CoV-2 vaccine-induced immunity, with glucocorticoids and B cell depletion therapy more severely impeding optimal responses.
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http://dx.doi.org/10.1101/2021.04.05.21254656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043473PMC
April 2021

Targeting Nerve Growth Factor for Pain Management in Osteoarthritis-Clinical Efficacy and Safety.

Rheum Dis Clin North Am 2021 May 12;47(2):181-195. Epub 2021 Mar 12.

Division of Rheumatology, Department of Internal Medicine, UC Davis Health, 4625 Second Avenue, Sacramento, CA 95917, USA.

Nerve growth factor (NGF) is a neurotrophin that mediates pain sensitization in pathologic states, including osteoarthritis. In clinical trials, antibodies to NGF reduce pain and improve physical function due to osteoarthritis of the knee or hip and have a long duration of action. Rapidly progressive osteoarthritis is a dose-dependent adverse event with these agents, and additional joint safety signals, such as subchondral insufficiency fractures and increased rates of total joint replacement, are reported. The effects on pain and potential mechanisms behind these joint events both are of considerable importance in the consideration of future use of anti-NGF therapies for osteoarthritis.
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http://dx.doi.org/10.1016/j.rdc.2020.12.003DOI Listing
May 2021

Proinflammatory cytokines and ARDS pulmonary edema fluid induce CD40 on human mesenchymal stromal cells-A potential mechanism for immune modulation.

PLoS One 2020 6;15(10):e0240319. Epub 2020 Oct 6.

Departments of Medicine and Anesthesiology, Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America.

Human mesenchymal stem/stromal cells (hMSCs) are a promising therapy for acute respiratory distress syndrome (ARDS) and other inflammatory conditions. While considerable research has focused on paracrine effects and mitochondrial transfer that improve lung fluid balance, hMSCs are well known to have immunomodulatory properties as well. Some of these immunomodulatory properties have been related to previously reported paracrine effectors such as indoleamine-2,3-dioxygenase (IDO), but these effects cannot fully account for cell-contact dependent immunomodulation. Here, we report that CD40 is upregulated on hMSCs under the same conditions previously reported to induce IDO. Further, CD40 transcription is also upregulated on hMSCs by ARDS pulmonary edema fluid but not by hydrostatic pulmonary edema fluid. Transcription of CD40, as well as paracrine effectors TSG6 and PTGS2 remained significantly upregulated for at least 12 hours after withdrawal of cytokine stimulation. Finally, induction of this immune phenotype altered the transdifferentiation of hMSCs, one of their hallmark properties. CD40 may play an important role in the immunomodulatory effects of hMSCs in ARDS and inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240319PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537876PMC
December 2020

Lower PDL1, PDL2, and AXL Expression on Lung Myeloid Cells Suggests Inflammatory Bias in Smoking and Chronic Obstructive Pulmonary Disease.

Am J Respir Cell Mol Biol 2020 12;63(6):780-793

Medical Service, San Francisco Veterans Affairs Healthcare System, San Francisco, California.

Lung myeloid cells are important in pulmonary immune homeostasis and in the pathogenesis of chronic obstructive pulmonary disease (COPD). Multiparameter immunophenotypic characterization of these cells is challenging because of their autofluorescence and diversity. We evaluated the immunophenotypic landscape of airway myeloid cells in COPD using time of flight mass cytometry. Cells from BAL, which were obtained from never-smokers ( = 8) and smokers with ( = 20) and without ( = 4) spirometric COPD, were examined using a 44-parameter time of flight mass cytometry panel. Unsupervised cluster analysis was used to identify cellular subtypes that were confirmed by manual gating. We identified major populations of CD68 and CD68 cells with 22 distinct phenotypic clusters, of which 18 were myeloid cells. We found a higher abundance of putative recruited myeloid cells (CD68 classical monocytes) in BAL from patients with COPD. CD68 classical monocyte population had distinct responses to smoking and COPD that were potentially related to their recruitment from the interstitium and vasculature. We demonstrate that BAL cells from smokers and subjects with COPD have lower AXL expression. Also, among subjects with COPD, we report significant differences in the abundance of PDL1 and PDL2 clusters and in the expression of PDL1 and PDL2 across several macrophage subtypes suggesting modulation of inflammatory responses. In addition, several phenotypic differences in BAL cells from subjects with history of COPD exacerbation were identified that could inform potential disease mechanisms. Overall, we report several changes to the immunophenotypic landscape that occur with smoking, COPD, and past exacerbations that are consistent with decreased regulation and increased activation of inflammatory pathways.
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http://dx.doi.org/10.1165/rcmb.2020-0085OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790144PMC
December 2020

Non-catalytic ubiquitin binding by A20 prevents psoriatic arthritis-like disease and inflammation.

Nat Immunol 2020 04 16;21(4):422-433. Epub 2020 Mar 16.

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

A20 is an anti-inflammatory protein that is strongly linked to human disease. Here, we find that mice expressing three distinct targeted mutations of A20's zinc finger 7 (ZF7) ubiquitin-binding motif uniformly developed digit arthritis with features common to psoriatic arthritis, while mice expressing point mutations in A20's OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20 cells exhibited prolonged IκB kinase activity that drove exaggerated transcription of late-phase nuclear factor-κB response genes in vitro and in prediseased mouse paws in vivo. In addition, mice expressing double-mutant A20 proteins in A20's ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20's ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.
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http://dx.doi.org/10.1038/s41590-020-0634-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195210PMC
April 2020

Age-related changes to macrophages are detrimental to fracture healing in mice.

Aging Cell 2020 03 25;19(3):e13112. Epub 2020 Feb 25.

Department of Orthopaedic Surgery, School of Medicine, Orthopaedic Trauma Institute, Zuckerberg San Francisco General Hospital, University of California San Francisco, San Francisco, CA, USA.

The elderly population suffers from higher rates of complications during fracture healing that result in increased morbidity and mortality. Inflammatory dysregulation is associated with increased age and is a contributing factor to the myriad of age-related diseases. Therefore, we investigated age-related changes to an important cellular regulator of inflammation, the macrophage, and the impact on fracture healing outcomes. We demonstrated that old mice (24 months) have delayed fracture healing with significantly less bone and more cartilage compared to young mice (3 months). The quantity of infiltrating macrophages into the fracture callus was similar in old and young mice. However, RNA-seq analysis demonstrated distinct differences in the transcriptomes of macrophages derived from the fracture callus of old and young mice, with an up-regulation of M1/pro-inflammatory genes in macrophages from old mice as well as dysregulation of other immune-related genes. Preventing infiltration of the fracture site by macrophages in old mice improved healing outcomes, with significantly more bone in the calluses of treated mice compared to age-matched controls. After preventing infiltration by macrophages, the macrophages remaining within the fracture callus were collected and examined via RNA-seq analysis, and their transcriptome resembled macrophages from young calluses. Taken together, infiltrating macrophages from old mice demonstrate detrimental age-related changes, and depleting infiltrating macrophages can improve fracture healing in old mice.
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http://dx.doi.org/10.1111/acel.13112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059136PMC
March 2020

Osteoimmunology: entwined regulation of integrated systems.

Authors:
Mary C Nakamura

Semin Immunopathol 2019 09;41(5):547-549

Department of Medicine, Division of Rheumatology, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA, 94143, USA.

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http://dx.doi.org/10.1007/s00281-019-00763-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309165PMC
September 2019

Female-Specific Role of Progranulin to Suppress Bone Formation.

Endocrinology 2019 09;160(9):2024-2037

Endocrine Unit, San Francisco VA Medical Center, San Francisco, California.

Progranulin (PGRN) is best known as a glial protein for which deficiency leads to the most common inherited form of frontotemporal dementia. Recently, PGRN has been found to be an adipokine associated with diet-induced obesity and insulin resistance. Therefore, PGRN may have homeostatic effects on bone because PGRN is reported to promote the differentiation of bone-resorbing osteoclasts. We investigated the actions of PGRN on bone using PGRN gene (Grn) knockout (KO) mice and transgenic mice with PGRN mutation and surprisingly found that loss of PGRN prevented the bone loss in female mice induced by aging and estrogen deficiency, whereas it had no effect on male bones during aging. Strikingly, bone formation was increased in female (but not male) PGRN KO mice. We also found that loss of PGRN inhibited bone resorption and osteoclastogenesis in both male and female mice and promoted the production of osteogenic factors in osteoclast lineage cells. These results indicate that PGRN serves to uncouple bone turnover in female mice by promoting bone resorption and suppressing bone formation. Furthermore, we demonstrated that microglial cells/macrophages, but not adipocytes, are an important source of PGRN in producing negative skeletal effects in females. Targeting PGRN production by microglial cells/macrophage-lineage cells may provide a therapeutic approach for the treatment of osteoporosis in females.
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http://dx.doi.org/10.1210/en.2018-00842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691684PMC
September 2019

NF-κB/MAPK activation underlies ACVR1-mediated inflammation in human heterotopic ossification.

JCI Insight 2018 11 15;3(22). Epub 2018 Nov 15.

Division of Endocrinology and Metabolism, Department of Medicine, and the Institute for Human Genetics, UCSF, San Francisco, California, USA.

Background: Inflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear.

Methods: We examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-β activated kinase 1 (TAK1), and NF-κB pathways.

Results: FOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-β production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages.

Conclusions: Abnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.
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http://dx.doi.org/10.1172/jci.insight.122958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302947PMC
November 2018

Brain trauma elicits non-canonical macrophage activation states.

J Neuroinflammation 2016 05 24;13(1):117. Epub 2016 May 24.

Department of Medicine, Division of Rheumatology, University of California, San Francisco, 4150 Clement St. 111R, San Francisco, CA, 94121, USA.

Background: Macrophage polarization programs, commonly referred to as "classical" and "alternative" activation, are widely considered as distinct states that are exclusive of one another and are associated with different functions such as inflammation and wound healing, respectively. In a number of disease contexts, such as traumatic brain injury (TBI), macrophage polarization influences the extent of pathogenesis, and efforts are underway to eliminate pathogenic subsets. However, previous studies have not distinguished whether the simultaneous presence of both classical and alternative activation signatures represents the admixture of differentially polarized macrophages or if they have adopted a unique state characterized by components of both classical and alternative activation.

Methods: We analyzed the gene expression profiles of individual monocyte-derived brain macrophages responding to TBI using single-cell RNA sequencing. RNA flow cytometry was used as another single-cell analysis technique to validate the single-cell RNA sequencing results.

Results: The analysis of signature polarization genes by single-cell RNA sequencing revealed the presence of diverse activation states, including M(IL4), M(IL10), and M(LPS, IFNγ). However, the expression of a given polarization marker was no more likely than at random to predict simultaneous expression or repression of markers of another polarization program within the same cell, suggesting a lack of exclusivity in macrophage polarization states in vivo in TBI. Also unexpectedly, individual TBI macrophages simultaneously expressed high levels of signature polarization genes across two or three different polarization states and in several distinct and seemingly incompatible combinations.

Conclusions: Single-cell gene expression profiling demonstrated that monocytic macrophages in TBI are not comprised of distinctly polarized subsets but are uniquely and broadly activated. TBI macrophage activation in vivo is deeply complex, with individual cells concurrently adopting both inflammatory and reparative features with a lack of exclusivity. These data provide physiologically relevant evidence that the early macrophage response to TBI is comprised of novel activation states that are discordant with the current paradigm of macrophage polarization-a key consideration for therapeutic modulation.
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http://dx.doi.org/10.1186/s12974-016-0581-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4879757PMC
May 2016

A Comprehensive Review of Immunoreceptor Regulation of Osteoclasts.

Clin Rev Allergy Immunol 2016 Aug;51(1):48-58

Division of Rheumatology, Department of Medicine, University of California, San Francisco, CA, USA.

Osteoclasts require coordinated co-stimulation by several signaling pathways to initiate and regulate their cellular differentiation. Receptor activator for NF-κB ligand (RANKL or TNFSF11), a tumor necrosis factor (TNF) superfamily member, is the master cytokine required for osteoclastogenesis with essential co-stimulatory signals mediated by immunoreceptor tyrosine-based activation motif (ITAM)-signaling adaptors, DNAX-associated protein 12 kDa size (DAP12) and FcεRI gamma chain (FcRγ). The ITAM-signaling adaptors do not have an extracellular ligand-binding domain and, therefore, must pair with ligand-binding immunoreceptors to interact with their extracellular environment. DAP12 pairs with a number of different immunoreceptors including triggering receptor expressed on myeloid cells 2 (TREM2), myeloid DAP12-associated lectin (MDL-1), and sialic acid-binding immunoglobulin-type lectin 15 (Siglec-15); while FcRγ pairs with a different set of receptors including osteoclast-specific activating receptor (OSCAR), paired immunoglobulin receptor A (PIR-A), and Fc receptors. The ligands for many of these receptors in the bone microenvironment remain unknown. Here, we will review immunoreceptors known to pair with either DAP12 or FcRγ that have been shown to regulate osteoclastogenesis. Co-stimulation and the effects of ITAM-signaling have turned out to be complex, and now include paradoxical findings that ITAM-signaling adaptor-associated receptors can inhibit osteoclastogenesis and immunoreceptor tyrosine-based inhibitory motif (ITIM) receptors can promote osteoclastogenesis. Thus, co-stimulation of osteoclastogenesis continues to reveal additional complexities that are important in the regulatory mechanisms that seek to maintain bone homeostasis.
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http://dx.doi.org/10.1007/s12016-015-8521-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867136PMC
August 2016

Triggering receptor expressed on myeloid cells 2 (TREM2) deficiency attenuates phagocytic activities of microglia and exacerbates ischemic damage in experimental stroke.

J Neurosci 2015 Feb;35(8):3384-96

Departments of Neurology and Medicine, University of California, San Francisco, and San Francisco Veterans Affairs Medical Center, San Francisco, California 94121, and

Clearing cellular debris after brain injury represents an important mechanism in regaining tissue homeostasis and promoting functional recovery. Triggering receptor expressed on myeloid cells-2 (TREM2) is a newly identified receptor expressed on microglia and is thought to phagocytose damaged brain cells. The precise role of TREM2 during ischemic stroke has not been fully understood. We explore TREM2 in both in vitro and in vivo stroke models and identify a potential endogenous TREM2 ligand. TREM2 knockdown in microglia reduced microglial activation to an amoeboid phenotype and decreased the phagocytosis of injured neurons. Phagocytosis and infarcted brain tissue resorption was reduced in TREM2 knock-out (KO) mice compared with wild-type (WT) mice. TREM2 KO mice also had worsened neurological recovery and decreased viable brain tissue in the ipsilateral hemisphere. The numbers of activated microglia and phagocytes in TREM2 KO mice were decreased compared with WT mice, and foamy macrophages were nearly absent in the TREM2 KO mice. Postischemia, TREM2 was highly expressed on microglia and TREM2-Fc fusion protein (used as a probe to identify potential TREM2 binding partners) bound to an unknown TREM2 ligand that colocalized to neurons. Oxygen glucose deprivation-exposed neuronal media, or cellular fractions containing nuclei or purified DNA, but not cytosolic fractions, stimulated signaling through TREM2. TREM2-Fc fusion protein pulled down nucleic acids from ischemic brain lysate. These findings establish the relevance of TREM2 in the phagocytosis of the infarcted brain and emphasize its role in influencing neurological outcomes following stroke. Further, nucleic acids may be one potential ligand of TREM2 in brain ischemia.
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http://dx.doi.org/10.1523/JNEUROSCI.2620-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4339351PMC
February 2015

CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury.

J Neurotrauma 2014 Oct 21;31(20):1677-88. Epub 2014 Jul 21.

1 Immunology Section, San Francisco VA Medical Center , San Francisco, California.

Traumatic brain injury (TBI) provokes inflammatory responses, including a dramatic rise in brain macrophages in the area of injury. The pathway(s) responsible for macrophage infiltration of the traumatically injured brain and the effects of macrophages on functional outcomes are not well understood. C-C-chemokine receptor 2 (CCR2) is known for directing monocytes to inflamed tissues. To assess the role of macrophages and CCR2 in TBI, we determined outcomes in CCR2-deficient (Ccr2(-/-)) mice in a controlled cortical impact model. We quantified brain myeloid cell numbers post-TBI by flow cytometry and found that Ccr2(-/-) mice had greatly reduced macrophage numbers (∼80-90% reduction) early post-TBI, compared with wild-type mice. Motor, locomotor, and cognitive outcomes were assessed. Lack of Ccr2 improved locomotor activity with less hyperactivity in open field testing, but did not affect anxiety levels or motor coordination on the rotarod three weeks after TBI. Importantly, Ccr2(-/-) mice demonstrated greater spatial learning and memory, compared with wild-type mice eight weeks after TBI. Although there was no difference in the volume of tissue loss, Ccr2(-/-) mice had significantly increased neuronal density in the CA1-CA3 regions of the hippocampus after TBI, compared with wild-type mice. These data demonstrate that Ccr2 directs the majority of macrophage homing to the brain early after TBI and indicates that Ccr2 may facilitate harmful responses. Lack of Ccr2 improves functional recovery and neuronal survival. These results suggest that therapeutic blockade of CCR2-dependent responses may improve outcomes following TBI.
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http://dx.doi.org/10.1089/neu.2013.3252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545982PMC
October 2014

Bone and the innate immune system.

Curr Osteoporos Rep 2014 Mar;12(1):1-8

Department of Medicine, Division of Rheumatology, Allergy and Immunology, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA, 02115, USA,

The immune system and bone are intimately linked with significant physical and functionally related interactions. The innate immune system functions as an immediate response system to initiate protections against local challenges such as pathogens and cellular damage. Bone is a very specific microenvironment, in which infectious attack is less common but repair and regeneration are ongoing and important functions. Thus, in the bone the primary goal of innate immune and bone interactions is to maintain tissue integrity. Innate immune signals are critical for removal of damaged and apoptotic cells and to stimulate normal tissue repair and regeneration. In this review we focus on the innate immune mechanisms that function to regulate bone homeostasis.
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http://dx.doi.org/10.1007/s11914-014-0195-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992341PMC
March 2014

Triggering Receptor Expressed on Myeloid Cells-2 Correlates to Hypothermic Neuroprotection in Ischemic Stroke.

Ther Hypothermia Temp Manag 2013 Dec;3(4):189-198

Department of Neurology, University of California , San Francisco, San Francisco Veterans Affairs Medical Center, San Francisco, California.

Hypothermia is neuroprotective against many acute neurological insults, including ischemic stroke. We and others have previously shown that protection by hypothermia is partially associated with an anti-inflammatory effect. Phagocytes are thought to play an important role in the clearance of necrotic debris, paving the way for endogenous repair mechanisms to commence, but the effect of cooling and phagocytosis has not been extensively studied. Triggering receptor expressed on myeloid cells-2 (TREM2) is a newly identified surface receptor shown to be involved in phagocytosis. In this study, we examined the effect of therapeutic hypothermia on TREM2 expression. Mice underwent permanent middle cerebral artery occlusion (MCAO) and were treated with one of the two cooling paradigms: one where cooling (30°C) began at the onset of MCAO (early hypothermia [eHT]) and another where cooling began 1 hour later (delayed hypothermia [dHT]). In both groups, cooling was maintained for 2 hours. A third group was maintained at normothermia (NT) as a control (37°C). Mice from the NT and dHT groups had similar ischemic lesion sizes and neurological performance, but the eHT group showed marked protection as evidenced by a smaller lesion size and less neurological deficits up to 30 days after the insult. Microglia and macrophages increased after MCAO as early as 3 days, peaked at 7 days, and decreased by 14 days. Both hypothermia paradigms were associated with decreased numbers of microglia and macrophages at 3 and 7 days, with greater decreases in the early paradigm. However, the proportion of the TREM2-positive microglia/macrophages was actually increased among the eHT group at day 7. eHT showed a long-term neurological benefit, but neuroprotection did not correlate to immune suppression. However, hypothermic neuroprotection was associated with a relative increase in TREM2 expression, and suggests that TREM2 may serve a beneficial role in brain ischemia.
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http://dx.doi.org/10.1089/ther.2013.0020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868297PMC
December 2013

CIITA: a master regulator of adaptive immunity shows its innate side in the bone.

Authors:
Mary C Nakamura

J Bone Miner Res 2014 Feb;29(2):287-9

Department of Medicine, Division of Rheumatology, University of California, San Francisco, San Francisco, CA, USA; Arthritis/Immunology Section, Veterans Affairs Medical Center, San Francisco, CA, USA.

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http://dx.doi.org/10.1002/jbmr.2161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852740PMC
February 2014

Traumatic brain injury induces macrophage subsets in the brain.

Eur J Immunol 2013 Aug 5;43(8):2010-22. Epub 2013 Jun 5.

San Francisco VA Medical Center, San Francisco, CA, USA.

Traumatic brain injury (TBI) elicits innate inflammatory responses that can lead to secondary brain injury. To better understand the mechanisms involved in TBI-induced inflammation, we examined the nature of macrophages responding to TBI in mice. In this model, brain macrophages were increased >20-fold the day after injury and >77-fold 4 days after injury in the ipsilateral hemisphere compared with sham controls. TBI macrophage subsets were identified by using a reporter mouse strain (YARG) that expresses eYFP from an internal ribosome entry site (IRES) inserted at the 3' end of the gene for arginase-1 (Arg1), a hallmark of alternatively activated (M2) macrophages. One day after TBI, 21 ± 1.5% of ipsilateral brain macrophages expressed relatively high levels of Arg1 as detected by yellow fluorescent protein, and this subpopulation declined thereafter. Arg1(+) cells localized with macrophages near the TBI lesion. Gene expression analysis of sorted Arg1(+) and Arg1(-) brain macrophages revealed that both populations had profiles that included features of conventional M2 macrophages and classically activated (M1) macrophages. The Arg1(+) cells differed from Arg1(-) cells in multiple aspects, most notably in their chemokine repertoires. Thus, the macrophage response to TBI initially involves heterogeneous polarization toward at least two major subsets.
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http://dx.doi.org/10.1002/eji.201243084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210355PMC
August 2013

Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function.

J Clin Invest 2012 Dec 1;122(12):4592-605. Epub 2012 Nov 1.

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

Increased osteoclastic bone resorption leads to periarticular erosions and systemic osteoporosis in RA patients. Although a great deal is known about how osteoclasts differentiate from precursors and resorb bone, the identity of an osteoclast precursor (OCP) population in vivo and its regulatory role in RA remains elusive. Here, we report the identification of a CD11b(-/lo)Ly6C(hi) BM population with OCP activity in vitro and in vivo. These cells, which can be distinguished from previously characterized precursors in the myeloid lineage, display features of both M1 and M2 monocytes and expand in inflammatory arthritis models. Surprisingly, in one mouse model of RA (adoptive transfer of SKG arthritis), cotransfer of OCP with SKG CD4+ T cells diminished inflammatory arthritis. Similar to monocytic myeloid-derived suppressor cells (M-MDSCs), OCPs suppressed CD4+ and CD8+ T cell proliferation in vitro through the production of NO. This study identifies a BM myeloid precursor population with osteoclastic and T cell-suppressive activity that is expanded in inflammatory arthritis. Therapeutic strategies that prevent the development of OCPs into mature bone-resorbing cells could simultaneously prevent bone resorption and generate an antiinflammatory milieu in the RA joint.
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http://dx.doi.org/10.1172/JCI60920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3533532PMC
December 2012

PSTPIP2 deficiency in mice causes osteopenia and increased differentiation of multipotent myeloid precursors into osteoclasts.

Blood 2012 Oct 24;120(15):3126-35. Epub 2012 Aug 24.

Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Missense mutations that reduce or abrogate myeloid cell expression of the F-BAR domain protein, proline serine threonine phosphatase-interacting protein 2 (PSTPIP2), lead to autoinflammatory disease involving extramedullary hematopoiesis, skin and bone lesions. However, little is known about how PSTPIP2 regulates osteoclast development. Here we examined how PSTPIP2 deficiency causes osteopenia and bone lesions, using the mouse PSTPIP2 mutations, cmo, which fails to express PSTPIP2 and Lupo, in which PSTPIP2 is dysfunctional. In both models, serum levels of the pro-osteoclastogenic factor, MIP-1α, were elevated and CSF-1 receptor (CSF-1R)-dependent production of MIP-1α by macrophages was increased. Treatment of cmo mice with a dual specificity CSF-1R and c-Kit inhibitor, PLX3397, decreased circulating MIP-1α and ameliorated the extramedullary hematopoiesis, inflammation, and osteopenia, demonstrating that aberrant myelopoiesis drives disease. Purified osteoclast precursors from PSTPIP2-deficient mice exhibit increased osteoclastogenesis in vitro and were used to probe the structural requirements for PSTPIP2 suppression of osteoclast development. PSTPIP2 tyrosine phosphorylation and a functional F-BAR domain were essential for PSTPIP2 inhibition of TRAP expression and osteoclast precursor fusion, whereas interaction with PEST-type phosphatases was only required for suppression of TRAP expression. Thus, PSTPIP2 acts as a negative feedback regulator of CSF-1R signaling to suppress inflammation and osteoclastogenesis.
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http://dx.doi.org/10.1182/blood-2012-04-425595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471520PMC
October 2012

Expression of A20 by dendritic cells preserves immune homeostasis and prevents colitis and spondyloarthritis.

Nat Immunol 2011 Oct 23;12(12):1184-93. Epub 2011 Oct 23.

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

Dendritic cells (DCs), which are known to support immune activation during infection, may also regulate immune homeostasis in resting animals. Here we show that mice lacking the ubiquitin-editing molecule A20 specifically in DCs spontaneously showed DC activation and population expansion of activated T cells. Analysis of DC-specific epistasis in compound mice lacking both A20 and the signaling adaptor MyD88 specifically in DCs showed that A20 restricted both MyD88-independent signals, which drive activation of DCs and T cells, and MyD88-dependent signals, which drive population expansion of T cells. In addition, mice lacking A20 specifically in DCs spontaneously developed lymphocyte-dependent colitis, seronegative ankylosing arthritis and enthesitis, conditions stereotypical of human inflammatory bowel disease (IBD). Our findings indicate that DCs need A20 to preserve immune quiescence and suggest that A20-dependent DC functions may underlie IBD and IBD-associated arthritides.
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http://dx.doi.org/10.1038/ni.2135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419270PMC
October 2011

Effects of inflammation on bone: an update.

Curr Opin Rheumatol 2011 Jul;23(4):389-95

University of California, Department of Medicine, San Francisco VA Medical Center, San Francisco, CA, USA

Purpose Of Review: To present an updated summary of the relationship between inflammation and localized and generalized bone loss in the rheumatic diseases.

Recent Findings: In addition to the well established role of inflammatory cytokines in promoting enhanced osteoclast function and bone loss, recent work has discovered the cytokine milieu may also inhibit osteoblast function and bone repair. The WNT and bone morphogenetic protein pathways provide molecular links between inflammation and altered bone homeostasis in chronic inflammatory states. These pathways and others have been the targets of emerging therapies for the management of inflammatory bone loss.

Summary: Inflammation and bone loss are linked through a number of molecular pathways. Both of these processes need to be addressed when designing an effective treatment strategy for the rheumatic diseases.
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http://dx.doi.org/10.1097/BOR.0b013e3283474dbeDOI Listing
July 2011

Assessment of bone remodelling in childhood-onset systemic lupus erythematosus.

Rheumatology (Oxford) 2011 Mar 23;50(3):611-9. Epub 2010 Nov 23.

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

Objective: To identify predictors of bone remodelling in children and young adults with SLE.

Methods: Ninety subjects with SLE aged 8-22 years underwent yearly measurements of height, bone age, bone turnover markers, serum Type I IFNs, SLEDAI and BMD. Predictors of bone turnover were examined using serum osteocalcin as a marker of bone formation and both serum tartrate-resistant acid phosphatase (TRAP) and urine N-telopeptide (NTx) as markers of bone resorption.

Results: Subjects demonstrated short stature, high BMI and bone age delay. A spine BMD Z-score of less than -2.0 was seen in 16.1% of subject visits. Serum osteocalcin was negatively correlated with glucocorticoid dose (Spearman rank correlation coefficient R = -0.34, P < 0.0001) but was not associated with SLEDAI after adjustment for confounders. Serum TRAP was negatively associated with SLEDAI, even after controlling for confounders (P = 0.04). Similar results were obtained for urine NTx. There was a negative association between TRAP and serum IFN-β (P = 0.03).

Conclusions: In this population of children and young adults with moderate lupus disease activity, glucocorticoid dose was a negative predictor of bone formation, whereas lupus disease activity was not. Interestingly, lupus disease activity was a negative predictor of bone resorption, suggesting that lupus disease activity is not the primary factor contributing to the bone deficits of childhood-onset SLE. The potential protective role of IFN-β and the effects of SLE treatment on bone loss require further study.
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http://dx.doi.org/10.1093/rheumatology/keq307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3042255PMC
March 2011

The loss of Cbl-phosphatidylinositol 3-kinase interaction perturbs RANKL-mediated signaling, inhibiting bone resorption and promoting osteoclast survival.

J Biol Chem 2010 Nov 17;285(47):36745-58. Epub 2010 Sep 17.

Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

Cbl is an adaptor protein and an E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and is phosphorylated by Syk and Src family kinases. Phosphorylated Cbl Tyr(737) creates a binding site for the p85 regulatory subunit of PI3K, which also plays an important role in the regulation of bone resorption by osteoclasts. To investigate the role of Cbl-PI3K interaction in bone homeostasis, we examined the knock-in mice (Cbl(YF/YF)) in which the PI3K binding site in Cbl is ablated due to the mutation in the regulatory tyrosine. We report that in Cbl(YF/YF) mice, despite increased numbers of osteoclasts, bone volume is increased due to defective osteoclast function. Additionally, in ex vivo cultures, mature Cbl(YF/YF) osteoclasts showed an increased ability to survive in the presence of RANKL due to delayed onset of apoptosis. RANKL-mediated signaling is perturbed in Cbl(YF/YF) osteoclasts, and most interestingly, AKT phosphorylation is up-regulated, suggesting that the lack of PI3K sequestration by Cbl results in increased survival and decreased bone resorption. Cumulatively, these in vivo and in vitro results show that, on one hand, binding of Cbl to PI3K negatively regulates osteoclast differentiation, survival, and signaling events (e.g. AKT phosphorylation), whereas on the other hand it positively influences osteoclast function.
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http://dx.doi.org/10.1074/jbc.M110.124628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978603PMC
November 2010

Binding and uptake of H-ferritin are mediated by human transferrin receptor-1.

Proc Natl Acad Sci U S A 2010 Feb 4;107(8):3505-10. Epub 2010 Feb 4.

Department of Medicine, Veterans Administration Medical Center, San Francisco, CA 94121, USA.

Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.
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http://dx.doi.org/10.1073/pnas.0913192107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840523PMC
February 2010

A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia.

J Neurochem 2009 May 19;109(4):1144-56. Epub 2009 Mar 19.

University of California, San Francisco, and the San Francisco VA Medical Center, San Francisco, California, USA.

Following neuronal injury, microglia initiate repair by phagocytosing dead neurons without eliciting inflammation. Prior evidence indicates triggering receptor expressed by myeloid cells-2 (TREM2) promotes phagocytosis and retards inflammation. However, evidence that microglia and neurons directly interact through TREM2 to orchestrate microglial function is lacking. We here demonstrate that TREM2 interacts with endogenous ligands on neurons. Staining with TREM2-Fc identified TREM2 ligands (TREM2-L) on Neuro2A cells and on cultured cortical and dopamine neurons. Apoptosis greatly increased the expression of TREM2-L. Furthermore, apoptotic neurons stimulated TREM2 signaling, and an anti-TREM2 mAb blocked stimulation. To examine the interaction between TREM2 and TREM2-L in phagocytosis, we studied BV2 microglial cells and their engulfment of apoptotic Neuro2A. One of our anti-TREM2 mAb, but not others, reduced engulfment, suggesting the presence of a functional site on TREM2 interacting with neurons. Further, Chinese hamster ovary cells transfected with TREM2 conferred phagocytic activity of neuronal cells demonstrating that TREM2 is both required and sufficient for competent uptake of apoptotic neuronal cells. Finally, while TREM2-L are expressed on neurons, TREM2 is not; in the brain, it is found on microglia. TREM2 and TREM2-L form a receptor-ligand pair connecting microglia with apoptotic neurons, directing removal of damaged cells to allow repair.
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http://dx.doi.org/10.1111/j.1471-4159.2009.06042.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087597PMC
May 2009

Glucocorticoid excess in mice results in early activation of osteoclastogenesis and adipogenesis and prolonged suppression of osteogenesis: a longitudinal study of gene expression in bone tissue from glucocorticoid-treated mice.

Arthritis Rheum 2008 Jun;58(6):1674-86

University of California at Davis Medical Center, Sacramento, CA 95817, USA.

Objective: Glucocorticoid (GC) excess induces alterations in bone metabolism that weaken bone structure and increase fracture risk. The aim of this study was to identify genes associated with bone metabolism in GC-treated mice, by performing a microarray analysis.

Methods: Long bones from mice exposed to GC excess were collected after 0, 7, 28, and 56 days of treatment, to measure bone microarchitecture and extract RNA for microarray analyses.

Results: Bone loss in this animal model was confirmed by changes in bone turnover markers as well as bone architecture, as measured by microfocal computed tomography. GC excess induced an early up-regulation of genes involved in osteoclast activation, function, and adipogenesis, which peaked on day 7. The expression of genes associated with osteoclast cytoskeletal reorganization and genes associated with matrix degradation peaked on day 28. On day 28 and day 56, the expression of genes associated with osteoblast activation and maturation was decreased from baseline, while the expression of Wnt antagonists was increased. In addition, the expression of genes expressed in osteocytes associated with bone mineralization was significantly higher at the later time points, day 28 and day 56. Reverse transcription-polymerase chain reaction confirmed the results of microarray analysis in selected genes.

Conclusion: GC excess is associated with early activation of genes associated with osteoclastogenesis and adipogenesis and a later suppression of genes associated with osteogenesis and mineralization. Novel interventions with agents that modulate either Wnt signaling or mineralization may be effective in GC-induced osteoporosis.
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http://dx.doi.org/10.1002/art.23454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3892702PMC
June 2008

The innate immune response to Salmonella enterica serovar Typhimurium by macrophages is dependent on TREM2-DAP12.

Infect Immun 2008 Jun 7;76(6):2439-47. Epub 2008 Apr 7.

Department of Medicine, University of California-San Francisco, San Francisco, California 94143, USA.

Macrophage recognition of Salmonella enterica serovar Typhimurium leads to a cascade of signaling events, including the activation of Src family and Syk kinases and the production of reactive oxygen species (ROS), which are critical for host innate defense during early stages of bacterial infection. ROS production depends on the NADPH oxidase, but little is known about the innate immune receptors and proximal adapters that regulate Salmonella-induced ROS. Herein, we demonstrate that serovar Typhimurium induces ROS through a pathway that requires both triggering receptor expressed on myeloid cells 2 (TREM2) and DAP12. This pathway is highly analogous to the pathways utilized by Fc receptors and integrins to regulate ROS production. Oral infection of mice with serovar Typhimurium demonstrates that the DAP12-dependent pathway regulates cecal colonization during early stages of Salmonella infection. Thus, DAP12 is an important regulator of Salmonella-induced ROS production in macrophages, and TREM2 is essential for linking DAP12 to the innate response to serovar Typhimurium.
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http://dx.doi.org/10.1128/IAI.00115-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423085PMC
June 2008

Osteoclasts - the innate immune cells of the bone.

Autoimmunity 2008 Apr;41(3):183-94

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

Osteopenia and periarticular bony erosion are consequences of chronic inflammatory autoimmune disease due to an imbalance of osteoclast activity relative to new bone formation. Osteoclasts, which are specialized as the only bone resorbing cell type, are differentiated from hematopoietic myeloid precursor cells. Inflammatory signals mediated by multiple types of immune cells and cytokines have significant influence over osteoclast differentiation and function through direct effects on osteoclast precursors and indirect effects via osteoblasts and other cells in the bony microenvironment including synovial cells, stromal cells, osteocytes and chondrocytes. Recent studies have demonstrated that osteoclasts themselves express a number of immune receptors and are regulated similarly to macrophages and dendritic cells, closely related cells in the innate immune system. Though we are only beginning to understand the roles of innate immune receptors in osteoclasts, some of these receptors have been shown to be critical regulators of differentiation and function of osteoclasts. Osteoclasts likely function as the innate immune cells of the bone, thus are highly regulated to appropriately respond to stress and inflammatory changes in their microenvironment.
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http://dx.doi.org/10.1080/08916930701693180DOI Listing
April 2008

Bone microenvironment specific roles of ITAM adapter signaling during bone remodeling induced by acute estrogen-deficiency.

PLoS One 2007 Jul 4;2(7):e586. Epub 2007 Jul 4.

Department of Medicine, VA Medical Center, University of California San Francisco, San Francisco, California, United States of America; Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, United States of America.

Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcepsilon receptor Igamma chain (FcRgamma) have been shown to be critical for osteoclast differentiation and maturation under normal physiological conditions. Their function in pathological conditions is unknown. We studied the role of ITAM signaling during rapid bone remodeling induced by acute estrogen-deficiency in wild-type (WT), DAP12-deficient (DAP12-/-), FcRgamma-deficient (FcRgamma-/-) and double-deficient (DAP12-/-FcRgamma-/-) mice. Six weeks after ovariectomy (OVX), DAP12-/-FcRgamma-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRgamma-/- mice had significant LVB bone loss. In contrast, all ITAM adapter-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes. Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFbeta and TNFalpha, were able to induce osteoclastogenesis independent of RANKL in BMMs from WT but not DAP12-/- and DAP12-/-FcRgamma-/- mice. FSH stimulated RANKL-induced osteoclast differentiation from BMMs in WT, but not DAP12-/- and DAP12-/-FcRgamma-/- mice. Our study demonstrates that although ITAM adapter signaling is critical for normal bone remodeling, estrogen-deficiency induces an ITAM adapter-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1895921PMC
July 2007