I am a board-certified gastroenterologist and physician-scientist with a broad research background in neuroimmunology. I am currently applying my knowledge of neuroimmunology and inflammation to advancing our understanding of gastroparesis and other functional motility disorders. Through translational clinical trials and laboratory research, my long-term career goal is to understand the neuroimmune basis of functional motility gut disorders and provide improved diagnostics and treatments to patients.
Instructor, Medicine - Gastroenterology & Hepatology
Boards, Advisory Committees, Professional Organizations
Member, American Neurogastroenterology & Motility Society (2016 - Present)
Member, American Gastroenterology Association (AGA) (2015 - Present)
PhD, The Rockefeller University, NY, NY, Neuroimmunology and Neuroendocrinology (2007)
GI-Challenge Study for Gastroparesis Patients and Healthy Controls
Gastroparesis Patients and Healthy Controls ages 20-49 will be asked to participate in an observational study measuring vagal activity following food ingestion in order to establish parameters of autonomic nerve/vagal function in healthy human subjects compared to those with gastroparesis. Information generated from this study may be used in the future to establish what is normal and abnormal enteric vagal tone and how much vagal nerve stimulation treatment may be required to help patients with gastroparesis.
Vagal Nerve Stimulation for Gastroparesis
This study is investigating a new form of treatment for a digestive disorder called gastroparesis. Gastroparesis is thought to be caused by a mix of inflammation and neural dysfunction. The vagal nerve is a large nerve originating from the brain that regulates digestive function. Patients with gastroparesis have what is a called a low vagal tone which results in gastrointestinal motility problems and inflammation; therefore, investigators hypothesize that increasing vagal tone through a hand-held vagal nerve simulator will reduce inflammation and gastrointestinal motility problems in gastroparesis patients. Investigators will evaluate this hypothesis through the use of upper endoscopy testing, breath testing, and blood, stool, urine, heart rate variability, and saliva testing before and after 4 weeks of vagal nerve stimulation (VNS) treatment. There are 6 research visits Visit 1 and visit 2 may take up to 8 weeks (screening/baseline) Visit 3 and visit 4 will take 4 weeks (VNS treatment) visit 5 and 6 will take approximately 4 weeks (VNS followup/washout) Consequently, it is possible that if a patient were to be at the farthest ends of visit windows, they could potentially be in the study for approx 16 weeks. Visit 1 and 2 may be less than 8 weeks which would shorten the patient's overall involvement in the study. The treatment phase of the study will always be 4 weeks with an additional 4 week washout phase. Use of the VNS device takes 4 weeks. Endoscopy and blood work are taken before and after the treatment period.
Stanford is currently not accepting patients for this trial. For more information, please contact Brandon Lam, BS, 650-725-0226.
Transitional dendritic cells are distinct from conventional DC2 precursors and mediate proinflammatory antiviral responses.
High-dimensional approaches have revealed heterogeneity amongst dendritic cells (DCs), including a population of transitional DCs (tDCs) in mice and humans. However, the origin and relationship of tDCs to other DC subsets has been unclear. Here we show that tDCs are distinct from other well-characterized DCs and conventional DC precursors (pre-cDCs). We demonstrate that tDCs originate from bone marrow progenitors shared with plasmacytoid DCs (pDCs). In the periphery, tDCs contribute to the pool of ESAM+ type 2 DCs (DC2s), and these DC2s have pDC-related developmental features. Different from pre-cDCs, tDCs have less turnover, capture antigen, respond to stimuli and activate antigen-specific naive T cells, all characteristics of differentiated DCs. Different from pDCs, viral sensing by tDCs results in IL-1beta secretion and fatal immune pathology in a murine coronavirus model. Our findings suggest that tDCs are a distinct pDC-related subset with a DC2 differentiation potential and unique proinflammatory function during viral infections.
View details for DOI 10.1038/s41590-023-01545-7
View details for PubMedID 37414907
Rapid recruitment and IFN-I-mediated activation of monocytes dictate focal radiotherapy efficacy.
2023; 8 (84): eadd7446
The recruitment of monocytes and their differentiation into immunosuppressive cells is associated with the low efficacy of preclinical nonconformal radiotherapy (RT) for tumors. However, nonconformal RT (non-CRT) does not mimic clinical practice, and little is known about the role of monocytes after RT modes used in patients, such as conformal RT (CRT). Here, we investigated the acute immune response induced by after CRT. Contrary to non-CRT approaches, we found that CRT induces a rapid and robust recruitment of monocytes to the tumor that minimally differentiate into tumor-associated macrophages or dendritic cells but instead up-regulate major histocompatibility complex II and costimulatory molecules. We found that these large numbers of infiltrating monocytes are responsible for activating effector polyfunctional CD8+ tumor-infiltrating lymphocytes that reduce tumor burden. Mechanistically, we show that monocyte-derived type I interferon is pivotal in promoting monocyte accumulation and immunostimulatory function in a positive feedback loop. We also demonstrate that monocyte accumulation in the tumor microenvironment is hindered when RT inadvertently affects healthy tissues, as occurs in non-CRT. Our results unravel the immunostimulatory function of monocytes during clinically relevant modes of RT and demonstrate that limiting the exposure of healthy tissues to radiation has a positive therapeutic effect on the overall antitumor immune response.
View details for DOI 10.1126/sciimmunol.add7446
View details for PubMedID 37294749
Metalloendopeptidase ADAM-like Decysin 1 (ADAMDEC1) in Colonic Subepithelial PDGFRalpha+ Cells Is a New Marker for Inflammatory Bowel Disease.
International journal of molecular sciences
2022; 23 (9)
Metalloendopeptidase ADAM-Like Decysin 1 (ADAMDEC1) is an anti-inflammatory peptidase that is almost exclusively expressed in the gastrointestinal (GI) tract. We have recently found abundant and selective expression of Adamdec1 in colonic mucosal PDGFRalpha+ cells. However, the cellular origin for this gene expression is controversial as it is also known to be expressed in intestinal macrophages. We found that Adamdec1 mRNAs were selectively expressed in colonic mucosal subepithelial PDGFRalpha+ cells. ADAMDEC1 protein was mainly released from PDGFRalpha+ cells and accumulated in the mucosal layer lamina propria space near the epithelial basement membrane. PDGFRalpha+ cells significantly overexpressed Adamdec1 mRNAs and protein in DSS-induced colitis mice. Adamdec1 was predominantly expressed in CD45- PDGFRalpha+ cells in DSS-induced colitis mice, with only minimal expression in CD45+ CD64+ macrophages. Additionally, overexpression of both ADAMDEC1 mRNA and protein was consistently observed in PDGFRalpha+ cells, but not in CD64+ macrophages found in human colonic mucosal tissue affected by Crohn's disease. In summary, PDGFRalpha+ cells selectively express ADAMDEC1, which is localized to the colon mucosa layer. ADAMDEC1 expression significantly increases in DSS-induced colitis affected mice and Crohn's disease affected human tissue, suggesting that this gene can serve as a diagnostic and/or therapeutic target for intestinal inflammation and Crohn's disease.
View details for DOI 10.3390/ijms23095007
View details for PubMedID 35563399
MiR-10b-5p Rescues Diabetes and Gastrointestinal Dysmotility.
BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) and pancreatic beta cells require receptor tyrosine kinase (KIT) to develop and function properly. Degeneration of ICCs is linked to diabetic gastroparesis. The mechanisms linking diabetes and gastroparesis are unclear, but may involve miRNA mediated post-transcriptional gene silencing in KIT+ cells.METHODS: We performed miRNA-seq analysis from isolated ICCs in diabetic mice and plasma from patients with idiopathic and diabetic gastroparesis. miR-10b-5p target genes were identified and validated in mouse and human cell lines. For loss-of-function studies, we used KIT+ cell-restricted mir-10b knockout mice and KIT+ cell depletion mice. For gain-of-function studies, a synthetic miR-10b-5p mimic was injected in multiple diabetic mouse models. We compared the efficacy of miR-10b-5p mimic treatment vs. antidiabetic and prokinetic medicines.RESULTS: miR-10b-5p is highly expressed in ICCs from healthy mice, but drastically depleted in ICCs from diabetic mice. A conditional knockout of mir-10b in KIT+-cells or depletion of KIT+-cells in mice leads to degeneration of beta cells and ICCs, resulting in diabetes and gastroparesis. miR-10b-5p targets the transcription factor Kruppel-like factor 11 (KLF11), which negatively regulates KIT expression. The miR-10b-5p mimic or Klf11 siRNAs injected into mir-10b knockout mice, diet-induced diabetic mice, and TALLYHO polygenic diabetic mice rescues the diabetes and gastroparesis phenotype for an extended period of time. Furthermore, the miR-10b-5p mimic is more effective in improving glucose homoeostasis and GI motility as compared with common antidiabetic and prokinetic medications.CONCLUSIONS: miR-10b-5p is a key regulator in diabetes and gastrointestinal dysmotility via the KLF11-KIT pathway. Restoration of miR-10b-5p may provide therapeutic benefits for these disorders.
View details for DOI 10.1053/j.gastro.2020.12.062
View details for PubMedID 33421511
- Serotonin is elevated in COVID-19-associated diarrhoea. Gut 2021
Serotonin Deficiency is Associated with Delayed Gastric Emptying.
Gastrointestinal (GI) motility is regulated by serotonin (5-hydroxytryptamine, 5-HT), which is primarily produced by enterochromaffin (EC) cells in the GI tract. However, the precise roles of EC cell-derived 5-HT in regulating gastric motility remain a major point of conjecture. Using a novel transgenic mouse line, we investigated the distribution of EC cells and the pathophysiological roles of 5-HT deficiency in gastric motility in mice and humans.We developed an inducible, EC cell-specific Tph1CreERT2/+ mouse, which was used to generate a reporter mouse line, Tph1-tdTom, and an EC cell-depleted line, Tph1-DTA. We examined EC cell distribution, morphology, and subpopulations in reporter mice. GI motility was measured in vivo and ex vivo in EC cell-depleted mice. Additionally, we evaluated 5-HT content in biopsy and plasma specimens from patients with idiopathic gastroparesis (IG).Tph1-tdTom mice revealed EC cells were heterogeneously distributed throughout the GI tract with the greatest abundance in the antrum and proximal colon. Two subpopulations of EC cells were identified in the gut: self-renewal cells located at the base of the crypt and mature cells observed in the villi. Tph1-DTA mice displayed delayed gastric emptying, total GI transit, and colonic transit. These gut motility alterations were reversed by exogenous provision of 5-HT. Patients with IG had a significant reduction of antral EC cell numbers and 5-HT content, which negatively correlated with gastric emptying rate.The Tph1CreERT2/+ mouse provides a powerful tool to study the functional roles of EC cells in the GI tract. Our findings suggest a new pathophysiological mechanism of 5-HT deficiency in IG.
View details for DOI 10.1053/j.gastro.2021.02.060
View details for PubMedID 33662386
Gastrointestinal symptoms and healthcare utilization have increased among patients with functional gastrointestinal and motility disorders during the COVID-19 pandemic.
Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society
The coronavirus disease 2019 (COVID-19) pandemic has led to unprecedented disruptions in healthcare. Functional gastrointestinal and motility disorders (FGIMD) are associated with significant healthcare utilization. The clinical implications of these healthcare disruptions due to the COVID-19 pandemic on clinical outcomes in patients with FGIMD are unclear.We performed a retrospective study of patients with three common FGIMD (irritable bowel syndrome [IBS], gastroparesis, functional dyspepsia [FD]) tested for SARS-CoV-2 to describe alterations in gastrointestinal symptoms, medication use, and healthcare utilization during and before the pandemic and factors associated with COVID-19.The prevalence of COVID-19 during the pandemic (03/2020-09/2020) was 3.20% (83/2592) among patients with FGIMD, 3.62% in IBS (57/1574), 3.07% in gastroparesis (23/749), and 2.44% in FD (29/1187) at our institution. Patients with FGIMD had increased abdominal pain, nausea/vomiting, diarrhea, constipation, and weight loss (p < 0.001) along with increased proton pump inhibitor, H2 blocker, and opioid use (p < 0.0001). Both inpatient hospitalizations and outpatient visits (p < 0.0001) and number of diagnostic tests including cross-sectional imaging (p = 0.002), and upper and lower endoscopies (p < 0.0001) were significantly higher during the pandemic as compared to 6 months prior. Diarrhea-predominant IBS was positively (OR 2.37, 95% CI 1.34-4.19, p = 0.003) associated with COVID-19, whereas functional dyspepsia was negatively (OR 0.46, 95% CI 0.27-0.79, p = 0.004) associated.Patients with common functional gastrointestinal and motility disorders have reported more gastrointestinal symptoms during the COVID-19 pandemic with concurrent increased medication use and healthcare utilization.
View details for DOI 10.1111/nmo.14243
View details for PubMedID 34378840
Gastric Mucosal Immune Profiling and Dysregulation in Idiopathic Gastroparesis.
Clinical and translational gastroenterology
2021; 12 (5): e00349
It is unclear how immune perturbations may influence the pathogenesis of idiopathic gastroparesis, a prevalent functional disorder of the stomach which lacks animal models. Several studies have noted altered immune characteristics in the deep gastric muscle layer associated with gastroparesis, but data are lacking for the mucosal layer, which is endoscopically accessible. We hypothesized that immune dysregulation is present in the gastroduodenal mucosa in idiopathic gastroparesis and that specific immune profiles are associated with gastroparesis clinical parameters.In this cross-sectional prospective case-control study, routine endoscopic biopsies were used for comprehensive immune profiling by flow cytometry, multicytokine array, and gene expression in 3 segments of the stomach and the duodenal bulb. Associations of immune endpoints with clinical parameters of gastroparesis were also explored.The gastric mucosa displayed large regional variation of distinct immune profiles. Furthermore, several-fold increases in innate and adaptive immune cells were found in gastroparesis. Various immune cell types showed positive correlations with duration of disease, proton pump inhibitor dosing, and delayed gastric emptying.This initial observational study showed immune compartmentalization of the human stomach mucosa and significant immune dysregulation at the level of leukocyte infiltration in idiopathic gastroparesis patients that extends to the duodenum. Select immune cells, such as macrophages, may correlate with clinicopathological traits of gastroparesis. This work supports further mucosal studies to advance our understanding of gastroparesis pathophysiology.
View details for DOI 10.14309/ctg.0000000000000349
View details for PubMedID 33979305
Effects of processing conditions on stability of immune analytes in human blood.
2020; 10 (1): 17328
Minimizing variability in collection and processing of human blood samples for research remains a challenge. Delaying plasma or serum isolation after phlebotomy (processing delay) can cause perturbations of numerous analytes. Thus, a comprehensive understanding of how processing delay affects major endpoints used in human immunology research is necessary. Therefore, we studied how processing delay affects commonly measured cytokines and immune cell populations. We hypothesized that short-term time delays inherent to human research in serum and plasma processing impact commonly studied immunological analytes. Blood from healthy donors was subjected to processing delays commonly encountered in sample collection, and then assayed by 62-plex Luminex panel, 40-parameter mass cytometry panel, and 540,000 transcript expression microarray. Variance for immunological analytes was estimated using each individual's baseline as a control. In general, short-term processing delay led to small changes in plasma and serum cytokines (range-10.8 to 43.5%), markers and frequencies of peripheral blood mononuclear cell phenotypes (range 0.19 to 3.54 fold), and whole blood gene expression (stable for>20K genes)-with several exceptions described herein. Importantly, we built an open-access web application allowing investigators to estimate the degree of variance expected from processing delay for measurements of interest based on the data reported here.
View details for DOI 10.1038/s41598-020-74274-8
View details for PubMedID 33060628
Clinical and immunomodulatory effects of transcutaneous vagal nerve stimulation for idiopathic gastroparesis
View details for Web of Science ID 000521974900325
Noninvasive vagal nerve stimulation for gastroenterology pain disorders.
Abdominal pain continues to be a major challenge and unmet need in clinical practice. Normalization of bidirectional gut-brain signaling has generated much interest as a therapeutic approach to treat chronic abdominal pain. Vagal nerve stimulation (VNS) is emerging as a potential non-pharmacologic strategy for the treatment of abdominal pain. In this review paper, we will summarize the etiologies of chronic pain in gastrointestinal disorders and discuss the rational for VNS as a therapeutic approach to chronic abdominal pain, with particular emphasis in the gammaCore stimulator which allows for noninvasive VNS.
View details for DOI 10.2217/pmt-2020-0067
View details for PubMedID 33111642
Open-label pilot study: Non-invasive vagal nerve stimulation improves symptoms and gastric emptying in patients with idiopathic gastroparesis.
Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society
BACKGROUND: Gastroparesis, a chronic motility disorder characterized by delayed gastric emptying, abdominal pain, nausea, and vomiting, remains largely unexplained. Medical therapy is limited, reflecting the complex physiology of gastric sensorimotor function. Vagus nerve stimulation is an attractive therapeutic modality for gastroparesis, but prior methods required invasive surgery. In this open-label pilot study, we aimed to assess the benefit of non-invasive vagal nerve stimulation in patients with mild to moderate idiopathic gastroparesis.METHODS: Patients self-administered the gammaCore vagal nerve stimulator for 4weeks. The gastroparesis cardinal symptom index daily diary (GCSI-dd) was assessed during a two-week run-in period, ≥4weeks of therapy, and 4weeks after therapy was completed. Gastric emptying and autonomic function testing were also performed. The primary endpoint was an absolute reduction in CGSI-dd of 0.75 after nVNS.RESULTS: There was a total improvement in symptom scores (2.56±0.76 to 1.87±1.05; P=.01), with 6/15 (40%) participants meeting our primary endpoint. Therapy was associated with a reduction in gastric emptying (T1/2 155 vs 129minutes; P=.053, CI -0.4 to 45). Therapy did not correct autonomic function abnormalities, but was associated with modulation of reflex parasympathetic activity.CONCLUSIONS: Short-term non-invasive vagal nerve stimulation led to improved cardinal symptoms and accelerated gastric emptying in a subset of patients with idiopathic gastroparesis. Responders had more severe gastric delay at baseline and clinical improvement correlated with duration of therapy, but not with improvements in gastric emptying. Larger randomized sham-controlled trials of greater duration are needed to confirm the results of this pilot study.
View details for DOI 10.1111/nmo.13769
View details for PubMedID 31802596
7 alpha-Hydroxylation of dehydroepiandrosterone does not interfere with the activation of glucocorticoids by 11 beta-hydroxysteroid dehydrogenase in (EC)-C-t cerebellar neurons
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
2013; 138: 290-297
The neuroprotective action of dehydroepiandrosterone (DHEA) in the absence of a known specific receptor has been attributed to its metabolism by different cell types in the brain to various steroids, with a preference to its 7-hydroxylated products. The E(t)C cerebellar granule cell line converts DHEA almost exclusively to 7α-hydroxy-DHEA (7α-OH-DHEA). It has been postulated that DHEA's 7-OH and 7-oxo metabolites can decrease glucocorticoid levels by an interactive mechanism involving 11β-hydroxysteroid dehydrogenase (11β-HSD). In order to study the relationship of 7-hydroxylation of DHEA and glucocorticoid metabolism in intact brain cells, we examined whether E(t)C cerebellar neurons, which are avid producers of 7α-OH-DHEA, could also metabolize glucocorticoids. We report that E(t)C neuronal cells exhibit 11β-HSD1 reductase activity, and are able to convert 11-dehydrocorticosterone into corticosterone, whereas they do not demonstrate 11β-HSD2 dehydrogenase activity. Consequently, E(t)C cells incubated with DHEA did not yield 7-oxo- or 7β-OH-DHEA. Our findings are supported by the reductive environment of E(t)C cells through expression of hexose-6-phosphate dehydrogenase (H6PDH), which fosters 11β-HSD1 reductase activity. To further explore the role of 7α-OH-DHEA in E(t)C neuronal cells, we examined the effect of preventing its formation using the CYP450 inhibitor ketoconazole. Treatment of the cells with this drug decreased the yield of 7α-OH-DHEA by about 75% without the formation of alternate DHEA metabolites, and had minimal effects on glucocorticoid conversion. Likewise, elevated levels of corticosterone, the product of 11β-HSD1, had no effect on the metabolic profile of DHEA. This study shows that in a single population of whole-cells, with a highly reductive environment, 7α-OH-DHEA is unable to block the reducing activity of 11β-HSD1, and that 7-hydroxylation of DHEA does not interfere with the activation of glucocorticoids. Our investigation on the metabolism of DHEA in E(t)C neuronal cells suggest that other alternate mechanisms must be at play to explain the in vivo anti-glucocorticoid properties of DHEA and its 7-OH-metabolites.
View details for DOI 10.1016/j.jsbmb.2013.07.001
View details for Web of Science ID 000327906300031
View details for PubMedID 23851218
- Recurrent skeletal muscle and abdominopelvic low-grade leiomyosarcoma INTERNATIONAL JOURNAL OF GYNECOLOGY & OBSTETRICS 2013; 120 (3): 289-290
Brain dendritic cells: biology and pathology
2012; 124 (5): 599-614
Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In their quiescent and mature form, the presentation of self-antigens by DC leads to tolerance; whereas, antigen presentation by mature DC, after stimulation by pathogen-associated molecular patterns, leads to the onset of antigen-specific immunity. DC have been found in many of the major organs in mammals (e.g. skin, heart, lungs, intestines and spleen); while the brain has long been considered devoid of DC in the absence of neuroinflammation. Consequently, microglia, the resident immune cell of the brain, have been charged with many functional attributes commonly ascribed to DC. Recent evidence has challenged the notion that DC are either absent or minimal players in brain immune surveillance. This review will discuss the recent literature examining DC involvement within both the young and aged steady-state brain. We will also examine DC contributions during various forms of neuroinflammation resulting from neurodegenerative autoimmune disease, injury, and CNS infections. This review also touches upon DC trafficking between the central nervous system and peripheral immune compartments during viral infections, the new molecular technologies that could be employed to enhance our current understanding of brain DC ontogeny, and some potential therapeutic uses of DC within the CNS.
View details for DOI 10.1007/s00401-012-1018-0
View details for Web of Science ID 000309863100001
View details for PubMedID 22825593
View details for PubMedCentralID PMC3700359
Accumulation of resident and peripheral dendritic cells in the aging CNS
NEUROBIOLOGY OF AGING
2012; 33 (4): 681-+
Dendritic cells (DC) are specialized antigen-presenting cells, responsible for peripheral immune responses. Recently, resident brain dendritic cells (bDC) were identified and functionally characterized in the young adult Itgax (CD11c) EYFP+ transgenic mouse brain. In the present study, we describe changes in number, phenotype, and source of bDC in the aging mouse brain. Immunohistochemistry and fluorescent activated cell sorting (FACS) analysis revealed an age-related increase in bDC with a concomitant rise in the expression of immune activation markers MHCII, CD80, and CD86. Quantification of immunolabeled bDC in the cortex, corpus callosum, and cerebellum of the aged brain revealed a 2- to 5-fold increase. In contrast, either no change or a decrease in bDC was noted in regions of adult neurogenesis. Chimeras (wild type host/EYFP+ bone marrow) suggest that the increase of EYFP+ cells in the aging brain is in part due to an accumulation of peripherally derived cells. Collectively, the numerical and phenotypic changes in bDC indicate these cells may serve as an important immune component in the functional and anatomic alterations associated with aging.
View details for DOI 10.1016/j.neurobiolaging.2010.06.007
View details for Web of Science ID 000301506800006
View details for PubMedID 20692074
Microglia Express Functional 11 beta-Hydroxysteroid Dehydrogenase Type 1
2010; 58 (10): 1257-1266
Glucocorticoids are potent regulators of inflammation exerting permissive, stimulatory, and suppressive effects. Glucocorticoid access to intracellular receptors is regulated by the activity of two distinct enzymes known as 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) Type 1 and Type 2, which catalyze the activation or deactivation of glucocorticoids. Although expression of these enzymes in major organ systems and their roles in the metabolic effects of glucocorticoids have been described, their role in the inflammatory response has only recently started to be addressed. In this report, we have studied the expression and activity of 11 beta HSD Type 1 and Type 2 in microglia cells. Microglia, the brain's resident macrophages, initiate and orchestrate CNS inflammatory responses. Importantly, activated microglia are implicated in most neurodegenerative conditions, making them key subjects of study. We found that microglia expressed 11 beta HSD-1, but not 11 beta HSD-2, both in ex vivo FACS-sorted adult cells and in vitro primary cultures. 11 beta HSD-1 expression was increased in LPS-activated microglia. Moreover, 11 beta HSD-1 catalyzed the metabolic conversion of 11-dehydro-corticosterone into corticosterone (CORT), which potently reduced cytokine production in activated microglia. We propose that 11 beta HSD-1 may provide microglia with an intrinsic mechanism to autoregulate and inhibit proinflammatory mediator production through CORT formation.
View details for DOI 10.1002/glia.21007
View details for Web of Science ID 000279389100012
View details for PubMedID 20544861
Brain dendritic cells in ischemic stroke: Time course, activation state, and origin
BRAIN BEHAVIOR AND IMMUNITY
2010; 24 (5): 724-737
The immune response to stroke is comprised of inflammatory and regulatory processes. One cell type involved in both innate and adaptive immunity is the dendritic cell (DC). A DC population residing in the healthy brain (bDC) was identified using a transgenic mouse expressing enhanced yellow fluorescent protein (EYFP) under the promoter for the DC marker, CD11c (CD11c/EYFP Tg). To determine if bDC are involved in the immune response to cerebral ischemia, transient (40 min) middle cerebral artery occlusion (MCAO) followed by 6, 24, or 72 h reperfusion was conducted in CD11c/EYFP Tg mice. Our results demonstrated that DC accumulated in the ischemic hemisphere at 24 h post-MCAO-reperfusion, particularly in the border region of the infarct where T lymphocytes accrued. To distinguish resident bDC from the infiltrating peripheral DC, radiation chimeras [1. wild type (WT) hosts restored with CD11c/EYFP Tg bone marrow (BM) or 2. CD11c/EYFP Tg hosts restored with WT BM] were generated and examined by immunocytochemistry. These data confirmed that DC populating the core of the infarct at 72 h were of peripheral origin, whereas those in the border region were comprised primarily of resident bDC. The brain resident (CD45 intermediate) cells of CD11c/EYFP Tg mice were analyzed by flow cytometry. Compared to microglia, bDC displayed increased major histocompatibility class II (MHC II) and co-stimulatory molecules following MCAO-reperfusion. High levels of MHC II and the co-stimulatory molecule CD80 on bDC at 72 h corresponded to peak lymphocyte infiltration, and suggested a functional interaction between these two immune cell populations.
View details for DOI 10.1016/j.bbi.2009.11.002
View details for Web of Science ID 000279125900005
View details for PubMedID 19914372
View details for PubMedCentralID PMC2885548
Acute in vivo exposure to interferon-gamma enables resident brain dendritic cells to become effective antigen presenting cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (49): 20918-20923
Dendritic cells (DC) are the professional antigen presenting cells (APC) that bridge the innate and adaptive immune system. Previously, in a CD11c/EYFP transgenic mouse developed to study DC functions, we anatomically mapped and phenotypically characterized a discrete population of EYFP(+) cells within the microglia that we termed brain dendritic cells (bDC). In this study, we advanced our knowledge of the function of these cells in the CD11c/EYFP transgenic mouse and its chimeras, using acute stimuli of stereotaxically inoculated IFNgamma or IL-4 into the CNS. The administration of IFNgamma increased the number of EYFP(+)bDC but did not recruit peripheral DC into the CNS. IFNgamma, but not IL-4, upregulated the expression levels of major histocompatibility class II (MHC-II). In addition, IFNgamma-activated EYFP(+)bDC induced antigen-specific naïve CD4 T cells to proliferate and secrete Th1/Th17 cytokines. Activated bDC were also able to stimulate naïve CD8 T cells. Collectively, these data reveal the Th1 cytokine IFNgamma, but not the Th2 cytokine IL4, induces bDC to up-regulate MHC-II and become competent APC.
View details for DOI 10.1073/pnas.0911509106
View details for Web of Science ID 000272553000066
View details for PubMedID 19906988
View details for PubMedCentralID PMC2791588
CD11c/EYFP transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain
JOURNAL OF COMPARATIVE NEUROLOGY
2008; 508 (5): 687-710
The CD11c enhanced yellow fluorescent protein (EYFP) transgenic mouse was constructed to identify dendritic cells in the periphery (Lindquist et al.  Nat. Immunol. 5:1243-1250). In this study, we used this mouse to characterize dendritic cells within the CNS. Our anatomic results showed discrete populations of EYFP(+) brain dendritic cells (EYFP(+) bDC) that colocalized with a small fraction of microglia immunoreactive for Mac-1, Iba-1, CD45, and F4/80 but not for NeuN, Dcx, NG2 proteoglycan, or GFAP. EYFP(+) bDC, isolated by fluorescent activated cell sorting (FACS), expressed mRNA for the Itgax (CD11c) gene, whereas FACS anlaysis of EYFP(+) bDC cultures revealed the presence of CD11c protein. Light microscopy studies revealed that EYFP(+) bDC were present in the embryonic CNS when the blood-brain barrier is formed and postnatally when brain cells are amenable to culturing. In adult male mice, EYFP(+) bDC distribution was prominent within regions of the CNS that 1) are subject to structural plasticity and neurogenesis, 2) receive sensory and humoral input from the external environment, and 3) lack a blood-brain barrier. Ultrastructural analysis of EYFP(+) bDC in adult neurogenic niches showed their proximity to developing neurons and a morphology characteristic of immune/microglia cells. Kainic acid-induced seizures revealed that EYFP(+) bDC responded to damage of the hippocampus and displayed morphologies similar to those described for seizure-activated EGFP(+) microglia in the hippocampus of cfms (CSF-1R) EGFP mice. Collectively, these findings suggest a new member of the dendritic cell family residing among the heterogeneous microglia population.
View details for DOI 10.1002/cne.21668
View details for Web of Science ID 000255722000002
View details for PubMedID 18386786
Steroid hormone receptor expression and function in microglia
2008; 56 (6): 659-674
Steroid hormones such as glucocorticoids and estrogens are well-known regulators of peripheral immune responses and also show anti-inflammatory properties in the brain. However, the expression of steroid hormone receptors in microglia, the pivotal immune cell that coordinates the brain inflammatory response, is still controversial. Here we use real time RT-PCR to show that microglia, isolated from adult fms-EGFP mice by FACS, express glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and estrogen receptor alpha (ERalpha). GR was the most abundant steroid hormone receptor transcript in microglia. The presence of GR and ERalpha immunoreactivity was further confirmed in vivo at the ultrastructural level. To understand the role of steroid hormone receptors during the inflammation process, we evaluated the expression of steroid hormone receptors after inflammatory challenge and found a significant down-regulation of GR, MR, and ERalpha in microglia. Finally, we tested the immunomodulatory properties of estrogens and glucocorticoids. Estradiol benzoate did not have any significant impact on the inflammatory profile of ex vivo sorted microglia, either in resting conditions or after challenge. Furthermore, corticosterone was a more consistent anti-inflammatory agent than 17beta-estradiol in vitro. Our results support the hypothesis that adult microglia are a direct target of steroid hormones and that glucocorticoids, through the predominant expression of GR and MR, are the primary steroid hormone regulators of microglial inflammatory activity. The down-regulation of steroid hormone receptors after LPS challenge may serve as a prerequisite to suppressing the anti-inflammatory actions of endogenous steroid hormones on the immune system, and contribute to a sustained activation of microglia.
View details for DOI 10.1002/glia.20644
View details for Web of Science ID 000254816000007
View details for PubMedID 18286612
Brain microglia express steroid-converting enzymes in the mouse
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
2008; 109 (1-2): 96-107
In the CNS, steroid hormones play a major role in the maintenance of brain homeostasis and it's response to injury. Since activated microglia are the pivotal immune cell involved in neurodegeneration, we investigated the possibility that microglia provide a discrete source for the metabolism of active steroid hormones. Using RT-PCR, our results showed that mouse microglia expressed mRNA for 17beta-hydroxysteroid dehydrogenase type 1 and steroid 5alpha-reductase type 1, which are involved in the metabolism of androgens and estrogens. Microglia also expressed the peripheral benzodiazepine receptor and steroid acute regulatory protein; however, the enzymes required for de novo formation of progesterone and DHEA from cholesterol were not expressed. To test the function of these enzymes, primary microglia cultures were incubated with steroid precursors, DHEA and AD. Microglia preferentially produced delta-5 androgens (Adiol) from DHEA and 5alpha-reduced androgens from AD. Adiol behaved as an effective estrogen receptor agonist in neuronal cells. Activation of microglia with pro-inflammatory factors, LPS and INFgamma did not affect the enzymatic properties of these proteins. However, PBR ligands reduced TNFalpha production signifying an immunomodulatory role for PBR. Collectively, our results suggest that microglia utilize steroid-converting enzymes and related proteins to influence inflammation and neurodegeneration within microenvironments of the brain.
View details for DOI 10.1016/j.jsbmb.2007.12.013
View details for Web of Science ID 000255301200012
View details for PubMedID 18329265
View details for PubMedCentralID PMC2423427
Characterization of a cerebellar granule progenitor cell line, (EC)-C-t.1, and its responsiveness to 17-beta-estradiol
2007; 1186: 29-40
Mouse cerebellar development occurs at late embryonic stages and through the first few weeks of postnatal life. Hormones such as 17-beta-estradiol (E2) have been implicated in cerebellar development, through the expression of E2 receptors (ER). However, the role of E2 in the development and function of cerebellar neurons has yet to be fully elucidated. To gain insight into E2's actions on the developing cerebellum, we characterized a cloned neuronal cell line, E(t)C.1, derived from late embryonic cerebellum for its neural properties and responsiveness to E2. Our results revealed that E(t)C.1 cells express markers characteristic of neural progenitor cells such as Nestin, Musashi, and Doublecortin (DCX), and of the granule cell lineage such as Math1 and Zipro1. The ER alpha and beta (ERalpha and ERbeta) were also identified in this cell line. Functionality of ERs was verified using an Estrogen Response Element (ERE)-Luciferase reporter plasmid. E2 modulated ERalpha, FMRP, and IL-6, which were expressed in these cells. However, E2 did not induce changes in neural proteins nor induce maturation of E(t)C.1 cells. CREB and ERK(1/2) protein kinases were not modulated by E2 either. Interestingly, E(t)C.1 expressed active p450 Aromatase (P450arom), which was confirmed by the aromatization of androstenedione (AD) to E2 and other estrogen metabolites. Collectively, our results show that the E(t)C.1 cell line may serve as a model to study early development of cerebellar progenitor granule cells, and their responsiveness to E2.
View details for DOI 10.1016/j.brainres.2007.08.071
View details for Web of Science ID 000252201500004
View details for PubMedID 17980864
Transcriptional activity of estrogen receptors ER alpha and ER beta in the (EC)-C-t.1 cerebellar granule cell line
2007; 1186: 41-47
Estrogen receptors alpha and beta (ERalpha and ERbeta) are expressed in the cerebellum throughout development and in the adult suggesting an important role of 17-beta-estradiol (E2) in this brain structure. In the present study, we have characterized the functionality of estrogen receptors (ERs) expressed in the immature cerebellar granule cell line E(t)C.1 by transfecting such cells with a luciferase reporter gene (ERE-Luc) coupled to an estrogen response element promoter. The induction of luciferase activity in E(t)C.1 cells by E2 and ER-subtype selective agonists was compared in normal cells and in cells overexpressing human ERalpha or ERbeta (hERalpha or hERbeta). E2-mediated transcription of the reporter gene was blocked by the ER antagonist ICI 182,780 (ICI), demonstrating the presence of functional native ERs. The selective agonist for ERalpha (PPT) showed a reduced response in luciferase induction compared to E2. Moreover, the ERbeta agonist (DPN) was unable to induce luciferase activity. E2-induced ERE-Luc transcription was not increased by overexpression of hERalpha. In contrast, hERbeta overexpression reduced the efficacy of E2 and abolished ERalpha-selective agonist activity. The ERbeta-specific agonist did not induce gene reporter activity unless hERbeta was overexpressed in the cells, suggesting that the endogenous ERbeta in E(t)C.1 cells is transcriptionally inactive. ICI inhibition of E2 responses was not affected by overexpression of the human ERs. The data suggest that ERalpha plays a predominant role in E2-mediated transcription in E(t)C.1 cells. Our data are discussed in view of other reports alluding to the complexity and cell-type specificity of E2-mediated transcription.
View details for DOI 10.1016/j.brainres.2007.10.033
View details for Web of Science ID 000252201500005
View details for PubMedID 18021758
Selective conversion by microglia of dehydroepiandrosterone to 5-androstenediol - A steroid with inherent estrogenic properties
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
2007; 107 (3-5): 156-162
The well-established neuroprotective effect of dehydroepiandrosterone (DHEA) has been attributed to its metabolism in the brain to provide estrogens known to be neuroprotective and to enhance memory and learning in humans and animals. However, our previous work showed that the conversion of DHEA to 4-androstenedione (AD), the precursor of estrone (E(1)) and estradiol (E(2)), is very low in several different types of neural cells, and that the main product is 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). In this study, we found that microglia are an exception and produce mainly 5-androstene-3beta,17beta-diol (Delta(5)-Adiol), a C(19) steroid with estrogen-like activity from DHEA. Virtually, no other products, including testosterone (T) were detected by TLC or HPLC in incubations of (3)H-labeled DHEA with the BV2 microglial cell line. Microglia are important brain cells that are thought to play a house-keeping role during the steady state, and that are crucial to the brain's immune reaction to injury and the healing process. Our findings suggest that the microglia-produced Delta(5)-Adiol might have a role in modulating estrogen-sensitive neuroplastic events in the brain, in the absence of adequate local synthesis of estrone and estradiol.
View details for DOI 10.1016/j.jsbmb.2007.04.004
View details for Web of Science ID 000250183600004
View details for PubMedID 17681749
Microglia derived from aging mice exhibit an altered inflammatory profile
2007; 55 (4): 412-424
Microglia play a critical role in neurodegenerative diseases and in the brain aging process. Yet, little is known about the functional dynamics of microglia during aging. Thus, using young and aging transgenic mice expressing enhanced-green fluorescent protein (EGFP) under the promoter of the c-fms gene for macrophage-colony stimulating factor receptor, we evaluated in vivo-induced inflammatory responses of EGFP-expressing microglia sorted by flow cytometry. Aging microglia were characterized by the presence of lipofuscin granules, decreased processes complexity, altered granularity, and increased mRNA expression of both pro-inflammatory (TNFalpha, IL-1beta, IL-6) and anti-inflammatory (IL-10, TGFbeta1) cytokines. Following lipopolysaccharide (LPS) challenge (1 mg/kg, 3 h), aging microglia exhibit increased basal expression of TNFalpha, IL-1beta, IL-6, and IL-10. Yet, the fold-over-basal LPS response remained constant across age, implying that the inflammatory machinery in aging microglia is functional and adjusted to the basal state. Gender differences were not overall observed across the treatments (age, LPS). The low but sustained production of pro-inflammatory cytokines by aging microglia may have a profound impact in the brain aging process.
View details for DOI 10.1002/glia.20468
View details for Web of Science ID 000243570300007
View details for PubMedID 17203473
Dehydroepiandrosterone (DHEA) metabolism in the brain: Identification by liquid chromatography/mass spectrometry of the delta-4-isomer of DHEA and related steroids formed from androstenedione by mouse BV2 microglia
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
2006; 98 (1): 41-47
Studies to elucidate the role of dehydroepiandrosterone (DHEA) metabolism in neuroprotection have compared its relative 7-hydroxylation against estrogen formation by way of 4-androstenedione (AD) in various rodent brain cell lines. In all cases, the 7alpha- and 7beta-hydroxy epimers of DHEA were found to be the dominant products with one notable exception. BV2 mouse microglia were virtually unable to hydroxylate DHEA at C-7 and converted AD to a major unknown metabolite not observed with mouse BHc hippocampal cells. In this paper, we describe the identification of this compound based on its physical properties and analysis by TLC and HPLC. Its identity as 3beta-hydroxy-4-androstene-17-one, the Delta(4)-isomer of DHEA, was confirmed by mass spectrometry (LC/MS), as well as by reverse isotope dilution analysis involving co-crystallization with the synthetic steroid. Possible mechanisms for the formation of this isomer of DHEA by BV2 microglia are proposed, together with that of other C-19 steroids detected which include testosterone (T), 5alpha-dihydrotestosterone and 5alpha-androstanedione.
View details for DOI 10.1016/j.jsbmb.2005.07.006
View details for Web of Science ID 000235292200006
View details for PubMedID 16203131
Metabolism of dehydroepiandrosterone by rodent brain cell lines: Relationship between 7-hydroxylation and aromatization
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
2005; 93 (1): 81-86
The rate of aromatization of 4-androstenedione (AD) and 7-hydroxylation of dehydroepiandrosterone (DHEA) by different neuronal cell lines from fetal rat and mouse brain was compared to that of embryonic rat hippocampal cells in primary culture. The (3)H-labeled steroids were incubated with the cells and the metabolites extracted and separated by thin layer chromatography (TLC), as well as analyzed by high-performance liquid chromatography (HPLC) for further identification. All cell types produced estrone (E(1)) and estradiol (E(2)) from [(3)H]AD but the rate of aromatization was lowest with the rat hippocampal cells in primary culture. With [(3)H]DHEA, BHc.2 mouse hippocampal cells and E(t)C.1 neurons behaved like the mixed cells from rat hippocampus, forming 7-hydroxy DHEA as the almost exclusive product. In contrast, mouse brain BV2 microglia were virtually unable to hydroxylate DHEA at C-7 and yielded estrogen and more testosterone (T) than other cell types tested. These experiments highlight the pivotal role of 3beta-hydroxysteroid dehydrogenase/ketoisomerase in the control of AD formation for its subsequent aromatization to estrogen. It raises the possibility that differences in metabolism of DHEA by certain brain cells could account for differences in their immunomodulatory and neuroprotective functions. Some could exert their effects by converting DHEA to its 7-hydroxylated form while others, like BV2 microglia, by converting DHEA primarily to other C-19 steroids and to estrogen by way of AD.
View details for DOI 10.1016/j.jsbmb.2004.11.008
View details for Web of Science ID 000227933000010
View details for PubMedID 15748836
Protein kinase C-mediated phosphorylation and desensitization of human alpha(1b)-adrenoceptors
EUROPEAN JOURNAL OF PHARMACOLOGY
1999; 385 (2-3): 263-271
Human alpha(1b)-adrenoceptors stably expressed (B(max) approximately 800 fmol/mg membrane protein) in mouse fibroblasts were able to increase intracellular Ca(2+) and inositol phosphate production in response to noradrenaline. Activation of protein kinase C desensitized the alpha(1b)-adrenergic-mediated actions but did not block the ability of the cells to respond to lysophosphatidic acid. Inhibition or downregulation of protein kinase C also blocked the action of the tumor promoter on the adrenergic effects. Photolabeling experiments indicated that the receptor has an apparent molecular weight of approximately 80 kDa. The receptors were phosphorylated in the basal state and such phosphorylation was increased when the cells were incubated with phorbol myristate acetate or noradrenaline. Incubation of the cells with phorbol myristate acetate or noradrenaline blocked noradrenaline-promoted [35S]GTP-gamma-S binding to membranes, suggesting receptor-G protein uncoupling. The results indicate that activation of protein kinase C blocked/desensitized human alpha(1b)-adrenoceptors and that such effect was associated to receptor phosphorylation.
View details for DOI 10.1016/S0014-2999(99)00629-9
View details for Web of Science ID 000084293000023
View details for PubMedID 10607885